Molecular and Cellular Endocrinology 313 (2009) 9–16

Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology

journa l homepage: www.e lsev ier .com/ locate /mce

Progesterone regulation of primordial follicle assembly in bovine fetal ovaries

Eric E. Nilsson, Michael K. Skinner !

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, WA 99163-4231, United States

a r t i c l e i n f o

Article history:Received 22 June 2009Received in revised form 1 September 2009Accepted 2 September 2009

Keywords:Embryonic developmentFetal ovaryOvarian morphogenesisCowFetal endocrinology

a b s t r a c t

Fertility in mammals is dependant on females having an adequate primordial follicle pool to supplyoocytes for fertilization. The formation of primordial follicles is called ovarian follicular assembly. In ratsand mice progesterone and estradiol have been shown to inhibit follicle assembly with assembly occur-ring after birth when the pups are removed from the high-steroid maternal environment. In contrast,primordial follicle assembly in other species, such as cattle and humans, occurs during fetal developmentbefore birth. The objective of the current study is to determine if progesterone levels regulate primordialfollicle assembly in fetal bovine ovaries. Ovaries and blood were collected from bovine fetuses. Interest-ingly, ovarian progesterone and estradiol concentrations were found to decrease with increasing fetalage and correlated to increased primordial follicle assembly. Microarray analysis of fetal ovary RNA sug-gests that progesterone membrane receptor and estrogen nuclear receptor are expressed. Treatment offetal bovine ovary cultures with a higher progesterone concentration significantly decreased primordialfollicle assembly. Observations indicate that progesterone affects ovarian primordial follicle assembly incattle, as it does in rats and mice.

© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Fertility in mammals is dependant on females having a sup-ply of oocytes for fertilization. Oocytes are stored in mammalianovaries in an arrested state in structures called primordial follicles.Each primordial follicle is composed of a single oocyte arrested inthe diplotene stage of the first meiotic division and surrounded bya single layer of squamous pre-granulosa cells (Hirshfield, 1991).The size (i.e. number) of the primordial follicle pool is one deter-minant of the reproductive lifespan of a female (Hirshfield, 1994).The formation of primordial follicles is a process known as ovar-ian follicular assembly. During follicle assembly groups or ‘nests’ ofoocytes, having completed mitotic proliferation and entered intomeiosis, separate to form individual primordial follicles (Pepling,2006). Many of the oocytes undergo apoptotic cell death duringnest breakdown (Pepling and Spradling, 2001; Tilly, 1996). Folli-cle assembly occurs after birth in rodents (Pepling, 2006; Skinner,2005), and during fetal development in other mammalian speciessuch as cattle (Sakai, 1955; Tanaka et al., 2001), sheep (Mauleon,1974; Sawyer et al., 2002) and humans (Baker, 1963). Cattle andhumans have similar gestation lengths. The corresponding age dur-ing human fetal gestation at which primordial follicle assembly isobserved varies in the literature (Abir et al., 2005; Hirshfield, 1991;Sforza and Forabosco, 1998; van Wagener and Simpson, 1965), but

! Corresponding author. Tel.: +1 509 335 1524; fax: +1 509 335 2176.E-mail address: skinner@wsu.edu (M.K. Skinner).

appears to occur between 16 and 19 weeks (112–133 days) gesta-tion. This is slightly later than the initiation of assembly observedin the current study of approximately 80 days in cattle, and likelyreflects the fact that calves are born at a more developed physicalstage than are human infants.

The factors that regulate follicle assembly are not well under-stood. Tumor necrosis factor alpha (TNF!) signaling is thought tobe important in promoting the oocyte apoptosis that accompaniesoocyte nest breakdown and follicle assembly (Marcinkiewicz etal., 2002; Morrison and Marcinkiewicz, 2002). Administration ofactivin A, a member of the transforming growth factor beta (TGF")superfamily of growth factors, during follicle assembly in miceresults in an increased pool of primordial follicles (Bristol-Gouldet al., 2006), indicating a role for this growth factor. Genetic studieswith knockout mice suggest that several more growth factors mayplay a role in follicle assembly. Growth and differentiation factor9 (GDF-9) (Yan et al., 2001), bone morphogenetic factor 15 (BMP-15) (Yan et al., 2001) and lunatic fringe (Lfng) (Hahn et al., 2005)knockout mouse adults have ovaries where some follicles havemultiple oocytes. Multiple oocyte follicles are thought to be theresult of faulty oocyte nest breakdown. Transgenic over-expressionof inhibin alpha also results in multiple oocyte follicles (McMullenet al., 2001). In addition, nerve growth factor knockout mice haveoocytes not enclosed in follicles at seven days of age, again suggest-ing faulty follicle assembly (Dissen et al., 2001).

Sex steroids have also been shown to play a role in follicle assem-bly. In rats progesterone, but not estrogen, was shown to inhibitfollicle assembly both in vitro and in vivo (Kezele and Skinner, 2003).

0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.mce.2009.09.004

10 E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16

Progesterone was also shown to decrease oocyte apoptosis duringfollicle assembly (Chen et al., 2009; Kezele and Skinner, 2003). Inmice estrogen as well as progesterone inhibit oocyte nest break-down (Chen et al., 2007), suggesting that there may be speciesdifferences in the regulation of follicle assembly. In fetal sheepovaries, steroidogenic cells have been identified and the specula-tion has been made that these steroids are important for formationof ovigerous cords (i.e. oocyte nests), and that a fall in steroid lev-els corresponds with entry of oogonia into meiosis (Juengel et al.,2002). In contrast, in some other species there are indications thatsteroid hormones, especially estrogen, promote follicle assembly.In baboons an experimental decrease in estradiol levels duringpregnancy resulted in the fetal ovaries having fewer assembledfollicles and an increase in un-assembled oocyte nests (Zachos etal., 2002). In hamsters the presence of estrogen promotes follicleassembly (Wang and Roy, 2007; Wang et al., 2008), although thiseffect decreases at higher estrogen doses.

The fact that female sex steroids inhibit follicle assembly inrats and mice, and that rodent follicle assembly occurs in the firstdays after birth, has led to the ‘endocrine model’ of follicle assem-bly (Kezele and Skinner, 2003). In this model, rodent embryos areexposed to high levels of maternal progesterone late in gestation,which inhibits follicle assembly. Right at birth the pups are removedfrom the high progesterone environment, levels of progesteronein the pups fall and follicle assembly commences. This endocrinemodel explains regulation of primordial follicle assembly in thosespecies in which assembly occurs shortly after birth. However, asmentioned above there are species, including cattle and humans,in which follicle assembly occurs during fetal development beforebirth. The objective of the current study was to determine if sexsteroid levels help regulate primordial follicle assembly in cattleprior to birth. The hypothesis tested was that progesterone inhibitsprimordial follicle assembly in bovine fetal ovaries in vitro and invivo. The timing of normal bovine fetal ovarian follicle assemblywas characterized, and levels of fetal progesterone and estrogenwere determined. Fetal bovine ovaries were also cultured in thepresence and absence of progesterone to test for an effect on folli-cle assembly. Understanding the mechanisms of follicle assemblyin cattle could give insights into how follicle assembly is regulatedin other species in which follicle assembly occurs prior to birth,including humans.

2. Materials and methods

2.1. Tissue collection and processing

Bovine fetal ovaries were collected from abattoirs by the Center for ReproductiveBiology, Animal Reproduction Core Laboratory at Washington State University. Theovaries were collected from fetuses ranging in size from 6.5 to 50 cm of crown-rumplength (CRL). This corresponds to fetuses estimated to be 60–170 days of gestation(Rexroad et al., 1974). From each fetus the ovaries were dissected free of surroundingbursal tissue, and one ovary was placed in Bouin’s solution for 5–8 h for fixation. Thesecond ovary was placed in a sterile plastic bag with a small amount of phosphatebuffered saline (PBS) and the bag filled with 50% oxygen/45% nitrogen/5% CO2 mixand placed on ice for transport (4–7 h) to the laboratory. Some of these ovaries wereused for steroid assays and others for organ culture experiments. Not all ovarieswere assayed for both progesterone and estrogen. Fetal blood was also collectedfrom each animal and transported on ice to the laboratory. Tissues were collectedin accordance with Washington State University Institutional Animal Care and UseCommittee approved protocols.

2.2. Morphological evaluation of follicle assembly

Bouin’s-fixed ovaries collected from bovine fetuses were paraffin embedded andsectioned at 5 #m thickness. Sections were made parallel to the longitudinal axis ofthe ovary and those sections with the largest cross-section (through the center of theovary) were chosen for morphometric evaluation. Sections were stained with hema-toxylin and eosin according to standard protocols. Oocytes or pre-meiotic germ cellswere recognized by morphological characteristics, including having a large roundnucleus surrounded by clear, non-basophilic cytoplasm. An oocyte that was sepa-rate from other oocytes and surrounded by granulosa cells was considered to have

undergone follicle assembly and to be part of a primordial or developing follicle. Tworegions in each ovary were evaluated, and each oocyte/germ cell in each region wasclassified as either un-assembled or assembled into a follicle. The regions evaluatedfor each ovary were, at minimum, strips within the plane of the chosen histologicsection that were 0.1 mm high by 1.0 mm wide and that ran perpendicular to thelong axis of the ovary from the ovarian surface to the center of the ovary. Two stripsmeeting the above criteria were located randomly within an ovary section. In smallerfetal ovaries 300–450 germ cells are present within two such strips. In larger ovariesfrom older animals oocyte density was lower, so larger portions of an ovary section(one-quarter section, one-half or entire ovary sections) were counted to ensure thatat least 200 oocytes were counted and categorized per ovary.

2.3. Steroid assays

Fetal blood samples were evaluated for their progesterone content, and fetalovaries were evaluated for their progesterone (P4) and estradiol (E2) content.Briefly, fetal whole blood was allowed to clot, then centrifuged and serum wascollected for assays. Fetal ovaries were weighed (wet weight), homogenized in phos-phate buffered saline (PBS), and submitted for assay. Both serum samples and fetalovary samples were subjected to an organic phase extraction procedure to con-centrate the samples. The extracted samples were re-constituted into the samebuffer as used with the assay standards. Both sample concentration procedures andsteroid radioimmunoassays were performed at the Assay Core Laboratory, Center forReproductive Biology, Washington State University. Steroid recovery was >95% andlinearity was within acceptable criteria for the Assay Core Laboratory. Intra assaycoefficient of variation (CV) for the progesterone assay (Kit DSL-3400, DiagnosticSystem Laboratories, Webster, TX) was 3.8%. For the estradiol assay (Kit DSL-39100,Diagnostic System Laboratories, Webster, TX) CV was 5.6%. Cross-reactivity data isavailable from the manufacturer.

2.4. Ovary culture

Ovaries from four bovine fetuses ranging from an estimated 73–117 days of ges-tation were shipped to the laboratory from abattoirs as described above and theneach dissected so as to separate the outer ovarian cortex from the inner medullarytissue after which the cortex was cut into pieces approximately 1 mm3. Ovary frag-ments were cultured as previously described (Wandji et al., 1996) on floating filters(0.4 #m Millicell-CM, Millipore, Bedford, MD, USA) in 0.5 ml Waymouth mediumcontaining 10% calf serum (Hyclone; Logan, UT, USA), 25 #g/ml pyruvic acid, one-half suggested concentration ITS-X (0.335 #g/ml sodium selenite, 0.5 mg/ml insulin,0.275 mg/ml transferrin, 0.1 mg/ml ethanolamine) (Invitrogen Corp., Grand Island,NY, USA), and 50 #g streptomycin sulfate and 75 #g penicillin/ml (Invitrogen Corp.,Grand Island, NY, USA). Alternatively, in one experiment ovaries were culturedin 50% Ham’s F12/50% DMEM (Dulbecco’s Modified Eagles’ Medium) (Gibco BRL,Gaithersburg, MD) containing 0.1% bovine serum albumin, 0.1% Albumax (Gibco BRL,Gaithersburg, MD) 27.5 #g/ml transferrin, 200 ng/ml insulin (human recombinant,Sigma) and 50 mg/ml l-ascorbic acid. There was no obvious difference in responseto culture for this experiment so results were combined. The culture experimentsoccurred on four different days. Ovaries were cultured in 0.5 ml medium for twodays at 37 "C in an incubator with 5% CO2. Treated ovaries received progesterone(P4) (Sigma) at 10#6 M (314 ng/ml). After culture, ovaries were fixed, sectioned at3 #m, and stained with hematoxylin/eosin for use in morphological analysis.

2.5. RNA isolation and microarray analysis

Ovaries from three bovine fetuses (67, 72 and 144 days gestation estimatedby crown-rump length, CRL) were collected and stored at #20 "C until the time ofRNA isolation. Total RNA was isolated after homogenizing ovaries in Trizol® reagent(Invitrogen, USA), using manufacturer’s protocols. RNA quality was evaluated byexamining a portion on an RNA gel. 18S and 28S bands were clear, and there waslittle smearing, indicating that quality was acceptable. However the 18S band wasnot as dark as expected, suggesting that some slight RNA degradation had occurred.RNA was used for microarray analysis with the Affymetrix (Affymetrix, Santa Clara,CA) bovine genome array chip. This chip contains probes for 23,000 genes. TheGenomics Core in the Center for Reproductive Biology at Washington State Uni-versity performed the analysis using standard Affymetrix protocols. The microarraywas scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., PaloAlto, CA). Microarray data were pre-processed using the Microarray Suite (MAS-5) statistical algorithm. All chips were scaled to target signal of 260 in order tokeep scaling factor to 1. Using Gene Chip Operating System (Affymetrix, Santa Clara,CA) an absolute analysis was performed to assess the relative abundance of thetranscripts based on signal and detection (present, absent, or marginal). MAS-5 pre-processed data were obtained from GCOS and imported into GeneSpring GX 7.3.Pre-processed data were used for analysis in GeneSpring. Quality measures includedevaluation of 3$-end degradation, which indicated that all three samples had similar3$-end degradation, and so are comparable to each other. The quantitative poly-merase chain reaction (PCR) procedure used was a Real-time procedure previouslydescribed (Nilsson et al., 2006). Primers were designed to amplify products thatcross intron–exon boundaries (except 3"HSD), and melting curve analyses wereperformed at the end of the real-time run. These melting curve analyses indicated

E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16 11

there was minimal secondary product generated. The primers used for CYP11A1were:

Set 1: Forward 5$AGA CGT GGC CCA TCT CTT CAA GTT-3$ ,Reverse 5$TTA TTG CCT CTG GAG CCA TCA CCT-3$;Set 2: Forward 5$-CGG AAA GTT TGT AGG GGA CA-3$ ,Reverse 5$-ACG TTG AGC AGA GGG ACA CT-3$;and for PGRMC1 were:Forward 5$-CAGGACCCGCGTATACTCAT-3$ ,

Reverse 5$-AGGAGTGAGGTCGGAAAGGT-3$;and for 3"HSD:Set 1: Forward 5$-AGA CAT TCT GGA TGA GCA GTG CCT-3$ ,Reverse 5$-ACA GCA GCT GGG TAC CTT TCA CAT-3$;Set 2: Forward 5$-ACC AGA AGT TCG GGA GGA AT-3$ ,Reverse 5$-TTC TAT GGT GCT GGT GTG GA-3$;And for house keeping gene, ribosomal protein S2, rat:Forward 5$-CTGCTCCTGTGCCCAAGAAG-3$ ,Reverse 5$-AAGGTGGCCTTGGCAAAGTT-3$ .

2.6. Statistics

Steroid concentration data from ovaries and fetal blood were plotted againstgestational age. Curve fitting suggested that an exponential decay formula provideda good fit of both progesterone and estrogen concentration data. In order to deter-mine if there is a significant decrease in steroid concentrations over time, the datawere linearized using the assumption that steroid concentrations followed an expo-nential decay curve, and the resulting linear regression was tested as to whether itsslope was significantly non-zero. A slope different from zero indicates a change insteroid concentration over time.

For the cultured ovary experiments morphometric data was collected that deter-mined the proportion of oocytes assembled into follicles versus those not assembled.Within each experiment the proportion of assembled follicles for each ovary frag-ment was divided by the proportion of assembled follicles in the control group.This normalized the data so that each control was equal to 1 and the proportions ofassembled follicles in the progesterone-treated ovary fragments were expressed asratios relative to their controls. Since all the control ovary values were now equalto 1, a one-sample t-test with relative mean equal to 1.0 was performed on cul-

Fig. 1. Fetal bovine ovarian follicle assembly and development is shown at different estimated gestational ages, indicated at left margin. Representative fetal ovaries werecollected at different gestational ages, sectioned and hematoxylin/eosin stained. The right column of images (B, D, F, H) are a higher magnification (see scale bars) of the leftcolumn (A, C, E, G). Arrows = oocyte nests. Arrowheads = individual assembled follicles.

12 E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16

Fig. 2. The percentage of oocytes in an ovarian cross-section that are assembledinto primordial follicles at different estimated gestational ages (days). Each pointrepresents one ovary. Regression line is from a sigmoidal dose–response equationwith variable slope (R2 = 0.91).

tured ovary follicle assembly data using GraphPad Prism version 4.0b for Macintosh,GraphPad Software, San Diego, CA, USA, www.graphpad.com.

3. Results

Ovaries were collected from 9 bovine fetuses measuringbetween 7 and 50 cm crown-rump length (CRL) in size, whichcorresponds to 62–170 days of gestation (Rexroad et al., 1974).Morphological evaluation of hematoxylin–eosin stained ovariesshowed that germ cells are arranged in clearly identifiable clusters(i.e. ovigerous cords) (Gondos, 1973; Pepling, 2006) by the timefetuses reached 75 days of age (12 cm CRL). Some isolated oocyteswere found assembled into primordial follicles by an estimated 75days of development in the central portion of the fetal ovary (Fig. 1).Both oocyte nests and primordial follicles were common at 98 daysof development. Developing primary follicles, that had undergoneprimordial to primary follicle transition, were present in ovariesestimated as 143 days, along with many primordial follicles andsome small oocyte nests (Fig. 1). The proportion of oocytes in fetalbovine ovary cross-sections that were assembled into primordialfollicles was determined morphometrically by microscopic exami-nation of hematoxylin/eosin stained ovaries from bovine fetuses ofdifferent ages. Fig. 2 shows the percent of assembled follicles as aproportion of total oocytes plotted against fetal gestational age. Theproportion of assembled primordial follicles rises with increasingfetal age as expected (Fig. 2).

Experiments were performed to determine the levels of proges-terone and estradiol present in fetal ovaries and progesterone infetal blood serum. Steroid assays were performed on whole fetalovaries from 60 to 103 days of gestation as described in Section2. Ovarian progesterone concentration (ng/g of wet tissue weight)was found to decrease with increasing fetal age (p = 0.002, see Sec-tion 2 for statistical procedure) (Fig. 3A). Ovarian estrogen levelsalso were found to decrease with increasing fetal age (Fig. 3B)(p % 0.0005). Since the ovaries from the youngest bovine fetuseswere very small, there was concern that inaccurate measurementof ovarian wet weight might give spurious results. Even withthese earliest ovaries excluded there was a significant decreasein progesterone (p = 0.006) and estrogen (p % 0.0005) concentra-tions (Fig. 3A and B, insets). Both progesterone and estrogen levelsappeared to decrease until about 80 days gestation. Fetal bloodserum progesterone levels were at the low limits of detectioneven after sample concentration (see Section 2), so results mustbe viewed with caution. However, there was no indication thatfetal serum progesterone levels changed with increasing fetal age

Fig. 3. The progesterone (A) and estradiol (estrogen) (B) concentration in fetalbovine ovaries (ng/g wet weight) taken from fetuses at different estimated ges-tational ages (days). Regression lines are fitted using exponential decay equations,R2 = 0.83 for progesterone regression, R2 = 0.99 for estrogen regression. The insetgraphs are included to show more clearly steroid concentrations in older ovaries.

(data not shown). Mean fetal serum progesterone concentrationwas 0.10 ± 0.015 ng/ml (mean ± standard deviation).

Bovine ovary organ culture experiments were performed todetermine experimentally if high levels of progesterone can reg-ulate the rate of primordial follicle assembly. Ovarian cortex pieceswere dissected out of fetal ovaries from four bovine fetuses rangingfrom about 73–117 days of gestation, and placed into a floating fil-ter culture system as described in Section 2. Ovarian cortex piecesfrom one of the ovaries from each fetus were cultured for twodays in either the presence or absence of 10#6 M progesterone.Histological examination determined the proportion of oocytes ineach ovary sample that had undergone follicle assembly to formisolated primordial or developing follicles. This was accomplishedby counting the number of oocytes assembled into follicles andthose un-assembled in each of two serial sections, and averagingthe counts (un-assembled vs. assembled) for each ovary. Since theovaries came from bovine fetuses that varied in age, the startingpercentage of primordial follicles prior to culture varied betweenovaries. To account for this variable each ovary was assessed forthe percentage of oocytes assembled into follicles after culture inthe absence or presence of control and progesterone treatment.The percentage of oocytes assembled into follicles was normalizedto the control culture percentage of assembled follicles by divid-ing by the percentage of assembled follicles in the control groupof that experiment. A one-sample t-test of these data determinedthat this progesterone treatment significantly (p < 0.05) reducedthe proportion of assembled follicles (Fig. 4D). The representativemorphology of control and progesterone-treated organ cultures isshown in Fig. 4A–C. There is nuclear condensation of some cells visi-

E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16 13

Fig. 4. Representative ovarian cortex pieces after two days of ovary culture showing oocyte nests and assembled follicles: (A) progesterone-treated ovary, (B) duplicate ofimage (A) with the addition of green shaded areas showing oocyte nests, and arrows pointing to assembled primordial follicles. (C) Control untreated cultured ovary. (D)The effect of progesterone on follicle assembly in cultured fetal bovine ovaries. Fetal ovary cortex pieces were cultured in the absence (control) or presence of progesterone(10#6 M) for two days. After culture the pieces were sectioned, stained and the proportion of oocytes assembled into follicles determined. The percentage of assembled follicleswas normalized to the control culture percentage of assembled follicles for each experimental replicate, for each fetal ovary (n = 4). Data are presented as the mean ± S.E.M.as described in Section 2. * indicates a statistical difference (p < 0.05) between treatment and control by one-sample t-test.

ble after culture in both treated and control ovary fragments, whichis likely a result of explant culture conditions. However, follicleassembly did occur. The decrease in primordial follicle assembly isreflected in the retention of oocyte nests and the decreased numberof primordial follicles in progesterone-treated cultures (Fig. 4D).

RNA isolated from fetal bovine ovaries was used for a microarrayanalysis in order to determine whether selected genes importantin steroid signaling and synthesis were expressed in these ovaries.Two ovaries (from fetuses estimated at 67 and 72 days gestation)were from the period of development when ovarian steroid con-centrations are higher, and one (from a fetus of 144 days gestation)from when ovarian steroid concentrations are low. The resultsindicate which gene transcripts are expressed (i.e. present) or not(i.e. absent), Table 1. The presence of nuclear progesterone recep-tor is equivocal. The membrane progesterone receptor PGRMC1 isstrongly present. Other membrane associated progesterone recep-tors including mPR!, mPR" and mPR$ were not represented onthe bovine microarray chip. Estrogen receptor ER1 (alpha) is not

expressed, but ER2 (beta) is present. These data suggest that thecomponents of steroid ligand–receptor signaling are present in fetalbovine ovaries. The presence of transcripts for certain steroido-genic enzymes was also investigated. CYP11A1 (i.e. P450scc), thatconverts cholesterol to pregnenolone, is present. 3"HSD, that con-verts pregnenolone to progesterone, is also present in the earlystage ovaries, but not present in the later stage ovary. Transcriptsfor other steroidogenic enzymes leading to estradiol formation (i.e.aromatase) were also present.

To confirm and extend the microarray analysis real-time PCRwas performed on seven additional fetal ovary samples rangingfrom 70 to 150 days gestation for expression of 3"HSD, CYP11A1and PGRMC1. Compared to the housekeeping gene ribosomal S2,expression of PGRMC1 was detectable and was at consistent lev-els across samples. CYP11A1 was also detectable, with expressionlevels quite variable between samples, and not correlated withfetal age. The detected expression level of 3"HSD was negligiblethroughout fetal ovary development (data not shown). Observa-

Table 1Presence/absence calls for selected genes from microarray analysis.

Common name Gene symbol Presence/absence (fetal ovary age)

67 days 72 days 144 days

Progesterone nuclear receptor (Genbank ID CK983148) PGR P A PProgesterone receptor membrane component 1 PGRMC1 P P PEstrogen receptor 1 (alpha) ER1 A A AEstrogen receptor 2 (beta) ER2 P P PSide-chain clevage enzyme CYP11A1 (P450scc) P M P3 Beta-hydroxysteroid dehydrogenase 3"HSD P P A17 Alpha-hydroxylase CYP17 (P450c17) P A AAromatase (Genbank ID NM 174305.1) CYP19 (P450aro) P P P17 Beta-hydroxysteroid dehydrogenase 17"HSD1 A A A

P = present, A = absent, M = marginal.

14 E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16

Fig. 5. Correlation of progesterone concentration in fetal bovine ovaries and thepercentage (%) of oocytes assembled into primordial follicles as a function of esti-mated gestational age (days). Follicle assembly is initiated as ovarian progesteroneconcentration declines.

tions confirm that some components of steroid ligand–receptorsignaling are expressed in fetal bovine ovaries.

4. Discussion

The objective of the current study is to determine if high proges-terone levels regulate primordial follicle assembly in cattle priorto birth. To determine this, experiments were performed usingfetal bovine ovaries collected from abattoirs. Abattoir ovary speci-mens were collected in central and western Washington State andcame from a mixed population of beef and dairy cattle breeds.There was an increase in assembled follicles with increasing fetalsize, as expected (Fig. 2). There is some variation in the literatureconcerning at what stage of bovine fetal development particularstages of oocyte development occur (Erickson, 1966; Kurilo et al.,1987; Marion and Gier, 1971; Mbassa, 1989; Russe, 1983; Sakai,1955; Tanaka et al., 2001; Vigier et al., 1976). However, in the cur-rent study the CRL (crown-rump length) sizes and embryonic agesat which oocyte nests and primordial follicle assembly were firstobserved fall within the range expected from previous investiga-tions. These observations indicate that the sample population usedis appropriate for performing the current study.

Experiments were performed to determine the levels of pro-gesterone and estrogen present in fetal ovaries and the levels ofprogesterone in fetal blood. Interestingly, the estrogen and proges-terone concentrations in the fetal ovaries decline with increasingfetal age (Fig. 3). This is consistent with the endocrine model offollicle assembly previously proposed (Kezele and Skinner, 2003),that higher levels of progesterone inhibit assembly and that fol-licles assemble as progesterone levels decline (Fig. 5). However,in cattle, humans and sheep, primordial follicle assembly is notsynchronous after birth as it is in rodents (Mbassa, 1989; Pepling,2006; Sakai, 1955; Sawyer et al., 2002; Tanaka et al., 2001). In thebovine fetal ovary there is an extended period of time during whichoocyte nests, primordial follicles and some developing follicles areall present. This suggests that progesterone is likely not the onlyregulatory factor involved in initiating follicle assembly. In fact, inprogesterone receptor knockout mice follicle assembly still occurs(Lydon et al., 1995). As mentioned, ovarian estrogen concentrationsalso decline, suggesting a role for estrogen in bovine follicle assem-bly. Cell–cell signaling via other locally produced growth factors(Pepling, 2006) likely also plays a role in the assembly process.

It is notable that fetal ovary progesterone levels decrease acrossa time period of gestation (estimated at 60–103 days) duringwhich bovine maternal blood progesterone levels do not change(Echternkamp et al., 2006). In addition, maternal blood proges-terone concentrations are at about 7 ng/ml between 50 and 100

days of gestation (Echternkamp et al., 2006), which is lower thanthe 20–150 ng/g progesterone concentration seen in fetal ovariesbetween 60 and 75 days of gestation. Previous literature has estab-lished that maternal and fetal progesterone levels are differentfrom each other and may be regulated independently in macaques(Ducsay et al., 1985; Thau et al., 1976), humans (Donaldson et al.,1991), sheep (Nicol et al., 1997) and elephants (de Villiers et al.,1989). This suggests that ovarian fetal progesterone is regulated tocontrol the initiation or progression of primordial follicle assembly,independent of maternal progesterone levels.

While fetal ovary progesterone levels were found to decreaseduring gestation, fetal blood progesterone levels were not corre-lated with fetal age. This observation suggests that fetal ovarianprogesterone concentrations are being regulated independentlyfrom those of fetal blood. Previous literature has shown that ovariantissue steroid concentrations change more rapidly than circulat-ing blood concentrations (Peters and McNatty, 1980). Certainly itis the case in the current study that fetal ovary progesterone con-centrations (10–175 ng/g wet weight) are different from the fetalserum concentrations (<1 ng/ml). Interestingly, past studies havealso indicated that fetal and adult erythrocytes are able to metabo-lize progesterone (Chen et al., 1987, 1989, Nancarrow et al., 1984;Vahdat et al., 1984). This may be one mechanism by which fetalblood can maintain a different progesterone concentration thanthat of the fetal ovary and maternal blood.

Microarray analysis and real-time PCR of fetal ovary RNA indi-cates that the transcript for the surface membrane progesteronereceptor PGRMC1 is present in the fetal ovary. This indicates thatthe actions of progesterone in regulating follicle assembly may bemediated, at least in part, through the surface membrane recep-tor. These data are consistent with findings in rat ovaries at thetime of follicle assembly (Kezele et al., 2002; Nilsson et al., 2006).In previous work, the classic nuclear progesterone receptor was notdetected in fetal sheep ovaries (Juengel et al., 2006). In the currentstudy, the presence of mRNA for the nuclear progesterone recep-tor was equivocal. The transcript for estrogen receptor 2 (ER2) ispresent, similarly to what has previously been shown for fetal cat-tle and sheep ovaries (Gaverick et al., 2005; Juengel et al., 2006).In the current study ER1 mRNA was not detected. This is differentfrom previous findings of ER1 in surface epithelial cells and somestromal cells of fetal sheep ovaries (Juengel et al., 2006). However,taken together, these data suggest that the components for steroidligand–receptor signaling are present in bovine fetal ovaries.

Previous research suggests that the progesterone and estradioldetected in fetal bovine ovaries is, at least in part, produced locallyin the ovaries. There have been several studies showing that mam-malian gonads around the time of sexual differentiation synthesizesteroids (George and Wilson, 1994). In ovary culture experiments,fetal bovine ovaries have been shown to produce both progesteroneand estradiol (Dominguez et al., 1988; Shemesh et al., 1978; Shoreand Shemesh, 1981; Tanaka et al., 2001). Both progesterone andestradiol have also been demonstrated to be produced by fetalsheep ovaries (Lun et al., 1998). In a recent study in which cul-tured fetal bovine ovaries were shown to produce estrogen andprogesterone (Yang and Fortune, 2008), it was shown that steroidproduction decreased with increasing fetal age, in concordancewith the results of the current study in which ovarian steroid con-centration decreased with age. Estradiol production by culturedovaries was also seen to drop after about 70 days gestation in previ-ous research (Dominguez et al., 1988; Shemesh et al., 1978). Tanakaet al. (2001) reported no change in total fetal ovary estradiol con-tent from 60 to 100 days of gestation, during which time the ovarianmass increases considerably (Tanaka et al., 2001). Therefore, estra-diol concentrations in these ovaries must decrease. In fetal sheepovaries both P450scc (CYP11A1) and 3"HSD have been shown to bepresent at the time of morphological sexual differentiation (Quirke

E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16 15

et al., 2001). In the current study, microarray analysis and real-timePCR show that CYP11A1, the enzyme that converts cholesterol topregnenolone is present in fetal ovaries.

A limitation of the microarray analyses of the current study isthat it was impractical to collect replicate RNA samples from ovariesof a particular developmental time point. This problem was inher-ent since the ovaries were from abbitoirs. This increases the chancesof receiving false positive and false negative presence/absencedeterminations from the microarray analyses. To address this issuethree genes were selected (i.e. 3"HSD, CYP11A1 and PGRMC1)to run real-time PCR on a larger number of ovaries and deter-mine whether these genes were expressed. Results confirmed thatPGRMC1 and CYP11A1 were expressed in fetal ovaries, althoughexpression levels of CYP11A1 were variable and did not corre-late well with fetal ovary age. The levels of 3"HSD detected werenegligible. These results do not match exactly with the results ofmicroarray analysis. This may reflect on the quality of the RNA sam-ples, or reflect the robustness of microarray analysis, where eachgene is evaluated with several probes, compared to real-time PCR,where each gene is evaluated with only one product. Although caremust be taken in interpreting these results, as mentioned aboveprevious research has demonstrated that fetal ruminant ovariesproduce sex steroids (Dominguez et al., 1988; Lun et al., 1998;Shemesh et al., 1978; Shore and Shemesh, 1981; Tanaka et al., 2001;Yang and Fortune, 2008). This previous literature in combinationwith supporting data from the current experiment suggest localovarian steroid production may be regulating primordial follicleassembly. Results indicate that progesterone receptor PGRMC1 iscontinuously expressed across the ovary development period ofthis study. A previous study confirmed the expression of PGRMC1 inprimordial follicle assembly as well (Kezele and Skinner, 2003). Thisraises the possibility that low progesterone levels may support con-tinued follicle development, even while high levels are inhibitoryto follicle assembly.

Fetal ovary estrogen concentrations also decreased with fetalage in the current study. Similar to progesterone, this occurs overa period of gestation during which both fetal and maternal bloodserum estrogen concentrations have been shown to be eitherunchanging or gradually increasing (Challis et al., 1974; Eley etal., 1979; Robertson and King, 1979). This observation suggeststhat bovine primordial follicle assembly may be regulated by pro-gesterone and estrogen. It is also possible that the progesteroneproduced in fetal ovaries acts only as a precursor for estrogen syn-thesis. In contrast to the rat (Kezele and Skinner, 2003), mousefollicle assembly appears to be regulated by both progesterone andestrogen (Chen et al., 2007), indicating that there may be speciesdifferences in control of follicle assembly. Previous research hasindicated that the presence of estrogen, and perhaps progesterone,are required for follicle assembly to occur normally in some species(Anand Kumar, 1968; Wang and Roy, 2007; Wang et al., 2008;Zachos et al., 2002). These findings are not necessarily in con-flict with the results of the current study. It is possible that highintro-ovarian steroid concentrations inhibit follicle assembly, whilelower basal concentrations support the assembly process. Furtherresearch is needed to clarify this issue. Wang and Roy (2007) foundin the hamster that higher estrogen doses did not promote follicleassembly as did lower doses. It is also possible that different speciesrespond differently to estrogen signaling.

In order to definitively determine if a high level of progesteronecan affect follicle assembly in fetal bovine ovaries, organ cultureexperiments were performed. It was found that the percentageof oocytes assembled into follicles was significantly decreased inprogesterone-treated ovary cultures compared to untreated con-trol cortex cultures from the same ovary (Fig. 4). This supportsthe hypothesis that progesterone at this dose inhibits primordialfollicle assembly in bovine fetal ovaries (Fig. 5). The dose of pro-

gesterone used in these organ culture experiments was 314 ng/ml(10#6 M). This is about twice the concentration seen in the youngestovary measured in these experiments, but is less than the pro-gesterone concentration found in follicular fluid. The effect of lowdoses of progesterone on bovine follicle assembly remains to bedetermined. The results of these studies also suggest that the abilityto respond to progesterone may still exist in fetal ovaries after ovar-ian progesterone levels drop. Progesterone has also been found toinhibit follicle assembly in organ cultures of mouse and rat ovaries(Chen et al., 2007; Kezele and Skinner, 2003). These observationssuggest that progesterone inhibition may be a widely used mech-anism of regulating follicle assembly across mammalian species.

The premature assembly and development of primordial fol-licles during gestation could cause an abnormal endocrine stateand/or the entry of oocytes into meiosis. Therefore, a mechanismto inhibit premature primordial follicle development appears tohave evolved. Since progesterone is essential for pregnancy andfetal development, high levels of progesterone provide an idealinhibitory factor for species where the completion of follicle assem-bly occurs after birth. The authors speculate that this endocrinemodel of follicle assembly is evolutionarily conserved. For thosespecies that evolved to complete follicle assembly before birth (i.e.cattle and humans), there also evolved an independent local reg-ulation of fetal ovarian steroids. As fetal and ovarian developmentproceeds, primordial follicle assembly is initiated and linked to thedecline in steroids in the ovary itself. This occurs in larger mammals(i.e. cattle and humans) during mid-gestation.

In summary, fetal ovarian progesterone and estrogen concentra-tions decreased during mid-gestation, at the same time as follicleassembly was initiated in the bovine ovary. This occurred at atime during which maternal blood progesterone and estrogen lev-els were stable. The mRNA transcripts for certain progesterone andestrogen receptors are present in fetal ovaries, as are the transcriptsfor enzymes to synthesize progesterone. In cultured fetal bovineovaries, treatment with high levels of progesterone significantlydecreased follicle assembly. These data indicate that progesteroneand possibly estrogen are important regulators of ovarian follicleassembly. Understanding these mechanisms in cattle could provideinsight into how these processes are regulated in other species inwhich follicle assembly occurs prior to birth, including humans.

Acknowledgements

We acknowledge the expert technical assistance of Dr. MarinaSavenkova in the Bioinformatics Core Laboratory and Ms. NadaCummings in the Animal Reproduction Core Laboratory, Center forReproductive Biology, Washington State University. We also thankMs. Heather Johnson for assistance in preparing the manuscript.This research was supported by a grant from the National Institutesof Health, NICHD to Michael K. Skinner.

References

Abir, R., Fisch, B., Jin, S., Barnnet, M., Ben-Haroush, A., Felz, C., Kessler-Icekson, G.,Feldberg, D., Nitke, S., Ao, A., 2005. Presence of NGF and its receptors in ovariesfrom human fetuses and adults. Mol. Hum. Reprod. 11, 229–236.

Anand Kumar, T., 1968. The effects of gonadal hormones on primordial germ cellpopulation in the slender loris. J. Reprod. Fertil. 16, 322–323.

Baker, T.G., 1963. A quantitative and cytological study of germ cells in human ovaries.Proc. R. Soc. Lond. B. Biol. Sci. 158, 417–433.

Bristol-Gould, S.K., Kreeger, P.K., Selkirk, C.G., Kilen, S.M., Cook, R.W., Kipp, J.L., Shea,L.D., Mayo, K.E., Woodruff, T.K., 2006. Postnatal regulation of germ cells byactivin: the establishment of the initial follicle pool. Dev. Biol. 298, 132–148.

Challis, J.R., Kim, C.K., Naftolin, F., Judd, H.L., Yen, S.S., Benirschke, K., 1974. The con-centrations of androgens, oestrogens, progesterone and luteinizing hormone inthe serum of foetal calves throughout the course of gestations. J. Endocrinol. 60,107–115.

Chen, Q.X., Nancarrow, C.D., Sweet, F., 1987. Isolation of 3 beta, 20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood. Steroids 49, 477–496.

16 E.E. Nilsson, M.K. Skinner / Molecular and Cellular Endocrinology 313 (2009) 9–16

Chen, Q.X., Rosik, L.O., Nancarrow, C.D., Sweet, F., 1989. Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examinedby affinity labeling with 16 alpha-(bromo[2$-14C]acetoxy)progesterone. Bio-chemistry 28, 8856–8863.

Chen, Y., Breen, K., Pepling, M., 2009. Estrogen can signal through multiple path-ways to regulate oocyte cyst breakdown and primordial follicle assembly in theneonatal mouse ovary. J. Endocrinol..

Chen, Y., Jefferson, W.N., Newbold, R.R., Padilla-Banks, E., Pepling, M.E., 2007. Estra-diol, progesterone, and genistein inhibit oocyte nest breakdown and primordialfollicle assembly in the neonatal mouse ovary in vitro and in vivo. Endocrinology148, 3580–3590.

de Villiers, D.J., Skinner, J.D., Hall-Martin, A.J., 1989. Circulating progesterone con-centrations and ovarian functional anatomy in the African elephant (Loxodontaafricana). J. Reprod. Fertil. 86, 195–201.

Dissen, G.A., Romero, C., Hirshfield, A.N., Ojeda, S.R., 2001. Nerve growth factor isrequired for early follicular development in the mammalian ovary. Endocrinol-ogy 142, 2078–2086.

Dominguez, M.M., Liptrap, R.M., Basrur, P.K., 1988. Steroidogenesis in fetal bovinegonads. Can. J. Vet. Res. 52, 401–406.

Donaldson, A., Nicolini, U., Symes, E.K., Rodeck, C.H., Tannirandorn, Y., 1991. Changesin concentrations of cortisol, dehydroepiandrosterone sulphate and proges-terone in fetal and maternal serum during pregnancy. Clin. Endocrinol. (Oxf.)35, 447–451.

Ducsay, C.A., Stanczyk, F.Z., Novy, M.J., 1985. Maternal and fetal production rates ofprogesterone in rhesus macaques: placental transfer and conversion to cortisol.Endocrinology 117, 1253–1258.

Echternkamp, S.E., Vonnahme, K.A., Green, J.A., Ford, S.P., 2006. Increased vascu-lar endothelial growth factor and pregnancy-associated glycoproteins, but notinsulin-like growth factor-I, in maternal blood of cows gestating twin fetuses. J.Anim. Sci. 84, 2057–2064.

Eley, R.M., Thatcher, W.W., Bazer, F.W., 1979. Hormonal and physical changesassociated with bovine conceptus development. J. Reprod. Fertil. 55, 181–190.

Erickson, B.H., 1966. Development and radio-response of the prenatal bovine ovary.Reprod. Fertil. 10, 97–105.

Gaverick, A., Juengel, J., Smith, P., Heath, D., Burkhart, N., Schenk, J., Perry, G., Smith,M., McNatty, K., 2005. mRNA and protein expression of P450 aromatase (AROM)and estrogen receptors (ER) ! and " during early development of bovine fetalovaries. Biol. Reprod. T467, 184, Special Issue.

George, F., Wilson, J., 1994. Sex determination and differentiation. In: Neill, E.K.J.(Ed.), The Physiology of Reproduction, second edn. Raven Press, New York.

Gondos, B., 1973. Germ cell degeneration and intercellular bridges in the humanfetal ovary. Z. Zellforsch. Mikrosk. Anat. 138, 23–30.

Hahn, K.L., Johnson, J., Beres, B.J., Howard, S., Wilson-Rawls, J., 2005. Lunatic fringenull female mice are infertile due to defects in meiotic maturation. Development132, 817–828.

Hirshfield, A.N., 1991. Development of follicles in the mammalian ovary. Int. Rev.Cytol. 124, 43–101.

Hirshfield, A.N., 1994. Relationship between the supply of primordial follicles andthe onset of follicular growth in rats. Biol. Reprod. 50, 421–428.

Juengel, J.L., Heath, D.A., Quirke, L.D., McNatty, K.P., 2006. Oestrogen receptor alphaand beta, androgen receptor and progesterone receptor mRNA and protein local-isation within the developing ovary and in small growing follicles of sheep.Reproduction 131, 81–92.

Juengel, J.L., Sawyer, H.R., Smith, P.R., Quirke, L.D., Heath, D.A., Lun, S., Wakefield, S.J.,McNatty, K.P., 2002. Origins of follicular cells and ontogeny of steroidogenesisin ovine fetal ovaries. Mol. Cell. Endocrinol. 191, 1–10.

Kezele, P., Skinner, M.K., 2003. Regulation of ovarian primordial follicle assemblyand development by estrogen and progesterone: endocrine model of follicleassembly. Endocrinology 144, 3329–3337.

Kezele, P., Nilsson, E., Skinner, M.K., 2002. Cell–cell interactions in primordial follicleassembly and development. Front. Biosci. 7, d1990–1996.

Kurilo, L.F., Tepliakova, N.P., Lavrikova, G.V., 1987. Development of ovaries in bovinefetuses. Ontogenez 18, 500–506.

Lun, S., Smith, P., Lundy, T., O’Connell, A., Hudson, N., McNatty, K.P., 1998. Steroid con-tents of and steroidogenesis in vitro by the developing gonad and mesonephrosaround sexual differentiation in fetal sheep. J. Reprod. Fertil. 114, 131–139.

Lydon, J.P., DeMayo, F.J., Funk, C.R., Mani, S.K., Hughes, A.R., Montgomery Jr.,C.A., Shyamala, G., Conneely, O.M., O’Malley, B.W., 1995. Mice lacking proges-terone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 9,2266–2278.

Marcinkiewicz, J.L., Balchak, S.K., Morrison, L.J., 2002. The involvement of tumornecrosis factor-alpha (TNF) as an intraovarian regulator of oocyte apoptosis inthe neonatal rat. Front. Biosci. 7, d1997–2005.

Marion, G.B., Gier, H.T., 1971. Ovarian and uterine embryogenesis and morphologyof the non-pregnant female mammal. J. Anim. Sci. 32 (Suppl. 1), 24–47.

Mauleon, P., 1974. The establishment of the primordial follicle reserve in the sheepembryo studied by labelling of oocytes with tritiated thymidine. Eur. J. Obstet.Gynecol. Reprod. Biol. 4, S133–139.

Mbassa, G.K., 1989. Studies on the ovarian development in zebu cattle (Bos indicus).Anat. Histol. Embryol. 18, 143–149.

McMullen, M.L., Cho, B.N., Yates, C.J., Mayo, K.E., 2001. Gonadal pathologies intransgenic mice expressing the rat inhibin alpha-subunit. Endocrinology 142,5005–5014.

Morrison, L.J., Marcinkiewicz, J.L., 2002. Tumor necrosis factor alpha enhancesoocyte/follicle apoptosis in the neonatal rat ovary. Biol. Reprod. 66, 450–457.

Nancarrow, C.D., Connell, P.J., Stevens, D., 1984. Relationship between glucose and 20alpha-hydroxysteroid dehydrogenase in fetal sheep erythrocytes. J. Endocrinol.101, 131–139.

Nicol, M.B., Hirst, J.J., Walker, D., Thorburn, G.D., 1997. Effect of alteration of mater-nal plasma progesterone concentrations on fetal behavioural state during lategestation. J. Endocrinol. 152, 379–386.

Nilsson, E.E., Detzel, C., Skinner, M.K., 2006. Platelet-derived growth factor modu-lates the primordial to primary follicle transition. Reproduction 131, 1007–1015.

Pepling, M.E., 2006. From primordial germ cell to primordial follicle: mammalianfemale germ cell development. Genesis 44, 622–632.

Pepling, M.E., Spradling, A.C., 2001. Mouse ovarian germ cell cysts undergo pro-grammed breakdown to form primordial follicles. Dev. Biol. 234, 339–351.

Peters, H., McNatty, K.P., 1980. The Ovary. A Correlation of Structure and Functionin Mammals. University of California Press, Berkeley and Los Angeles.

Quirke, L.D., Juengel, J.L., Tisdall, D.J., Lun, S., Heath, D.A., McNatty, K.P., 2001.Ontogeny of steroidogenesis in the fetal sheep gonad. Biol. Reprod. 65, 216–228.

Rexroad, C.E., Cassida, L.E., Tyler, W.J., 1974. Crown-rump length of fetuses in pure-bred holstein-friesian cows. J. Dairy Sci. 57, 346–347.

Robertson, H.A., King, G.J., 1979. Conjugated and unconjugated oestrogens in fetaland maternal fluids of the cow throughout pregnancy. J. Reprod. Fertil. 55,463–470.

Russe, I., 1983. Oogenesis in cattle and sheep. Bibl. Anat. 24, 77–92.Sakai, T., 1955. Studies on the development of the embryonic ovary in swine, cattle

and horse. Jpn. J. Vet. Res. 3, 183–194.Sawyer, H.R., Smith, P., Heath, D.A., Juengel, J.L., Wakefield, S.J., McNatty, K.P., 2002.

Formation of ovarian follicles during fetal development in sheep. Biol. Reprod.66, 1134–1150.

Sforza, C., Forabosco, A., 1998. A morphometric approach to the study of humanovarian organogenesis. Ital. J. Anat. Embryol. 103, 51–62.

Shemesh, M., Allenberg, M., Milaguir, F., Ayalon, N., Hansel, W., 1978. Hormonesecretion by cultured bovine pre- and postimplantation gonads. Biol. Reprod.19, 761–767.

Shore, L., Shemesh, M., 1981. Altered steroidogenesis by the fetal bovine freemartinovary. J. Reprod. Fertil. 63, 309–314.

Skinner, M.K., 2005. Regulation of primordial follicle assembly and development.Hum. Reprod. Update 11, 461–471.

Tanaka, Y., Nakada, K., Moriyoshi, M., Sawamukai, Y., 2001. Appearance and num-ber of follicles and change in the concentration of serum FSH in female bovinefetuses. Reproduction 121, 777–782.

Thau, R., Lanman, J.T., Brinson, A., 1976. Declining plasma progesterone concentra-tion with advancing gestation in blood from umbilical and uterine veins andfetal heart in monkeys. Biol. Reprod. 14, 507–509.

Tilly, J.L., 1996. Apoptosis and ovarian function. Rev. Reprod. 1, 162–172.Vahdat, F., Seguin, B.E., Whitmore, H.L., Johnston, S.D., 1984. Role of blood cells in

degradation of progesterone in bovine blood. Am. J. Vet. Res. 45, 240–243.van Wagener, G., Simpson, M., 1965. Embryology of the Ovary and Testis HomoSapi-

ens and Macaca mulatta. Yale University Press, New Haven, CT.Vigier, B., Prepin, J., Jost, A., 1976. Chronology of development of the genital tract of

the calf fetus. Arch. Anat. Microsc. Morphol. Exp. 65, 77–101.Wandji, S.A., Srsen, V., Voss, A.K., Eppig, J.J., Fortune, J.E., 1996. Initiation in vitro of

growth of bovine primordial follicles. Biol. Reprod. 55, 942–948.Wang, C., Roy, S.K., 2007. Development of primordial follicles in the hamster: role

of estradiol-17beta. Endocrinology 148, 1707–1716.Wang, C., Prossnitz, E.R., Roy, S.K., 2008. G protein-coupled receptor 30 expression is

required for estrogen stimulation of primordial follicle formation in the hamsterovary. Endocrinology 149, 4452–4461.

Yan, C., et al., 2001. Synergistic roles of bone morphogenetic protein 15 and growthdifferentiation factor 9 in ovarian function. Mol. Endocrinol. 15, 854–866.

Yang, M.Y., Fortune, J.E., 2008. The capacity of primordial follicles in fetal bovineovaries to initiate growth in vitro develops during mid-gestation and is associ-ated with meiotic arrest of oocytes. Biol. Reprod. 78, 1153–1161.

Zachos, N.C., Billiar, R.B., Albrecht, E.D., Pepe, G.J., 2002. Developmental regulationof baboon fetal ovarian maturation by estrogen. Biol. Reprod. 67, 1148–1156.