Species-specificity of growth-promoting effects of prolactin ...

J. Anat. (1998) 192, pp. 1–12, with 8 figures Printed in the United Kingdom 1

Species-specificity of growth-promoting effects of prolactin

during rat embryogenesis

AHMET KAGAN KARABULUT1 AND MARGARET K. PRATTEN2

"Department of Anatomy, University of Selcuk, Konya, Turkey and #Department of Human Anatomy and Cell Biology,

Queen’s Medical Centre, Nottingham, UK

(Accepted 29 July 1997)

In the early stages of embryonic development, many growth-promoting molecules must be provided by the

maternal system. The molecules involved in growth processes may be either hormones or growth factors, or

molecules that interact with such factors. The pregnancy related hormone, prolactin (PRL, MW 23 kDa) has

been implicated in the control of embryonic growth. The growth-promoting potential of PRL and its

species-specificity was investigated by culturing 9±5 d rat embryos in vitro for 48 h in depleted serum in the

presence and absence of PRL from 3 different species. The growth-supporting capacity of the serum was

reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with

a molecular weight exclusion of 30 kDa. This method provided a ‘semidefined’ medium (retenate) in which

embryonic growth and development was significantly reduced, demonstrating that the low molecular weight

fraction of serum may contain some growth-promoting factors. Addition of PRL (0±4–25±6 ng}ml) from

different species (human, sheep and rat) to retenate significantly improved embryonic growth and

development, suggesting that the developing embryo may utilise PRL. Amongst PRLs, rat PRL was found

to be active at much lower concentrations than either of the other molecules, and human PRL had more

effect in low concentrations than sheep PRL suggesting a species-specificity for this hormone. It may be that

the PRL receptors of the rat embryos have greater affinity for the rat hormone as different responses for

hormones from different species have been shown. These findings suggest that embryos may be able to

utilise maternally derived PRL during organogenesis.

Key words : Whole embryo culture ; rat ; organogenesis ; development.

It has been well known that a variety of hormones

play an important role in the development of the

mammalian embryo, from the moment of conception.

The hormonal milieu which bathes the embryo is the

product of a maternal–placental–embryonic inter-

action. The hormones that play a critical role in the

regulation of postnatal growth, such as growth

hormone, thyroid hormone, glucocorticoids and sex

steroids, appear to be less important in embryonic and

fetal growth, since the absence or deficiency of these

hormones is not accompanied by fetal growth failure

(Freemark & Handwerger, 1989). Since growth hor-

Correspondence to Dr Margaret K. Pratten, Department of Human Anatomy and Cell Biology, Queen’s Medical Centre, Nottingham, NG7

2UH, UK. Tel : 44-115 9709429; fax: 44-115 9709732; e-mail : Margaret.Pratten!nottingham.ac.uk

mone (GH) appears to be of minimal importance for

the stimulation of embryonic and fetal growth, GH-

like peptides synthesised by the placenta such as

placental lactogen (PL) and prolactin (PRL) have

been postulated to play roles in the regulation of

embryonic and fetal growth processes due to their

circulation in fetal serum in mid-late gestation (Ogren

& Talamantes, 1988), direct metabolic effects in fetal

tissues (Freemark & Handwerger, 1984a, b) and

receptor expression in embryonic and fetal tissues

(Underwood & D’Ercole, 1984; Hill et al. 1988; Hill,

1992; Fowlkes & Freemark, 1993; Freemark et al.

1993). However, the role of these hormones in early

embryonic development is still not clear.

Several studies demonstrated the importance of the

hormone for fetal development. Treatment of preg-

nant sheep with bromocriptine, a well known an-

tagonist for PRL, reduces both maternal and fetal

PRL levels, resulting in decreased fetal weight (Taka-

hashi et al. 1979), thus suggesting that there is a

possible link between PRL and fetal growth. PRL has

also been postulated as an important embryological

and neonatal growth hormone (Nicoll, 1978; Under-

wood & D’Ercole, 1984). It has been suggested that

hormones with lactogenic activity play an important

role in the enhancement of insulin secretion by

pancreatic islets of Langerhans that occurs during

pregnancy. Recent in vitro studies have confirmed

that both PLs and PRLs, but not GHs, are potent

stimulators of both insulin secretion and beta cell

proliferation in pancreatic islets of Langerhans iso-

lated from mice, rat, and humans (Nielsen, 1982;

Sorenson et al. 1987; Brelje & Sorenson, 1988, 1991;

Brelje et al. 1989, 1993, 1994). The importance of

insulin for growth and development of the embryo

and fetus has been demonstrated (Travers et al. 1989,

1992).

Another possible mechanism for the effect of

prolactin may be in the regulation of fetal insulin-like

growth factor (IGF) concentrations during pregnancy

(Francis & Hill, 1975; Hill et al. 1977; Underwood &

D’Ercole, 1984). It is known that the IGFs are anabolic

hormones with both proliferative and differentiative

functions (D’Ercole et al. 1980; D’Ercole & Under-

wood, 1981; Adams et al. 1983a, b ; Hill et al. 1985;

Sara & Hall, 1990).

It is clear that PRL may be involved in the

regulation of embryonic and fetal growth. However,

apart from a limited study (Calvert, 1985; Calvert et

al. 1986), there are no published data observing the

direct effect of PRLs on embryonic growth and

development using the embryo culture system.

Prolactin is a single 23 kDa polypeptide chain of

199 amino acid residues with 3 intramolecular

disulphide bridges. The sequence identity of human

prolactin (hPRL) with PRL of sheep (sPRL), pig

(pPRL) and rat (rPRL) is 73, 77 and 60% respectively,

and it shows 16% homology to GH. Of the 199 amino

acid residues of prolactins 99 residues are identical in

sequence position in all 4 species (Basudev & Parlow,

1977). There is also 63% overall homology between

rat and sheep prolactin (Nicoll et al. 1986). Therefore,

considering the species-specificity, we investigated the

influence of the addition of wide range concentrations

of human (hPRL), sheep (sPRL) and rat (rPRL)

prolactin to depleted serum on early embryonic

growth and development. Depletion of serum was

achieved by prolonged filtration of serum in a

centrifugal concentrator, which has been shown to

reduce its growth-supporting capacity by removing

low molecular weight molecules (Karabulut &

Pratten, 1995). We have also shown that depletion

of whole rat serum by prolonged filtration does not

have any detrimental effect on serum components as

reconstitution of the retenate (high molecular weight

fraction) with the filtrate (low molecular weight

fraction) completely restored the growth supporting

capacity of the serum molecules (Karabulut & Pratten,

1995).

Chemicals

Rat prolactin was supplied by Biogenesis Ltd (Poole,

Dorset, UK). Human and sheep PRL, and all other

chemicals were supplied by Sigma Chemical Company

(Poole, Dorset, UK).

Animals

Wistar rats (Rattus norvegicus) were used in this study

and were obtained from the breeding colony of the

Queen’s Medical Centre, University of Nottingham.

Male and female rats were placed together in the

evening (1 pair per cage), and the presence of a vaginal

plug the following morning was taken to indicate

mating had occurred. The female rat would be

considered to be 0±5 d pregnant at noon on that day,

since copulation was assumed to have occurred within

2 h either side of midnight.

Explanation and the culture of embryos

Female Wistar rats were anaesthetised, using ether, on

d 9±5 and the conceptuses explanted according to a

standard method of embryo culture (New, 1978). The

uterus was quickly removed from pregnant rats and

transferred to a dish containing Hanks’ balanced salt

solution. It was then opened with jeweller’s forceps to

expose pear-shaped masses of decidual tissue which

were teased away from the contained egg cylinder

under the dissecting microscope. Reichert’s membrane

was removed, and the 9±5 d egg cylinders were placed

in a sterile glass bottle which contained 1 ml of

immediately centrifuged, heat-inactivated rat serum

per embryo (New, 1978). The bottles containing

medium were then stoppered and placed horizontally

on rollers in an incubator at 37 °C at 30–40 rev}min

for 48 h. Embryos were gassed immediately after

2 A. K. Karabulut and M. K. Pratten

explantation with a gas mixture containing 5% O#,

5% CO#, 90% N

#. After 24 h this was replaced with

20% O#, 5% CO

#, 75% N

#, and after 44 h with 40%

O#, 5% CO

#, 55% N

#. Embryos were harvested at

48 h.

Endpoint measurement

After 48 h of culture, the growth and development of

embryos was evaluated under a dissecting microscope

using the morphological scoring system of Van Maele-

Fabry et al. (1990). In this system the following

endpoint measurements were assessed. For growth,

crown-rump length was measured; for differentiation,

somite number was recorded; and for development,

the flexion of the embryos, as well as the development

of the fore, mid and hind brain, caudal neural tube,

optic, otic and olfactory systems, maxillary and

mandibular processes, branchial bars, and fore and

hind limbs were examined. In addition the size and

vascularisation of the yolk sac and development of

allantois were also recorded. Protein assay (Lowry et

al. 1951) was performed to assess the total protein

content of yolk sac and embryo, using bovine serum

albumin as reference protein.

Filtration of serum

Filtration of homologous heat inactivated serum was

achieved using a Macrosep centrifugal concentrator

with a nominal molecular weight exclusion of 30 kDa

(Filtron, Northborough, USA). Pooled serum (15 ml)

was added to each concentrator and centrifuged for a

total of 8 h at 3500 rpm at 4 °C, with 5 ml retained as

control serum for each experiment. After 2 h of

centrifugation the low molecular weight filtrate was

removed and stored at ®20 °C. The high molecular

weight fraction retained by the filter membrane

(retenate) was resuspended with distilled water to

15 ml to prevent it becoming too viscous to be filtered

efficiently. Serum was recentrifuged and the above

procedure repeated every 2 h. At the end of the

procedure the filtrate was removed and stored. During

the filtration retenate loses many of the low molecular

weight molecules it originally contained; therefore, it

has a very low osmolarity. The retenate was recon-

stituted with double-strength Hanks’ balanced salt

solution (600 mOsm) to a final volume of 15 ml. The

osmolarity of the retenate was then corrected to

within the normal range for whole rat serum (290–330

mOsm) with distilled water. Also the pH was adjusted

to 7±8 with 0±1 sodium hydroxide or 0±1 hy-

drochloric acid if required. Prior to use the retenate

was sterile filtered with a Minisart NML disposable

syringe filter holder, pore size 0±2 µm (Sartorius AG,

Go$ ttingen, Germany) and supplemented with 2 mg}ml glucose and 10 µl}ml minimal essential medium

(MEM) vitamin solution (Sigma, Poole, UK).

Experiments undertaken

In order to investigate the influence of PRL from

different species on mammalian embryonic growth

and development rat embryos were cultured in

retenate in the presence of human, sheep and rat PRL.

Human and sheep PRL were used at concentrations

between 0±4 and 25±6 ng}ml. In order to determine the

effect of rat PRL a wider range of concentrations

(0±1–25±6 ng}ml) of hormone was added to retenate.

In each experiment retenate (supplemented with

glucose and MEM vitamins) and whole rat serum

controls were included. At least 10 embryos were used

for each experimental condition. Embryos were then

cultured for 48 h with appropriate gassing, assessed

morphologically and embryonic and yolk sac protein

contents determined.

Statistics

Differences between experimental and control groups

were analysed statistically as follows. The yolk sac

diameter, crown–rump length and protein contents of

the embryos and yolk sacs were analysed using 1-way

analysis of variance (ANOVA) to determine the

presence of significant difference within the data, and

a parametric Duncan’s multiple range test was

performed in order to demonstrate where the exact

significant differences lay. Analysis of differences in

morphological score and somite number was per-

formed using a nonparametric Kruskal–Wallis test to

demonstrate the presence of a significant difference

within the data and this was followed by subsequent

Mann–Whitney U tests as the use of parametric tests

on such data was inappropriate. All tests were

performed on the statistics package, SPSS-X. Signi-

ficance was assumed at P! 0±05, P! 0±01, and

P! 0±001.

The embryos cultured in retenate supplemented with

glucose and vitamins showed severe growth retar-

dation in all scoring criteria (total morphological

Effects of prolactin during rat organogenesis 3

Fig. 1. Effects of supplementation with human PRL (0±4–25±6 ng}ml) on the growth supporting capacity of retenate. WRS, whole rat serum;

30 KR, retenate. Results are the mean of the values³... for at least 10 embryos. *, **, *** Significantly different from retenate control

values at P! 0±05, P! 0±01, P! 0±001.


Fig. 2. 11±5 d rat embryos following 48 h culture (a) in normal rat

serum, (b) in serum depleted of low molecular weight molecules

(MW! 30 kDa). Note severe growth retardation in embryo grown

in retenate. Bars, 500 µm. Bb, branchial bars ; H, hindbrain; He,

heart ; F, forebrain; M, midbrain; O, optic system; Ot, otic system;

S, somites.

score, yolk sac diameter, crown–rump length, somite

number, protein contents of embryo and yolk sac)

compared with those cultured in whole rat serum

(Figs 1, 2).

The addition of hPRL to retenate caused an

increase in embryonic development when compared

with development of embryos cultured in retenate

supplemented with glucose and vitamins only (Fig. 1).

Morphological score and somite number were signifi-

cantly increased in the presence of 0±4 and 0±8 ng}ml

hPRL. Addition of higher levels (1±6–25±6 ng}ml) of

hPRL significally improved all parameters, especially

morphological score and somite number, and embryo

protein content when 6±4 ng}ml and higher concen-

Fig. 3. A left lateral view of an 11±5 d rat embryo following 48 h

culture in the presence of 12±8 ng}ml human PRL. Bar, 500 µm.

trations of hPRL were added (Fig. 3). Indeed, there

was no significant difference between the values

obtained for somite number at 12±8 ng}ml and for

yolk sac protein content at 1±6–25±6 ng}ml and those

observed in whole rat serum.

All embryos cultured in the presence of low

concentrations (0±4–1±6 ng}ml) of sPRL did not show

any significant difference in growth and development

in all parameters compared with retenate control

embryos. Significant improvement was seen when

retenate was supplemented with higher levels (3±2–25±6ng}ml) of sPRL in morphological score, somite

number, crown–rump length, embryo and yolk sac

protein contents, and yolk sac diameter when 3±2 and

12±8 ng}ml sPRL was added (Fig. 4). The extent of

growth and development of the embryos in this group

did not reach that of those grown in whole rat serum

at any concentration of sPRL (Fig. 5).

The outcome of all parameters of growth and

development was significantly improved on the ad-

dition of 0±4–25±6 ng}ml rPRL compared with ret-

enate only (Figs 6, 7). There was no significant

difference between the results obtained with rPRL-

supplemented retenate and those obtained for whole

rat serum for embryonic protein (0±4–3±2 ng}ml), yolk

sac protein (1±6–6±4 ng}ml), and somite number

(0±8–1±6 ng}ml). It was noticeable that at higher

concentrations both yolk sac and embryo protein

levels tended to be decreased. It was observed that the

growth-promoting effects of rPRL started at very low

concentrations and increased dose dependently (Fig.

8).


Fig. 4. Effects of supplementation with sheep PRL (0±4–25±6 ng}ml) on the growth-supporting capacity of retenate. WRS, whole rat serum;


values at P! 0±05, P! 0±01, P! 0±001.


Fig. 5. A left lateral view of an 11±5 d rat following 48 h culture in

the presence of 12±8 ng}ml sheep PRL. Bar, 500 µm. Addition of

sheep PRL to the retenate improved embryonic growth compared

with retenate, but this improvement did not reach the level of those

cultured in whole rat serum.

Embryonic growth and development were decreased

in all parameters (total morphological score, somite

number, yolk sac diameter, crown–rump length,

protein content of embryo and yolk sac) in those

embryos cultured in retenate compared with whole rat

serum. These results support our previous observa-

tions (Karabulut & Pratten, 1995). Many growth-

promoting factors such as epidermal growth factor

(EGF; 6 kDa), basic fibroblast growth factor (bFGF;

17–19 kDa), transforming growth factor α (TGF-α ;

7±5 kDa) and β (TGF-β ; 25 kDa), insulin (6 kDa),

prolactin (PRL; 23 kDa), placental lactogen (PL;

21±5 kDa), insulin-like growth factors I (7±6 kDa)-II

(7±5 kDa), platelet derived growth factor II (PDGF-

II ; 28 kDa) are of low molecular weight, and therefore

the removal of such molecules may cause the reduction

in the growth-supporting capacity of serum.

This study shows that there is improvement of

embryonic growth and development on the addition

of prolactin from different species to the retenate.

Because the filtration of serum to reduce its growth-

supporting capacity by removal of low molecular

weight molecules is a nonspecific method, the growth-

promoting effects of PRL on the addition to retenate

observed in this study may be a compensatory effect

for other growth factors depleted from the serum.

However, depletion of serum has previously been used

to investigate the potential of maternally derived

growth-promoting factors. It has been demonstrated

that the embryos undergoing organogenesis are

capable of utilising the maternally derived factors

such as EGF, transferrin and insulin when cultured in

‘exhausted serum’ (Pratten et al. 1988). It has also

been shown that when embryos are cultured in

depleted serum in the presence of some growth factors,

there is an improvement in embryonic growth, e.g.

EGF (Andrews, 1986), IGF-I (Calvert, 1985), FGF,

platelet-derived endothelial cell growth factor (PD-

ECGF) and vascular endothelial growth factor

(VEGF) (Ulger et al. 1996, 1997; Ulger, 1997). Other

growth factors have no effect, such as platelet derived

growth factor (PDGF) (Khan, 1995) and nerve growth

factor (NGF) (Calvert, 1985).

Previously, Calvert et al. (1986) showed that

addition of hPRL to dialysed serum at a concentration

of 4 ng}ml led to significant increases in morpho-

logical score and somite number only. In our study the

effects of additional hPRL were observed when much

lower concentration 0±4 ng}ml augmented the mor-

phological score and somite number, and also other

parameters when 1±6 ng}ml and higher concentrations

were added. This slight discrepancy could be explained

by the inclusion in their results of batches of poorly

dialysed serum, which seems to mask the beneficial

effects of hPRL addition. We have observed that rat

prolactin was active at much lower concentrations

than either of the other molecules, and human

prolactin had more effect in low concentrations than

sheep prolactin. It may be that the prolactin receptors

of rat embryos are more specific for the rat hormone

or show different responses for hormones from

different species. These results suggest that embryos

undergoing organogenesis may be able to utilise

maternally derived PRL which supports the hypoth-

esis that PRL is an important embryological and

neonatal growth hormone (Nicoll, 1978; Underwood

& D’Ercole, 1984).

There are a few mechanisms by which PRLs may

affect embryonic and fetal growth such as increasing

the insulin secretion from the islets of Langerhans

during pregnancy, stimulating the synthesis and

secretion of IGFs and}or direct effects on the

developing embryo via its own receptors.

The effect of PRLs on embryonic development via

increasing the insulin secretion is not the explanation

in our study. First, the studies demonstrated the

effects of PRLs in increasing the insulin secretion from

islets have been carried out in neonatal tissues, whilst

the rat embryo in our culture system is unable to

produce its own insulin since at this time the pancreas

has not yet developed. It is therefore totally dependent


Fig. 6. Effects of supplementation with rat PRL (0±4–25±6 ng}ml) on the growth-supporting capacity of retenate. WRS, whole rat serum;


values at P! 0±05, P! 0±01, P! 0±001.


Fig. 7. A left lateral view of an 11±5 d rat embryo following 48 h

culture in the presence of 12±8 ng}ml rat PRL. Bar, 500 µm.

Development of the embryo is very similar to that found with whole

rat serum.

on maternal insulin levels. The effects of PRL

observed in our study may be upon the embryo and

the hormone may use its own receptors for a direct

effect on embryonic growth and}or induce the

synthesis and secretion of the IGFs, as do some other

hormones and growth factors.

Because embryonic and fetal growth is largely

independent of both fetal and maternal growth

hormone, it has been suggested by several authors

that the lactogenic hormone PRL may achieve its

growth-promoting effect through stimulating IGF

production and this functions as a skeletal growth

hormone (Francis & Hill, 1975; Underwood &

D’Ercole, 1984).

Critical analysis of the literature confirms a close

relationship between IGFs and embryonic and fetal

growth. It is well demonstrated that as well as their

receptors, both IGF I and II are present in most

embryonic tissues in many species including human,

mouse, rat and chick, and these factors have been

shown to affect embryonic and fetal development by

their mitogenic and differentiative actions at both cell

and tissue levels (reviewed by Han & Hill, 1992).

High levels of PRL in amniotic fluid have been

suggested to be important for fetal development and

the PRL concentrations in human amniotic fluid

exceed maternal and fetal serum levels 10–20-fold at

midgestation (Clements et al. 1977), and this may be

produced by the chorion (Freisen et al. 1972). Several

other investigators demonstrated that the human

decidual cells synthesise and release decidual prolactin

(dPRL), immunologically and biochemically identical

to human pituitary PRL (Clements et al. 1977;

Riddick & Kusmin, 1977; Golander et al. 1978, 1979;

Riddick et al. 1978; Braverman et al. 1984; Wu et al.

1991). The PRL secreted by decidua may have an

influence on the maintenance of IGFs during preg-

nancy (Daughaday & Kapadia, 1978).

Although it is known that some high molecular

weight molecules such as IgG (Huxham & Beck, 1981)

and transferrin (Cumberland & Pratten, 1992) can

cross the yolk sac placenta of the rat, in general the

placenta acts as a barrier. Proteins are usually taken

up by the yolk sac endoderm and broken down in

lysosomes, and the resultant amino acids are sub-

sequently utilised by the embryo and visceral yolk sac

to synthesise several proteins (Freeman et al. 1981). It

is clear that the large molecular weight hormones

(glycoproteins and polypeptides, such as PL, PRL) do

not cross the placental barrier ; the embryo must

therefore depend on its own or the placental con-

tribution. Since there is no decidual tissue present in

the whole embryo culture system, the developing

embryo can only utilise the PRL from the culture

medium.

It is possible that PRL in the in vitro culture system

may influence embryonic growth and development

directly via its own receptors. Freemark et al. (1993)

demonstrated expression of PRL receptor mRNA

transcripts in fetal rat adrenal, kidney, small intestine,

liver, lung, and brain on d 20 of gestation and in the

whole conceptus on d 9 and 10. Recently the cellular

distribution and developmental expression of the PRL

receptor in the late gestational fetal rat (d 17±5–20±5)

have been examined by in situ hybridisation, immuno-

histochemistry, and radioligand binding (Royster et

al. 1995). They showed the widespread expression of

mRNA encoding the 2 isoforms of receptor in tissues

derived from all 3 germ layers. Immunohistochemical

studies using monoclonal anti-PRL receptor anti-

bodies demonstrated that the distribution of PRL

receptor immunoractivity was found to be similar to

that of PRL receptor mRNA.

The widespread expression of the PRL receptor in

fetal rat and uteroplacental tissues, the presence of

PRL receptor mRNA in whole conceptus as early as

9–10 d of gestation (Freemark et al. 1993), the

induction of receptor expression in late gestation, and

the effects of PRLs from different species on rat

embryonic growth and development in culture as

observed in the present study suggest an important

growth-promoting function for PRL in embryonic

and fetal development. However, it is still not clear


Fig. 8. Effects of supplementation with lower concentrations of rat PRL (0±1–0±4 ng}ml) on the growth-supporting capacity of retenate.

WRS, whole rat serum; 30 KR, retenate. Results are the mean of the values³... for at least 10 embryos. *, **, *** Significantly different

from retenate control values at P! 0±05, P! 0±01, P! 0±001.


what exact role PRL plays in tissue development, and

the mechanism of action of PRL is poorly understood.

Ahmet K. Karabulut acknowledges the generosity of

the University of Selcuk, Turkey for the financial

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Species-specificity of growth-promoting effects of prolactin ...

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