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INTRODUCTIONDuring the period from birth to puberty, mammary
gland
undergoes critical metamorphosis and obtains its future
potentialto produce milk (1). Mammogenesis is allometric during
thisinterval. At 3 months, the mammary gland starts a constant
growthphase, much more rapidly than other parts of the body. At
puberty,the proliferation of adipose tissue accompanies this rapid
growthof the milk ducts. The first pre-pubertal stage is
characterized byelongation and strengthening of the canals in the
center of thefreshly tissue (2). This is extremely important to
supply thesupport and space necessary to develop canals and
ultimately, tosupport the frame on which the alveolar system is
based. Onwardspuberty, the mammary gland follows an isometric
growth (3). Atthe start of gestation, the growth of the mammary
gland becomesallometric, because of an exponential increase in the
numbers ofcells and canals. Following the elongation of the canals,
thelobulo-alveolar system puts itself in place,
progressivelysubstituting the adipose tissue that is in the process
of regressing.In the last days of gestation, the epithelial cells
increase in volumeand acquire specific structures to synthesize
protein, to allowintense secretion. Many investigators have focused
their studiesduring the prepubertal period (4, 5). During this
critical stage, theincrease in mammary gland weight is correlated
to the live weightgain (LWG) due to the adipose tissue increase (6,
7). On the otherhand, mammary parenchyma development is
inverselyproportional to the live weight gain. Harrisson et al. (8)
shown thatmammary parenchyma was permanently impaired by high rates
ofLWG in the first year of life. This negative effect does not
occurafter the puberty. Thereby, every alteration of
mammogenesis
during the prepubertal using low/high feeding levels or
hormonalmanipulations has an impact on the subsequent milk
productionduring the first lactation.
In humans and rodents, evidence for role of estrogen inmammary
gland development is considerable. The obligate roleof estradiol in
mammogenesis has been underlined in ERαknocked out mice (9). In
their experiment, authors demonstratedthat mammary glands from
these mice were rudimentary withlimiting branching ducts and that
this malformation couldn't bereversed by estradiol injection. Other
studies showed that ERα isapproximately expressed in 15-30% of
luminal epithelial cellsand not in other cells within the mammary
gland (10, 11). Thissuggests that ERα is a key mediator of
estradiol action in thenormal mammary gland. Berry et al. (12) have
demonstrated thatovariectomy in prepubertal heifers caused a
dramatic reductionin mammary gland development and in mammary
epithelial cellproliferation. They have reported that ERα
expression wasrestricted to mammary epithelial cells. Moreover,
growthhormone (GH) injection can stimulate mammary
epithelialproliferation, but no changes in the proportion of
ERα-positivecells were found. Finally, their observations that
mammarydevelopment was more severely affected in
heifersovariectomized before 6 weeks of age implied that there is
acritical period of ovarian stimulation during the first 2 month
ofage. Ellis et al. (13) also concluded that parenchymal
mammarydevelopment in the prepubertal lamb is largely unaffected
byovariectomy, through roughly 3 months of age. They
proposedovarian independence for prepubertal ovine
mammarydevelopment and suggested that the endocrine control of
ovinemammogenesis is distinctly different than in other
species.
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 3,
127-133www.jpp.krakow.pl
F. DESSAUGE1, L. FINOT1, S. WIART1, J.M. AUBRY1, S.E. ELLIS2
EFFECTS OF OVARIECTOMY IN PREPUBERTAL GOATS
1INRA, Agrocampus Ouest, UMR1080 Dairy Production, Saint Gilles,
France; 2Animal and Veterinary Sciences Department, Clemson
University, Clemson, SC 29634, USA
The objectives of this study were to determine the effects of
ovariectomy on mammary gland development in prepubertalgoats and to
validate this model to study mammogenesis in young dairy ruminants.
In this experiment, 3 months of agedgoats were ovariectomized (ovx)
while shammed goats played as surgery controls (sham). Thereafter,
sham and ovx goatswere slaughtered at 7 months of age to provide
tissue for the assays. Results demonstrated that proliferation of
mammaryof mammary epithelial cells was significantly lower in
ovariectomized goats compared to control goats. In ovx
animal,epithelium structures were completely overstretched and
epithelial ducts were undeveloped with limited branching
whereascontrol animals had classical complex arborescent units with
multiple round ductules and limited stroma. Concerning ERα(estrogen
receptor α), PR (progesterone receptor) and P450 (aromatase)
expression, results showed number of ERα, PRand P450 positive cells
was higher in shammed goats compared to ovariectomized goats. All
this results suggested that goatmammogenesis and ovarian control
are similar to prepubertal heifers and that young goats are a good
model to studymammary gland development in ruminants. In
conclusion, we demonstrated that ovariectomy of prepubertal
goatsdecreased proliferation of mammary epithelial cells with a
profound alteration of cell adhesion molecules.
K e y w o r d s : mammary gland, ovariectomy, prepubertal,
goats, estrogen receptor
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Mammary gland development in goats during theprepubertal period
has not been extensively studied. The purposeof the present study
was to increase the understanding ofbiological mechanisms
underlying mammary growth anddevelopment as to elucidate the role
of ovary during earlyprepubertal mammogenesis.
MATERIAL AND METHODSAnimals and tissue preparation
Mammary tissues used in this study were obtained from sixAlpine
goats assigned to one of 2 treatments: shammed (sham)or
ovariectomized (ovx). Surgery on animals was performed 3months
after birth. Ovaries resection (ovx) concerned half goatsof the
experiment whereas the other half was only opened andstitched
(sham). Samples of mammary tissue were obtained atautopsy from
animals at 7 months of age, under generalanaesthesia with
subsequent euthanasia (Rompun i.v. 1 ml,Dolethal, i.v. 25 ml). The
mammary glands from goats wereremoved within 20 minutes of
slaughter. Freshly dissected tissuesegments were cut into small
pieces (1 g) and frozen in liquidnitrogen for protein extraction or
were fixed forImmunohistochemistry study. All procedures used in
this studywere approved by French National Institute of
AgriculturalResearch and by French Animal Care and Use
Committee.
AntibodiesThe following antibodies were used for western
blotting and
immunohistology: ERα (sc-787, Santa Cruz Biotechnology),P450
(sc-25270, Santa Cruz Biotechnology), PR (SM5025,Acris Antibodies
GmbH), Cadherin-Catenin antibody samplerkit (#9961, Cell Signaling
technology), PCNA (M0879,DakoCytomation), β-Actin (A5441,
Sigma-Aldrich, France),anti-rabbit HRP-conjugated antibody (#7074,
Cell Signalingtechnology), anti-mouse HRP-conjugated antibody
(31450,Pierce, Perbio Science, Belgium) and goat anti-mouse
FITC-conjugated antibody (F5387, Sigma-Aldrich, France).
Protein extractionTotal proteins were extracted from frozen
mammary glands
tissues using the tissue protein extraction reagent T-PER
(78510,Pierce, Perbio Science, Belgium). Mammary tissue sample
(100mg) was ground in liquid nitrogen with a mortar and a pestle
and 1ml of T-PER reagent was added to the powder.
Afterhomogenization, the mixture was centrifuged at 10 000g for
5minutes at 4°C and the supernatant containing total proteins
wasrecovered. The protein concentration was determined by the
Lowrymethod (14) using the DC Protein Assay kit (500-0111,
BioRad,Marnes-la-Coquette, France). Then, lysates were combined
withsample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 0.1%bromophenol
blue, 20% glycerol and 5% b-mercaptoethanol),boiled for 5 minutes
at 95°C and resolved by SDS-PAGE.
Western blot analysisProteins (30 µg per lane) were separates on
10% SDS-
polyacrylamide gels, blotted to PVDF membranes
(BioRad,Marnes-la-Coquette, France) and incubated with
blockingsolution (5% dry skimmed milk dissolved in TBS-T (50 mM
Tris-HCl pH: 8.6, 150 mM NaCl and 0.1% Tween) for 30 minutes. Aset
of prestained molecular mass standards (SM0671, Fermentas,St
Remy-les-chevreuses, France) was run in each gel.Membranes were
incubated overnight at 4°C with the appropriate
dilution of the primary antibodies. Then, membranes werewashed
with TBS-T before incubation with horseradishperoxidase-conjugated
anti-mouse or anti-rabbit secondaryantibodies for 1 hour at room
temperature. To detect theperoxidase activity, the enhanced
chemiluminescence detectionsystem ECL (RPN2109, GE Healthcare
Europe Gmbh, Saclay,France) was used. Membranes were then exposed
to hyperfilm(Curix ortho HT-G films, Agfa, Mortsel, Belgium) and
imagesgenerated were scanned at 16-bit/600dpi resolution with
aCanoscan D1250 U2, saved as tiff files and calibrated to anoptical
density scale. The integrated optical density of bands
wasquantified using the ImageJ software. Each sample wasnormalized
to β-actin content, a constitutively protein expressed.
Immunohistochemistry for proliferation and apoptosis assaysTo
determine mammary epithelial cells (MEC) in
proliferation in the mammary gland, we carried
outimmunohistochemical staining for PCNA (Proliferating CellNuclear
Antigen). Indeed, proliferating mammary epithelialcells (MEC) were
identified in mammary tissue as cellsexpressing the PCNA antigen as
previously reported (15).Briefly, mammary gland tissues were fixed
in PBS 4%paraformaldehyde for 24 h at 4°C, cryoprotected in 20%
sucrosefor 48 h at 4°C, frozen in isopentane bath, cooled on dry
ice, andstored at -80°C until use. Seven micrometer-thick
cryosectionswere mounted onto Superfrost/Plus slides (Prolabo,
Bondoufle,France) then quenched in PBS 3 % hydrogen peroxide
(H2O2)and 10% methanol for 30 min. The sections were
thoroughlywashed in PBS, permeabilized with PBS 1% SDS for 5
min,washed three times and pre-incubated in PBS 1% BSA for 1 h
atroom temperature. The slides were incubated in the presence
orabsence of the primary antibody for 1 night at 4°C. Afterwashing
in PBS 1% BSA, samples were incubated with a secondantibody for 1 h
at room temperature. Subsequently, themammary gland sections were
counterstained for 3 min with 33µg/mL 4',
6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich, France)
then 3 min with propidium iodide at 333 µg/mL(P4864, Sigma-Aldrich,
France).
Apoptosis induction in mammary cells was determined bythe
terminal deoxynucleotidyltransferase (TdT)-mediated dUTPnick-end
labeling (TUNEL) staining method based on DNAfragmentation
detection. Seven micrometer-thick cryosectionsmounted onto
3-aminopropyltriethoxysilane (Sigma-Aldrich)treated slides, were
unfrozen and incubated 30 min at 70°C in 10mM citrate sodium 0.1%
triton solution. The slides were washedin PBS then incubated 30 min
at 37°C in 200 ng/µL proteinaseK solution (V3021, Promega France,
Charbonnieres, France)followed by incubation with the reagents of
the DeadEndTMFluorometric TUNEL System (G3250, Promega
France)according to the manufacturer's instructions. Mammary
glandsections were counterstained with DAPI after TUNEL
reaction.The slides were then mounted with Vectashield
(Valbiotech,Paris, France), examined under fluorescence microscopy
usingEclipse E400 Nikon microscope (Nikon France, Le Pallet,France)
and pictures were captured by a Digital Still CameraDXM 1200
(Nikon). Height microscopic fields (magnification x200, area 0.14
mm2 per microscopic field) were examined foreach tissue sample.
PCNA positive epithelial cells weredetermined thanks to IP (iodide
propidium) staining whichoutlined the acini. Number of cells were
detected and countedusing ImageJ software.
Immunohistochemistry for ERα, PR and P450 expressionCellular
expression of ERα, PR and P450 were evaluated by
immunohistochemistry as described by Berry et al. (12).
Briefly,
128
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mammary gland tissues were fixed in PBS 4% paraformaldehydefor
24 h at 4°C and embedded in paraffin using standardprotocols. Five
micrometers sections were mounted on positivelycharged slides,
deparaffinized in xylene, rehydrated andfollowing rehydration,
endogenous peroxidases of tissue sectionswere quenched in 3% H2O2.
To unmask antigen sites, the slideswere microwaved for 3×5 min in
400 ml of 10 mM citrate buffer,pH 6.0. Slides were then cooled for
30 min, washed 3×2 min inPBS and blocked in 5% nonimmune serum for
30 min. Sectionswere incubated with 100 µl of primary antibodies
overnight at4°C. Subsequently, slides were washed in PBS for 3×2
min anddetection of the primary antibody was performed using
HRPimmunostaining kit (KP50L, Clinisciences, Montrouge,
France).Slides were incubated with streptavidin-peroxidase
(HRP)conjugate for 10 min, washed 3×2 min in PBS before
visualizingthe antibody-HRP complex by incubation with
diaminobenzidinefor 5 min. Finally, slides were dehydrated and
mounted withPermount (Fisher Scientific, Pittsburgh, PA).
Statistical analysisData were analyzed by ANOVA using the mixed
procedure of
SAS (SAS Institute, 1999) with repeated statement. The effect
ofovariectomy was tested. Data were expressed as the mean ±
SEM.
RESULTSHistology of mammary gland
Adipose tissue was widely represented compared tosecretory
tissue (parenchyma) in ovariectomized goats (Fig. 1).Morphological
analysis indicated that mammary parenchymaarea was very affected by
ovariectomy. In ovx animals,epithelium structures were completely
overstretched withundeveloped epithelial ducts with limited
branching. In a sametime, control animals had classical complex
arborescent unitswith multiple round ductules and limited stroma
(adipocytesand connective tissue). Moreover, like bovine
mammarystructures, there were three layers of cells: luminal cells,
basalcells and embedded cells. We observed, in shammed animals,that
these layers of epithelial cells were organized in ductules.Within
the mammary gland from ovx animals, parenchymatissue is rudimentary
and we observed a single layer ofepithelial cells.
Blood samples were collected weekly from birth to slaughterto
measure concentration of estradiol. We found cyclicconcentration of
circulating estrogen in control animals and theaverage of estrogen
concentration was decreased 5-fold inovariectomized animals
compared to control (data not shown).
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Fig. 1. Representative images of eosinstaining of parenchymal
tissue fromshammed (A) and ovariectomizedgoats (B). Left bar
represents 20 µm.
Fig. 2. Expression of ERα in mammary epithelial cells. A)High
magnification (bar represents 20 µm) of ductularstructures from
shammed goat. Note expression of ERα inthe nucleus of epithelial
cells only (brown staining). B) Highmagnification of ductular
structures from ovariectomizedgoat. Note lower expression of ERα in
the nucleus ofepithelial cells only (brown staining). C) Western
blotanalysis of ERα in goat mammary gland extracts. Notedecreased
expression of ERα in ovariectomized (OVX) goatscompared to shammed
goats. Actin was used as control ofloading.
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These results suggest that estradiol produced by ovaries has
adirect effect on mammary gland development.
Effect of ovariectomy on ERα, PR and P450
aromataseexpression
It is well established that estrogen and progesterone
areabsolutely essential for mammary epithelial proliferation
anddifferentiation. In order to better understand the implication
of
estrogen and progesterone during mammogenesis, we decided
toinvestigate the distribution and expression pattern of
estrogenreceptors (ERα) and progesterone receptors
(PR).Immunolocalisation for the ERα revealed a positive staining
inmammary epithelial cells of shammed animals and
exclusivelylocalized in the nucleus of these cells (Fig. 2).
Interestingly, verylow expression of ERα was found in ovx animals.
Myoepithelialcells, adipocytes and cells from vascular system
wereconsistently negative. Results obtained by Western Blot for
ERα
130
Fig. 3. Expression of PR in mammary epithelial cells. A)Low
magnification (bar represents 10 µm) of ductularstructures from
shammed goat. Note expression of PR in thenucleus of epithelial
cells only (brown staining). B) Lowmagnification of ductular
structures from ovariectomizedgoat. Note lower expression of PR in
the nucleus of epithelialcells only (brown staining). C) Western
blot analysis of PR ingoat mammary gland extracts. Note decreased
expression ofPR in ovariectomized (ovx) goats compared to
shammedgoats. Actin was used as loading control.
Fig. 4. Expression of P450 aromatase in mammary epithelialcells.
A) High magnification (bar represents 20 µm) ofductular structures
from shammed goat. Note expression ofP450 in epithelial cells
(brown staining). B) Highmagnification of ductular structures from
ovariectomizedgoat. Note slightly lower expression of P450 in
epithelialcells only (brown staining). C) Western blot analysis of
P450in goat mammary gland extracts. Note slightly
decreasedexpression of P450 in ovariectomized (ovx) goats
comparedto shammed goats. Actin was used as control of loading.
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in Fig. 2 confirmed that ERα (from mammary gland extracts)was
higher expressed in shammed animals than in ovx animals.
Concerning progesterone receptor (PR) localization, wefound it
localized in the nuclei of epithelial cells within themammary gland
of shammed animals in a manner comparablewith that described for
ER? expression (Fig. 3). Western blotresults indicated that PR
expressed is stronger expressed inshammed animals compared to ovx
animals.
Then we decided to identify the localization and theproduction
of P450 aromatase within the mammary gland.Surprisingly, we
observed a production of P450 aromatase in the
mammary epithelial cells at a high level both in shammed andovx
animals (Fig. 4). P450 aromatase quantification by Westernblot
showed more precisely that ovariectomy slightly affected
itsexpression.
Effect of ovariectomy on epithelial cell proliferation
andapoptosis
Proliferating cell nuclear antigen (PCNA) is highlyconserved
auxiliary protein for DNA polymerase and is greatlyincreased in
proliferation cells as compared with mitotically
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Fig. 5. A) Proliferation of mammary epithelial cells asmeasured
by positive PCNA staining into nucleus.Numbers of proliferating
cells are expressed as apercentage of total epithelial cells. B)
Apoptosis ofmammary epithelial cells as measured by TUNEL
staininginto DNA. Numbers of apoptotic cells are expressed as
apercentage of total epithelial cells. *, P
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quiescent cells (16, 17). Wolf et al. (18) demonstrated a
linearcorrelation between PCNA indices and S-phase fractions
asdetermined by bromodeoxyuridine incorporation in
regeneratingliver. Using PCNA staining, we observed that
proliferation ofmammary epithelial cells (Fig. 5A) was decreased
7-fold in ovxgoats compared to control goats (7.22 vs. 1.49%; P
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activation of numerous transcription factors and
thetransactivation of target genes including that which
encodeimportant regulators of growth, survival and differentiation.
Wehypothesized that β-catenin-Wnt pathway could be
inactivatedduring mammary gland development in ovx animals.
In conclusion, all this results suggested that goatmammogenesis
and ovarian control could be compared toprepubertal heifers and
that young goats are really good modelto study mammary gland
development in ruminants. The modelis less expensive than heifers
and animals manipulations aremore manageable.
Acknowledgements: Authors would like to thank MichelChorho and
Eric Siroux for animal assistance and knowledgeabout goats rearing.
We thank also Marion Boutinaud for criticalreading of the
manuscript.
Conflict of interest statement: None declared.
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November 5, 2008A c c e p t e d : April 15, 2009Author's address:
Frederic Dessauge, INRA, Agrocampus
Ouest, UMR1080 Dairy Production, F-35590 Saint Gilles,France;
e-mail: [email protected]
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