SUBTYPE EXPRESSION AND MYOMETRIUM IN

CORTICOTROPIN-RELEASING HORMONE RECEPTOR SUBTYPE 1 AND SUBTYPE 2 mRNA EXPRESSION AND PROTEIN LOCALIZATION IN THE

MYOMETRIUM IN PREGNANCY

Mata Yvette Stevens

A thesis submitted in c o n f o d t y with the requirements for the degree of Masters of Science Graduate Department of Physiology

University of Toronto

8 Copflght by Miata Yvette Stevens 1998

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ou autrement reproduits sans son autorisation,

Corticotropin-Releasing Hormone Reeeptor Sobtype 1 And Subtype 2 mRNA

Expression And Protein Locaiization in The Myometrium In Pregnnncy

Masters of Science 1998

Miata Yvette Stevens

Department o f Physiology, University of Toronto

ABSTRACT

The human placenta secretes increasing concentrations of corticotropin-

releasing hormone (CRH) in late pregnancy and in labour. CRH has been implicated in

the regulation of myometnal contractility. We hypothesized that CRH receptoa, CRH-

R1 and CRH-R2, mRNA in the rnyometrium would be upregulated in labour.

Myometrial samples were collected from nonpregnant, pregnant and laboring patients

nom the upper and lower uterine segment. CM-RI mRNA and protein were

downregulated with pregnancy and significantly upregulated at labour. This rise

appeared to be exclusive to the lower segment. Cm-W mRNA did not change.

Rats have been used extensively to shidy the regulation of CRH receptoa. We

examined CRH receptor mRNA in rat myomeûium. CRH-RI rnRNA was

undetectable. CRH-R2 mRNA was significantly increased at labour concomitant with a

nse in cornexin 43 mRNA, a gap junction protein associated with labour. In

conclusion, at the time of labour CRH-R1 mRNA is upregulated in the human but in

the rat CRH-EU mRNA is upregulated.

Dedicated to my mother, my inspiration.

Let earth and sky be your yardstick and eterniîy your nceasuremenL There is no height that you cannot accomplisi by &g the active intelligence of your mind

The Hon. Marcus Mosaih Garvey

iii

1 am forever indebted to my s u p e ~ s o r Dr. John Challis for giving me the

opportunity to experience research. Research, with al1 its highs and lows. I thank John for

his guidance, his support and his understanding throughout the time I have known him.

1 would like to thank my Advisory Cornmittee members Dr. Steve Lye, Dr. Steve

Matthews and Dr Neil MacLusky for their helpful cooperation throughout my degree and

for their invaluable assistance in editing and revising this thesis.

I thank al1 my lab members for the fond mernories of early morning meetings and

late night research always interlaced with good partying techniques. 1 would particularly

like to thank Dr. Mhoyra Fraser, Dr. Alison Holloway, Dr. Wendy Whittle, Dr. Vicky

Clifion, Debbie Sioboda and Fa1 Patel for their constant source of technical and

emotional support. A very special thanks to the "Lye Lab" especially to Dr. Isabella

Caniggia, Dr. Daniel MacPhee, Dr. Ryan Ou and Lindsay McWhirter for their readiness

to teach me and help me with invaluable techniques.

Special thanks to Boris, Rebecca and Alex. Your love, your support and the

wondemil strong bond we share makes me remember, every day, how blessed I am to

have siblings. Extreme gratitude to rny "twinie", Yvonne, completing a Masters of

Science concornitantly with you has made us share laughter, tears and a world of

understanding. My heartfelt thanks to my Mom who taught me everything 1 need to know

about being a powerful , intelligent and motivated woman. Infinite love and thanks to

Toke Petersen- my love, my heart, my best fiiend. You make life, love and success worth

living for.

TABLE OF CONTENTS

TITLE PAGE ..............................-.....o..a....................a........*..o...-.*..........a........................ i

1.1 Overview ................................................................................................ 1

1.2 The Placenta And Fetai Membranes ....................... ... ................................ 3

1 -3 The Uterus ..... ......... .... ......... ...... ., . . . ................ . ............ . . . . . . 5

1 -4 Stimulation Of Myometnal Activity .......... .. .................. .. .--..a.-... ..... ... ............ .... .. -6

1.5 Progesterone And Estrogen In Labour ............................... ... ..--..............-.... ......... - 8

1.6 Corticotropin Releasing Hormone ..... . .. ... .. .. . .. ... .... ...... .. . . . . . . . . . . . . . . . . . . 1 O

1.6.1 CRH-Related Peptides ... .... .... ................ .. ........... . . . . . . . . . ....... 12

1 -7 Human Placental C M ...... .. .. . ... . ... .. . .. . ..... . . .. ..... ....... . . . ....--.-.. .. . .. .. . .. ... .-. .. . . . . 12

1.8 CM-Binding Protein ... . . .. . ... . . .. .. .. . ... ... .. ... ... ......... . . . . . . . . . . . . . . . . . . . . . . 1 6

1.9 ûther Regdators Of CRH.. . .... .. .. .. . . .. .. ......... .. .. ... .... ..... . . . .. .. .... . ..... ...... ... .. ... . . ... 1 7

1 .10 Myometrial Contractility .......... ....... . ...... .. ....... .. ............................ . . 17

1.1 1 Other Roles For CRH ................................................................................ 18

1.12 CRH Receptors .................................. ................................................... 19

CHAPTER TWO: RATIONALE A , ! EYPOTHESIS .......................................... 44

CHAPTER THREE: CRE-Rl mRNA IS SIGNIFIC-Y UPREGULATED IN TEE HUMAN MYUMETRI[UM AT THE OF LABO UR .................... .47

3.1. Introduction ................................................................................................. .. 47

. ............................. 3 -2 Matends And Methods ... 4 8 3 .2.1 Biopsy Samples ............................................................................................. 48

....................... ...................................*............. 3 2.2 Total RNA E.xtraction .,,. 5 0 3.2.3 Reverse Transcription Polymerase Chain Reaction (RT-PCR) .................... 51 . . 3.2.4 Semi-Quantitative PCR ............................. .... ............................. 5 3 3.2.5 Immunohistochemistry ................................................................................ 5 4 3.2.6 Protein Extraction ............................... ., ................................................ 56 3.2.7 Western Blots ............................................................................................... 57 3.2.8 Data Anaiysis ............................. ... ..................................................... 5 8

...................................................................................... ................... 3.3 Results ... 5 9 3.3.1 CRH Receptor Expression in the Myometrium of Nonpregnant Patients .... 59

3.3.2 CRH-RI mRNA and CRH-R2 mRNA Expression in Myornetrium Pregnant

................................................................................................................... Patients 60

3.3 -3 Regional Expression of CRH-R 1 in Myometriurn From Nonpregnanf

Pregnant, Pregnant in Labour and Post-Pamim Patients ....................................... 61

3.3.4 CRH-RI mRNA Expression in the Fetai Membranes and Decidua ............. 62

.................................. 3.4 Discussion .-. ............................................................... 62

CHAI'TF,R 1: THE RAT- A SUITABLE MODEL TO STUDY THE REGULATION OF CRH RECEPTOR EXPRESSION IN THE

....................................................................................................... WOME- ? 95

4.1 Introduction .......................................................................................................... 95

4.2 Materials And Methods ..................................................................................... 9 6 4.2.1 Animais ................... ... ................................................................................. 9 6 4.2.2 Total RNA Extraction ............................................................................... ,... 97 4.2.3 Reverse-transcription-PCR ........................................................................... 98 4.2.4 Semiquantitative-PCR .............................................................................. 100 4.2.5 Data andysis ......................................................................... ................ 100

4.3 Results .............. ,....... ........................................................................................ 101 4.3.1 CRH-RI mRNA And CRH-R2 mRNA Expression in Rat Myometrium On

Day 15. Day 20. Day 2 1. Day 22. At Labour And 1 Day Postpammi ................. 101

4.3.2 Cx 43 Expression In Rat Myomeûium On Day 15. Day 20. Day 2 1. Day 22.

At Labour And 1 Day Postpamim ...................................................................... 102

.......................................................................................................... 4.4 Discussion 102

C W T E R 5: F7PiA.L DISCUSSION... .................................................................... 114

vii

LIST OF TABLES

Table 1.1 DiEerent Species Homologs of CRH-RI ......................................................... 42

......................... Table 1.2 DifZerent Species Hornologs of CM-EU ...................... .. 43

Table 3.1 CRH-R2 mRNA Expression Prior to and at the Onset of Labour in Term and

Preterm Patients -94 .............................................................................................................

viii

LIST OF FIGURES

Figure 1.1 . Schematic Diagram of the Structures W ithin the Uterus ..................................... 33

Figure 1.2. Schematic Diagram Demonstrating the Change in the S ù e and Shape of the

Uterus in Term Pregnancy and in Labour. ...................................................................... 35

Figure 1.3. Schematic Diagrarn Depicting the Upper Active Segment and the Lower Passive

..................................................................................................................... Segment. 37

Figure 1.4. Schematic Diagram Comparing the Amino Protein Sequence of CRH and CRH-

...................................................................................................... Related Peptides. ...... 39

Figure 1 S. Schematic Diagrarn Depicting the Seven Trammembrane Structure and the .

Arnino Acid Sequence Of the CRH Receptor Subtypes ............................ .. ........... 4 1

Figure 3.1. Photograph of a PCR Gel Showing C M - R I cDNA Enzyme Digestion ..........A9

Figure 3.2. Photograph of a PCR Gel Showing the Detection of CRH-RI mRNA and CRH-

R2 mRNA in Myometnum frorn Nonpregnant Women ............................................... 7 1

Figure 3.3. Photograph Showing Irnmunostaining for CRH-RI Protein in Human

Myometrium Smooth Muscle ......................................................................................... -73

Figure 3.4. Photograph Showing Immunostaining for CM-W Protein in Human

................................................................................................. Myometrium Smooth Musc le 75

Figure 3.5. Photograph Showing Immunostaining for CRH-RI Protein in the Smooth

Muscle Layer of the Uterine Vascuiature .............................................................................. -.77

Figure 3.6. Photograph Showing Immunostaining for CRH-R2 Protein in the Smooth

Muscle Layer of the Uterine Vasculature .......................... ... ............................................. -.79

Figure 3 -7. Photograph of CRH-R 1 and CRH-R2 Protein Immunostaining in the

Sheep Pituitary .................... .. ................................................................................................ 8 1

Figure 3.8. Photograph of PCR Gel Showing the Detection of CRH-RI mRNA

in Human Myometrium in Term and Preterm Patients ........................................................... 83

Figure 3.9. Analysis Of Sq-PCR for CRH-RI mRNA In Human Myornetrium in Tem

Patients .......................................................................................................................... 85

Figure 3.10. Analysis Of Sq -PCR for CRH-RI mRNA In Human Myometriurn in

................................................................................................................... Pretenn Patients .-..87

Figure 3.1 1. CRH-RI mRNA Expression in the Human Myometrium from

32 Weeks to 39 Weeks of Gestation and at the Time of Labour

in Preterm and Term Pregnancies ....................................................................................... 89

Figure 3.12. Photograph of a PCR gel and analysis of Sq-PCR showing

the regional detection of CRH-RI mRNA in human myometrium in

nonpregnant, pregnant, labourhg and postpartum patients .................................... .... ........... 9 1

Figure 3.13. Analysis Of Sq-PCR for CRH-R1 mRNA Expression in the Chorion and the

Decidua in Term Pregnancy and at the Time of Labour ............................................... 93

........... Figure 4.1. Photograph of a PCR Gel Showing CRH-EU cDNA Enzyme Digestion 107

Figure 4.2. Photograph Of A Sq-PCR Gel Showing the Detection of CM-R2 mRNA and

........... Cx 43 mRNA in Rat Myometrium in Pregnancy, at Delivery and Post-Partum 109

Figure 4.3. Analysis of Sq-PCR for CRH-R2 mRNA Expression in Rat Myometrium

in Pregnancy, at Delivery and Post-Partum ....................................... .. ............................... 1 1 1

Figure 4.4. Analysis of Sq-PCR for Cx 43 mRNA Expression in Rat Myometrium

in Pregnancy, at Delivery and Post-Partum ...................................... .. ............................... 1 13

ACTH ANOVA

bp BSA

CAMP cDNA Cx CRH CRH-BP C M - R I CRH-R2

DHEAS

MGA mRNA MW

PCR PGs PGE2 PGF2a PGHS

adrenocortico trophin hormone analysis of variance

base pairs bovine serum albumin

cyclic adenosine 3',5-mono phosphate complementary deoxyribonucleic acid connexin corticornopin releasing hormone cortico tropin-releasing hormone binding pro tein corticotropin releasing hormone receptor subtype- 1 corticotropin releasing hormone receptor subtype-2

dehydroepiandrosterone sulphate

estradiol

human placentai CRH human rat CRH

radioiabeled iodine immunoreactive

kilobase dissociation constant kilodaltons

rnean gestational age messenger ribonucleic acid molecuiar weight

oxytocin ovine CRH

polymerase chah reaction prostaglandins prostaglandin E2 prostaglandin F2-alpha prostaglandin H synthase

PKA PKC PLC POMC PTL PVN

RT RT-PCR

SDS sq-PCR

protein kinase A protein kinase C phosphotipase C pro-opiomelanocorlln pre-term labour paraventricular nucleus

reverse transcriptase reverse transcription polymerase chain reaction

sodium dodecyl sulfate semi-quantitative polymerase chah reaction

xii

CELWlXR Ir INTRODUCTION

Human parturition aises as a result of complex interactions within the matemal-

placental-fetal unit Spontaneous term human labour is defined as Zhe onset of reguiar

uterine contractions of increasing intensity and fkquency associated with progressive

effacement and dilatation of the cerWt following the completion of 38 weeks of

gestation" (Cunningham et al., 1993) and is the initial stage in a series of events leading

to birth. An understanding of the mechanisms regulating human parturition is vital for the

prevention and treatrnent of labour complications including dy stocia pre-term labour

(PTL) and post-term pregnancy each of which is associated with increased morbidity,

rnortaiity, health care costs and emotional distress. PTL is the most fiequent complication

of pregnancy and occurs in about 7% of a l i pregnancies; however, despite the advances in

perinatal medicine over the past 20 years littie change has been made in the incidence of

PTL, and delivery (Cunningham et ai., 1993). Given the potential threats of labour

complications on the health and survival of both the mother and the fenis, there exists a

crucial need for scientists to study the mechanisms involved in the onset, the progress and

the termination of human labour.

There are currently several hypotheses regarding the mechanisms involved in the

omet of labour. These include the progesterone withdrawal theory and the myometrium

activation-stimulation theory. The progesterone withdrawal theory

proposes that progesterone maintains uterine quiescence throughout pregnancy and

that a decrease in maternai plasma concentrations of progesterone close to term triggers

uterine contractility. W e this theory applies to the sheep mode1 of parturition

(Thorburn and Chailis, 1979), midies in humans have fded to observe a decrease in

circdating concentrations of progesterone prior to the onset of labour (TulchinsIq et

al., 1972). The myometrium activation-stimulation theory forwarded by Chaliïs and

Lye (1994) proposes that with the approach of labour the myomeaium is activated

(increased muscle excitability), by the synthesis of "contraction -associated proteins",

that renders the myometrium more responsive to stimulation by uterotonic agents.

Severai factors, produced by the placenta and the fetal membranes, such as

corticotroph-releasing hormone (CRW) (Riley et al., 1991), have the ability to

stimulate the myometnum (Quartero et al., 1989) The human placenta synthesizes and

secretes increasing concentrations of placental CRH (hCRH- pl) into the materna1

circulation in the third triinester of pregnancy and throughout labour (Goland et al.,

1986; Okamoto et al., 1989). The physiologicai significance of hCRH-pl in human

pregnancy still rernains to be established CRH exerts its actions via specific G-protein

coupled receptors (Chen et al., 1993; Lovenberg et al., 1995a) of which two subtypes

have been identifie4 CRH-RI and CRH-R2 (Chen et al., 1993; Lovenberg et al.,

1995a). CRH-RI messenger ribonucleic acid (mRNA) and CRH-R2 mRNA are

expressed in the myometnum of both nonpregnant and pregaant women (Rodriguez-

Limes et al., 1998). CRK receptors have been linked to secondary pathways

associated with both relaxation and contraction in pregnanc y (Grammatopoulos ., 1 994)

suggesting that hCRH-pl may potentially have differential effects on the human

myornetrium.

As of yet no studies have examined the mRNA expression and protein levels of

CRH-RI and CRH-R2 at the time of labour. Given the increase in circuiating levels of

hCRH-pl in the m a t e d circulation at that tirne and the presence of CRH recepton in

human myometnum it has been suggested hCW-pl may play a role in prepancy

and/or labour. One of the overall objectives of our lab is to understand the role of

hCRH-pl in the initiation a d o r progression of labour. The specinc aims of the present

study were a) to determine the expression of CRH-RI mRNA and CRH-R2 mRNA in

the myometrium both in pregnancy and at the time of labour and b) to establish a

mammalian mode1 to study the regulation of CRH receptor expression in the

myometrium.

1.2 THE PLACENTA AND FETAL MEMBRANES

The human placenta is classified as a hemochorioendothelial placenta The

materna1 blood does not directly contact the fetal blood and the exchange of gases,

nutrients and wastes occurs via a chorioendotheliai membrane. Shortiy following

fertilization, the ceUs of the early blastocyst differentiate into the inner cell mass and

the trophoblasts. The blastocy st descends into the uterus and the tropho blasts

differentiate into the outer syncytiotrophoblast layer and the inner proliferative

cytotrophoblast. The syncytiotrophoblast layer results from the fusion of

cytotrophoblast cells. The cytotrophoblasts and the synctial layer form the primary

placental villi. The placentai villi are the fûnctional mits of the human placenta and are

classined as prïmary, secondary or tertiary. Prirnary villi become secondary in structure

following the invasion of mesenchymai celis flom the extraembryonic rnesenchyma

Tertiary villi result fiom angiogenesis within the mesenchyme of the secondary vilh

and consist of a centrai core of fetal blood vessels surrounded by the cytotrophoblasts

and sync ytio tropho blast lay er. With advancing gestation, the ratio of cytotropho blasts

to the syncytiotrophoblast component decreases progressively, until the syncytial layer

is the dominant trophobiastic compogent at term (Petraglia et al., 1996a)Xhe

syncytiotrophoblast layer is a major site of protein and steroid hormone synthesis

within the human placenta (Petraglia et al., 1996a).

The amnion and the chorion are the fetal membranes and together form the

amniotic sac which holds the fetus and the amniotic fluid. The amnion is a thin

avascular membrane comprised of a single layer of epithelial cells on a loose

connective matrix. The chorionic membrane is composed of an inner connective tissue

layer and an outer epithelial layer. It is in direct contact with the decidua except at the

site of placenta implantation. The decidua is composed of the decidua basalis; the

region directiy beneath the site of implantation of the embryo, the decidua capsuloris;

the region surroundhg the embryo and the decidua vera which lines the remainder of

the uterus. The three layers of the decidua represent a vascular anatmnical interface

that ailows communication between the fetd membranes and the uterine myometnum

(see Figure 1.1) (Peûaglia et al., l996a).

13 THE UTERUS

The utem is a muscular, pear shaped organ located in the pelvic cavity between

the bladder and the rectum (Pritchard et al., 1985). The size and the shape of the utenis

vary markedly, dependent on age and parity. The u tem is composed of four regions:

the fundus, the corpus, the isthmus, and the cervix. The fundus is the uppermost and

widest region followed by the corpus, the isthmus and the narrow cervix.

The wail of the utenis is composed of three layea: the outer perimetriurn, the rniddle

myometrium and the inner endomemum. The myometrium is comprised of an outer

layer of longitudinal muscle fibers parallel to the long axis of the uterus and an inner

layer of circular muscle fibers perpendicuiar to the long axis of the utem (Pritchard, ef

al., 1 985). The longitudinal and the cucular muscle fibers of the myometrium originate

from the subserosd comective tissue and the paranephrie ducts. respectively (see rev.

Challis and Lye., 1994).

The rnyometrium undergoes sporadic, low pressure minimal intensiq

contractions kaown as Brmton-Hicks connacrions throughout pregnancy. Labour is

charac terized by high-fiequency , high amplitude, intense contractions of the

myometnum. At the time of labour, the human uterus differentiates into an actively

contracthg upper segment and a relatively quiescent lower segment (see Figure 1.2).

Uterine contractions of hi& intensity and long duration are present in the funda1

segment when compared to the activity of the lower segment (see Figure 1.3). The

development of regions of opposing contractile activity within the uterus aiiows for the

effective and forcehi expulsion of the fetus fiom the uterus.

Challis and Lye (1994) hypothesized that the activity of the myometrium, in

pregnancy, was fkely regulated by the synthesis of "contraction-associated proteias"

and the balance of stimuiatory versus inhibitory factors acting on the myornetrium.

Lye coined the term "contraction-associated" proteins to refer to the proteins involved

in increasing the excitability of the myometriai smooth muscle including the gap

j unctions and the uterotonic agonist receptors.

Gap junctions are specialized regions of the ceil membrane through which

direct cell-to-cell contact is maintained. They are formed fiom the aggregation of

hundreds or thousands of hydrophilic protein charnels (connexins) on the plasma

membranes of adjacent cells. Gap junctions provide the structural basis necessary for

coordinated synchronized rnyometriai contraction (Mackenzie and Garfield, 1 98 5 ;

Kilarski et al., 1994). Petroceiii and Lye, (1993) reported that, one of the gap junction

proteins, cornexin 43 (Cx 43) mRNA and protein levels were low in rat myometriurn

in pregnancy and increased markedy prior to labour attaining a peak during delivery.

Oxytocin (OT) is a nonapeptide hormone that stimulates uterine smooth muscle

contraction. This hormone is thought to be vital for the initiation of labour in humans,

but maternal plasma OT concentrations do not rise pnor to labour. In addition, serial

studies showed that the maternal plasma concentrations of oxytocin only increased

during the expulsive phase of labour (Leake et al., 198 1). Fuchs et al., (1982) reported

a signincant conelation between the concentration of OT receptors and the sensitiviw

of the uterus to OT. This led to the proposition that the concentrations of OT receptors

could be a possible regdatory mechanism mediating the effects of OT. However,

despite a reported increase in oxytocin receptor numben with the onset of labour

(Fuchs et al., 1994)- it is not clear whether OT is imporîant for the initiation of labour.

It has been suggested that increased OT receptor nurnbes facilitate the effects of OT

on an already "activatedm myometrium (see rev. Challis and Lye. 1994).

Prostaglandins (PGs) play a central role in stirnulating myometnal contractility

in many species ( see rev. Challis and Lye, 1994). A role for PGs in labour is suggested

by the demonstration that prostaglandin E2 (PGE3 and prostaglandin F2 alpha (PGF2a

stimulate the contraction of myometrium collected fkom pregnant women (Embrey and

Momson, 1970), that the concenaations of PGF2, and PGEz in amniotic fluid and of

their metabolites in matemal plasma and urine increase at labour (see rev. Challis and

Lye, 1994) and that the inhibition of PG synthesis suppresses uterine activity and

prolongs the length of pregnancy (Anderson et al., 1981).

PGs are forrned fkom arachidonic acid, that is liberated fiom cellular

phospholipids by phospholipase C (PLC) and PLAz enzyme activity. Arachidonic acid

is metabolized by the cyclooxygenase activity of prostaglandin H synthase to the

unstable endoperoxide, PGG2, that is reduced to PGH2. PGHz is unstable and

isomerizes readily to PGE,, PGF2, and PGD2. PGs are metabolized by an oxidized

fom of nicotinamide adenine dinucleotide type 1 15-hydroxyprostaglandin

dehydrogenase to form 15-keto compounds. The 15-keto compounds are reduced by

A ~ ~ ' ~ ~ reductase to 13- 14 dihydro- 15 keto derivatives that are oxidized and excreted in

urine.

1.5 PROCESTERONE AND ESTROGEN IN LABOUR

The placenta plays an essential role as an endocrine organ of pregnancy and is

actively involved in the synthesis and secretion of progesterone and estrogen. The

effects of progesterone and estrogen are complimentary, as well as, antagonistic to each

other (Roy and hdkumara, 1991). In addition to other effects , both steroids regulate

the formation of gap junction proteins and the synthesis of OT, and therefore have

critically important roles in regulating the key physiological events vital to the

initiation and progression of labour.

Progesterone inhibits the appearance of gap junctions between rat myometrial

ceIls and reduces cell-to-ce11 coupling (Garfield et al., 1990). Specificaily, Petrocelli

and Lye (1993) have shown that progesterone blocks the expected rise in Cx 43

mRNA at the Mie of labour in rat myomeaium. The myometnum under progesterone

dominance is characterized by its rehctoriness to stimulation by OT and PGF2,

(Neulen and Breckwoldt, 1994). These bioactive effects of progesterone are necessary

for the maintenance of human quiescence (see rev. Challis and Lye, 1994).

Matemd plasma progesterone concentrations increase corn the sixth week of

gestation on through to term (Tulchinslq et al., 1972). Progesterone is formed fkom

circulating low-density lipoproteins (LDL) Iûiked to cholesterol. LDL bind to specific

membrane receptors on the syncytiotropho blast and are internaiized by endocytosis.

Within the ceIl cytoplasm, LDL fuse with lysosomes and are hydrolyzed to amino

acids and cholesterol esten. Cholesterol esters are m e r hydroiyzed to fatty acids and

cholesterol and cholesterol is cleaved by the side chain cleavage (P,,,scc) eiuyme

to pregnenolone (C21) that is hydroxylated by the mitochondrial placental type 1 3L1-

hydroxysteroid dehydrogenase: A 5 4 isomerase e n y m e (30HSD) to progesterone.

Estrogens accelerate the biosynthetic pathways involved in placentai

progesterone production (Pepe and Albecht, 1993). They upregulate LDL receptor

expression and increase the expression of P4SOSCC in the baboon syncytiotrophoblast

layer. However, estradiol (E2) stimulates the development of myometrial gap junctions

in the pregnant rat (Mackenzie and Garfield, 1985) and Lye et al., ( 1993) have shown

that the rise in Cx 43 &A expression in labour is associated with an increase in the

plasma estr0gen:progesterone ratio. Estrogens also increase the responsiveness of the

myometrium to OT and PGFZ, (Leslie et al., 1994), therefore in general estrogens

promote myometrial contractility (see rev. Challis and Lye, L 994).

Matemal plasma concentrations of E2 rise throughout gestation (Tulchinsky et

al., 1972). The synthesis of estrogens in human pregnancy requires an obligatory

interaction between the placenta and the maternai and fetal adrenal glands. The human

placenta lacks the P45Ki7 enzyme and is therefore unable to convert C21 steroids

(progestins) directly to C 19 steroids (androgens). Estmgen (C 1 8) synthesis is

dependent on the production of dehydroepiandrosterone sulphate (DHEAS) in the

maternal and fetal adrenals. DHEAS is extracted fiom the fetal and maternai

circulations by the placenta. It is desuifonated to DHEA that is converted to

androstenedione. Androstenedione is readily aromatized to estrone and E2. The

majority of DHEAS produced in the fetal adrenal is converted in the liver to 16a

hydroxy-DHEAS before entering the placenta 16a-OH-DHEAS is cleaved to 16u-

OH-DHEA which is converted to 16a-OH-mdrostenedione and aromatized to estriol.

1.6 CORTICOTROPIN RELEASING HORMOIW

ln addition to steroid hormone synthesis, the human placenta the decidua and

the f e d membranes produce a large number of hypothalamic-like peptides which

ioclude luteinking hormone releasing hormone and CRH (Khodr and Siler-Khod.,

1978: Riley et al., 1991). These peptides which appear to be stnicturally and

bioactively identical to their hypothalamic counterparts. are thought to modulate

autocrine andlor paracrine mechanisrns within the uterus. This review will initially

discuss the properties of hypothalamic CRH. This will be followed by an in depth

explanation of the synthesis and secretion of hCRH-pl and discussion of the putative

roles of hCRH-pl in human pregnancy and labour.

CRH is a 41 amino acid peptide, initially characterized in the ovine

hypothalamus by Vale er al., (1 981). It is synthesized predominantly in the perikarya

of neurons within the medial pawocelldar region of the paraventricular nucleus (PVN)

and released into the hypophyseal portal system in response to stress (Vde et al., 198 1 ;

Antoni, 1983). The gene for prepro CRH, the biosynthetic precursor of CRH, was

isolated fkom a human genomic DNA library with an ovine CRH (oCRH)

complementary DNA (cDNA) probe (Shibahara et al., 1983). The deduced amino acid

sequence of human CRH exhibits seven amino acid substitutions when compared to the

ovine CRH (Vale et al., 1981): glutamic acid for glutamine (at position 2), alanine for

threorine (22), arginine for lysine (23), methionine for leucine (38), glutamic acid for

aspartic acid (39) and isoleucine for alanine (41). AU of these substitutions represent

changes in chemically sirnilar amino acids resulting fiom single nucleotide amino acid

substitutions except for the isoleucine/alanine substitution in the carboxyl terminus of

CRH (Shibahara et al., 1983: Vaie et al., 1981). The rat CRH (rCRH) gene has a

similar structurai organization to that of the human CRH gene and the deduced amino

acid sequence of human and rat CRH (&RH) appears to be identical (Rivier et al.,

1993). In addition, a well-conserved CRH amino acid sequence has also been isolated

and characterked in the mouse and the dog hypothalamus (Keegan et al., 1994; Mol et

al., 1994).

CRH acts on the corticotrophs of the pituitary to upregulate pro-

opiomelanocortin O M C ) mRNA expression and the release of adrenocoticotropin

hormone (ACTH). ACTH increases the expression of key enzymes involved in

corticosteroid synthesis in the fetal adrend gland (Fujieda et al., 1981) leading to an

increase in plasma glucocorticoid concentrations. Glucorticoids feedback on the

hypothalamus and the pituitary, via specific receptors to decrease ACTH release from

the pituitary (Yang et al., 1990; Lu et al., 1991).

1.6.1 CRB-Related Peptides

Sauvagine and urotensui 1 are 50 % homologous to hir CRH at the amino acid

level with hi& homology particularly in the carboxy tenninus (see Figure 1.4)

(Montecucchi and Henschen, 1981 ; Lederis et al., 1982; Ichikawa et al., 1982).

Sauvagine is a 40 amùio acid peptide, isolated fiom the skin of the frog Phylzomedusa

sauvogei (Montecucchi and Henschen, 198 1) and urotensin I is a 41 amino acid peptide

isolated from the caudal neurosecretory system of both Catostomus mersoni (sucker

fish) and C'yprinus curpio (carp fi&) (Lederis et al., 1 982; Ichikawa et al., 1 982). Both

sauvagine and urotensin I stimulate the release of ACTH from anterior pituitary ce11

cultures (Lederis et al., 1982; Tm. et al., 1990).

Recently, another CRH-related peptide has been isolated and characterized in

the rat midbrain and fiom a human genomic DNA library (Vaughan et al., 1995;

Donalcison et al., 1996). Urocortin is a 40 amino acid peptide that is highly

homologous to urotensin I (63%) and CRH (45%). This peptide also stimulates ACTH

release firom dispersed rat anterior pituitary cells in culture (Donalson et al.? 1996).

Urocortin mRNA and peptide have been identified in the human placenta and fetal

membranes (Petraglia et al., 1996b), corresponding to a site of CRH synthesis and

secretion.

1.7 HUMAN PLACENTAL CRH

Shibasaki et al., (1982) were the £kt to demonstrate CRH-like bioactivity in

human placenta. These investigators showed that CRH exûacted from purified

placental extracts, obtained fiom term spontaneous deliveries, was biologicaily active

in vifro, stimdated the release of ACTH and D-endorphin from cultured rat anterior

pituitary ceils. Later studies by Frim et al., (1 988) reveded that hCRH-pl mRNA was

expressed in human placental tissue in huma. placenta fiom the seventh week of

gestation. Human CRH-pl levels increased more than 20-fold during the last 5 weeks of

pregnancy, concomitantly with an increase in hCRH-pl peptide (Frim et al.. 1988).

These scientists reported that the expression of hCRH-pl mRNA and the leveis of

peptide were low in the nrst trimester of pregnancy, increased during mid-gestation and

increased exponentially during the 5 weeks prior to labour (Frim et al., 1988). Sasaki

et al., (1988) reported the presence of three molecular species with CRH

immunoreactivity in extracts of human terni placenta. While, the major species eiuted

with KRH the other two had apparent molecular weights of 18 kiloclaltons @Da) and 8

kDa, respectively. These two higher molecular weight species did not stimulate ACTH

release fkom cultured rat anterior pihiitary cells (Sasaki ei al., 1988) and are thought to

represent a precursor and a biosynthetic intermediate. The CRH gene is expressed in

human placenta and hCRH-pl mRNA is the same size (1.3 kilobases; kb) as

hypothalamic CRH (Grino et al., 1 987)

Jones et al., (1989b) showed that hCRH-pl was synthesized in human p lacen~

decidua and fetal membrane ce11 cultures. These investigatoa shawed that significantly

more CRH was produced in placental and fetal membrane tissues collected afier

spontaneous term labour than tissues obtained at elective cesarean sections. It is now

generally accepted that CRH immunoreac tivity is present on the syncytiotro phoblast of

term placenta (Riiey et al., 1991). Earlier studies by Saijonmaa et ai., (1988) and

Petraglia et al., (1987) had localized immunoreactive (ir)-hCRH-pl on the

cytotrophoblasts in early and tem human placenta, respectively. However, Riley et al.,

(1991) and showed that ir-hCRH-pl is present on the syncytiotrophoblast layer but that

it was undetectable in the cytotrophoblasts (Cooper et al., 1994; Warren and Silverman,

1 995; Perkins and Linton, 1 995). Ir-hCRH-pl staining on the syncytiotropho blast layer

increased fkom the ninth week of gestation onwards. Positive staining for ir-hCRH-pl

was also present on the intemediate trophoblasts cells, the chorionic tmphoblasts and

the epithelial cells of the amnion in term placenta (Riley et al., 199 1; Warren and

Silverman, 1995; Perkins and Linton, 1995). Ir-hCRH-pl is also present in the decidua

and increases with gestational age (Petmglia et al., 1992).

Human CRH-pl is secreted into the matemal and fetal circulation and released

into the d o t i c tluid throughout human pregnancy (Sasaki et al., 1987; Goland et al.,

1988). Plasma concentrations of ir-hCRH-pl in the matemal and fetal circulation

increase in p d e l to hCRH-pl levels in the placenta (Frim et al., 1987). In the first

trimester of pregnancy plasma concentrations of h-CRH-pl are barely detectable (5.9 f

0.8 pg/mL) (Sasaki et al., 1987; Goland et al., 1986; Maser-Gluth et al., 1985;

Campbell et al., 1987). Plasma ir-hCRH-pl concentrations rise progressively from the

second trimester of pregnancy (35.4 f 5.9 pg/mL) (Sasaki et al., 1988) to term

(- 1 O00 pg/mL) (Camp bel 1 et al., 1 987; Sasaki et al., L 987). Wiîh a significant increase

throughout labour (- 4000 pg/mL) (Okarnoto et al., 1989; Petraglia et al., 1990).

h e d i a t e l y post-partum matemal plasma hCRH-pl concentrations decrease rapidly to

barely detectable levels (Sasaki et al., 1987; Campbell et al., 1987; Schdte and Healy

1987; Goland ef al., 1988; Stalla et a[., 1 989). No information has been reported on the

concentrations of the other CRH mokcular weight species.

Hunan CM-pl is thought to be secreted predominantly into the matemal

circulation. At the time of delivery, mean umbilical cord hCRH-pl concentrations are

1000 fold lower than t h s e reported in the maternal circulation (Sasaki et al., 1988).

The umbilical venous plasma hCRH-pl concentration (50.6 + 6.1 pg/mL ) was

signiticantly greater than that in simultaneouslysbtahed umbilicai arterial plasma

(41.8 t 4.9 pg/mL) (Schulte and Healy, 1987). The decrease in matemal plasma

hCRH-pl concentrations post-partum and the elevated concentrations of hCRH-pl in

the umbilical vein when compared to the urnbilical artery c o n f i the placenta as a

source of hCRH-pl. Human CRH-pl concentrations, in the amniotic fluid increase

progressively throughout gestation (Maser-Gluth et al., 1 987; Campbell et al., 1 987:

Laatikainen et al., 1988). Contrary to plasma hCRH-pl concentrations, however,

amniotic fiuid concentrations of hCRH-pl are not altered during labour (Petraglia et al.,

1990).

M a t e d plasma hCRH-pi concentrations are significantly elevated in PTL

patients in comparison to gestational-age matched controls. This rise is apparent prior

to the onset of labour (Linton et al 1987; Warren et al., 1992; McLean et al., 1 995).

CRH binding protein (CRH-BP) is a 37 kDa peptide that binds hCRH-pl and

inhibits its ACTH-releasing activity in anterior pituitary cultures (Orth and Mount,

1987; Linton et al., 1988). In addition CRH-BP decreases PG release fkom cultured

matemal decidua and dampens the potentiated contractile activity of CRH and PGF2,

on human myometnum in vitro. CRH-BP is predominantly secreted by the matemal

Iiver (Potter et al., 1991) but has also been identified on the syncytiotrophoblast, the

intermediate trophoblast cells, the stroma1 celIs of the decidua and the epithelial celIs of

the amnion (Petmglia et al-, 1993; Rarnirez et al., 1995).

Matemal plasma CRH-BP concentrations (- 5 nrnol/L) fa11 sigrilficantly (- 2

nmoVL) in late pregnancy retuming to basal concentrations 48 hours postpartum

(Linton et al., 1993). In pregnancies complicated with PTL the decrease in plasma

CRH-BP concentrations is more pronounced when compared with gestationai-age

matched controls (Orth, 1992). Mean CRH-BP concentrations, in fetal plasma also

decrease pnor to term (Linton et al., 1993). The mechanisms controlling the decrease

of CRH-BP concentrations, in both pre-term and term pregnancies are d l under

investigation. Woods et al., (1994) suggested that the association of CRH-BP with

CRH in blood may triggers the clearance of CRH-BP h m the circulation. A decrease

in plasma CRH-BP in both the matemal and the fetal circulations, would result in a net

increase in the circulating concentrations of "biologically active" hCRH-pl (Linton et

al., 1988), primarily because the majorïty of hCRH-pl in both the matemal and fetal

circulations is bound to CRH-BP (Salminen-Lappalainen and Laatikainen, 1 990).

1.9 OTHER RIZGULATORS OF CRB

It is well established that glucocorticoids inhibit hypothalamic CRH release in

humans (Vale and Rivier, 1977; Owens and Nemeroff., 1991 ; Orth, 1992). However,

in human pregnancy hCRH-pl concentrations nse paradoxically with plasma

concentrations of cortisol (Fencl et al., 1 980; Cam et al., 1 98 1). Robinson et al., ( 1 988)

and Jones et ai., (1989b) have shown that dexamethasone, a synthetic glucocorticoid,

stimdates hCRH-pl synthesis and secretion from prhary cultures of term human

placen-ta, decidua, amnion and chorion. In addition, the neurotransmitters.

norepinephrine and acetycholine, stimulate hCRH-pl output (Petraglia et al., 1989). in

agreement with the mechanisms regdating hypothalamic CRH secretion interleukin-

la, neuropeptide Y, angiotensin II ( h g II), arginine vasopressin and OT increase the

release of hCRH-pl in vitro from cultured trophoblast cells (Petraglia et ai., 1991). In

contrast, progesterone and nitric oxide (NO) inhibit hCRH-pl output fiom tenn human

placenta, decidual, amnion and chorion (Jones er al ,1989b; Karialis and Mazoub,

1994; Sun et al., 1994).

1.10 MYOMETRIAL CONTRACTILITY

CRH does not have the intrinSic ability to stimulate human myometrial activity.

Quartero and Fry (1 989) have shown that CRH has both a priming and potentiating

effect on the myometrial contractile response to OT. In addition, hCRH-pl stimdates a

3 to 4 fold increase in ir-OT release fiom placental cells in vitro (Florio et a l , 1996).

The rise in circulating concentrations of hCRH-pl in pregnancy coincides with an

increase in the sensitivity of the myometrium to OT (Caldeyro-Barcia and Serono,

196 1 ; Fuchs et al., 1994) and may niggest a role for h-CRH in the regulation of OT

receptor nurnbers. McLean et al., (1994) showed a positive association between

matemal plasma hCRH-pl concentrations and the fkquency of uterine contractions in

women who entered labour following OT infusion.

CRH also enhances PGF, -stimulated myometrial activity (Benedetto et al.,

1994) and stimulates PGF2, and PGk release fiom primary cell cultures of placenta,

chorion and amnion (Jones and Challis, 1989a). A role for hCRH-pl in the initiation of

labour is also suggested by the presence of CRH binding sites in the myomehium

(HilIhouse et al., 1993) and the recent identification of CRH receptor mRNA

(Rodriguez-LinZres et al., 1998). This is central to our study and will be discussed

later.

1.11 OTHER ROLES FOR CRH

McLean et al., (1995) proposed that the materna1 plasma concentrations of

hCRHpl at 16-20 weeks of human pregnancy could be an indicator of the timing of

delivery and a determinator of patients at risk of PTL. The role of CRH as a rnarker of

PTL has been controversial, because of wide interpatient variations in plasma CRH

concentrations and the stability of hCRH-pl in plasma samples (Warren et al., 1992).

CRH-pl is a potent vasodilator in the human placenta vasculature and mediates

its vasoactive effects via NO (Clifton et al., 1994). in addition, it has been

hypothesized that hCRH-pl might contribute to the activation of the fetal pituitary

adrenai axis in late pregnancy, thus indirectiy contributing to prenatal lmg maturation

(Petraglia et al., 199 1 ) . F W y , it has k e n suggested that there is a local CRH-POMC-

ACTH axis within the placenta responsible for the increased concentrations cortisol in

matemal plasma and urine (Cam et al., 198 1 ; Goland et al., 1986; Margioris et al.,

1988). This suggestion was made based on the presence of POMC mRNA and POMC-

denved peptides in the placenta (Margioris et al., 1988).

1.12 CRET RECEPTORS

CRH binding sites have been identified in several tissues including the pihutary

gland (De Souza et al., 1984), the brain (De Souza et al.. 1985), the placenta (Petraglia

et al., 1990; Clifton et al., 1995) and the myomehium (HiIlhouse et al., 1993) by

radioligand bhding, autoradiographicai and in situ hybridization techniques. There are

at Ieast two major classes of mammalian CRH receptos, CRH-R 1 and C W R 2 .

Chen et al., (1993) recently reported the cloning of a cDNA encoding a CRH-

R1 fiom a human corticotropic tumour library. The CRH receptor is comprised of

seven putative membrane-spanning domains and belongs to the calcitonin/vasoactive

intestinal peptidelgrowth hormone releasing hormone subfamily of G-protein coupled

receptors. The cloned CRH-RI cDNA encoded two -apparent spliced forms of the

receptor, CRH-Rla and CM-RI P (Chen et al., 1993). CRH-Rla encodes a 41 5

amino acid peptide and appears to be the predominant form ( see rev. Dieterich et al.,

1997). CRH-RlP encodes a 444 amino acid peptide which has not been identified in

any other species or specific tissue other than the human piniitary. Recently, cDNA's

encoding CRH-RI homologs have been isolated and characterized in the human cortex,

braiostem, hippocampus and testis, mouse pituitary (Vita et al., 1993) and rat brain

(Perrin et al., 1993). Two other spliced variants of CRH-R1 have been reported; a

receptor with a 40 amino acid deletion in the amino terminal domain (Ross et a[.. 1994)

and a truncated receptor that contains a fhmeshift that only encodes the fïrst 185 amino

acids of CRH-Rla (Chang et al., 1993) (see Table 1.1). Al1 species homologs of the

CRH-RI are of comparable size and are 98% homologous to one another.

CM-RI is a glycoproteh (Grigonadis and D e S o w 1989) and contains five

potential N-linked glycosylation sites in the N-terminal extracellular domain (Chen et

al., 1993). While the amino acid sequence of rat brain and rat pituitary CRH-R1 are

97% identical differences have been reported in their molecular weights which appear

to be due to differentiai tissue specinc glycosylation of CRH-R1 (Grigoriadis and De

Souza, 1989a). In addition, five potential protein kinase C (PKC) phosphorylation sites

have also been identified in the first and second intracellula. loops and in the

C-tenninal tail (Chen et al., 1993) Both a casein kinase II and a protein kinase A

(PKA) phosphorylation site are present in the thud intracellular loop (Chen et al., 1993;

Perrin et al., 1993).

CRH-R2 is encoded by a separate and distinct gene This receptor subtype has

been cloned fiom rat brain (Lovenberg et al., 1995a), human (Liaw et al., 1996), mouse

heart and skeletal muscle (see Table 1.2) (Kishimoto et al., 1995; Perrin et al., 1995;

Stenzel et al., 1995). Two alternatively spliced forms of the receptor, CRH-RZa and

CRH-WP, encoding a 41 1 amino acid peptide and a 43 1 amino acid peptide

respectively, have been identified. Their amino acid numbers cliffer in that the fïrst 34

amino acids in the N-tenninal of CRH-R2a are substituted with a unique sequence of

54 amino acids in the CRH-R2B protein (see Figure 1.5). CRH-R2a and CRH-WP

have been identified in distinct anatomical zones. (Lovenberg et al., 1995b). CRH-R2a

mRNA is abundantly expressed in rat brain and CRH-R2P mRNA is predorninantly

expressed in the rat heart and skeletal muscle. In the human CRH-R2a is the prevailing

CRH-R2 subtype in the heart and skeletal muscle (Vaidenaire et al., 1997).

The CRH-R2 variants ais0 have 5 potential N-linked glycosylation sites,

identical to those in CRH-RI, as well as potential PKC phosphorylation sites

(Lovenberg et ai., 1995a). Both rat and human CRH-R2a share 70% sequence identity

to CRH-Rla with the clifference being the insert of amino acids in the first cytoplasmic

loop (Lovenberg et al., 1995a). The receptors show high homology particularly in

respect to the complete identity of the third intracellular domain (see rev. Dieterich et

al., 1997). It has been reported that a major determinant of the coupling of G-proteins

receptors to adenylate cyclase is located in the third intraceIlular loop (Okamoto et al.,

1991). This is consistent with the demonstrated abilities of both CRH-RI and CRH-R2

to increase intracelluiar CAMP (Chen et al., 1993; Lovenberg et al., 1995) and suggests

conservation of second messenger function between the CRH-R1 and the CRH-EU

receptor genes. Nabhan et al., (1 999, however, have reported that CRH-R.2 is not well

coupled to the G-protein in LLCPK-1 ceils and requires elevated levels of CRH to

stimulate intracellular CAMP levels. The CRH-RI gene is present in the 17q12-q22

interval of the long arm of chromosome 17 (Polymeropuoulos et al., 1995) but the

CRH-Rî gene has not yet been locaiized.

CRH binds to its receptor to fom a complex that subsequently binds and

activates a guanosine 4' triphosphate stimulatory binding protein (Gs), causing GDP to

dissociate nom the inactive G protein, and GTP to bind the alpha (a) subunit of the G

protein. The G protein a subunit dissociates fiom the py subunits, binds and stimulates

the adenylate cyclase enzyme, which catalyses the intracelldar CAMP synthesis fiom

ATP. The released CAMP can bind CAMP dependent PEU. It is well documented that

CRH binds its receptor with high affinity and stimulates adenylate cyclase activity

leading to increased intracellular cyclic adenosine 3'5' monop hosphate (CAMP) in the

rat pituitary (Labrie et al., 1982; Aguilera et al., 1983; Bilezikjian and Vale. 1983), the

brain (Battaglia et al., 1987; Chen et al., 1986) and the myometrium (Grammatopodos

et al., 1994). The EC, values in stimulating CAMP production in the above rnentioned

tissues are similar (ES, = 0.2 - O.5nM). In addition, the binding of 125~-radiolabeled

oCRH ('"1-OCRH) to the receptor i s inhibiteci by guanyl aucleotides, confirming that

CRH receptors are coupled to the adenylate cyclase pathway by a guanyl nucleotide

regdatory protein (Aguilera et al., 1987).

It has been suggested that optimal CRH receptor expression requires both the

PKC and the adenylate cyclase pathway (Lutz-Bucher et al., IWO). The PKC pathway

appears to be an important modulator of CRH function. A role for PKC in regulating

CRH function is substantiated by the ability of arginine vasopressin and angiotensin II,

which use PKC as their secondary messenger, to potentiate CRH-stimulated ACTH

from the pituitary (Abou-Sambra et al., 1986) and the placenta (Petraglia et al., 199 l),

and the demonstrated ability of direct activators of the PKC pathway to stimulate

ACTH secretion and CAMP production in comcotrophs (see rev. Dieterich et al.,

1997). Possible cross taik between PKC and adenylate cyclase is of interest as in

certain sites, such as the rat Leydig ceus, CRH receptoa are aot directly coupled to a

G-protein, but mediate their actions via the direct or indirect actions of PKC (Dufau et

al., 1989). Activation of several receptors fkom the 7 transmembrane receptor family

has shown that they are able to couple to PLC and stimulate phosphoinositol hydrolysis

when in the presence of high receptor or Ligand concentrations (Jelinek et al., 1993;

Spengler et al., 1993; Usdin et al., 1993; Nabhan et al., 1995).

The pharmacological properties of CRH binding sites are usually determined by

the relative ability of a varîety of CM-related and unrelated peptides to displace

specifically bound 12*1 oCRH or I2*l ratmuman CRH (rh CRH) ( see rev. Dieterich et

al., 1997). The binding of 1 2 S ~ ~ ~ ~ to specific sites in the CNS and the periphery

appears to be both saturable and reversible (De Souza es al., 1984; 1985; Hillhouse et

al., 1993; Hatzoglou et al., 1996). In addition, CRH and CRH d o g s bind these sites

with high &ty bkding, an apparent dissociation constant (K.,& of 0.2 - 0.8 nM. The

simila. Kd of CRH and CRH fragments suggest that similar structurai ligand

requirernents are shared by CRN receptors in the pituitary, the brain, the placenta and

the myornetrium. Recent work by Liaw et al., (1997) has found three regions in a

chimeric receptor construct of CRH-Rla and CRH-ma, that were required for the

optimal binding of ' 2 S ~ - r h CRH andor receptor activation.

Increased exposure of target tissues to peptide hormones is u d y Linked to the

downregulation and desensitization of the homologous receptors. Desensitization

reflects either a short term, (affinity change, conformational alteration of the receptor

protein, uocoupling fiom the corresponding G-protein) or a long-term (downregulation

of the receptor) adaptation process (Dietench et al., 1997). CRH regulates the

expression of its receptor in a tissue-specific manner, such that exogenous C RH

decreases CRH receptor concentrations in the rat anterior pituitary both in vivo and in

vitro (Wynn et al., 1983; 1988; Hauger and Aguilera., 1993; Lu et PI.. 1994; Sakai et

al 1996; Pozzoli et al., 1996) whereas intracerebroventncular administration of CRH

stimulates a significant increase in CRH-RI mRNA in the PVN (Mansi et al.

1996).The expression of CRH receptors are also regulated by other factors such as

stress, adrenaiectomy and glucocorticoids. Stress leads to a rise in circuiating C M ,

causing a decrease in C M receptor numbers in the hypothalamus (Makino et al.,

1995). Adrenaiectomy resufts in an increase in cuculating hypothalamic C M , due to

withdrawai of adrenal cortisol negative feedback. This increase in plasma CRH causes

a û-ansient decrease in CRH-RI mRNA expression in the anterior piniitary and an

increase in CR,-RI rnRNA expression in the PVN (Luo et al., 1995). This effect was

removed with exogenous glucocorticoid administration (Wynn et al., 1983). High

circuiating concentrations of glucocorticoids are known to decrease the expression of

CRH receptors in the PVN, the anterior pinlltary and the brain (Makino et al., 1992)

and it has been proposed that hi& concentrations of glucocorticoids act synergisticaily

with CRH to decrease CRH receptor mRNA in the anterior pituitary (Makino et al.,

1995). Recent studies by Makino et al., (1997) have shown that CRH-Rla mRNA and

CRH-R2a &A are differentially regulated in the rat hypothalamic PVN after

adrenalectorny and glucocorticoid administration: CRH-Rla mRNA was demeased in

foiIowing these treatments whereas CM-R2 mRNA Ievels were unaltered. This

suggested that the expression of CRH-RI mRNA and CRH-R2 mRNA is differentially

regulated in the rat brain. Ttis may also be reflected at other sites that express both the

receptor subtypes.

CRH binds CRH-R1 with a higher affinty than CRH-R2 (Lovenberg et al..

1995a) whereas urotensin 1 and sauvagine bind CRH-R2a with a higher aanity than

CRH in transfected rnouse Ltk- ceUs and in rat heart and muscle cells (Lovenberg et

al., 1995a; Perrin et al., 1995). It has been suggested that CRH is not the preferential

ligand of CRH-W implying the possible existence of another endogenous ligand for

this receptor subtype. A possible candidate is urocortin which has been proven to bind

the CRH-FU, with a higher e t y than CRH (Vaughan et al., 1995).

Despite an understanding of some of the phannacological and second

messenger characteristics of CRH receptors, the mechanisms involved in the

differential post-translational processing of CRH receptors remain to be elucidared.

DBerences have been reported in the molecular weight of CRH receptors present in

the rat pituitary (58 H a ) (Grigoriadis and De Souza 1988; 1989b), the rat brain and the

human placenta (75 kDa; Grigoriadis and DeSouza, 1989a; De Souza et al., 1985;

Grigonadis et al., 1993; Clifton et al., 1995) and the human myometriurn (40 and 45

ma; Castro et al., 1996). Grigoriadis et al., (1989a) deglycosylated the carbohydrate

moiety nom the rat brain and pituitary CRH receptor and found that it was

predomimmtiy composed of N-acetyl@ucosamine a d o r terminal sialic acid residues,

as well as some hi& mannose c h a h . The native CRH receptor protein revealed, f i e r

deglycosylation, had molecuiar weight of 40 - 45 kDa This was consistent with the

molecular weight of CRH-Rl (Chen et al., 1 993).

Differences can occur in the CRH receptors at the transcriptional, the post-

transcriptional, the translational and the post-translational level. To understand the

mechanisms functioning at these each of these stages it is important to determine the

site of expression of CRH receptor mRNA and the protein levels. The regionai and

cellular expression of CRH receptor message bas been characterized extensively in the

rat brain and the rat pihiitary (Potter et aï., 1994; Chalmers et al., 1995:). These studies

report that CM-RI mRNA expression is abundant in the cerebrd cortex, the

subcortical limbic structures. the amygdala and in the anterior and intemediate lobe of

the pituitary. CRH-R2 &A expression was found in distinct subcortical regions of

the rat brain including the lateral septal nucleus, the venûomedial hypothalamic

nucleus and the choroid plexus (Chaimers et al., 1995). It was also measured at lower

levels in the olfactory bdb, amygdaioid nuciei, the paraventricular and supraoptic

nuclei of the hypothalamus, as well as in the cerebrai arteries throughout the brain.

Based on the separate pattern of distribution of CRH-R1 mRNA and CRH-R2 mRNA

in the brain it has been proposed that CRH-RI is pRmarily a neuroendocrine receptor

mediating the bioactive effects of CRH in the pituitary and that CRH-R2 is involved in

the hypothalamic nemendocrine, aidonomic and behavioral actions of central CRH

(see rev. Dietench et al., 1997).

The major* of the work reported on CRH receptor expression has been in the

brain. However the pleiotropic effects of CRH in the human (Quartero and Fry, 1989;

Clifton et ai., 1994) suggests the presence of putative CRH recepton at multiple other

sites. Recentty CRH receptors have been identified within the structures of the human

utems including the placenta and the myometnum. This wodd suggest CRH may play

a role in the mechanisms leading to parturition.

i 25 1-oCRH binding sites, have been localized almost exclusively on the

syncytiotrophoblast of human placenta (Hatzoglou et al., 1996) and are thought to

represent a single population of CRH receptos (Clifton et al., 1995; Hatzoglou er al.,

1996). The buiding of L 2 5 ~ - o ~ ~ ~ and CRH analogs to the placenta is dependent on

t h e , temperature, pH and the presence of divalent ions and reversed on addition of

excess oCRH (Clifton er al., 1995; Hatzoglou et al., 1996; Saeed et al., 1997).

Interestingly, the number of ' 2 5 ~ - o ~ ~ binding sites in placenta obtained d e r vaginal

delivery was increased compared to placenta delivered by elective cesarean section.

This suggests an upregulation in CRH receptor numbers in the placenta at the time of

labour in human pregnancy (Petraglia et al., 1990; Clifion et al., 1995). Clifton et

a1.,(1995) also reported that in vaginally delivered placenta, the CRH binding sites had

a much lower aanity than in elective cesarean delivercd placenta, they suggested that

the high concentrations of hCRH-pi in the maternai circulation at term could regulate

the binding properties of the CRH receptor. Despite the apparent presence of CRH

receptors in the human placenta, no midies have, as of yet, examined the expression of

the CRH receptor sub~rpes in this tissue or detemiined if these mbtypes are

differentiaüy regulated with pregnancy andor labour.

Specific '%CRH binding sites are present in human rnyornetrium (Willhouse

et al., 1993). These bhding sites display similar properties to those in the placenta

(Clifton et al., 1995). A majoriv of the studies investigating the binding and second

messenger properties of the CRH receptor in the human myometrium have been

performed by the group of Hiilhouse and Grammatopoulos. These investigaton have

used myometnal samples solely fiom the lower uterine segment, have failed to

differentiate between the existence of C M - R I and/or CRH-R2 and have not studied

receptor concentrations or regdation throughout the time of labour.

Recently CRH-R1 mRNA and CRH-R2 mRNA expression have been

characterized in myometrium fiom nonpregnant and pregnant women (Rodriguez-

Linares et al., 1998). Hillhouse et al., (1993) had initially identified the presence of a

single population of CRH recepton in the human myometrium. Subsequently they

reported the presence of multiple isoforms of the CRH receptor in the rnyometrium

collected fiom nonpregnant and pregnant women (Grammatopoulos et al., 1995). The

CRH receptor is reported to have a higher afnnity in the myometrium of pregnant

women, when compared to the myometriurn of nonpregnant wornen. While this

suggests a role for CRH in myometrial function in pregnancy, it fails to s p e c e

whether there is a differential change in the affinity of al1 the CRH receptor isoforms. It

appears that other binding properties of CRH binding sites are in altered in pregnancy.

In the myomeeiurn of nonpregnant women, the binding of '%OCRH mse with

hcreasing concentrations of myometrial membranes, then stabilized at a plateau, while

in the rnyometriurn of pregnant wornen. the binding of 1 2 5 ~ - o ~ ~ myometrid

membranes increased to a peak, and then decreased (Grammatopouios and Hillhouse,

1994). The authors suggest that the presence of an inhibitor of CRH bindkig exists in

the myometrium in t e m pregnancy. This is in contmst with the previous report fkorn

this group that demonsîrated that the CRH receptor increased in affinity for CRH in

late pregnancy (Hillhow et al., 1993). However this is in agreement with recent work

from this group where they suggest that in human term pregnancy OT is able to

desensitize the CRH receptors in the myometrium by stimulating PKC-mediated

phosphorylation of the receptor (Grammatopouios et al., 1997). A role for CRH in

regulating myometrial tone in pregnancy is evidenced by the ability of CRH to

potentiate contractions of human myomeaium in the presence of OT and PGF2,

(Quartero and Fry, 1989; Benedetto et al., 1994). Despite the demonstrated ability for

CRH to act on human myometrium and the rise in plasma hCRH-pl concentrations

throughout labour (Okamoto er al., 1989) no studies have examined the change in CRH

receptors at the time of labour in the human myometrium.

The ability of CRH recepton to mediate the actions of CRH is dependent

largely on the secondary rnessenger to which the receptor is linked. CRH activates a

dose-dependent increase of both CAMP and PGEz fkom hurnan myometrial membranes

obtahed in term pregnancies. This indicates that CRH recepton in the myornetrium in

term pregnancies are coupled to both the adenylate cyclase and the cyclo-oxygenase

secondary messenger pathways (Grammatopoulos et al., 1994). The ability of the CRH

receptor to stirnulate an increase in iniracellular CAMP, via the adenylate cyclase

pathway, is reduced at the end of pregnancy concurrent with the ability of C M to

stimulate PG synthesis (Grammatopoulos et al., 1994: 1996). This would suggest a shift

tiom the relaxation sbte of the myometrium, which is associated with increased

intracellular CAMP, to the contractile state, associated with cyclooxygenase activity at

term. However, no studies have examined the secondary messenger pathway

mechanisms associated with the CRH receptors at the time of labour or post-parnim.

This review has focused on the roles and mechanisms of CRH and the CRH

receptor subtypes in human pregnancy . The human placenta secretes increasing

concentrations of hCRH-pl into the matemal circulation in the Iast trimester of

pregnancy and throughout labour, suggesting a d e for this hormone in the

mechanisms of parturition. The presence of CRH receptors in the human myometrium,

suggests a local effect of CRH on the muscular component of the utenis. The

demonstrated ability of CRH to stimulate PG synthesis and to enhance the contractile

activity of the human rnyometrium, suggests CRH plays an active role in human

parturition. To potentiate the reported roles of myometrial CRH receptors in the

regdation of myometrial tone in labour, the mRNA expression and peptide

concentrations of the CRH receptor subtypes have to be d e t e d e d both in the absence

and presence of labour. Furthennore, to d e t e d e the putative regdatory mechanisms

involved in myomeaial CRH receptor expression, it is important to establish a suitable

mammalian model. Determining the presence and regdation of CRH recepton in the

human myometrium at the tirne of labour in pre-term and term pregnancies is important

to fùrther our knowiedge in the roie of CRH in the mechanisms of parturition.

Figure 1.1 Schematic di- of the intrauterine environment showing the position of the matemalànd the fetal tissues in pregnancy. A. The membranes (left to right): the myometrium, the decidua w u , the chorion and the amnion. B. The membranes at the site of implantation (righ? to lefi): the perimetrium, the myometrium, the decidua basalis, the placentai villi, the chorion and the amnion (Modiûed fiom Pritchard et ai., 1995).

Figure 1:2 Schematic d i a p m showing the change in the size and shape of the utem in term pregnancy and the development of the upper active and the lower passive segment at the time of Iabour (Modified from Pritchard et al., 1985).

Uterus in thc hnpregnant U'ornan Cterus at thc Time of Labour

Figure 1.3 Schematic diagram depicthg the high intensity and long duration uterine contraction tracings in the upper active segment and the relative absence of contractions in the lower passive segment (Modined fiom Pritchard et al., 1985)

Figure 1.4 Cornparison of the amino acid sequences of human C M , &og sauvagine, sucker urotensin 1 and carp urotensin 1. The one letter amino acid notation is used. The sets of identical residues are enciosed in solid lines (modified fiom Shibatiara ei al., 1983)

M 1 Methionine 1

G H

Glycine Histidine

0- 1 Glutamine 1

N P

Asparagine Proline

I

Y 1 Tyrosine

- - -

R S

Arginine Serine

Figure 1.5 Schematic diagram depicting the seven tranmiembrane structure and the amino acid sequence of CRH-R2a and CRH-R2P. The arrows point to the site in the N terminus where the sequence of CM-R2a and CM-R2P diverge (Modified fkom Chalmers et al., 1 996)

Symbol Identification

Yeilow shaded amino acid amino acid specific to CRH-R2a

Red shaded amino acid

1

1 'P 1 Potential sites for the phosphorylation of PKC 1

amino acid specific to CM-RZP

I

Blue shaded amino acid amino acid that differs between C M - R l and CRH-R2

h Potentiai N glycosylation sites

Table 1: Different Species Homolop of CRH-RI

Perrin et al., 1993

Vita et al., 1993

Ross et ai., 1994

Source of cDNA Humau corticotropic tumor library

Rat brain cDNA iibraiy

Mouse piniitary tumor celf (AtT2O) and human brain cDNA library Cloned cDNA based on identifieci regions amongst ali known rnernbers of 7-TMD Gs coupled receptors

Recep tor Expression Human pituïtary, rat pituitary and braîn

Rat brain

w

r

Mouse pituitary and human brain

Eiuman hippocampus and fetai brain cDNA library

Rat brain

h l m a n brain

Binds CRH to increase CAMP Uitracellular levels

- - - -

Receptor Pro perties 415 aa

Splice variant with 29 amino acid insert in h putative intracellular loop. Receptor with 12 diEerent aa in cornparison to CRH-Rla

Tnincated splice variant containing a

-- --

Second Messenger Pathway Binds rat/hman CRH with high atfinty to increase intracelluiar CAMP levels.

Binds C M to increase CAMP intraceUular levels

eameshift that only encodes the first 185 aa of CRH- R l a

- - -

The receptor has a 40 aa deletion in corn parison to CRH-R 1 a

Binds CRH with hi& affin@ to increase intracelluiar CAMP tevels.

Needs eievated concentrations of CRH to increase intracellular c.hW levels-

Table 2:Different Species Homologs of CRH-R2

Authors

Pemn et ai-, 1995

tovenbe rg et al., 1995

Klshimot O et ai., 1995

Liaw et al., 1996

Source of cDNA

Mouse heart cDNA library

Rat hypothal amus cDNA llibrary

Mouse heat-î cDNA l i brary

Mouse ha r t cDNA library

Hurnan front cerebral CO rt ex cDNA library

Receptor Expression

Mouse heart, GIT, epidydimis and bain

~redominantl y in rat brain, heart and skeletal muscle

Mouse heart and skeletal muscle

Mouse heart, bain and lung

Receptor Properties

431 aa 16 additional aa in the N- terminal in cornparison to CRH-Rla 41 1 aa

Splice variant with a different N-terminal dornain encoding a 431 aa

70 % homology with CRH- R1 a

Second Wessenger Pathway

Human front cerebral cortex

Binds sauvagine with a higher affinity than CRH to stimulate CAMP

41 1 aa

94% identical ta rat CRH- R2a

Sauvagine is > 10 fold more potent in stimulating cAMP levels Binds sauvagine with a higher affÏnity than CRH to stimulate CAMP

Nomenclature

CaAPTER 'IWO: RATIONALE AND HYPOTHESIS

2.1 STUDY 1

CRH-RI mRNA and CRH-R2 mRNA are expressed in the myometrium of

nonpregnant and pregnant women (Rodriguez-Linares et al., 1 998). CRH acts

synergisticaily with OT (Quartero and Fry, 1989) and PGF, (Benedetto et al., 1994) to

potentiate myometriai contractility suggesting it may contribute to the complex

mechanisms involved in the omet and progression of labour in humans. To substantiate

the reported d e s of CRH, in the myornetrium, the mRNA expression and the protein

concentrations of both the CRH-R1 and CRH-EU have to be detennined in the upper

fiuidal region and the lower segment of the utem both in the absence and presence of

labour. Furthemore, the expression of CRH-RI mRNA and CRH-RZ mRNA in human

myometrium have to be compared in pre-tem and term pregnancies to assess the

regulatory effects of gestational age and PTL on CRH receptor expression.

Based on the presence of elevated levels of bioactive hCRH-pl in the matemal

circulation at the time of labour, in both term and PTL pregnancies, the presence of CRH

receptos in the rnyometrium and the ability of CRH to enhance myometnai activity we

hypothesized that the expression of CRH-RI mRNA and/or CRH-R2 mRNA expression

and protein concentrations in the human myometrium would be upregulated at the tirne of

labour.

C h u specific aims were:

1. To estabIish the presence of CRH-RI mRNA and CRH-R2 mRNA and the

locaiization of their proteins in human myometrium h m both nonpregnant patients

and pregnant patients.

2. To detennine whether CRH-RI mRNA andor CRH-R2 mRNA expression in

human myometrium is upregulated at the t h e of labour in term andor in pretem

pregnancies.

3. To determine whether CRH-R1 mRNA expression is differentially expressed in the

myometrium fiom the upper fûndal and the lower segment in nonpregnant,

pregnmt, labouring and postpartum.

4. To determine whether CRH-RI mRNA andor C M - R 2 are expressed in human

decidua, chorion and amnion,

5. To determine ZCRH-RI &A and/or CRH-R2 mRNA expression was increased

at the time of labour in the decidua, chorion and amnion..

CRH receptors are abundantly expressed in rat brain and rat pituitary (Wynn et

al., 1983; Agudera et al., 1987). Extensive studies have been done in the rat brain and

pituitary to determine the factors regulating pituitary and brain CRH receptor

expression (Makino et al., 1995; Pozzoli et al., 1996; Sakai et al., 1996). CurrentIy, no

studies have examined the factors regulating the expression of CRH receptors in the

myometrium. Determinhg the endogenous factors regdating the transcription,

translation and /or postranslationd expression of CRH receptors in the myometrium is

a prerequisite to establish the role of CRH in pregoancy. Because of the obvious

diniculties of performing these studies in vivo it is crucial to identfi a mammalian

mode1 that expresses CRH-RI mRNA a d o r CRH-R2 mRNA in the myometrium.

The goal of our study was to establish a marnmalian mode1 to study the

regdation of CRH receptor expression in the myometrium in pregnancy, in labour and

post-partum. Based on the identical structure of the human and the rat CRH receptors

and the simila. bioactivity of human and rat CRH peptide we hypothesized that CRH-

R1 mRNA and CRH-EU mRNA expression would be increased at the time of labour in

rat myometrium.

Our specific aims were:

1. To detennine if CRH-RI mRNA and CRH-R2 mRNA are expressed in the rat

myometrium fiom day 15 of gestation to 1 day postpartum.

2. To determine if CRH-RI mRNA and CRH-R2 mRNA expression is increased in rat

at the time of labour.

3.1. INTRODUCTION

The human uterus differentiates into an actively contracthg upper segment and a

relatively quiescent lower segment at the time of labour (Pritchard et al., 1985). The

factors involved in this anatomic functional Merence of the uterus remain unclear, CRH

is a 41 amho acid peptide hormone, that has both a priming and potentiating effect on

myometrïal contractility (Quartero and Fry, 1989; Benedetto et al., 1994). Two distinct

subtypes of CRH receptors, CM-RI and CRH-R2 have been isolated and characterïzed

(Chen et al., 1993; Vita et al., 1993; Lovenberg et al., 1995; Liaw et al., 1996). The

cloned cDNA of CRH-RI was isolated fiom human pituitary (Chen et al., 1993) and rat

brain (Perrin et al., 1 993). Recently, the cDNA of CRH-R2 was isolated fkom rat

(Lovenberg et al., 19951; 1995b) and human (Liaw et al-, 1995; Valdenaire et al., 1997)

and encodes two anatomically distinct spliced forms of the receptor (Lovenberg et al.,

1995a; 1995b).

The human placenta secretes increasing concentrations of hCRH-pl into the

materna1 circulation in the third tnmester of pregnancy (Goland et al., 1987 reaching a

peak at the time of labour (Okamoto et al., 1989; McLean et al., 1995). Recently CRH-

RI mRNA and CRH-R2 mRNA have been idenaed in the myometnum of pregnant

women (Rodriguez-Linares et al., 1998). This strongly suggests that CRH may play an

important role in human parturition. We hypothesized that the expression of CRH-RI

mRNA a d o r CRH-R2 mRNA expression and protein concentrations in the

myometrium would be upreguiated at the time of labour in human pregnancies. We

examined the expression of CM-R1 mRNA and CRH-R2 mRNA and their respective

proteins in human rnyometrium in term and preterm pregnancies prior to at the t h e of

labour. Moreover, we disthguished between myometrium collected fiom th: upper

segment and nom the lower segment of the utem. Finally we examined the level of

expression of CRH-RI mRNA and CRH-W mRNA in the decidua and fetal

membranes in term pregnancies prior to and at the time of labour so to establish if the

regulation of CRH receptor expression at the time of labour was specific to the

myometrium.

3.2.1 Biopsy Samples

Myometrial samples were collected fiom 2 groups of women: a) nonpregnant

and b) pregnant. In the nonpregnant, premenopausal women (age 42 f 1.5 years; n = 4)

the myometrid samples were collected fiom the lower region of the uterus at

hysterectomy, performed due to fibroid invasion. Pregnant patients (age 3 1 + 2.4 years),

were divided into the following groups: preterm no labour (mean gestational age

(MGA) = 32 weeks; n = 3, PTL (MGA = 32 weeks; n = 6), terni no labour (MGA = 39

weeks; n = 7) and term in labour (MGA = 39 weeks; n = 7). The myometrial samples

were collected h m the incision Iule withui the lower segment. Regional myometrial

samples were also collected fiom the both the upper and the lower segment of the

utenis collected in the nonpregnant patients (aged 40 + 1.3 years; n = 4) at

hysterectomy performed as a result of fibroids. Upper segment and lower segment

myometrial samples were also collected fiom subjects (aged 32 k 2.1 years; a = 4) at

elective cesarean followed by a hysterectomy performed for progressive cervical cancer

and in active labour (aged 3 5 years; n = 1 ) at a classical cesarean section performed due

to remove conjoint twins. Finaily, a myometrîal sample was couected fkom the upper

and the lower uterine segment at a hysterectomy performed post-parturn [aged 34

years; n=l) for disseminated intravascdar haemorrhage. The pregnant patients were

not admùiistered giucorticoids or oxytocin. All tissue samples were immediately snap

frozen in liquid nitrogen and stored at -80°C.

Decidua (n=8), chorion (n = 4) and amnion (n = 4) were coliected fiom term

patients in the absence (MGA = 39 weeks) and the presence of labour (MGA = 39

weeks). The individual chorion and the decidua were separated by gentle scraping and

immediately individually snap fiozen in liquid nitrogen. Al1 tissue biopsies were

coliected at Mount Sinai Hospital in Toronto, Ontario. informed consent was obtained

&orn al1 the patients and ethical approval for the study was obtahed nom the Bioethic

Cornmittee of Mount Sinai Hospital and the University of Toronto.

To obtain positive and negative control tissues virgin fernale Wistar rats (250-

280 grarns; Charles River Canada, St. Constant, Quebec, Canada) were decapitated and

piNitaries and liver, respectively. Sheep pituitarîes were also coUected fkom fetuses at

term delivery (&y 145 gestation) following euthanasia with Euthanyl (5 mL) via

cardiac puncture to use as positive controls. AU tissue samples were immediately snap

fi-ozen in liquid nitrogen and stored at -80°C.

Al1 animal experimental protocols were approved by the Samuel Lunefeld

Institute Animal Care Cornmittee and Animal Care C o d t t e e of the University of

Toronto according to the Guidelines of the Canadian Council of Animal Care.

3.2.2 Total EWA Extraction

Total RNA was extracted fiom the samples of myometrium, decidua, chorion,

and amnion using the methods desmibed by Chomcynski and Sacchi (1987). Bnefly,

fkozen tissue samples (2- 5 mg) were powdered under liquid nitrogen and homogenized

in 1 mL of a denaturing solution (4M guanidinium thiocyanate, 25 m M sodium citrate,

0.5% sodium lauroylsarcosine, 0.1M LJ-mercaptoethanol (v/v)) using an ULTRA-

TübUUC homogenizer (Janke & Henkel, IKA-Labortechnik, ON, Canada). Sodium

acetate b&er (0.1 mL of 2M ; pH 4) was added to the tissue homogenate, followed by

phenol (water-saturated) ( 1 ml) , and a chloroform-isoarnyl alcohol mixture (0.2 mL ;

49: 1). Each addition was foilowed by thorough rnixing . The samples were incubated on

ice for 15 minutes then cenaifuged (Sorvall RC-SB, Du Pont Instruments, MA, USA)

at 6,500g for 40 minutes at 4OC. The supernatant was tmnsferred to a fresh

polypropylene tube (12 mL; Becton and Dickinson, New Jersey, USA) and mixed with

isopropanol(1 mL) and incubated for 1 hou at -20°C to allow RNA precipitation. The

samples were centrifuged again at 6,500g for 1 hour 4°C. The redting RNA pellet was

dissolved in the same denaturing solution (see above; 1 mL), transferred to an

eppendorf tube (1.5 mL, Diamed, Ont, Canada) and precipitated with an equal volume

of isopropanol ovemight at -20°C. The samples were then centrifbged for 15 minutes at

4°C. The RNA pellet was resuspended in 70% ethanol, (1 mL) vacuum dried and

redissolved in double distilled water (ddH20) with 0.1% diethylpyrocarbonate (DEPC)

water. The total RNA purity and recovery for each sample was determined with a UV

spectrophotometer (Mode1 DU-64, Beckman Instnunents. Inc., CA, USA) at 260 and

280 nanometers.

3.23 Reverse Transcription Poiymerase Chain Reaction (RT-PCR)

Total RNA fkom myomeûium, decidua, chorion, amnion, rat liver and rat

pituitary was converted by reverse transcription into cDNA. The reverse transcription

reaction mix consisted of 1 pg of total RNA, 1 x PCR b s e r (10 mM Tris-HC1,50 m M

KCI, Perkin Eimer, Cetus), 5 mM MgC12 (Perkin Elmer, Cetus), 1 m M each of the

dNTP (dATP, dCTP, dGTP, dTTP, Pharmacia), 5 ng/ pL random (Pharmacia), 1 U/ pL

Mase inhibitor (Boeh~ge r Mannheim) and lOOU Moloney Murine Leukemia V i m

Reverse Transcriptase (MMLV-RT; GibcoBRL, Gaithersburg, MD) in 21 pL DEPC

water. Negative controls were prepared as above but without RNA, to cab there

was no contamination of the PCR reagents. The reaction mixture was incubated at

25°C for 10 minutes, then at 42OC for 30 minutes and finally at 99OC for 5 minutes.

The resuitant cDNA (reverse transcriptase; RT) mixture was stored at -20°C until it

was used.

PCR was performed using the RT mixture. The PCR mixture consisted of 10

pL of the RT mixture, 0.25 mM dNTP, 50 ng of each specific PCR primer (ACGT

Corporation, Toronto), 2.5 U Taq polymerase (Boehringer Mannheim) in 1.25 mM

MgCl,, 50 mM K I , 10 mM Tris-HC1 (pH 8.3) in 24.5 jL DEPC water. Each PCR

reaction undenirent an amplification regime characterized by a pre-incubation stage

(95OC, 5 minutes), a denaturation stage (94°C. 30 seconds), a primer annealing stage

(62OC, 30 seconds), an extension stage (72OC, 30 seconds) and a long extension stage

(72OC, 8 minutes) in a thennal cycler (MJ Research Inc, Mass, USA). PCR reactions

were also performed using RNA that had not been reverse transcribed to establish the

extent of genomic DNA contamination in the RNA. PCR products were

electrophoresed on a 2 % agarose gel, stained with 1.5 % ethidiurn brornide in Tris-

acetatd EDTA b a e r to allow visualization of the PCR product under a

transilliuninator (Lighttools Research, On& Canada).

Specific primers were used to identi& a 333 base pair (bp) product for CRH-RI

in human myometrium, decidua, and fetal membranes (Slorninski et al., 1995). A sense

primer 5' GCC CTG CCC TG€ CTT ï"TT CTA 3' and an antisense primer 5' GCT

CAT GGT TAG CTG GAC CA 3' corresponding to positions 235-255 and 549-568,

respectively were used (Accession number L23 332) (Chen et al., 1993). Similady,

primers were designed to identifjr a 781 bp product for CRH-R3 in human

myometrium, decidua and fetai membranes. A sense primer 5' GGC ATC AAG TAC

AAC ACG AC 3' and an antisense primer 5' CAT CCA GTA CAG GAA GGC AG 3'

corresponding to positions 423-442 and 1 184-1 204, respectively, were used (Accession

number U 1 6253) (Lovenberg et al., 1 995a). p-actin gene expression (intemal control)

was also determined in d sarnples to assess the integrity of the RNA. Prirners were

designed to idenhfy a 2 18 bp product for eactin in ail the samples. A sense primer 5'

AAG AGA GGC ATC CTC ACC CT 3' and an antisense primer 5' TAC ATG GCT

GGG GTG T G AA 3' correspondhg to positions 222- 241 and 420- 439, respectively

were used (Accession number M10278). The designed primers do not flank intron

segments.

3.2.4 Semi-Quantitative PCR

Further anaiysis using semiquantitative PCR (sq-PCR) was performed to compare

myornetrium obtained after different treatments (Kinoshita et al., 1992). To obtain

measurements in the Linear range of C M - R I cDNA we used 28,30, and 32 cycles and

in the decidua and fetal membranes we used 31, 33 and 35 cycles. To obtain

measurements in the linear range of CRH-W cDNA in human myometrium we used

3 1,33, and 35 cycles. The cycles used for p-actin were 16, 18,20. The relative intensity

of cDNA signals was quantified fiom negatives using a computerized image analysis

(Imaging Research Inc., St. Catherine, Ont, Canada).

The identity for the PCR product for CRH-RI was c o b e d using LOU of the

restriction enzymes Alu 1 (GibcoBRL) and BSR 1 (New England Biolabs) in the

appropriate b&er (10 PL) Restriction bufTer A, (GibcoBRL) and B e e r 3 (New

England Biolabs). The PCR product (0.5 - 2 pg cDNA) was incubated at 37'C (Alu 1)

or at 65°C @SR I) for 2-3 hours. The Alu I enyme digest was inactivated at 65°C for

10 minutes then products were nui on a 2% agarose gel to allow visualkation.

33.5 Immunohistochemistry

Myometnal samples from nonpregnant patients (n = 4) and term pregnant

patients in the absence (n = 4) and the presence of labour (n = 4) were removed and

k e d in 4% parafomaldehyde and 0.2% glutaldehyde for 24 hours following tissue

collection. The samples were then washed twice daiIy in phosphate-buEered saline

(PBS, 0.01 M, p H 7.4) for t h e days and stored at in 70% ethanol at 4°C. The samples

were embedded in par& by the Pathology labs at Toronto General Hospital

(Toronto, Ontario, Canada). Sections (5 pm) were cut on a microtome (Leica RM

2035, Nussloch, Gennany) and placed on glass slides coated with 2%

ambopropyltnethoxy-silane (APTEX; Sigma Chernical Co., Missouri, USA) in

acetone and dried for 24 hours at 37°C. The slides were deparafnnized with 3 washes

of xylene substitute (EM Diagnostic Systems, NJ, USA) for 5 minutes each, then

rehydrated in a series of washes 2 minutes each, in 100% ,95%, 70% and 50% ethanol,

followed by washing in 0.01 M PBS (pH 7.4). Endogenous peroxidase activity was

quenched by incubation in 3% hydrogen peroxidase (in PBS) followed by incubating

the sections with 10% normal rabbit s e m to inhibit nonspecific staining.

The slides were incubated with the primary antibodies for CRH-RI and CRH-

R 2 at 4°C ovemight. The primary antibody for CM-RI (Santa Cruz Biotechnology

hc., CA, USA). was a polyclonal antibody raised in a goat against a peptide

corresponding to amino acids 425- 444 of the human and rat CRH-RI (Chen et al.,

1993). The primary antibody for CRH-R2 (Santa Cruz Biotechnology Inc., CA, USA)

is a poiyclonal antibody raised in a goat against a peptide corresponding to amino acids

47-66 of the rat CRH-R;! Covenberg et al., 1995). The CRH-RI antibody cross-reacts

with CRH-R2. Therefore, in some sections we pre-absorbed the CRH-RI d b o d y with

an excess of CRH-R2 peptide (1 pM) (Santa Cruz Biotechnology inc., CA, USA) to

determine CRH-R1 stainuig. The primary antibodies were diluted to 1: 175 in antibody

dilution bmer (lg bovine senun albumin, 0.02 g sodium azide in 100 mL PBS; pH

7.5). M e r 16 hours of incubation with the primary antibody, the slides were washed

twice in PBS for 5 minutes each and incubated with biothylated secondary antibody

(1 : 500; Vectastalli ABC kit; Vector Laboratones, CA, USA) at room temperature for 2

hours. The sections were washed twice in PBS for 5 minutes and incubated with the

avidïn-biotin-peroxidase complex (ABC; Vectastain) in PBS for 2 hours at room

temperature. To visualize antibody staining the sections were incubated with 3',3-

diaminobenzidine tetrahydrochloride dihydrate (DM; Sigma Chernical Co., Miss,

USA) for 10 minutes. nie DAB solution was made by dissolving 50 mg of DAB in

200 mL PBS and adding 2 drops of hydrogen peroxide Mmediately prior to use. The

slides were washed in ddH20 water, counterstained with Carrazi's hematoxylin for 45

seconds, rinsed in ddH20, dehydrated in an alcohoi series; 50%, 70%, 95%, 100% for 2

minutes each and incubated in xylene for 3 washes of 5 minutes each. The slides were

then mounted with Permount (Fisher Scientific, Ontario, Canada) and viewed with a

microscope (Leica, DMRB, Nussloch, Gemany). Sheep pituitaries were used as

positive controls for CRH-RI. For the negative controls, the primary antibody was

preabsorbed with synthetic receptor peptide (1 pM; Santa Cruz Biotechnology Inc.,

CA, USA).

3.2.6 Protein Extraction

Protein was extracted fiom tissue with the use of RIPA lysis buffer. H m a n

myometrial tissue (n=8 term pregnancy and n=7 at the time of labour), rat brain and rat

heart (positive controls) and rat iiver (negative control) tissue (1-2 grams) was ground

in a pestle placed on dry ice. The ground tissue was transferred to a fiesh

polypropylene tube (12 mL; Becton and Dickinson, New Jersey, USA) and pulverized

(Janke & Henkel, MA-Labortechnik, ON, Canada) for approximately 30 seconds in

RIPA lysis buffer (50 mM Tris-HC1; pH 7.5, 150 m M NaCl, 1% Triton X-100; v/v, 1%

sodium deoxycholate; w/v, 0.1% sodium dodecyl sulphate (SDS); w/v, 100 p M Na

orthovanadate, 100 pg/pL leupeptin, 1 rnM phenyhethyl-sulfonyl fluoride while

rnaintained on ice. The tissue homogenates were spun in a microcentrifuge (Sorvall

RC-SB, Du Pont Instruments, MA, USA) at 8,500g for 15 minutes at 4OC. The

supernatant was coliected, aliquoted into eppendorf tubes (1.5 mL, Diamed, Ont,

Canada) and stored at -80°C until used.

Prior to use, a sample (10~1) of the protein was assayed to determine

concentration with a bicinchoninic acid (BCA) protein assay kit (Pierce, Illinois, USA).

A fiesh set of standards ranging fiom 200 pg/ mL to 3 pg/ 2 mL were made by diluting

2 m g l d bovine serum albumin @SA) stock standard in BCA working reagent (50

parts BCA reagent A with 1 part BCA reagent B). The standard or protein sample (10

pl) where added to the working reagent (same as above; 2 mL), in a borosillicate tube

and thoroughly mixed. The tubes where then maintained (45 minutes) in a water bath

(37°C). The protein in the tubes was meanired at absorbance 562 nm versus a water

reference. A standard c w e was prepared by plotting the 562 nm reading for each BSA

standard versus its concentration in pg/mL and used to determine the protein

concentration of the extracted samples.

3.2.7 Western Btots

Western blot gels where made using a miniprotean II ceIl (Bio-Rad. California

USA). A 10% seperating gel mixture was made in an erlmeyer flask with 1. 5 M Tris-

HCl (pH 8.8; 5 mL), 10% SDS, acrylamide/bis (37.5: 1; 3.2 mi.) and ddHzO (4 mL)

was deaerated for 15 minutes. Then* 10% fresh ammonium pesulfate (50 PL) and

N,N,N7&,-temethylenediamine (10 FL; Biorad, Clif, USA) were added. the gel was

poured and lefi to polymerise for 1 hour with a dW20 overlay. A 4% stacking gel was

prepared from 0.5 M Tris-HC1 (pH 6.8; 2.5 mL), acrylamidehis (37.5: 1;l.j mL), and

ddH20 (6.1 mL) and was deaerated for 15 minutes. Immediately afier 10% fresh

ammonium peadfate (50 PL) and N,N.N9,N,-tetramethylenediamine (10 PL) were

added, the gel was poured on top of the stacking gel, a 10 or 15 well comb was

inserted. The mixture was left to polymerise for 1 hour.

An aliquot of the protein samples was thawed and diluted with 0.5 M Tris-HCl

(pH 6.8) to a fmal concentration of 10 pglpL. The protein (100 pg) fiom each sample

was mked with 10 pL of loading b a e r (Tris-HCI; 0.15 g, glycerol; 1 mL, 1 O%SDS; 4

mL, 2-P-me~ptoethanol, 20 mg bromophenol blue and ddHîO; 4 mL) in an eppendod

(1.5 mL, Diarned Ont, Canada). The samples/Ioading b a e r where heated at 9S°C, in a

waterbath, for 4 minutes and cooled at room temperature. The gel apparatus was placed

into the b a e r chamber and the electrode buffer (Tris base; 9 g, glycine; 43.2 g, SDS; 3

g and ddH20; 600 mL) added. The wells were loaded and the gel was run at 100 volts

for 2 hours with a prestakted protein marker (Broad range, New England Biolabs). The

gel was transferred at 15 volts for 30 minutes at room temperature onto nitrocellulose

blotting membrane. The blot was washed 9 X 10 minutes) in ïTBS (1 50 m M NaCl and

50 m M Tris-HCI) with 0.05% Tween-20 and incubated in 4% BSA in TTBS with

0.05% Tween-20 for 30 minutes. The blots where incubated in the primary antibodies

for CRH-RI (same as above) and CRH-R2 (same as above) at 4OC overnight. The

primary antibodies were used at a range of dilutions (1 200 - 12000) to establish the

optimal antibody dilution.

The blot was washed (3 X 10 minutes) in TTBS with 0.05% Tween-70 and

incubated with biotinylated secondary antibody (1 : 1000 in TTBS; same as above) for 2

hours. The blot was washed again and incubated in the avidid biotin complex (1 : 1000

in Ti"E3S; Vectastain) for 2 hours. Finally the biots were developed in 12 mg DAI3

(Sigma Chernical Co., Miss, USA) in ddH20 from 30 seconds to a maximum of 5

minutes to ensure maximal staining of the blot.

3.2.8 Data Analysis

To correct for clifferences in the initial amount of RNA used for RT-PCR we

detemiined O-acM mRNA expression in al1 the samples. The ratio of the optical

densitometry reading measurements for the expression of CRH-RI mRNA or CRH-R2

mRNA (at 3 progressive amplincation cycles) to that of B-actin mRNA expression (at 3

progressive amplification cycles) was detennined for each sample. The mean of the

ratio was determined for each treatment from al1 the samples within that group. A

Mann-Whiîney Rank S u n Test was performed to judge the change in CRH receptor

expression at the rime of labour iri term and then in pretenn prepancies. To detemine

the difference amongst al1 the treatment groups (PTL, no labour, PTL in labour, term no

labour and term in labour) a Kruskal-Wallis One Way Analysis of Variance (ANOVA)

on Ranks was performed followed by an Al1 Painnse Multiple Cornparison Procedure

(Dunn's Method) to isolate the group or groups that differ f?om the othen.

3.3.1 C M Receptor Expression in the Myornetriurn of Nonpregnant Patients

Enzyme digestion of CRH-RI was used to confirm the identity of the receptor

(Figure 3.1) and yielded fragments of the expected size. Digestion with BSR I yielded a

244 bp product and digestion with Alu 1 produced a Both the expression of CRH-RI

rnRNA and CRH-R2 mRNA were present in the myometrium obtained fiom the non-

pregnant hysterectomy patients Figure 3.2). We identified the expected band of 333 bp

representing CRH-R1 (maximally expressed after 32 cycles) and the expected band of

781 bp representing CRH-R;! (maximally expressed after 35 cycles). CRH-RI

mRNAwas present at consistently high levels in di the samples studied whereas CRH-

R2 mRNA expression was variable. When using primers designed to idente CM-W

we observed a second band at 500 bp. We suggest this band represents a spiiced form

of the receptor. Bactin &A (maximally expressed after 20 cycles) expression was

present at similar levels in ai i the patients.

Both the CRH-RI and CRH-R2 protein were localized in the circular and

longitudinal smooth muscle bundles of the human myometrium (Figure H A and

Figure XJA, respectively? and in the smooth muscle of the myometrial vasculature

(Figure 3.5A and Figure MA, respectively). CRH-RI and CRH-R2 staining was

preabsorbed f?om the uterine smooth muscle (Figure M C and Figure 3 . K ) and nom

the smooth muscle of the vasculanire (Figure X5C) with an excess of peptide (1 FM).

CRH-RI was present in the sheep pituitary (Figure 3.7A and Figure 3.7B) and the

staining was pre-absorbed with CRH-RI peptide (Figure 3.7C).CRH-R2 was not

detected in the pituitary (Figure 3.7D). CRH-RI and CRH-R2 were not detected in the

human myometrium, the rat heart, brain and liver by Western blot.

3.3.2 CRH-R1 mRNA and CRH-R2 mRNA Expression in Myometrium Pregnant

Patients

We identified C M - R I mRNA expression in rnyometrium from the lower

segment of women in tenn and preterm pregnancies (Figure 3.8). CRH-R1 mRNA

expression was amplified at 28, 30, and 32 cycles in the sampies from term and preterm

myometrium to obtain linear measurements in the expression of the gene. CRH-RI

rnRNA expression was significantly upregulated @ < 0.03; Mann-Whitney Rank Sum

Test) at the time of labour in term pregnancies (MGA= 39 weeks; Figure 3.9). CRH-Rl

M A expression was also signincantiy upregulated @ c 0.01 ; Mann-Whitney Rank

Sum Test) at the thne of labour in preterm pregnancies (MGA= 32 weeks; Figure 3.10).

CRH-RI mRNA expression showed a trend towards rishg fYom 32 weeks to 39 week's

gestation and rose signincantly at the time of labour (p < 0.05; Al1 Painÿise Multiple

Cornparison Procedures) (Figure 3.1 1). CRH-RI protein was undetectable in human

myometrium at term (Figure 3.3B and Figure 3.5B) but was detected in the uterine

smooth muscle and the smooth muscle of the vasculanire at the time of labour (Figure

3.3D and Figure 3.5D).

We identified CRH-R2 mRNA in the myometnum of some (28%) of the

pregnant patients. We did not fïnd a significant change in CRH-R2 mRNA expression

at the time of labour (Table 3 .l) In the preterm pregnancy, CRH-R2 was present in the

myometrium of 3 of the patients not in labour (n = 5) and in 2 of the patients in labour

(n = 6 ). In term pregnancy CRH-R2 mRNA was not detectable in the myometrium of

the patients not in labour (n = 7) but was present in 2 of the patients in labour (n = 7).

CRH-R2 protein was not detectable in the myometrium of term patients (Figure 3.43

and Figure 3.6B) but was present in the myometrium of pregnant in labour patients

(Figure 3.4D and 3.6C) at the antibody concentrations used.

3.33 Regional Expression of CM-R1 in Myometrium From Noopregnant,

Pregnant, Pregnant in Labour and Post-Partum Patients

Samples were taken from the upper and the lower segments of the uterus to

examine differences in the regionai distribution of C M - R I in the myometriurn. CRH-

RI mRNA expression was present in the upper and lower segment of human

myometrium in nonpregnant (n = 4), pregnant (n = 4), pregnant in labour (n = 1) and in

post-parhm (n = 1) women (Figure 3.12) C M - R I mRNA expression was present at

simiiar levels in myometrium fiom the upper fundal and the lower segment of the

utenis nonpregoant and pregnant women as determined CRH-RI rnRNA/ B-actin

mRNA expression. CRH-R 1 mRNA expression was ap parent1 y higher in my omeûiurn

fiom the lower segment when compared to the upper segment in the at the time of

labour. CRH-RI mRNA expression was apparentfy decreased in the Lower segment

post-pamun and returned to levels sirnilar to that in the nonpregnant patients (Figure

3.12). CRH-RI mRNA expression appears to stay relatively constant in myomeniurn

fiom the fundal segment in the nonpregnant, the pregnant, the labouring and the post-

partum patients.

33.4 CRH-R1 mRNA Expression ui the Fetal Membranes and Decidua

CRH-KI mRNA expression was present in the decidua and the chorion but was

not significantly altered at the time of labour as determîned by anaiysis of sq-PCR

CRH-R 1 mRNA/B-actin rnRN A expression (Figure 3.1 2). C M - R I mRNA expression

was undetectable in the amnion. CRH-R? mRNA expression was undetectable in the

chorion, the amnion and the decidua after 40 amplification cycles.

3.4 DISCUSSION

Using RT-PCR we have shown that CRH-RI mRNA and CRH-R2 mRNA expression

and their proteins are present in the myometnum of both non-pregnant and pregnant

women. CRH-RI mRNA expression is signincantly upregulated at the time of labour

in both pretem and texm pregnancies. The nse in CRH-RI mRNA expression appeared

to be specific to the lower segment of the utem and disappeared immediately

postparhim. CM-R2 mRNA expression was not significantly changed and at the time

of labour in both term and pretem human pregnancies. CRH-RI protein was prevalent

in the myometrium fiom nonpregnant women, was undetectable in term pregnancy and

was iacreased at the t h e of labour in the uterine smooth muscle and the uterine

vascdature. CRH-R2 protein was present in the smooth muscle and the vasculature of

the uterine vessels in the nonpregnant myometrium, was undetectable in term

pregnancy and was modestly increased only in the smooth muscle at the time of labour.

The "classical section", a vertical incision into the body of the utenis fiom the

lower uterine segment to the upper fundal segment of the uterus is rarely used

(Pritchard et al., 1995), therefore making it difficult to obtain samples fiom the active

region of the uterus. Most always the incision is made in the lower uterine segment.

Moreover, because the size and shape of the uterus varies with age and parity it is

impossible to collect rnyometrial samples at the same site in al l the patients. We only

used nonpregnant patients undergohg hysterectomy for fibroidal growth so as to

eliminate the possible effect of this condition on gene expression. However, the tissue

may not be representative of normal tissue. CRH-RI mRNA expression in the

labouring patients was variable, possibly due to cisering stages of labour andor the

duration of labour prior to the surgery however, we no Longer have access to this

information.

We have used PCR to detect the expression of CRK-RI mRNA and CRH-R?

mRNA. There are diffculties associated with the use of this technique for quantitative

analysis of gene expression. To allow us to quantitate gene expression we we have

obtained the linear expression of the CRH-RI and CM-W gene by gradually

increasing the number of amplification cycles. We have used the ratio of the optical

densitometry readings obtained within the linear range of expression of CRH-RI

M A / CRH-EU mRNA to P-actin mRNA. Whereas this method is only semi-

quantitative it is sensitive and effective method for comparing genes expressed at low

levels.

The low levels of CRH-RI mRNA expression and the undetectable levels of

CRH-RI protein in pregnancy suggests there is a decrease in the transcription of the

CRH-RI gene which is associated with a decrease in the translation of CRH-Ri mRNA

in the human myomeûium in pregnancy. It is important to note that the CRH-RI

mRNA may be present in the huma. myometrium at tenn pregnancy but at levels

below the detection limits of the antibody concentration used. The rise in CM-RI

mRNA expression at the t h e of labour could suggest an increase in the transcription

rate of the CRH-RI gene or an increase in the stability of CRH-mRNA at the time of

labour. The significance of the upregulation CRH-RI gene transcription is not known

however it appears to be restncted to the myometrïurn fiom the lower uterine segment

conclusions with regards to the levels of CM-RI protein in the human myomenium.

We suggest that, at the time of labour, CM-RI helps to preserve the quiescence of the

lower utenne segment. In this way the low decreased amplitude and intensity of

myometrial contractions wiU be maintained. TES proposal is consistent with CRH

acting through a CAMP second messenger pathway (Labrie et al., 1982; Aguilera et

al., 1987).

In contrast, reports by Quartero and Fry, (1989) report that CRH acts

synergistically with OT and PGF2= to potentiate myometriai contractility and that this

synergistic effect appears to be mediated via a fdi in CM levels in the lower uterine

segment ( Q ~ e r o et al., 1992). Benedetto et al., (1994) were unable to obtain CRH-

OT potentiated myometrial contractility, however they showed that only when

myometrial strips where pre-incubated in CRH they demonstrated enhanced

myometnal contractility when stirnulated with PGF?,. No contractility was observed

with PGE,. These studies suggest a role for CRH in potentiating myometrial

contractility and are supported by Grammatopoulos et al., (1996). These investigatos

suggested that there was a modification in the coupling between CRH receptors and

adenylate cyclase in term pregnancy. This resulted in a reduction of CRH-stimulated

CAMP production at a time when the CRH receptor was coupled to the cyclooxygenase

pathway (Grammatopoulos et al., 1994). WhiIe this is an atiractive theory to explain

the shift from the relaxed utem at term to the contracthg uterus at the time of labour,

the authors only measured PGEz concentrations in their myometnal membrane media.

PGE2 has the paradoxical ability to mediate relaxation or stimulation dependent on the

receptor nibtype present or if it is converted to PGF?, (Nelen and Breckwoldt, 1994).

The authors fail to explain why CRH receptors in the myomeaium of nonprepant

women pathway are coupled to the cyclooxygenase pathway and not the adenylate

cyclase pathway. This data suggests the cyclooxygenase pathway rnay be constitutve in

the myometrium and possibly not be a major component in the omet of human labour.

In al1 the above mentioned studies the authon have used myometrium from the

lower uterine segment. Our data suggests that the regulation of CRH-RI mRNA

expression in the lower segment at the time of labour does not appear to reflect the

regulation of CRH-RI mRNA at this time in the funda1 region. Differentiaily regulated

CRH-RI rnRNA expression within the human uterus at the time of labour suggests

CRH may mediate different roles in these regions. This brings into question the ability

to assess the role of CRH at the time of labour from samples collected solely in the

lower uterine segment.

Mon of the past snidies in the u tem myometrium have failed to differentiate

between the CRH receptor subtypes, we are the fust group to compare CRH-RI mRNA

and CM-R2 mRNA expression in the hurnan myornetrium at the time of labour. The

presence of heterogeneous CRH receptors in the myorneviurn is not a novel concept,

Grammatopoulos et al., (1995) had previously identified heterogenous populations of

CRH receptors in myometrium. We show that CRH-RI mRNA and CM-R2 mRNA

expression are differentially regulated at the time of labour suggesting that these

receptor subtypes may mediate distinct functions in the hurnan myornetrium at the tirne

of labour. Differential regulation of the CRH receptor subtypes has previously been

reported by Mansi et al., (1 996) and Makino et al., (1 997).

Little is known about CRH receptors in the myometrium of nonpregnant

women. We have detected the expression of CRH-RI mRNA and CM-EU mRNA in

myometrium O btained fkom non-pregnant patients at hy sterectomy . This is in

accordance with Hillhouse et al, (1993) who identified the presence of specific CRH

binding sites on human rnyometrium We have shown that CRH-RI mRNA is the

predominant receptor subtype in the lower region of the utenis, and that it is present at

similar levels in the fundus and the isthmus region. We have Iocalised CM-R1 and

CRH-R2 protein on the u t e ~ e smooth muscle and on the smooth muscle layer of the

uterine vasculature in the myometrium of nonpregnant patients. We are the ftrst to

identiQ both CRH-RI and CRH-W protein in the smooth muscle of the myomeaial

vasculature. Based on the vasoactive effects of CRH (Clifton et al., 1994) we suggest

CRH may be involved in the regulation of myometrial blood flow, however, the

physiological significance of CRH in the myometnum of nonpregnant women remains

to be determined.

In conclusion, CRH-RI mRNA and CRH-EU rnRNA expression was present in

the human myometnum in nonpregnant women and in pregnancy. CRH-RI mRNA

was significantly upregulated in myometrium fiom the lower uterine segment, at the

t h e of labour. Our hding of CRH recepton in myometrium from pregnant women, is

in accordance with reported roles for CRH in the myometrium however the function of

CRH receptors in the myometrium are still unclear and requires further studies.

Figure 3.1 Enzyme digestion to c o d k m the identity of CRH-R1 cDNA: A. original 333 bp prodùct, B. digestion with Ah 1 (244 bp), C. digestion with BSR 1 (277 bp).

Figure 3.2 Photograph of a PCR gel showing the detection of CM-RI mRNA (expected - band size 333 bp; after 32 cycles) and CRH-R2 mRNA (expected band size

781 bp; d e r 35 cycles) in human myometrium (n = 4) obtained at hysterectomy. A 500 bp band was present in association with the 78 1 bp band in the CRH-IU PCR reactions. The detection of pactin mRNA (2 18 bp), the intemal controi is shown.

Figure 3 -3 Photopph showing immunostaining for CRH-R1 protein in the human myornetfium smooth muscle (SM) and blood vessels (BV).Magnified 200x

A. Myometnum in the nonpregnant B. Myometrium in term pregnancy C . Myometnum in nonpregnant (negative conlrol- preabsorbtion with CRH-R 1 antibody) D. Myometrium at the t h e of labour.

Figure 3 -4 Photograph showing immunostaining for CRH-EU protein in the human myomenium smooth muscle (SM). Magaified 200x

A. Myometrium in the nonpregnant B. Myometrium in tenn pregnancy C. Myometrium in nonpregnant (negative control- preabsorbtion with CRH-R2 antibody) D. Myometrium at the time of labour.

Figure 3.5 Photograph showing immunostaining for C M - R I in the smooth muscle layer of the vasculahire (indicated by the arrow). Magnified ZOûx.

A. Myometrium in the nonpregnant B. Myometrium in term pregnancy C. Myomeaium in nonpregnant (negative control- preabsorbtion with CRH-RI antibody) D. Myometnum at the t h e of labour (Magnified 400x).

Figure 3.6 Photograph showing immunostaining for CRH-R2 in the smooth muscle layer of the vascdature (indicated by the arrow). Magnined 200x

A. Myometnum in the nonpregnant B. Myometrium in term pregnancy C. Myometrium at the t h e of labour.

Figure 3.7 Photograph of CRH-RI imrnunosiaining in the sheep pituitary.

A. C M - R I in the region adjacent to the intemediate lobe (IL)(Mapnified 200X) B. CRH-R1 is absent in the IL, (Magnined 200X) C. Preabsorbtion of CRH-RI antibody in the region adjacent to the IL (Magnified 200X) D. CRH-W is absent in the intermediate lobe (Magnined 400X)

Figure 3.8 Photograph of a PCR gel showing the detection of CRH-RI mRNA (333 bp; d e r 32 cycles) in the human myometrïum in pregnancy (NL) and at the time of labour (L). The detection of pactin mRNA (21 8 bp), the intemal control is shown.

A. CM-RI mRNA detection in term pregnancy (n=7) and at the time of labour (n=7). B. CRH-RI mRNA detection in pre-term pregnancy (n = 6) and at the time of labour (~5).

Figure 3.9 Analysis of sq-PCR for CRH-R1 mRNA (333 bp; 28,30, and 32 cycles), in term pregnancy, in myometrium, fiom the lower segment prior to (M,) and after (L) the omet of labour. CRH-RI mRNA expression is significantly increased @ < 0.03) at the time of labour @ < 0.03), in human myomeûium, in term pregnancy.

No labour Labour

No Labour Labour p .= OA13

Figure 3.10 Andysis of sq-PCR for CRH-RI mRNA (333 bp; 28,30, and 32 cycles) in myometriurn, fiom the lower segment, pnor to (NL) and after (L) the onset of labour. CRH-RI mRNA expression is significantiy increased @ < 0.0I)at the time of labour, in the human myometnum, in pre-term pregnancy.

CRH-RI

No labour Labour

1 +- 333 bp

No Labour Labour

Figure 3.1 1 CRH-RI mRNA expression in the human myometrium increases fiom 32 weeks (n = 5) to 39 weeks (n = 7) of gestation and rises M e r to similar levels at the time of labour in pre-term (n = 6) and term (n = 7) pregnancies.

Figure 3.12 Photograph of a PCR gel showing the detection of CRH-RI mRNA (333 bp; after 32 cycles) in human myometrium from the funcial (F) and lower (L) segment of the human utem. CRH-R I mRNA expression is shown in the nonpregnant (n = 4), the pregnant (n = 4), the labouring (n = 1 ) and the postpartum (n = 1 ) patients. Analysis of sq-PCR for CM-RI in human myometrium fiom the F and L segment of the human utenis in the nonpregnant, the pregnant, the Iabouring and the postpamim patients. The detection of P-actin mRNA (218 bp; the interna1 control) is shown.

Figure 3.13 Anaiysis of sq-PCR for CRH-RI mRNA (333 bp; 3 1,33, and 3 5 cycles) in the chorion (n = 4)and the decidua (II = 4) in term pregnancy prior to (NL) and &er (L) the omet of labour.

No labour Labour Na labour Labour Chorion Decidua

NOTE TO USERS

Page(s) missing in num ber only; text follows. Page(s) were microfilmed as received.

UMI

CHAPTER 3: THE RAT- A SUITMU3 MODEL TO STUDY THE

REGULATION OF CRH RECEPTOR EXPRESSION IN

THE MYOMETRIUM?

4.1 INTRODUCTION

We and othen (Rodriguez-Linares et al., 1998) detected CRH-R1 mRNA and

CRH-R2 mRNA expression in the myometrium h m pregnant women. The presence of

CRH receptoa in the myometrium suggests CRH may be of physioiogical importance in

the initiation a d o r progression of human parturition. However, the mechanisms

involved in the regulation of CRH-RI mRNA and CRH-EU mRNA expression in the

myometrium remain unknown. In an effort to further our understanding of CM-mediated

responses, extensive studies have been conducted, in the rat to delineate the factors

involved in the regulation of CRH receptor expression in the pituitary (Wym et al., 1984;

Luo et al., 1995; Sakai et al., 1996) and the brain (Makino et al., 1995; Imaki et al..

1996).

The specific aim of this study was to establish a mamrnalian model in which the

regulation of CRH-RI mRNA and CRK-EU mRNA expression in the myomeû-ium

during pregnancy and at the time of labour could be snidied, in view of the presence and

distribution of remarkably similar CEU receptors in the hurnan and the rat (Perrin et al.,

1995, Liaw et al., 1995; Valdenaire et al., 1997) and the common pharmacological and

secondary messenger pathways of the human and rat CRH receptors, we suggested that

the rat could be a suitable model to snidy the regulation of

CRH receptor expression in the myometrium. We hypothesized that CM-RI mRNA

andor CRH-R2 rnRNA would be upregulated at the time of labour. The objectives of

the study were to determine whether CM-RI mRNA and CRH-R2 mRNA were

expressed in the rat myornetrium and to examine whether the expression of CRH-R1

mRNA andfor CRH-Et2 mRNA was altered at the time of labour. Measurernents were

made relative to Cx 43, mRNA because the increased expression of diis gap junction

protein has been weiI characterized (Lye et al., 1993; Chow and Lye 1994).

4.2 MATERIALS AND METHODS

4.2.1 Animals

Virgin fernale Wistar rats (250- 280 grams; Charles River Canada. S t Constant.

Quebec, Canada) were mated, and the day of the appearance of the vaginal plug was set

as 1 day post-coitum. The animais were housed individdy, under standard conditions

(14 hours lightness and 10 hours darkness; at 22OC) and fed Purina Lab Chow

(Ralston, Purina, , MO, USA) and water ad l i b i ~ m . These animals routhely gave binh

on the moming of day 23. The rats were decapitated and the myometrium removed on

day 15 (n = 4), day 20 (n = 9, day 31 (n = 4), day 22 (n = 4), labour (n = 6) and on 1

day post-partum (n=4 ). Rat pituitary and liver were also collected. Al1 tissue sarnples

were immediately snap fiozen in liquid nitrogen and stored at -80°C.

Al1 experirnents were approved by the Samuel Lunefeld Institute Animal Care

Cornmittee at the University of Toronto according to the Guidelines of the Canadian

Council on Animal Care.

4.22 Total RNA Extraction

Totai RNA was extracted nom the samples of rat myometrium, pituitary and

liver using the methods descnbed by Chomczynski and Sacchi (1987). Briefly, kozen

tissue samples (2- 5 mg) were powdered under liquid nitrogen and hornogenized in 1

mL of a denaturing solution (4M guanidinuim thiocyanate, 25 mM sodium citrate,

0.5% sodium lauroylsarcosine, O.IM f3-rnercaptoethanoi (v/v)) using an ULTRA-

TLlRAXX homogenizer (Janke & Henkei, IKA-Labortechnik, ON, Canada). Sodium

acetate b e e r (0.1 mL of 2M ; pH 4) was added to the tissue homogenate followed by

of phenol (water-saturated) (1 id), and by a chloroform-isoamyl alcohol mixture (0.1

mL ; 49:l). Each addition was followed by thorough rnixing. The samples were

incubated on ice for 15 minutes then centrifùged (Sorvall RCdB, Du Pont Instruments.

MA, USA) at 6,500g for 40 minutes at 4°C. The supernatant was transferred to a fresh

polypropylene tube (12 rnL; Becton and Dickinson. New Jersey, USA) and mixed with

isopropanoI(1 mL) and hcubated for 1 hour at -ZO°C to allow RNA precipitation. The

sarnples were centrifuged again at 6,500g for I hour 4OC. The resulting RNA pellet \vas

dissolved in the denaturing solution (1 mL), transferred to an eppendod tube ( 1.5 mL,

Diarned, Ont, Canada) and precipitated with an equal volume of isopropanol ovemight

at -20°C. The samples were then centrifuged for 15 minutes at 4OC. The RNA pellet

was resuspended in 70% ethanol, (1 d) vacuum dried and redissolved in ddH.20 with

0.1% DEPC water. The total RNA purïty and recovery for each sample was determined

with a W spectrophotometer (Mode1 DU-64, Beckrnan Instruments, Inc., CA, USA)

at 260 and 280 nanometers.

Total RNA fiom rat myometrium, piniitary and liver was reverse transcribed

into cDNA. The reverse transcription reaction mix comisted of 1 pg of total RNA, 1 x

PCR b a e r (10 mM Tris-HCI, 50 mM KCI, Perkin Eher, Cetus), 5 mM MgC12

(Perkin Elmer, Cetus), 1 mM each of the dNTP (dATP, dCTP, dGTP, dTTPy

Pharmacia), 5 ng/@ random hexamers (Pharmacia), 1 U/ pL RNase inhibitor

(Boehringer Mannheim) and 100 U MMLV-RT (GibcoBRL, Gaithersburg, MD) in 21

pL DEPC water. Negative controls were prepared as above but without RNA. The

reaction mixture was incubated at 25°C for 10 minutes, then at 42OC for 30 minutes

and finally at 9g°C for 5 minutes. The resultant cDNA RT mixture was stored at -

20°C until it was used.

PCR was performed using the RT mixture. The PCR mixture consisted of 10

PL of the RT mixture, 0.25 mM dNTP, 50 ng of each specific PCR primer (ACGT

Corporation, Toronto), 2.5 U Taq polymerase (Boehringer Mannheim) in 1.25 mM

MgCl,, 50 mM KCI, 10 mM Tris-HC1 (pH 8.3) in 24.5 pL DEPC water. Each PCR

reaction underwent an amplification regime characterized by a pre-incubation stage

(95OC, 5 minutes), a denaturation stage (94OC, 30 seconds), a primer annealing stage

(62"C, 30 seconds), an extension stage (72OCY 30 seconds) and a long extension stage

(72"C, 8 minutes) in a thermal cycler (MJ Research hc, Mass, USA). PCR reactions

were dso performed using RNA that had not been reverse transcribed to establish the

extent of genomic DNA contamination in the RNA. PCR products were

electrophoresed on a 2 % agarose gel, stained with 1.5 % ethidiurn bromide in Tris-

acetatel EDTA buffer to allow visualkation of the PCR product under a

transilluminator (Lighttools Research, Ont, Canada).

Specific primes were used to ident* CRH-RI, CRH-R2, Cx 43 and p-actin

gene expression in the rat rnyometrium. Primers were designed to detect a 333 bp

product for CRH-R1 in rat myometnum (Slominski et al., 1995) a sense primer 5'

GCC CTG CCC TGC C ï T TTT CTA 3' and an antisense primer 5' GCT CAT GGT

TAG CTG GAC CA 3' corresponding to positions 235-255 and 549-568? respectiveiy

were used (Accession number L23332) (Chen et al., 1993). Similariy, primers were

designed to identie a 509 bp product for CRH-R2 in rat myometrium. A sense primer

5' TAG TGC TGC GGA GTA TCC GC 3' and an antisense primer 5' CAT CCA GTA

CAG G U GGC AG 3' corresponding to positions 63 1-650 and 1 12 1-1 140.

respectively, were used (Accession number U 162%). Cx 43 expression was recognized

by primers were designed to recognize a 433 bp product. A sense primer 5' CCA AGG

AGT TCC ACC AAC TT 3' and an antisense primer 5' AGA CTG ACG GGG TCA

ACG TG 3' corresponding to positions 137- 156 and 551- 570, respectively, were used

(Accession number Ml93 17). p-actin gene expression (intemal control) was ais0

detennined in al1 samples to assess the integrity of the RNA. Primers were designed to

identiQ a 331 bp for B-actin in al1 the samples. A sense primer 5' CGT GGG CCG

CCC TAG GCA CCA 3' and an antisense primer 5' CCC CCC TGA ACC CTA AGG

CCA A 3' corresponding to positions 1343- 1363 and 1653- 1674 , respectively were

used (Accession number JO069 1).

4.2.4 SQ-PCR

Sq-PCR was performed with the CRH-R.2, Cx 43 and i3-actin cDNA to

compare the expression of these genes in rat myomeaium fiom day 15 of gestation

through to labour and 1 day post-parhun. To obtain a linear range in the expression of

CRH-R2 cycles 28, 30, and 32. Cycles 18,20 and 22 were used for Cx 43 and for the

B-actin gene cycles 16,18,20 were used. The relative intensity of cDNA signais was

quantified using computerized image analysis (Imaging Research inc., Ont, Canada).

Enzyme digestion was performed to CO- the identity of the CRH-R2 cDNA

product using 10 U of the restriction enzyme Taq I (Pharmacia) in One-Phor-All buffer

(Pharmacia) The PCR product (0.5- 2pg cDNA) was incubated at 6j°C for 2-3 hours

then the products were nin on a 2% agarose gel (1.5% ethidum bromide) to allow

visualization.

4.2.5 Data Analysis

To correct for differences in the initial amount of RNA used we deterrnined B-

a c h mRNA expression in al1 the samples. The ratio of the optical densitornetry

reading measurernents for the expression of CRH-R2 mRNA or Cx 43 mRNA (at 3

progressive amplification cycles) to that of f3-actin mRNA expression (at 3 progressive

amplification cycles) was detennined for each sample of rat myometrium. The mean of

the ratio was detennined for each treatment fkom d l the samples within that group. To

determine the difference in gene expression amongst the treatment groups (day 15, day

20, day 21, day 32, labour and IPP) Ail Pairwise Multiple Cornparison Procedures

(Student-Newman-Keuls Method; CRH-R2 mRNA and Bonferroni's; Cx 43 mRNA)

were used,

4 3 RESULTS

43.1 CRH-Ri mRNA And CRH-R2 mRNA Expression in Rat Myometrium On

Day 15, Day 20, Day 21, Day 22, At Labour And 1 Day Postpartum

Enzyme digestion (Taq i ) of CRH-R2 was used to confirm the identity of the

product and yielded the product of expected size (409 bp). We have identified CRH-Et2

(expected band of 509 bp) in the rat myornetnum fiom day 1 5-day 22 of gestation, at

the time of delivery and 1 day postpartum (Figure 4.1). The amplification of CRH-RZ

expression increased iinearly in 28, 30 and 32 cycles in al1 the samples studied. We

observed a sidcant nse (p < 0.05; Student-Newman-Keuls Method ) in CM-R2

mRNN 8-actin mRNA expression in rat myometrium at the t h e of labour, with a

significant decrease rise (p < 0.05; Student-Newman-Keuls Method) to pre-labour

levels 1 day post-partum as determineci by anaiysis of sq-PCR CRH-W mRNA / P-

actin mRNA expression (Fig 4.2). CRH-RI mRNA expression was undetectable in the

rat myometrium kom day 15-day 22 of gestation. at the t h e of labour and 1 day

postpartum. The amplification of LI-actin mRNA withui the linear expression of the

gene (cycles 16, 18,20).

432 CI 43 Expression In Rat Myometrium On Day 15, Day 20, Day 21, Day 22,

At Labour And 1 Day Postpartum

Cx 43 mRNA expression (expected band of 433 bp) was present in the rat

myometrium at day 15- day 22 of gestation, at the tirne of labour and 1 day postpartum

(Figure 4.1 ).Cx 43 mRNA expression increased linearly at 1 8, 20 and 22 cycles in al1

the samples shidied. We obsenred a significant rise (p < 0.05; Bonfernoni's method) in

Cx 43 mRNN D-actin mRNA expression in rat rnyo~etrïum at the time of labour and a

s i d c a n t decrease @ < 0.05; Bonferroni's method) to pre-labour leveis 1 day post-

partum as determined by analysis of sq-PCR CRH-R2 mRNA 1 p-actin mRNA

expression (Fig 4.3).

4.1 DISCUSSION

We report the presence of CRH-R2 mRNA expression in the rat rnyornchrn at

day 15- day 22 of gestation, at the time of delivery and 1 day postpartum. CRH-R2

mRNA expression increased significantly and concurrently with Cx 43 mRNA

expression at the time of delivery. We were unable to detect CRH-RI mRNA

expression in the rat myorneûium in pregnancy and post-parnim.

Based on the obvious inappropriate and potentially dangerous reasons for

manipulating the human myometriurn in vivo and the difficulty in obtaining

myometnal samples fiom pregnant women we have used a rat mode1 to study CRH

receptor expression in the myometrium. There is no evidence in the literature

supporting the presence of distinct fiinctional regions within the rat uterus. Hence, no

distinctions were made between regions of the uterine horn, instead we used al1 the

rnyornetnum fiom the hom to determine gene expression. We have used a semi-

quantitative technique to assess CRH-R2 mRNA expression in the rat myometrium.

We have established hear gene expression of the CRH-R2 gene and determined the

ratio of CM-R2 mRNA io the linear gene expression of the Cx 43 gene to evaiuate the

change in receptor expression. However, we have not as yet detexmined the localization

of CRH receptor protein in the rat myometrium in pregnancy and delivery and

examined if the upregulation of CRH-R2 mRNA expression was reflected at the

protein level.

We have previously reported that CRH-RI mRNA expression is significantiy

increased at the Ume of labour in rnyomenium f?om the lower se-ment of the human

uterus. This rise was observed in both terni and pre-texm pregnancies. whereas CRH-

R2 mRNA expression remains unaltered. Our results shows that CRH-RI rnRNA and

CRH-R2 rnRNA are differentially expressed in rat and human myometrium, in

pregnancy. C W R 2 mRNA is upregulated at the tirne of delivery in the rat

myometrium whereas in the human myometriurn CRH-R2 mRNA expression was only

slighrly increased. In contrast, CRH-RI mRNA increased significantly at the time of

labour in the human myornetrium but CRH-RI mRNA expression was undetectable in

the rat myometrium both in pregnancy and at the tirne of delivery. In both the rat and

the human, however the expression of CRH-R.2 m m and CRH-RI &NA,

respectively decreased immediately postpartum suggesting they were regulated by

labour specific mechanisms.

CRH-RI mRNA and CRH-EU mRNA have been reported at simila. sites in the

rat and the human (Chalmers et al., 1996; Perrin et d., 1995; Valdenaire et al.,

1997).However, it is important to keep in mind that the regdatory elements involved in

CRH receptor expression may be species-specific The mechanisms regulating CRH-EC2

mRNA expression in the rat myometrium and the time of labour are unknown. It is

well documented that CRH receptors in the rat pituitary and brain are regulated by

CRH (Wynn et al., 1983; Lu et al., 1994; Makino et al., 1995). Karialis and Mazoub,

(1994) have shown that the rat placenta does iiot produce C M . CRH mRNA

expression and irCRH are present in the rat endometrium in prePancy (Zoumakis et

al., 1996; Makrigiannakis et al., 1997) but the presence of CRH in the rat myometrium

has not been reported. It has been suggested that endornetrial CRH may play a local

role in the regulation of myometrial tone, but the mechanisms of this interaction would

need to be deterrnined. Interestingly, it has recently been suggested that urocortin, a

CRH related peptide, is the preferential ligand for CRH-R2 (Vaughan et al., 1995. In

view of the presence of CRH-R2 in the rat myornetrium it would be of interest to

determine urocortin mRNA expression in the rat myometrium in pregnancy and

delivery .

In conclusion we have detemüned that the rat is a suitable model to study the

factors regulating the expression of CRH-R7 mRNA in the utew. However, the rat is

not an appropriate model to study the regulation of CRH-RI mRNA, which is the

major subtype of the receptor in humau myometrium. A clear understanding of the

mechanisms involved in reguiating CRH receptor expression wiU help decipher some

of the mechanisms regdating CRH-mediateci actions.

Figure 4.1 Enzyme digestion to confïrrn the identity of CM-R2 cDNA: A. Taq 1 digestion (409 bp), B. original size product (509 bp).

Figure 4.2 Photograph of a sq-PCR gel showing the hear expression of CRH-RZ rnRNA, Cx 43 mRNA and P-actin mRNA. The picture shows 3 amplification cycles per sample/ gene: CM-R2 mRNA expression (expected band size 509 bp; cycles 28,30,32). Cx 43 mRNA expression (expected band size 433 bp; cycles 1820,22) and 0-actin mRNA expression (expected band size 33 1 bp: cycles 16, 18,20). The expression of the genes is shown in rat myometrium on day 15 (n = 3), day 20 (n =3), day 21 (n = 3), day 22 (n = 3) of gestation, at the time of labour (day 23; n = 3) and 1 &y postpartum (n = 3).

Figure 4.3 Analysis of sq-PCR for CRHR2 mRNA expression (cycles 28,30,32) in rat myometrium on day 15 (n = 4), &y 20 (n = S), day 21 (n = 4), day 22 (n = 4) of gestation, at the time of labour (&y 23; n = 6) and 1 day postpartum (n = 4). CRH-R2 mRNA expression is signincantiy upregulated at the t h e of labour in rat myometrium @ < 0.05).

dayl5 day2O day2l day22 Labour 1PP

Figure 4.4 Analysis of sq-PCR for Cx 43 mRNA expression (cycles 18,20,22) in rat rnyometrium on day 15 (n= 4), day 20 (n=5), day 21 (II= J), day 22 (n=4) of gestation, at the time of labour (day 23; n= 6) and 1 day postpam (n= 4). Cx 43 mRNA expression is significantly upregulated at the t h e of labour in rat myorneaiurn (p < 0.05).

day15 day20 day21 day22 Labour 1PP

CHAPTER 5: FINAL DISCUSSION

We have shown that CRH-RI mRNA and CRH-R2 mRNA expression and their

proteins are present in the myometrîum of both non-pregnant and pregnant women.

CRH-RI mRNA expression and peptide levels in myometrium are downreguiated with

pregnancy and signincantly upregulated at the time of labour. This rise in CRH-RI

mRNA expression appears to be specific to myometrium nom the lower segment of the

uterus. Moreover, CRH-R1 mRNA expression was increased in both in both pre-term

and term pregnaucies. CRH-RI mRNA expression appears to r e m to pre-labour

levels immediately postpartum. CRH-R2 mRNA expression was not significantly

changed at the t h e of labour in either term or pre-terni human pregnancies. We did not

observe a significant rise in CRH-RI mRNA expression in the chorion and the decidua.

CRH-RI mRNA expression was undetectable in the amnion whereas C M - W mRNA

expression was absent fiom the decidua, the amnion and the chorion.

CRH-RI and CRH-R2 protein was iocalized to the uterine smooth muscle and

to the smooth muscle Iayer of the uterine vessels in the myometrium fiom nonpregnant

women. CM-R1 protein staining was undetectable in the myometnum in term

pregnancy but was apparent once again, but to a lesser degree at the tirne of labour.

CRH-R2 staining was also undetectable in the myometrium in term pregnancy. At the

time of labour, we observed a modest increase of positive CRH-IU protein staining in

the uterine smooth muscle, however, CRH-R2 protein was undetectable in the uterine

vasculanire.

We also report the presence of CRH-R2 mRNA expression in the rat

myornetrium tiom &y 15 to day 22 of gestation, at the t h e of delivery and 1 day

postpartum. C M - R 2 mRNA expression increased signincantly and concomitantly

with Cx 43 M A at the tirne of labour in the rat myometrium. Whereas the nse in

CRH-EU mRNA expression in the myometrium was sudden, the rise in Cx 43 mRNA

expression was gradual and attained a peak at the time of labour. Interestingiy, the

expression of both genes decreased immediately post-parnim. We were unable to detect

CRH-RI mRNA expression in the rat myometnurn in pregnancy, at the time of

delivery and post-partum.

'The observed nse in CRH receptor expression at the time of labour is in

agreement with a physiological mle for CRH in human and rat parturition. The

apparent exclusiveness of this significant rise of CRH-R1 mRNA to the lower segment

of the human uterus rnay suggest hct ional differences for CRH in the fundal and the

lower segment of the utenis. As the lower segment of the uterus is relatively quiescent,

at the time of labour we have suggested that CRH mediates relaxation of this segment

probably via a CAMP-mediated pathway. A CAMP secondary messenger pathway for

CRH is in agreement with studies done by Labne et al., (1982) and Aguilera et al.,

(1987) who have shown CRH receptors are linked to this pathway in the brain and the

pituitary. The increase in CRH-RI mRNA expression, at the tirne of labour in both

pretemi and term pregnancies suggests that the regdation of CRH-RI mRNA

expression is involved in andor mediated by labour related rnechanisms. In addition,

the absence of a nse in CRH-RI mRNA expression in the decidua and fetal membranes

suggests that the upregulation of CRH-R1 mRNA expression is specinc to the human

myometrium.

We observed a nse in CRH-RI protein in the myometrium at the time of labour.

However this rise did not appear to reflect the significant hcrease in CRH-RI mRNA

message that we observed. We are a little cautious in our interpretatîon of this data

because of the antibody used. ui the literature only two CRH-RI antibodies have been

used. The group of E.A. Linton synthesized a CRH-RI antibody which they have used

in a wide array of immmofluorescence and western blot studies (Castro et al., 1996).

Interestingiy, they report different rnolecular weight CRH receptors within and

amongst tissues. The identified a 40 and a 45 kDa CRH-RI in the myometrium (Castro

et al., 1996). In very recent studies they showed a heterogeneous spread of CRH-R1

protein staining in the myometrium of nonpregnant aod pregnant women (Rodriguez-

Linares et al., 1998). We are the first group to our knowledge to use the CRH-RI

antibody in this study. We were unable to detect C M - R I protein in the myometrium

or in positive control tissues with Western blots. Hence we are unable to determine the

size of the product the antibody is detecting. Even though the adbody is pre-absorbed

with the CRH-RI peptide much rem* to be done to characterize the antibody.

Namely the afFinity and specificity of the antibody for the peptide has to be clearly

established. In addition, we need to detennine if the antibody recognizes Merent post-

translational spliced foms of the receptor or furthemore if the antibody cross-reacts

with the CRH-BP.

CRH-R2 mRNA expression was not signifiicantly increased at the time of

labour in human pregnancy. However, CRH-R2 mRNA was present in the

myometrium of more of the preterm patients. We suggest that whiie CRH-R2 mRNA is

not signincantly upreguiated at the time of labour, its expression may be associated to

gestational age andor premature activation of the utenis. The absence of CRH-R2

mRNA fiom the decidua and the fetal membranes suggests that the reported actions of

CRH in these tissues (Jones et al., 1989) may be mediated by CRH-Rl or a possible

unlaiown subtype of the CRH receptor.

We have shown that CRH-R2 mRNA expression is upregulated at the time of

delivery in rat myometrium. The rise in CRH-R2 mRNA was s h o w relative to the

previously reported increase in Cx 43 mRNA (Petrocelli and Lye, 1994) at the t h e of

delivery in the rat myometrium. The presence of CRH-IU mRNA in the rat

myometrium suggests CRH may play a role in delivery. However, CRH is not secreted

by the rat placenta (Robinson et al., 1989). As CRH has not been identified in the rat

myometrium, we suggest that endometrial CRH (Makrigiannakis et al., 1995) may act

via paracrine mechanisms on the rnyornetrial CRH receptor. We aiso suggest that the

CRH-R2 &A may not be associated with relaxation-mediated pathways. This is

because the evidence in the literature suggests the rat uterus is one conûacting entity at

the t h e of labour. In addition, CRH is not thought to be the preferential ligand of

CRH-RZ based on the demonstration that urocortin binds CM-W with higher affinity

than CRH (Lovenberg et al., 1995a). However urocortin mRNA expression has not

been determined in the rat myometrium. The absence of CRH-RI mRNA expression in

the rat myornetriurn is consistent with studies by Chalmers et al., (1995) Lovenberg et

al., (199%) that have shown CRH-RI mRNA is restricted to the brain regions and that

CM-R2 mRNA is expressed in the brain and muscle.

The differential rise of CRH-Rl mRNA and CRH-R2 mRNA expression at the

time of labour in the human and the rat myometriurn, respectively, suggests that

perhaps the receptors are involved in mediahg different actions within this tissue. An

attractive proposal could be that CRHRl mediates relaxation whereas CRH-R2 is

involved in myometrial contractility. The rise in CRH-Rl mRNA levels rise in the

myometriurn nom the lower segment of the human utenis is in agreement with for the

effective expulsion of the fehis fiom the uterus. In the literature, only the genes of

uterotonin receptor mRNA expression have been shown to be tumed on at the time of

labour in the rat myometrium (Fukai et al., 1984). We suggest that CRH is a uterotonic

agent in the rat myometrium and that it mediates its effects via the CRH-R2 receptors.

Future interesthg studies could be to observe the effects of progesterone on CRH-W

mRNA expression. Progesterone is weli-known in its ability to induce refiactoriness of

the myometnum to OT and PGF2, hence leading to a prolongation of pregnancy. We

could examine CRH-W mRNA expression in the rat myometnum, following a)

progesterone infusion, b) progesterone infusion stopped after a period to ailow the rats

to deliver and c) pregnant ovariectomised (progesterone withdrawal) rats.

In this study we predominantly used PCR techniques to assess the level of gene

expression. We are aware of the difnculties in quantitatively assessing the data. We

have shown a linear comlation between the expression of the gene of interest and p-

actin mRNA (an internai controt gene). We have used the ratio of optical densitornetry

w i t b the linear expression of the gene of interest to the linear expression of p-actin

mRNA to assess gene expression. The validity of this technique is well established

(Kinoshita et al., 1 992).

Based on the presence of CRH-W mRNA expression on the rat myometrium

and the upregulation of the CRH-R2 gene expression at delivery, we have determined

that rat is a suitable mammalia. model to study the regulation of CRH-R2 mRNA at

the tirne of labour. However another mammaiim model has to be established to study

the regulation of the expression of CRH-RI mRNA in the myometrium. CRH-RI

mRNA is the predominant CRH receptor subtype in the human myometrium and

elucidating the factors regulating CM-Rl mRNA expression may help decipher the

role of this receptor in human pregnancy. We suggest that the expression of CRH

receptors could also be studied in myometrial cells via culturing or transfection

techniques. However, while these in viîro experiments may M e r our understanding

of the factors regulating CRH receptor expression in the myometrium the inherent

Limitations of in vitro studies prevail.

In conclusion, we have s h o w that CRH-RI mRNA expression is signincantly

upregulated in the human rnyometnum at the t h e of labour, whereas, in the rat CRH-

R2 mRNA expression is upregulated at the tune of delivery. The upregulation of both

CRH-RI mRNA and CRH-R2 mRNA expression in the human and the rat,

respectively, are similarly mediated by labour-related mechanisms, as suggested by the

decrease in the expression of the receptor subtypes immediately postpartum.

REFERENCES

Abou-Sambra, A.B., Catt. K J . and Aguilera, G. (1986) Involvement of Protein Kinase C in the Regdation of Adrenocorticotropin Releasing from Rat Antenor Pituitary Cells. Endocrinology 118: 2 12-2 17.

Aguilera, G., Harwood, J.P., Wilson, J.X., Morell, J., Brown, J.H. and Catt, K.J. (1983) Mechanism of Action of Cortïcotropin-Releasing Factor and Other Reguiatos of Coaicotropin Release in Rat Pituitary Cells. .i Bi01 Chem 258: 8039-8045.

Aguilera, G., Millan, M.A., Hauger, R.L. and Catt, K.J. (1987) Corticotropin-Releasing Factor Recepton: Distribution and Regdation in Brain, Piniitary, and Peripheral Tissues. Ann N Y Acad. Sci 512: 48-66.

Anderson, G.F., Kawarabayashi, T. and Marshall. J.M. ( 1 98 1) E ffect o f Indomethacin and Aspirin on Utrerine Activity in Pregnant Rats: Cornparison of Circular and Longitudinal Muscle. Bi01 Reprod 24: 359-372.

h t o n i , F.A., Holmes, M.C. and Jones, M.T. (1983) Oxytcin as well as Vasopressin Potentiate Ovine CRH In Vitro. Peptides 4: 41 1415.

Battaglia, G.. Webster, E.L. and De Souza, E.B. (1987) Characterization of Corticotropin- Releasing Factor Receptor-Mediated Adenylate Cyclase Activity in the Rat Central Nervous System. Synapse 1: 572-58 1.

Benedeîto, C., Petmglia, F., Marozïo, L., Chiarolini, L., Flono, P., GenaPani, A.R. and Massobrio, M. (1 994) Corticotropin-Releasing Hormone Increases Prostaglandin F2( Activity on Hurnan Myometrium In Vitro. Am J Obstet Gynecol 171: 126- 13 1.

Bilezikjian, L.M. and Vale, W.W. (1983) Glucocorticoids Inhibit Corticotropin-Releasing Factor-Induced Production of Adenosine 3',5' Monophosphate in CUltured Antenor Pituitary Cells. Endocrinology 113: 657-662.

Caldeyro-Barcia, R. & Serono, J. A. (1 96 1) In Oxytocin. Proceedings of a National Symposuim (Eds) Caldeyro-Barcia R. & Heller, R. pp 1 17. Oxford: Pergamon Press.

Campbell, E.A., Linton, E.A., Wolfe, C.D.A., Scraggs. P.R., Jones, M.T. and Lowry, P.J. (1 987) Plasma Corticotropin-Releasing Hormone Concentrations During Pregnancy and Parturition. J. Clin Endocrino Metab 64: 1054- 1059.

Carr, B.R., Parker Jr. CR., Madden, J.D., MacDonald, P.C. and Porter, J.C. (1 98 1) Materna1 Plasma Adrenocorticotropin and Cortisol Relationships Throughout Human Pregnancy. Am J Obstet Gynecol 139: 416-422.

Castro M.G., Momson, E.. Perone, MJ., Brown, O.A., Murray, C.A., Ahmed L, Perkuis, A.V., Europe-Finner G., Lowe~stein, P R and Linton, E.A. (1 996) Corticotropin-Releasing Hormone Receptor Type 1 : Generation and C haracterization of Polyclonal Antipeptide Antibodies and their Localization in Pituitary Cells and Cortical Neurones In Vitro. J. Neuroendocrinol8: 52 1 -53 1.

Challis, J. KG. And Lye, S. Parturition InThe Physiology Of Reproduction 2nd Ed, Edited By Knobil, E. And Neill, J.D. New York, Usa:: Raven Press. 1994, P 985- 1040.

Chalmers, D.T., Lovenberg, T.W. and De Souza. E.B. (1 995) Localization of Novel Corticotropin-Releasing Factor Receptor (C RF?) mRNA Expression to Specific Subcortical Nuclei in Rat Brain: Cornparison with CRF 1 Receptor mRNA Expression. J Neurosci 15: 6340-63 50.

Chalmers, D.T., Lovenberg, T.W., Grigoriadis, D.E., Behan, D.P. and De Souza E.B. (1 996) Corticotrophin-Releasing Factor Recepton: From Molecular Biology to Drug Design. Trends Pharmacol Sci 17: 166- 1 72.

Chang, C.P., Pearse, R-V., O'Comell, S. and Rosenfeld, M.G. (1993) Identification of a Seven Transmembrane Helix Receptor for Corticotropin-Releasing Factor and Sauvagine in Marnmalian Brain. Neuron 1 1 : 1 1 8% 1 1 95.

Chen, F.M., Bileiikjian, L.M., Perrin, M.H., Rivier, J. and Vale, W. W. (1 986) Corticoûopin-Releasing Factor Mediated Stimulation of Adenylate Cyclase Activity in Rat Brain. Brain Res 8 1 : 49-57.

Chen, R., Lewis, K.A., Perrin, M.H. and Vale, W.W. (1993) Expression of a Human Corticotropin-Releasing Factor Receptor. Proc. Natl. Acad. Sci USA 90: 8967-8971.

Chomczynski, P. and Sacci, N. (1987) Single-Step Method of RNA Isolation by Acid Guandiniurn Thiocyanate-Phenol-Chloroform Extraction. And Biochem 162: 1 56- 1 59

Chow, L. and Lye, S. (1994) Expression of Gap Junction Protein Connexin-43 is Increased in the Human Myometrium Towards Term and with the Omet of Labour. Am J Obstet. Gynecol170: 788-795.

Clifton, V.L., Read, M.A., Leitch, LM., Boura, A.L. Robinson, P.J. and Smith, R. (1 994) Corticotropin-Releasing Hormone-Induced Vasodilatation in the Human Fetal Placental Circulation. J Clin Endocrino1 Metab 79: 666-669.

Clifton, V.L., Owens, P.C., Robinson, P.J. and Smith, R. (1995) identification and Characterization of Corticoeopin-Releasing Hormone Receptor in Human Placenta. Eur J Endocrinol133: 59 1-597.

Cooper, ES, Brooks, A.N., Miller, M-R and Greer, LA. (1994) Corticotmphin- Releasing Factor bunostaining is Present in Placenta and Fetal Membranes fiom the Fim Trimester Onwards and is Not ABFected by Labour or Administration of Mifepristone. C h Endocrinol (Oxf) 41: 677-683.

Cunningham, MacDonald, P.C., Gant, N.F. Levrano and Gilstrap Williams Obstetrics, 19th Ed Conn USA: Appleton and Lange Publisher, 1993, p 220-240.

Dautzenberg, F.M., Dieterich, K., Palchaudhuri, M.R and Speiss, J. (1997) Identification of Two Corîicotropin-Releasing Factor Receptors fiom Xenopus laevis with High Ligand Selectiviv: U n d Pharmacology of the Type 1 Receptor. J Neurochem 69: 1640- 1649.

De Souza, E.B., Perrin, M.H., Rivier, J., Vale, W. W. and Kuhar, M.J. (1 984') Corticotro pin-Releasing Factor Receptors in Rat pituitary Gland: Autoradiographic Localkation. Brai. Res 296: 202-207.

DeSouza, E.B., Insel T.R. Perrin, M.H., Rivier, J., Vale. W. W. and Kuhar. M. J. ( 1985) Corticotrophin-Releasing Factor Receptors are Widely Distributed Within the Rat central nervous System. An autoradiographic Study, I. NeuroSci 5: 3 189- 3203.

Dietench, K.D., Lehnert, H. and De S o w E.B. (1997) Corticotropin-Releasing Factor Receptors: An O v e ~ e w . Exp Clin Endocrinol Diabetes 105: 65-82.

Donaldson, C.J., Sutton, S.W., Perrin, M.H., Comgan, A.Z., Lewis. KA., Rivier, J.E., Vaughan, LM. and Vaie, W. W. (1 996) Cloning and Characterization of Human Urocortin. Endocrinology 137: 2 L 67-2 1 70.

Dufau, M.L., Ulisse, S, Khanurn, a., Buczko, E., Kitarnm M., Fabbri, A. and Narniki, H. (1989) LH Action in the Leydig Cell: Modulation by Angiotensin II and Corticotropin-Releasing Hormone, and regdation of P450 (1 7) alpha mRNA. J steroid Biochem 34: 205-2 1 7.

Embrey, M.P. and Mornim, D.L. (1968) The Effect of Prostaglandins on Human Pregnant Myomeû-ium In Vitro. J Obstet Gynaecc Brit Commonw 75: 829-832.

Fencl M., Stillman, R.J.., Cohen, J. and Tulchinsky, D. (1980) Direct Evidence of Sudden Rise in Fetal Corticoids Late in Human Gestation. Nature 287: 225.

Flores, M., CarvalIo, P. and Aguilera, G. (1 990) Physicochemicai Characterization of Corticotropin-Releasing Factor Receptor in Rat Pituitary and Brain. Life Sci 47: 203 5- 2040.

Florio, P., Gallo, L.R., Di Carlo, C., Sutton, S., Genazzani, A.R. and Petmglia, F. (1 996) Activin A, Corticotropin-Releasing Factor and Prostaglandin FZa Increase Immunoreactive Oxytocin Release fkom Cultured Human Placental CeiIs. Placenta 17: 307-3 1 1 -

Frim, D.M., Exnanuel, R.L., Robinson, B.G., Smas, C.M., Alder, G.K. and Majzoub, J.A. (1 988) Characterization and Gestational Regdation of Corticotropin-Releasing Hormone Messenger RNA in Human Placenta. J Clin Invest 82: 287-292.

Fuchs, A. -R., Fuchs, F., Husslein, P., Soloff, M.S. and Femstrom, M.J. (1982) Oxytocin Receptors and Human Parturition. A Dual Role For Oxytocin in the Initiation of Labor. Science 215: l396-I398.

Fuchs, A.R., Fuchs, F., Husslein, P. and Soloff, M.S. (1984) Oxytocin Receptors in the Human Utem During Pregnancy and Parturition. Am J Obstet Gynecol 150: 734-741.

Fujieda, K., Faiman, C., Reyes, FA, and Winter, J.S. (1 98 1) The Control of Steroidogenesis by Human Fetai Adrenal Cells in Tissue Cdîure. 1. Responses to Adrenocorticotopin. J Clin Endocrinol Metab 53: 34-38.

Fukai, H., Den, K., Sakamoto, H., Kodaira, H., Uchida, F. and Takagi, S. (1984) Study of Oxytocin Receptors: Oxytocin and Prostaglandin F? Alpha Receptors in Human Myometria and Amnion-Decidua Complex During Pregnancy and Labor. Endocrin01 Jpn 31: 565-570.

Garfield, R.E. (1 990) Intercellular Coupling and Modulation of Uterine Contractility. In: Uterine Contractility, edited by Garfield RE, MA, USA: Serono Symposia, 1990. p 2 1-40.

Goland, R.S., Wardaw, S.L., Blum, M., Trooper, P.J. and Stark, R.I. (1988) Biologically Active Corticotropin-Releasing Hormone in Matemal and Fetal Plasma During Pregnancy. Am J Obstet Gynec 159: 884-890.

Goland, R.S., Wardaw, S.L., Stark, R.I., Brown Jr., L.S. and Frantz, A.G. (1986) High Levels of Corticotropin-Releasing Hormone Immunoactivity in Matemal and Fetal Plasma during Pregnancy. J Clin Endocrinol Metab 63: 1 199-1 203.

Grammatopoulos, D ., Milton N.G.N. and Hillhouse E. W. (1 994) The Human Myometrial CRH Receptor: G Proteins and Second Messengers. Mol Cell Endocrinol. 99: 245-250.

Gram~~topoulos, D., Thompson, S. and Hillhouse, E. W. (1 995) The Human Myometrium Expresses Multiple Isofomis of the Corticotropin-Releasing Homone Receptor. J Clin Endocrinol Metab 80: 2388-2393.

Grammatopoulos, D., Stirrat, G.M., Wiiams, S.A. and Hillhouse E. W. (1 996) The Biologicd Activity of the Corticotropin-ReIeasing Hormone Receptor-Adenylate Cyclase Complex in Human Myometrium is Reduced at the End of Pregnancy. J Clin Endocrino1 Metab 81: 745-75 1 -

Grigoriadis, D.E. and De Souza, E.B. (1988) The Brain Corticotropin-Releasing Factor (CE) Receptor is of Lower Apparent Molecular Weight than the CRI Receptor in Antenor Pituitary. Evidence fiom Chernicd Cross-Linking Shldies. J. Biol. Chem. 263: 10927-1093 1.

Grigoriadis, D.E. and De Souza E.B. (1 989a) Heterogeneity Between Brain and Pituitary Corticotropin-Releasing Factor Recepton is Due to Differentiai Glycosylation. Endocrinology 125: 1 877- 1 888.

Grigoriadis. D.E. and De Souza, E.B. ( 1 989b) Corticotropin-Releasing Factor (CM) Recepton in Intermediate Lobe of the Pituitary: Biochemicai Characterization and Autoradiographic Localization. Peptides 10: 1 79- 1 88.

Grigoriadis, D.E., Herow, J. A. and De Souza E.B. (1993) Characterization and Regdation of Corticotropin-Releasing Factor Recepton in the Central Nervous. Endocrine and Immune Systems. Ciba Found Symp 172: 85-101.

Grino, M., Chrousos, G.P. and Margions, A.N. (1 987) The Corticotropin-Releasing Hormone Gene is expressed in Human Placenta. Biochem Biophys Res Comm 148: 1208-1214.

Hatzoglou A.. Margions, A.N., Bakogeorgou, E., Gravanis, A. and Castanas, E. (1996) Identification, Characterization, and LocaIization of Corticotropin-Releasing Hormone Receptors in Human Placenta. Life Sci 59: 1 87 1 - 1 879.

Hauger, R.L. and Aguilera, G. (1993) Regdation of Pituitary Corticotropin Releasing Hormone (CRH) Receptors by CRH: interaction with Vasopressin. Endocrinology 133: 1708-1714.

Hillhouse, E.W., Grammatopoulos, D., Milton, N.G.N. and Quartero, H. W.P. (1 993) The Identification of a Human Myometrial Corticotropin-Releasing Homone Receptor that Increases in AfEnity During Pregnancy. I Clin Endocrinol Metab 76:736-76 1.

Ichikawa, T., McMaster, D., Lederis, K. and Kobayashi, H. (1 982) Isolation and Amino Acid Sequence of Urotensin 1, a Vasoactive and ACTH-Releasing Neuropeptide, fiom the Carp (Cyprinus curpio) Urophysis. Peptides 3: 859-867.

Imaki, T., Nanise, M., Harada, S., Chikada, N., Imaki, J., Onodera, H., Demura, H. and Vale, W. (1 996) Corticotropin-Releasing Factor Up-Regdates its Own Receptor

mRNA in the Paraventricular Nucleus of the Hypothalamus. Mol Brain Res 38: 166- 1 ?O.

Jelinek, L.J., Lok S., Rosenbberg, G.B., Smith, R.A., Grant, F J.. Biggs, S.. Bensch, P. A., Kuijper, J.L., Sheppard, P.O., Sprecher, C.A. O'Hara, P J., Foster, D., Walker, KM., Chen, L.H.J., McKernan, PA. and Kindsvogel, (1993) Expression Cloning and Sigmiing Properties of the Rat Glucagon Receptor. Science 259: 1614-16 16.

Jones, S.A. and Challis J.R.G. (1989a) Local stimulation of Prostaglandin Production by CRF in Human Fetai Membranes and Placenta. Biochem Biophys Res Comm 137: 780-785.

Jones, S.A., Brooks, A.N. and Challis, J.R.G. (1989b) Steroids Modulate Corticotropin-Releasing Homione Production in Human Fetal Membranes and Placenta. J Clin Endocrinol Metab 68: 825

Karialis, K. and Majzoub, J.A. (1994) Regdation of Placental Corticotropui-Releasing Hormone (CRH) Gene by Progesterone. in Programs of the 76th Annual iMeeting of the Endocine Society, Anaheim, CA Abstract 803

Keegan, C.E., Herma, J.P., Karolyi, LJ., OTShea, K.S.. Camper, S.A. and Seashola. A.F. (1 994) Differential Expression of Corticotropin-Releasing Hormone in Developing Mouse Embryos and Adult Brain. Endocrinology 134: 2547-2555.

Keirse, M.J.N.C. (1979) In Huma Parturition, edited by Keirse, A.B.M. Anderson, J.B. Gravehorst, Eds Leiden; University Press, Lciden, p 10 1

Khodr, G.S. and Siler-Khodr, T.M. (1 978) Localization of Luteinizing Hormone- ReIeasin Factor in the Human Placenta. Fertil Steril29523-530.

Kilarski. W-M., Rezapour, M., Backstrom, T., Roomens, G.M. and Ulmstem, U. (1994) Morphometric Analysis of Gap Junctions Density in Human Myometrium at Term. Act Obs Gyn Sca 73: 377-384.

Kinoshita, T., hamura, J., Nagai, H. and Shimotohno, K. (1992) Quantification of Gene Expression Over a Wide Range by the Polymerase Chain Reaction . Anai Biochern 206: 23 1-235.

Kishimoto, T., Pearse, R-V., Lin, C.R. and Rosenfeld, M.G. (1 995) A SauvaginelCodcotropin Releasing Factor Receptor is expressed in Heart and Skeletal Muscle. Proc. Nat. Acad. Sci. USA 92: 1 108-1 1 12.

Laatikainen, T.J., Raisanan, U and Salminen, KR. (1 988) Corticotropin-Releasing Homone in Amniotic Fluid During Gestation and Labor and in Relation to Fetd Lung Maîuration. Am J Obstet Gynec 159: 89 1-895.

Labrie, F., Veilleux, R., Lefevre, G., Coy, DM., Sueira-Diaz, J. and SchaUy, A.V. (1982) Coticotropin-Releasing Factor Stimulates Accumulation of Adenosine j7,5' - Monophosphate in Rat Piniitary Corticotrophs. Science 216: 1007- 1008.

Leake, R.D. Weitmian, R.E., Glatz, T.H. and Fisher, D.A. (1 98 1) Plasma Oxytocin Concentrations in Men, Noopregnant Women Before and During Spontaneous Labor. J C h Endocrino1 Metab 53: 730-733.

Lederis, K., Letter, A., McMaster, D., Moore, G. and Schlesinger D. (1992) Complete Amino Acid Sequence of Uroteosin 1, a Hypotensive and Cortico~ropin-releasing Peptide fiom Catostomus. Science 218: 162- 164.

Leslie, K.K., Zuckeman, DL, Schruefer, J., Burchel, M., Smith, J. and Albertson. B.D. (1994) Oestrugen Modulation with Parturition in Human Placenta. Placenta 15: 79-88.

Liaw, C.W., Lovenberg, T.W. Barry, G., Oltendorf, T., Grigoriadis. D.E. and De Sowa, E.B. (1 996) Cloning and C haracterization of the Corticotropin-Releasing Factor2 Receptor Complementary Deoxyribonucleic Acid. Endocrinoiogy 137: 72-77.

Liaw, C.W., Grigoriadis, D.E., Lovenberg, T.W., De Souza, E.B. and Maki, R.A. (1 997) Locaiization of Ligand-Binding Domains of Human Corticotropin-Releasing Factor receptor: A Chimerk Receptor Approach. Mol Endocrinol 11: 980-985.

Linton, E.A., Wolfe, C.D.A., Behan, D.P. and Lowry, P.J. (1988) A Specific Carrier Substance for Human Corticotropn-Releasing Factor Which Could Mask the ACTH- Releasing Activity. C h Endocrinol (Oxf) 28: 3 15-322.

Linton, E.A., P e r b , A.V., Woods, R.J., Eben, F., Wolfe, C.D.A., Behan, D.P., Potter, E., Vale, W. W. and Lowry, P.J. (1 993) Corticotropin-Releasing Hormone-Binding Protein (CRH-BP): Plasma Levels Decrease During the Third Trimester of Normal Human Pregnancy. J Clin Endocrinol Metab 76: 260-262.

Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmea, D.T., De Souza, E.B. and Oltersdorf, T. (1 995a) CIooing and Characterization of a Functionally Distinct Cortïcotropin-Releasing Factor Receptor Subtype fkom Rat Brain. Proc Nat1 Acad USA 92: 836-840.

Lovenberg, T.W., Chahers, D.T., L i4 C. and De Souza, E.B. (1995b) C W a and CRH2P Receptor mRNAs are Differentiaily Distributed Between the Rat Central Nervous System and Peripheral Tissues. Endocrinology 136: 41 39-4 142.

Lu, F ., Yang, K. and Challis, J-RG. ( 1 99 1) Characteristics and Developmental Changes of Corticotrohin-Releasing Hormone Binding Sites in the Fetal Sheep Anterior Pituitary Gland. J Endo 130: 223-229.

Luo, X., Kiss, A., Rabadan-Diehl, C. and Aguilera, G. (1 995) Reguiation of Hypothalamic and Pituitary Corticotropin-Releasing Hormone Receptor by Adrenalectomy and Glucocorticoids. Endocrhology 136: 3 877-3 883.

Lutz-Bucher, B., Felix, LM. and Koch B. (1990) Activation of Protein Kinase A Dinerentially Regulates Corticotropin-Releasing Factor-Stimulated Peptide Secretion and Cyclic AMP Formation of Intermediate and Antenor Pituitary cells in Culture. Peptides 11: 1183-1 189.

Lye, S.J., Nicholson, B.J., Mascarerhas, M., Mackenzie, L. and Petrocelli, T. (1993) Increased Expression of Cornexin 43 in the Rat Myornetrium During Labour is Associated with an Increase in the Progesterone: Estmgen Ratio. Endocrinology 132: 2380-2386.

Mackenzie, L. W. and Garfield, RG. (1 985) Effects of 17P-Estradiol on Myometrial Gap Junctions and Regnancy in the Rat. Can J Physiol Pharmacol 64: 462-466.

Makino, S., Schulkin, J., Smith, M.A., Pacak, K., Palkovits, M. and Gold, P.W. (1995) Regulation of Corticotropin-Releasing Hormone Receptor Messenger Ribonucleic Acid in the Rat Brain and Piniitary by Glucocorticoids and Stress. Endocrinology 136: 45 17- 4525.

Makino, S., Takemura, T., Asaba, K., Nishiyama, M., Takao, T. and Hashunoto, K. (1 997) Differentid Regulation of Type- 1 and Type-2 Alpha Corticotropin-Releasing Hormone Receptor mRNA in the Hypothalamic Paraventncular Nucleus of the Rat. Brain Res Mol Brain Res 47: 170- 176.

Makrigiannakis, A., Zoumakis, E., Margions, A.N., Theodoropoulos, P., Stournaras, C. and Gravanis A. (1995) The Corticotropin-Releasing Hormone (CRH) in Normal and Tumoral Epithelial Cells of Human Endometrium. J. Clin. Endocrinol. Metab 80: 185- 189.

Mansi, J.A., Rivest, S. and Drolet, G. (1996) Regulation of Corticotropin-Releasing Factor Type 1 (CRF 1) Receptor Messenger Ribonucleic Acid in the Paraventricular Nucleus of Rat Hypothalamus by Exogenous CRF. Endocrinology 137: 461 9-4629.

Margirons, A.N., Grino, M., Protos, A., Gold, P.W. and Chrousos, G.P. (1988) Cortico~phin-Releasing Hormone and Oxytocin Stimulate the Release of Placentai Proopiomelanocortin Peptides. J Clin Endocrinol Metab 66: 922-926.

Maser-Gluth, C., Lorenz, U. and Vecsei, P. (1985) In Pregnancy, Corticotropui- Releasing Factor in Matemd Blood and Amniotic FIuid Correlates with Gestational Age. Acta Endocrinol. 270: 4 2 4 .

McLean, M., Thompson, D., Zhang, H-P, Brinsrnead, M. and Smith, R. (1994) Corticotrophin-Releasing Hormone and P-Endorphin in Labour. Eur J Endocrinol 131 : 167- 172-

McLean, M., Bistis, A., Davies, I., Woods EL, Lowry, P. and Smith R. (1995) A Placental Clock Controlling the Length of Human Pregnancy . Nature Medicine 1 : 460- 463.

Mol, J.A., van Wolferen, M., Kwant, M. and Meloen, R. (1 994) Predicted Primary and Antigznic Structure of Canine Corticotropin Releasing Hormone. Neuropeptides 27: 7- 13.

Montecucchi, P.C. and Henschen, A. (1 98 1) Amino Acid Composition and Sequence Analysis of Sauvagine, a New Active Peptide From the Skin of Phyllomedusa Sauvagei. Int J Protein Res 18: 1 13- 120.

Nabhm, C., Xiong, X., Xie, L.Y. and Abou-Samra, A.B. (1995) The Altemativefy Spliced Type II Corticotropin-Releasing Factor Receptor, Stably Expressed in LLCPK- 1 Cells, is Not Well Coupied to the G-Protein (s). Biocheml Biophys Res Commun 212: 1015-1021.

Neden, J. and Breckwoldt, M. (1994) Placental Progesterone, Prostaglandins and Mechanisms Leading to Initiation of Parturition in the Human. Exp Clin Endocrinol 102: 195-202.

Okamoto, E., Takagi, Te, Makino, T., Sata, H., Iwata, E., Nishino, E., Mitsuda, N., Sugita, N., Otsuki, Y. and Tanizawa, O. (1989) immunoreactive Corticotmpin- Releasing Hormone, Adrenocorticotropin and Cortisol in Human Plasma during Pregnancy and delivery and Postpartum. Hom Merabol Res 21: 566-572.

Okamoto, E., Takagi, T., Anima, C., Kimura, T., Tokugawa, Y., Mitsuda, N., Saj i, F. and Tanizawa, 0. (1990) Expression of the Corticotropin-Releasing Hormone (CRH) Gene in Human Placenta and Amniotic Membrane. Hom Metab Res : 3 94-397.

Okamoto, T., Murayama, Y., Hayashi, Y, Inagaki, M., Ogata, E. and Nishimoto, 1. (199 1) Identification of a Gs Activator Region of the Beta 2-Adrenergic Receptor that is Autoregulated via Protein Kinase A-Dependent Phosphorylation. Ce11 67: 723-730.

Orth, D.N. (1992) Corticotropin-Releasing Hormone in Humans. Endocrine Rev 13: 164-191.

Orth, D.N. and Mount, C.D. (1987) Specific High A£finïty Binding Protein for Human Corticotropin Releasing Hormone in Normal Human Plasma. Biochem Biophys res Comm 143: 41 1-417.

Owens, M.J. and Nemeroff. (1 99 1) The Physiology anf Pharmacology of Corticotropin-Releasing Factor (CRF). Pharmacol Rev 43: 425-473.

Pepe, G.J. and Albrecht, E.D. (1995) Actions of Placental and Fetal Adrenal Steroid Hormones in Primate Pregnancy. Endocine Reviews 16: 608-648.

Perkins, A.V. and Linton, E.A. (1 995) Identification and Isolation of Corticotrophin- Releasing Hormone-Positive Cells fiom the Human Placenta. Placenta 16: 233-234.

Perrin, M., Donaldson. C., Chen, R., Blount, A., Berggren, T., Bilezikjian, L., Sawchenko, P. and Vale W., (1995) Identification of a Second Corticotropin-Releasing Factor Receptor Gene and Characterization of a cDNA Expressed in Heart. Proc Nat1 Acad Sci USA 92: 2969-2973.

Perrin, M.H., Donaidson, C.J., Chen, R.. Lewis, K.A. and Vale. W.W. (1993) CLoning and Functionai Expression of a Rat Brain Corticotropin-Releasing Factor (CM) Receptor. Endocrinology 133: 3058.306 1.

Petraglia, F., Sawchenko, P.E., Rivier, J. and Vaie, W. (1987) Evidence for Local Stimulation of ACTH Secretion by Corticotropin-Releasing Factor in Human Placenta. Nature 328: 7 17-7 19.

Petraglia, F., Sutton, S. and Vaie, W. (1989) Neurotransmitters and Peptides Modulate the Release of Immunoreactive Corticotropin-Releasing Factor from C ultured Human Placental Cells. Am J Obstet Gynecol 160: 247- 25 1.

Petmglia, F., Giardino, L., Coukos, G., Calza, L., Vale, W. and Genazzani, A.R. (1 990) Corticotropin-Releasing Factor and Parturition: Plasma and Amniotic Fluid levels and Placental Binding sites. Obstet Gyneco175: 784.

Fetraglia, F., Coukos, G., Volpe, A. and Genanani, A.R. (1991) Involvement of Placental Neurohormones in Human Parturition. Ann N Y Acad Sci 622: 33 1-340.

Peaaglia, F., Tabanelli, S., Galassi, M.C., Garuti, G.C., Mancini, A.C ., Genazzani, 4 . R and Gurpide, E. (1992) Human Decidua and In Vitro Decidualized Endometrial Stromal Cells at T e m Contain Immunoreactive Corticotropin-Releasing Factor (CRF) and CRF Messenger Ribonucleic Acid. I Clin Endocrinol Metab 74: 1427- 143 1.

Peaaglia F., Potter, E., Carnerun, V., Sutton, S., Behan. D.P., Woods, R.J., Sawchenko, P.E., Lowry, P.J. and Vale, W. (1 993) Corticotropin-Releasing Factor-Binding Protein is Produced by Human Placenta and htrauterine tissues. J Clin E n d o c ~ o l Metab 77: 9 1 9-924-

Petraglia, F.. Benedetto, C., Flono, P., D7Ambrogio, G.. GenaPani, A.D., Marozio, L. and Vale, W. (1 995) EEects of Corticotropin-Releasing Factor-Binding Protein on Prostaglandin Release fiom Cultured Matemal Decidua and on Contractile Activity of Human Myometrium In Vitro. J Clin Endocrinol Metab 80: 3073-3076.

Petraglia, F., Florio, P., Nappi, C. and Genazzani, A.R. (1 996a) Peptide Signaling in Human Placenta and Membranes: Autocrine, Paracrine and Endocrine Mec hanisms. Endocrine Reviews 17: 1 56- 1 86.

Petraglia, F., Flono, P., Gallo, G., Simoncini, T., Saviozzi, M., Di Blasio, A.M., Vaughan J. and Vale, W. ( 1996b) Human Placenta and Fetai Membranes Express Urocortin mRNA and Peptide. J Clin Endocrinol81: 3807-38 10.

Petrocelli, T. and Lye, S. (1993) Regdation of Transcripts Encoding the Myometrial Gap Junction Protein, Connexin 43, by Estrogens and Progesteroue. Endocrinology 133: 284-290.

Polymeropoulos, M.H., Torres, R., Yanvski, I.A., Chandrasekharappa, S.C. and Ledbetter, D.H. (1 995) Corticotropin-Releasing Factor Receptor ( C m ) Gene Maps to Chromosome 17q 12-q22. Genomics 28: 123- 124.

Potter, E., Behan, D.P., Fischer, M.H., Linton, E.A., Lowry, P.J. and Vale, W.W. (1 99 1) Cloning and Characterization of the cDNA for Human and Rat Corticotropin Releasing Factor-Binding Protein. Nature 349: 423-426.

Potter, E., Sutton, S., Donalcison, C., Chen, R., Perrin, M., Lewis, K., Sawchenko, P.E. and Vale, W. (1994) Distribution of Corticotropin-Releasing Factor Receptor mRNA Expression in the Rat Brain and Pi tu i tq . Proc Nat1 Acad Sci USA 91: 8777-878 1.

Pozzoli, G., Bilenkjian, L.M., Perrin, M.H., Blount, A.L. and Vale, W. W. (1 996) Corticotropin-Releasing Factor (CRF) and Glucocorticoids Modulate the Expression of Type 1 CRF Receptor Messenger Ribonucleic Acid in Rat Anterior Pituitary ce11 Cultures. Endocrinology 137: 65-7 1.

Pritchard, LA., MacDodd, P.C. and Gaat, N.F The Placenta and Fetai Membranes and Maternai Adaptation to Pregnancy- In: Williams Obstetrics 17th Ed, COM. USA: Appleton Century Croh, 1985 p 97- 1 15 and p 18 1 - 184.

Quartero. H.W.P. and Fry, C.H. (1989) Placental Corticotropin-Releasing Factor may Moduiate Human Parturition. Placenta 10: 439-443.

Quartero, H.W.P., Srivatsa, G. and GiIlham, B. (1992) Role for Cyclic Adenosine Monophosphate in the Synergistic Interaction Between Oxytocin and Corticotrophin- Release in Factor in Isolated Human Gestational Myometrium. Clin EndocMol 36: 141-145.

Rarnirez, M.iM., Matthews, S.G., White, C., Delange, S. and Challis, J.RG. (1995) Localization and Expression of Placental Corticotropin-Releasing Hormone Buiding Protein in Term and Preterm Labour. 42nd Annual Meeting of the Society for Gynecologic Investigation, Abstract No. 14 1.

Riley, SC.. Walton, J.C., Herlicks, LM. and Challis, J.R. (1991) The localization and Distribution of Co rtïcotro pin-Releasing Hormone in the Human Placenta and Fetal membranes Throughout Gestation. J Clin Endocrino1 Metab 72: 100 1 - 1007.

Rivier, J., Speiss, J. and Vaie, W.W. (1983) Chernical and Biological Characterization of Rat HypothaIamic Corticotropin-Releasing Factor Proc Natl. Acad. Sci USA 80: 485 1-4855.

Robinson, B.G., Emanuel, R-L., Frim, D.M. and Mazoub, J.A. (1988) Glucocorticoid Stimulates Expression of Corticotropin-Releasing Homone Gene in Human Placenta- Proc Nati. Acad. Sci. USA 85: 52444248.

Rodriguez-Linares B., Linton, E.A. and Phaneuf, S. (1998) Expression of Corticotropin-Releasing Hormone Receptor mRNA and Protein in the Huma. Myometxiurn. J Endo. 156: 1 5-2 1.

Ross, P.C., Kostas, C.M. and Ramabhadran, T.V. (1 994) A Variant of the Human Corticotropin-Releasing Factor (C FU) Receptor: Cloning, Expression and Pharmacology. Biochem Biophys Res Commun 205: 1 836- 1 842.

Roy, A.C. and Adkumaran, S. (1991) Pharmacology of Parturition. Ann Acad Med Singapore 20: 7 1-77.

Saeed, B.O., Weighmian, D.R and Self, C.H. (1997) Characterization of Corticotropin- Releasing Hormone Binding Sites in the Human Placenta. J Recept Signai Transduct Res 17: 647-666.

Sakai, K., Horiba, N., Sakai, Y.. Tozawa, F., Demura, H. and Suda T. (1996) Regdation of Corticotropin-Releasing Factor Receptor Messenger Ribonucleic Acid in Rat Anterior Pituitary. Endocrinology 137: 1 758-1 763.

Salminen-Lappalainen, K. and Laatikainen, T. (1990) Binding of Corticotropin- Releasing Homone (0 in Matemal and Fetal Plasma and in Amniotic Fluid. Clin Chim Acta 195: 57-66.

Sasaki, A., Shinkawa, O., Margions, A.N., Liotta, AS., Sato. S.. Murakami, O., Go, M., Shimizu, Y., Hanew, K. and Yoshinaga, K. (1987) Immunoreactive Corticotropin- Releasing Hormone in Human Plasma During Pregnancy, Labor, and Delivery. J Clin Endocrinol Metab 64: 224-229.

Sasaki, A., Tempst, P., Liotta, A.S.. Margions, A.N.. Hood, L.E., Ke, S.B., Sato, S., Shînkawa, O., Yoshinaga, K. and Krieger, D.T. (1 988) Isolation and Characterization of a Corticotropin-Releasing Hormone-Like Peptide kom Human Placenta. J Clin Endocrinol Metab 67: 768.

Schuite. H.M. and Healy, D.L. (1987) Corticotropin-Releasing Hormone and Adreno- Corticotmpin-Like Imrnunoreactivity in Human Placenta, Peripherai. and Uterine Vein Plasma. Hormone Metab Res Suppi 16: 44-46.

Shibahara, S., Morimoto, Y., Furutani, Y., Notake, M., Takahashi, H., Shimim S., Horikawa, S. and Numa, S. (1983) Isolation and Sequence Analysis of the Human Corticotropin-Releasing Factor Precursor Gene. EMBO J 5: 775-779.

Shibasaki, T., Odagiri, E., Shizume, K. and Ling N. (1 982) Corticotropin-Releasing Factor-Like Activity in Human Placental E.xtracts. .J Clin Endocrinol Metab 55: 384- 386.

Slominski, A., Ermak, G., Hwang, J., Chakraborty, A., Mazu~kiewicz, J.E. and Mihm, M. (1 995) Proopiornelanocortin, Corticotropin Releasing Hormone and Corticotropin Releasing Hormone Receptor Genes are Expressed in Human skia FEBS Lett 374: 113-1 16.

Souza, E.B. (1987) Corticotropin-Releasing Factor Receptors in the Rat Centrai Nervous System: Characterization and regional Distribution. J. Neurosci. 7: 88-100.

Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeberg, P. H. and Journot , L. (1993) DifTerential Signal transduction by Five Splice Variants of the PACAP Receptor. Nature 365: 170- 175.

Stalla, G.K., Bost, H., Stalla, J., Kaliebe, T., Dom, H.G., Pfeiffer, D,, von Werder, K. and Muller, O.A. (1 989) Human Corticotropin-Releasing Hormone During Pregnancy . Gynecol Endocrinol3: 1 - 1 0.

Stemel, P., Kesterson, R, Yeung, W., Cone, RD., Rittenberg, M.B. md Stenzel-Poore, M.P. (1995) Identincation of a Novel Murine Receptor for Corticotropin-Releasing Hormone Expressed in the Heart. Mol Endocrinol9: 637-645.

Sun, K., Smith, R and Robinson, P.J. (1 994) B a d and KCI-Stimulated Corticotmpin- Releasing Hormone Release fiom Human Placental Syncytiotrophoblasts is Inhibited by Soduim Nitroprusside. J. Clin Endocrinol. Metab. 79: 5 19-524.

Thorbum, G.D. And Challis, J.RG (1979) Control Of Parturition. Physiol Rev 59:863- 918.

Tran, TN., Fryer, J.N., Lederis K. and Vaudry, H. (1990) CRF, Urotensin and Sauvagine Stimulate the Release of POMC-Derived Peptides fkom Goldfish Neurointermediate 10 be Celis. Gen Comp Endocrinol 78: 3 5 1 -3 60.

Tulchioslq, D., Hobel, C.J., Yeager, E. and Marshall, J-IL (1972) Plasma Estrone, Estradïol, Progesterone and 17-Hydroxyprogestereone in Human Pregpancy. 1. Normal Pregnancy. Am J Obstet Gynecol112: 1095-1 100.

Usidin, T.B., Mezey, E., Button, D.C., Brownstein, M.J. and Borner, T.I. (1 993) WC Inhibitory polypeptide Receptor, A Member of the Secretin-Vasoactive Intesfinal Peptide Receptor Family, is Widely Distnbuted in Peripheral Organs and the Brain. Endocrinology 133 : 286 1 -70.

Valdenaire, O., Giller, T., Volker, B., Gottowik, J. and Kilpatrick, G. (1 997) A New Functionai Isoform of the Human CRF2 for Cortico~opin-Releasing Factor. B iochim Biophys Act 1352: 129-132.

Vale, W. and Rivier, C. (1977) Substances Moddatùig the Secretion of ACTH by Cultured Antenor Pituitary Cells. Fed Proc 36: 2049-2099.

Vaie, W., Speiss, J., Rivier, C. and Rivier, J. (198 1) Characterization of a 4 1-Residue Ovine Hypothalamic Peptide That Stimulates Secretion of Corticotropïn and p- Endorphin. Science 213: 1394-1 397.

Vaughan, J., Donalcison, C., Bittencourt, J., Perrin, MM., Lewis, K., Sution, S., Chan, R., Tumbuil, A.V., Lovejoy, D., Rivier, C., Rivier, J., Sawchenko, P. and Vale, W.W. (1 995) Urocortin, a Mammalian, Neuropeptide Related to Fish Umtensin I and to CRH. Nature 378: 287-292.

Vita, N., Laurent, P., Lefort, S., Chalon, P., Lelias, J.M. Kaghad, M., Le Fur, G., Caput, D. and Ferma, P. (1993) Primary Structure and Fuctional Expression of

Mouse pituitary and Human Brain Corticotmpin-Releasing Factor Receptoa. FEBS Lett 335: 1-5

Warren, W.B. and Süverman, A.J. (1 995) Cellular Localization of Corticotropin- Releasing Hormone in the Human Placenta, Fetal membranes and Decidua. Placenta 16: 147- 1 56.

Warren, W.B., Patrick, S.L. and Goland, RS. (1992) Elevated Plasma Corticotropin- Releasing Hormone Levels in Pregnancies Complicated by Pretem Labor. Am J Obstet Gynec 166: 1 198-1207.

Woods, RJ., Grossman, A., Saphier, P., Kennedy, k, Ur, E., Behan, D., Potter, E., Vale, W. and Lowry P.J. (1 994) Association of Human Corticotropin-Releasing Hormone to its Binding Protein in Blood May Tngger Clearance of the Complex. J Clin Endocrinol. Metab 7%: 73-76.

W~M, P.C., Aguilera, G., Momll, J. and Catt, K.J. (1983) Properties and Regulation of Hi&-Affinity Pituitary Receptors for Corticotropin-Releasing Factor. Biochem Biophys Res Commun 110: 602-608.

Wynn, P.C., Harwood, J.P., Catt, K. J. and Aguilera G. (1 98 8) Corticotropin-Relwing Factor (CRF) induces Desemitization of the Rat Pitu i tq CRF Receptor-Adenylaîe Cyclase Complex. Endocrinology 122: 35 1-358.

Wynn, P.C., Hauger. RL., Holmes, M.C., Milian, M.A., Catt, ICJ. and Aguilera, G. (1 984) Brais and Pi tu im Receptors for Corticotropin Releasing Factor: Localization and DBerentid Regulation Mer Adrextalectomy. Peptides 5: 1077- 1084.

Yang, K, Jones. S.A. and Challis, J.R.G. (1990) Changes in Glucocorticoid Receptor Numba in the Hypothalamus and Pituitary of the Sheep Fetus with Gestational Age and After Adrenocorticotropin Treatment. Endocrinology 126: 1 1-1 7.

Zhou, Y., Spangla, R., LaForge. ICS., Maggos, C.E., Ho, A. and Kreek, M. J. (1 996) Modulation of CRH-RI mRNA in Rat Anterior Pituitary by Dexamethasone: Correlation with POMC mRNA. Peptides 17: 43 5-44 1.

Zoumakis, E.. MMgiannakis, A., Margions, A., Stouniaras, C. and Gravanis , A. (1996) Corticotropin-Releasing Hormone (CRH) in Normal and Pregnant Utenis: Physiological Implications. Front Biosci 1: El -E8.

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