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BIOLOGY OF REPRODUCTION 72, 241–255 (2005)Published online before print 15 September 2004.DOI 10.1095/biolreprod.104.032060

Alterations in the Ovarian Transcriptome During Primordial Follicle Assemblyand Development1

Phillip R. Kezele, Jacquelyn M. Ague, Eric Nilsson, and Michael K. Skinner2

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University,Pullman, Washington 99164-4231

ABSTRACT

The assembly of the developmentally arrested primordial fol-licle and subsequent transition to the primary follicle are poorlyunderstood processes critical to ovarian biology. Abnormal pri-mordial follicle development can lead to pathologies such aspremature ovarian failure. The current study used a genome-wide expression profile to investigate primordial follicle assem-bly and development. Rat ovaries with predominantly unassem-bled, primordial, or primary follicles were obtained. RNA fromthese ovaries was hybridized to rat microarray gene chips, andthe gene expression (i.e., ovarian transcriptome) was comparedbetween the developmental stages. Analysis of the ovarian tran-scriptome demonstrated 148 genes up-regulated and 50 genesdown-regulated between the unassembled and primordial folli-cle stages. Observations demonstrate 80 genes up-regulated and44 genes down-regulated between the primordial and primaryfollicle stages. The analysis demonstrated 2332 genes commonamong the three developmental stages, 146 genes specific forthe unassembled follicles, 94 genes specific for the primordialfollicles, and 151 genes specific for the primary follicles. Ste-roidogenic genes are up-regulated between unassembled andprimordial follicles, and then many are again down-regulatedbetween primordial and primary follicles. The hormones inhibinand Mullerian inhibitory substance (MIS) display a similar pat-tern of expression with the highest levels of mRNA in the pri-mordial follicles. Several novel unknown genes that had dra-matic changes in expression during primordial follicle develop-ment were also identified. Gene families/clusters identified thatwere up-regulated from unassembled to primordial follicles in-clude growth factors and signal transduction gene clusters,whereas a down-regulated gene family was the synaptonemalcomplex genes associated with meiosis. Gene families/clustersthat were up-regulated between primordial and primary folliclesincluded immune response genes, metabolic enzymes, and pro-teases, whereas down-regulated gene families include the glob-ulin genes and some steroidogenic genes. The expression of sev-eral growth factors changed during primordial follicle develop-ment, including vascular endothelial growth factor and insulin-like growth factor II. Elucidation of how these changes in geneexpression coordinate primordial follicle assembly and the pri-mordial to primary follicle transition provides a better under-standing of these critical biological processes and allows selec-tion of candidate regulatory factors for further investigation.

1Supported by NIH grants to M.K.S. P.R.K. and J.M.A. contributed equallyto the manuscript and study.2Correspondence: Michael K. Skinner, Center for Reproductive Biology,School of Molecular Biosciences, Washington State University, Pullman,WA 99164-4231. FAX: 509 335 2176; e-mail: [email protected]

Received: 12 May 2004.First decision: 2 June 2004.Accepted: 30 August 2004.Q 2005 by the Society for the Study of Reproduction, Inc.ISSN: 0006-3363. http://www.biolreprod.org

follicle, follicular development, gene regulation, growth factors,ovary

INTRODUCTION

The assembly and subsequent development of the de-velopmentally arrested primordial follicle are poorly un-derstood processes that are necessary for normal femalereproduction. These processes are critical because they setthe size of the primordial follicle pool. Primordial folliclenumbers do not proliferate or increase once formed. Theprimordial follicle pool present at birth represents the totalnumber of primordial follicles available to a female duringher reproductive life [1]. Although a recent observation ledto speculation that a female germ-line stem cell may exist,further research is needed to confirm this observation andidentify a regenerating follicle pool [2]. Primordial folliclesare the female’s sole source of gametes and critical ste-roidogenic tissue. All of the primordial follicles will even-tually be lost by ovulation or by oocyte atresia. When theprimordial follicle pool is depleted, menopause occurs [3].Abnormal primordial follicle development can cause path-ological conditions such as premature ovarian failure [4].The current study was designed to provide insight into themolecular and cellular control of primordial follicle devel-opment.

Recent studies using ovarian organ cultures or null mu-tant knockout mice have begun to elucidate the cell-cellinteractions that coordinate primordial follicle assemblyand the primordial to primary follicle transition. The pro-cess of follicular assembly begins at mid gestation in largemonovulators such as humans [5] and at birth in rodents[6]. Follicular assembly is the apoptotic breakdown ofgroups of recently proliferating gametes called oocyte nests[7]. This apoptosis is coordinated by apoptotic factors suchas tumor necrosis factor alpha (TNFa) [8]. High progester-one concentrations in utero repress oocyte apoptosis andfollicular assembly in rodent pups until birth when the pupsexperience a dramatic decline in steroid concentrations [9].

The cell-cell interactions that occur during the primor-dial to primary follicle transition are better characterizedthan those of primordial follicle assembly. The primordialto primary follicle transition is coordinated by a combina-tion of positive and negative regulatory factors. The epi-thelial granulosa cells produce kit ligand [10] and leukemiainhibitory factor [11] that act on the oocyte and the sur-rounding stroma to promote the primordial to primary fol-licle transition. Specifically, kit ligand is involved in therecruitment of mesenchymal theca cells to the follicle [12].Basic fibroblast growth factor is produced by the oocyteand acts on the granulosa and theca to promote the pri-mordial to primary follicle transition [13]. Nerve growthfactor acts on the granulosa to promote the primordial toprimary follicle transition [14]. Bone morphogenic protein

242 KEZELE ET AL.

FIG. 1. Top: Schematic of follicular assembly and the primordial to primary follicle transition. The oocytes start as clusters of oocytes surrounded bybands of stromal tissue. Certain oocytes go through an apoptotic process and the ones remaining become primordial follicles that are an oocytesurrounded by a layer of squamous pregranulosa cells. At the primordial to primary follicle transition, the granulosa take on a cuboidal appearanceand begin proliferating. The oocyte grows in size and theca is sequestered from the surrounding stroma. Bottom: Light micrographs of follicles at theabove stages. Unassembled oocyte nests are surrounded by bands of stroma tissue. Primordial follicles, an oocyte surrounded by a layer of squamousgranulosa, are indicated by arrows. Primary follicles, an oocyte surrounded by a layer of proliferating granulosa, are also indicated by arrows. Originalmagnification 3200.

4 produced by the theca and stroma cells acts as a folliclesurvival factor [15]. Insulin acts as an endocrine factor topromote the primordial to primary follicle transition [16].Mullerian inhibitory substance (MIS) produced by largerfollicles represses the primordial to primary follicle tran-sition in adjacent follicles [17].

The current model of cell-cell interactions in primordialfollicle assembly and the primordial to primary follicletransition is based primarily on the investigation of growthfactors that have been shown to be important in cell-cellinteractions of the better characterized large antral follicles.A number of the growth factors known to coordinate antralfollicle growth have been shown to be involved in primor-dial follicle development. Additional factors will also likelybe involved in the development of primordial follicles. Thecurrent study is a gene discovery approach to identify newfactors that may coordinate primordial follicle assemblyand the primordial to primary follicle transition. This genediscovery project investigates the transcriptomes of ovariesthat contain predominately unassembled, primordial, or pri-mary follicles. Primordial and primary follicles are toosmall to dissect or isolate from the ovary, and it is notpossible to isolate individual cell types from them. The cur-rent study uses rat ovaries from different developmentalstages that have predominately one follicle population.Postnatal Day 0 ovaries contain only unassembled follicles.Postnatal Day 4 ovaries contain predominately primordialfollicles. Postnatal Day 0 ovaries cultured for 1 wk containpredominately primary follicles [9]. Comparing the globalgene expression profiles (transcriptomes) from ovaries at

these developmental stages allows a comparison betweenunassembled, primordial, and primary follicles.

MATERIALS AND METHODS

Ovary Dissection, Organ Culture, and RNA ExtractionOvaries from Postnatal Day 0 Sprague-Dawley rat pups were dissected

for both immediate RNA extraction and organ culture. Postnatal Day 4 ratovaries were dissected for immediate RNA extraction. Postnatal Day 0 ratovaries were also cultured for 7 days. Whole ovaries were cultured aspreviously described [10] on floating filters (0.4 mm; Millicell-CM; Mil-lipore, Bedford, MD) in 0.5 ml Dulbecco modified eagle medium(DMEM)–Ham F-12 medium (1:1, vol/vol) containing 0.1% bovine serumalbumin (BSA; Sigma, St. Louis, MO); 0.1% albumax (Gibco BRL, Gai-thersburg, MD); 2.75 mg/ml transferrin; 0.05 mg/ml L-ascorbic acid (Sig-ma); and 1 mg/ml insulin (bovine; Sigma) in a four-well culture plate(Nunc plate; Applied Scientific, South San Francisco, CA). Medium wassupplemented with penicillin, streptomycin, and gentamicin to preventbacterial contamination. RNA was extracted using the TRIZOL reagent(Invitrogen, Grand Island, NY) per the manufacturer’s instructions. About20 ovaries were used for each RNA extraction. All procedures were ap-proved by the Washington State University (WSU) Animal Care and UseCommittee.

Microarray AnalysisRNA was hybridized to the Affymetrix U34A 8799 gene chip (Affym-

etrix, Santa Clara, CA). The Genomics Core in the Center for Reproduc-tive Biology at Washington State University performed the analysis aspreviously described [18, 19]. Briefly, RNA from whole ovaries was re-verse transcribed into cDNA, and cDNA was transcribed into biotin-la-beled RNA. Biotin-labeled RNA was then hybridized to the AffymetrixU34A 8799 gene chips. Each gene set was composed of 16 pairs of 24-oligomer oligonucleotides, with one sense strand specific for the gene and

243FUNCTIONAL GENOMICS OF OVARIAN PRIMORDIAL FOLLICLE DEVELOPMENT

FIG. 2. A) Kinetics of folliculogenesis in vivo. Data displayed as percentunassembled, primordial, and primary follicles in the ovaries of the neo-natal and the Postnatal Day 4 rat. At birth (Postnatal Day 0) the majorityof oocytes are unassembled and are not in follicles. At Postnatal Day 4the majority are primordial follicles. B) Kinetics of folliculogenesis in vitro.Data displayed as percent unassembled, primordial, and primary folliclesin the neonatal ovary at 0 and 6 days of rat ovary culture. After 6 daysin culture the majority of the oocytes are in primary follicles. All data arerepresentative of a minimum of three different experiments done in rep-licate and presented as mean 6 SEM.

FIG. 3. Venn diagram of unassembled, primordial, and primary follicleovary stages. Similarities and differences in genes expressed are repre-sented. An additional 5755 genes on the microarray chip were not de-tected with a minimum relative signal of 100 used for each gene. Numberof stage-specific genes and genes expressed in common are indicated.

TABLE 1. Summary of follicle stage transcriptome changes.

Unassembled to primordial Primordial to primary

Total genes (.23)Total genes (.23)

80 genes up-regulated44 genes down-regulated

148 genes up-regulated50 genes down-regulated

Up-regulated gene clusters 5 steroidogenic genes3 zona pellucida genes6 hormones and growth factors11 signal transduction genes

17 immune response genes23 metabolism enzymes10 proteases

Down-regulated gene clusters 2 synaptonemal complex genes 3 globin genes3 steroidogenic genes

one antisense strand with single point mutations for use as a comparativenegative control. The oligonucleotides spanned the gene, so 59 and 39regions contributed to the final signal obtained. Biotinylated RNA wasthen visualized by labeling with phycoerythrin-coupled avidin. The mi-croarray was scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA). Two microarray chips from two differentRNA samples were analyzed for each of the predominate unassembled,primordial, and primary follicle ovary preparations.

Bioinformatics and Microarray StatisticsMicroarray output was examined visually for excessive background

noise and physical anomalies. The default Microarray Suite (Affymetrix,Santa Clara, CA) statistical values were used for all analyses. An absoluteanalysis using Microarray Suite was performed to assess the relative abun-dance of the transcripts based on signal and detection (present, absent, ormarginal) for the 16 different oligonucleotides per gene and comparisonfor analysis. The absolute analysis from Microarray Suite was importedinto GeneSpring 5.1 software (Silicon Genetics, Redwood City, CA). Thedevelopmental time course data were normalized within GeneSpring usingthe default/recommended normalization methods. These included the set-ting of signal values below 0.01 to 0.01, total chip normalization to the50th percentile, and normalization of each gene to the median. These nor-

malizations allowed for the visualization of data based on relative abun-dance at any given time point, rather than compared with a specific controlvalue. Data restrictions and analytical tools in GeneSpring were appliedto isolate noteworthy and possibly important patterns of gene expressionduring the course of testicular development and spermatogenesis. Tran-scripts expressed differentially at a statistically significant level were de-termined using a one-way ANOVA parametric test with variances notassumed equal and a P-value cutoff of 0.05. This was applied to all threedevelopmental stage samples and considered all transcripts represented onthe arrays. Two repeats for each developmental stage was performed andallowed a 2 3 2 factorial comparison in the experiment. Subsequently,expression restrictions were applied to the transcripts expressed in a sig-nificant manner. These restrictions were designed so that the remainingtranscripts met the following requirements in addition to being expressedin a significant manner: 1) each transcript must have a signal value of atleast 100 in at least one of the three developmental stages, and 2) had anaverage fold change of two or greater in signal intensity between devel-opmental stages. The resulting transcripts were screened using Excel (Mi-crosoft, Redmond, WA) for redundant UniGene entries. Transcripts thatpassed these restrictions were considered for further analysis. Cluster anal-ysis and patterns of gene expression were identified using unsupervisedcluster analysis within the set of differentially expressed transcripts thatmet the requirements detailed previously. Clustering algorithms allow forthe separation of distinct patterns of expression based on the similarity ofexpression profiles between different genes [20]. In this analysis, a hier-archical clustering algorithm using a smooth correlation with the default

244 KEZELE ET AL.

FIG. 4. Representative microarray data clustered into six distinct patterns of expression using the Genespring software. Each line represents a singlegene. Data are displayed as normalized signal intensity of each gene at unassembled, primordial, and primary follicle developmental stages. Notegenes with the greatest change are only expressed in one developmental stage (B, D, F).

parameters was used to isolate distinct, nonrepetitive patterns of expressionwithin the time course. A nonphylogenetic gene tree that illustrates themajor expressional patterns within the differentially expressed transcripts(determined through statistical analysis) in a continuous fashion was pro-duced from this analysis. Pathway Assist (Stratagene Inc., La Jolla, CA)software was used for detailed pathway analysis and gene associations.Previous studies have shown that microarray data correlates well with real-time quantitative PCR and Northern analysis [18, 21]. Therefore, microar-ray data does not need to be confirmed as previously suggested [19].However, a selected gene (i.e., vascular endothelial growth factor) wasused in a real-time quantitative PCR procedure as previously described[22] to help confirm the microarray procedure.

RESULTSThe current study investigates the transcriptomes of un-

assembled, primordial, and primary follicles as part of agene discovery and functional genomics analysis. RNA wastaken from freshly isolated Postnatal Day 0, freshly isolatedPostnatal Day 4, and Postnatal Day 0 ovaries cultured for1 wk. The percentage of each follicle type in these ovarieshas been previously determined by morphological analysis[9]. Briefly, unassembled follicles are defined as groups ofoocytes adjacent to each other surrounded by a layer of


FIG. 5. Dendrogram cluster analysis ofthe total (all genes, .23) ovary transcrip-tome in the unassembled, primordial, andprimary follicle developmental states. Eachline represents a single gene, and its rela-tive expression is indicated as increased(red) or decreased (blue) according to thecolor key provided. Selected gene set clus-ters for hormones, growth factors and cy-tokines, metabolic genes, and signal trans-duction genes are shown.

stromal tissue. Primordial follicles are defined as solitaryoocytes surrounded by a layer of squamous epithelial cells.Primary follicles are oocytes surrounded by a layer of pro-liferating cuboidal epithelial cells (Fig. 1). Postnatal Day 0ovaries contain 95% unassembled follicles (Fig. 2). Post-natal Day 4 ovaries contain approximately 70% primordialfollicles. Cultured Postnatal Day 0 ovaries contain greaterthan 75% primary follicles (Fig. 2). Ovaries from thesethree developmental stages were taken for analysis andRNA was collected.

The RNA from two different preparations of each ovar-ian stage and follicle type were analyzed separately usingthe Affymetrix U34A rat gene chips. This gene chip has14 000 genes for analysis. The global gene expression pro-file comparison between unassembled, primordial, and pri-

mary follicles is shown in Figure 3. The ovarian transcrip-tome analyzed is a combination of somatic cell and oocytecontributions. The Venn diagram demonstrates 2332 genesexpressed in all three developmental stages with 146 genesspecific to unassembled follicles, 94 genes specific to pri-mordial follicles, and 151 genes specific to primary follicles(Fig. 3). Similar numbers of genes were also in commonbetween two different developmental stages in the analysis(Fig. 3). The principle genes of interest were those thatchange expression levels between the developmental stages.Specific genes were categorized using a relative hybridiza-tion signal limit minimum of 100 and a minimum changegreater than twofold. The cutoff 100 for signal and greaterthan twofold change demonstrated that over 5000 genes didnot change expression levels and/or had very low levels of

246 KEZELE ET AL.

TABLE 2. Unassembled to primordial up-regulated genes.*

AccessionPrimordial

mean signal 6 SEMFold

change

S63167M33986AI235576J00713AB000778

3 b-hydroxysteroid dehydrogenase (HSD)Aromatase p450, subfamily 19Hydroxy-d-5-steroid dehydrogenaseCarboxypeptidase A1Phospholipase D gene 1

627.35109.15272.05132.15396.40

356.5523.45

151.8560.3563.50

13.2010.017.787.246.03

M12492M36453AB000929S35751AB006007

Protein kinase, cAMP dependent regulatory, type II bInhibin, a subunitZona pellucida 2 glycoprotein3 a-HSD; 3 a-hydroxysteroid dehydrogenaseSteroidogenic acute regulatory protein

181.301659.701624.35196.20100.65

66.60310.60157.9515.0736.75

5.875.255.234.904.89

AB012214AF045564J03190AJ005396M32754

DNA cytosine 5 methyltransferaseNeu development-related proteinAminolevulinic acid synthase 1Procollagen, type XI, a 1Inhibin a-subunit gene, exon 1

835.80389.45177.60104.50

1163.05

211.2050.5551.1013.8084.45

4.774.464.244.123.63

AB016800U90887AB000928Y10823U02320

7-Dehydrocholesterol reductaseArginase 2Zona pellucida glycoprotein 1Zona pellucida glycoprotein 3Neuregulin 1

296.75189.10245.15

1774.55124.35

77.3514.6622.75

307.8510.58

3.523.423.383.273.24

M91466AF035951M64733AA875411AF054826

Adenosine A2B receptorKinesin-related protein KRP1 (KRP1)TRPM-2 geneSuppressor of K1 transport defect 3Vesicle-associated membrane protein 5

114.35127.15474.60111.05123.80

12.4518.9537.6510.1721.50

3.063.033.022.992.87

S53987U60416AA685974U38253AA998164

Nicotinic receptor a 7 subunitMyosin 5BPlectinEukaryotic translation initiation factor 2B, subunit 3Cyclin B1

173.80275.10786.35227.80249.10

14.5026.0063.4819.4229.50

2.822.802.722.682.66

AI178971X16555AA892897A03913U02315

Hemoglobin, a 1Phosphoribosyl pyrophosphate synthetase 2Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2Glia-derived neurite-promoting factor (GdNPF)ndf40 neu differentiation factor

151.35243.30379.25489.65116.4

42.3596.0079.5540.773.6

2.662.652.592.592.53

AB009999S98336

CDP-diacylglycerol synthaseMIS; anti-Mullerian hormone

156.85487.55

20.8598.15

2.412.40

D32209AA891527AA892511AI639118H31604

Acidic nuclear phosphoprotein 32 family, member A4.5 LIM domains 2Calcium binding protein P22Calcium-dependent actin-binding proteinBudding uninhibited by benzimidazoles

243.80197.40114.60138.80140.30

196.5017.2020.4016.0048.30

2.382.342.342.312.24

D82363D16237X76489D49363M64301

Presenilin 1Cell division cycle 25BCD9 antigen (p24)Perchrolic acid soluble proteinMitogen-activated protein kinase 6

186.60160.25700.60580.80244.55

16.7328.75

164.3058.8035.95

2.232.212.202.192.17

S72505AJ011606X73411M34253AF016503

Glutathione S-transferase Yc1 subunitDNA polymerase alpha subunit IIsnRNP-associated polypeptide NInterferon regulatory factor 1Procollagen C-proteinase enhancer protein

492.75147.20291.75108.30686.15

45.2113.5924.9226.2061.50

2.142.142.142.132.12

U10188AF076619U16359AI230228U23769

Polo-like kinase homolog (Drosophila)Growth factor receptor bound protein 14Nitric oxide synthase genePhosphoserine aminotransferase mRNAPDZ and LIM domain 1

344.45143.45113.40214.60264.35

31.4817.9512.5420.1150.75

2.122.122.112.082.06

AF033109S61865L10326AI171630AA891445

Syntaxin 8Heparan sulfate proteoglycan core proteinGNAS complex locusMitogen activated protein kinase 14Suppressor of K1 transport defect 3

253.55555.00136.10281.10101.75

22.6896.8025.5026.109.75

2.062.062.062.052.05

J02773AB017912U38253AA848421M60322

Fatty acid binding protein 3MAD homolog 2 (Drosophila)Eukaryotic translation initiation factor 2B, subunit 3Uracil-DNA glycosylaseAldose reductase gene

369.05306.25277.05198.45

1141.00

138.5527.4037.5527.75

113.50

2.032.032.032.022.01

AI236601 Heat-shock protein 346.80 39.60 2.01

* ESTs not included and relative minimum signal of 100 used for at least one of the developmental stages.


TABLE 3. Unassembled to primordial down-regulated genes.*

AccessionUnassembledmean signal 6 SEM

Foldchange

X75785D83349Y13275AI176456X59864

Synaptonemal complex protein 3 (SCP3)Cadherin 22Transmembrane 4 superfamily member 3Metallothionein IIORF; Rat ASM15

242.30292.60487.65991.35643.90

57.7082.2047.35

361.35235.20

25.7224.1823.7623.6323.62

M23566AI014091X17012L19998AA800851

a 2-macroglobulin geneCbp/p300-interacting transactivatorInsulin-like growth factor IISulfotransferase family 1A, phenol-preferring, 1Carboxylesterase 3

2328.80296.55

1633.80170.90125.60

866.4083.55

373.3048.9017.10

23.5723.4823.4623.1923.03

D28562AI029920M10934J04488D12769

Solute carrier family 2, member 5Insulin-like growth factor–binding protein 5Retinol-binding protein (RBP)Prostaglandin D synthaseKruppel-like factor 9

188.50108.10257.45138.55223.50

16.3610.4060.2513.26

141.90

22.9322.6122.5122.4722.42

X81449D84336AI103238M83143X81449

Keratin complex 1, acidic, gene 19d-like homolog (Drosophila)Protein phosphatase 2Sialyltransferase 1Keratin complex 1, acidic, gene 19

1117.20582.25107.30146.45586.05

102.43173.8510.7113.6554.43

22.4022.3622.3622.3522.29

AA894092AA852004U67908D00036AB010999

Osteoblast-specific factor 2Glutamine synthease 1Chymase 1Phospholipase A2, group 1BPeptidyl arginine deiminase, type 4

1489.00100.75343.55293.85185.70

127.2945.15

135.5528.9517.79

22.2922.2822.2422.1922.18

Z46614M83680D89730U67911M77694

CaveolinGTPase Rab14T16 mRNAMast cell protease 8Fumarylacetoacetate hydrolase

195.65117.25115.95141.30106.85

50.8563.9521.6514.2910.72

22.1522.1522.1422.1122.09

AA965147Z35138H31813

Heterogeneous nuclear ribonucleoprotein A1Fibroblast growth factor receptor 2bHypothetical protein DKFZp586B1621.1

483.65126.20117.25

71.7512.9913.85

22.0422.0222.01


expression. Analysis of primordial follicle assembly dem-onstrated that 80 genes are up-regulated and 44 genes aredown-regulated between unassembled and primordial fol-licles. Analysis of the primordial to primary follicle tran-sition demonstrated that 148 genes are up-regulated and 50genes are down-regulated between primordial and primaryfollicles (Table 1).

Genes were grouped by expression pattern into six geneclusters (Fig. 4). Half of these clusters displayed dramaticchanges in gene expression (Fig. 4, B, D, and F). The tran-scriptomes of each developmental point were unique. Theother three clusters displayed smaller changes in gene ex-pression (Fig. 4, A, C, and E). The first displayed up-reg-ulation at primordial and primary stage follicles (Fig. 4A).The second displayed up-regulation at the primary folliclestage (Fig. 4C). The third displayed up-regulation at theunassembled and primary follicle stage (Fig. 4E). A den-drogram cluster analysis for all of the genes changing great-er than twofold between the developmental stages is shownin Figure 5. The genes with increased (red) and decreased(blue) expression between the stages are grouped and showunique clusters at each developmental stage. Specific ex-amples of functional gene clusters of metabolic, signaltransduction, and hormones/growth factors/cytokines arealso shown in Figure 5. Each developmental stage has geneclusters increasing and decreasing between the develop-mental stages. The specific genes examined are listed bychange in signal in Tables 2–5.

Of the 80 genes up-regulated during primordial follicleassembly (Tables 1, 2, and 6), a number were steroidoge-

netic enzymes. Also up-regulated was the hormonal factorinhibin and the known repressor of the primordial to pri-mary follicle transition MIS. All three zona pellucida genes,which code for the thick protein coat of the oocyte, wereup-regulated. Members of the neu differentiation factorfamily were also up-regulated (Tables 2 and 6).

Of the 44 genes down-regulated during follicular assem-bly (Tables 3 and 6), two were synaptonemal complex fam-ily genes involved in meiosis. A growth factor stronglydown-regulated between the unassembled and primordialfollicle stage was insulin-like growth factor II (IGFII), aswas an IGF binding protein–2 (IGFBP2).

Of the 148 genes up-regulated during the primordial toprimary follicle transition (Tables 1, 4, and 7), 17 wereimmune or inflammatory response–related genes such ascytokines. In addition, 10 proteases (e.g., cathepsins) wereup-regulated. Twenty-three of the genes were metabolic en-zymes. Also up-regulated were the IGFII growth factor andthe IGFBP2 (Fig. 6C). Interestingly, vascular endothelialgrowth factor (VEGF) was also found to be up-regulated(Fig. 6C). To confirm the microarray analysis, the VEGFgene expression change was also assessed with a real-timequantitative PCR procedure and found to give the samerelative increase in expression (data not shown). Absentfrom this list are growth factors known to promote the pri-mordial to primary follicle transition—such as kit ligand(data not shown)—primarily due to low levels (i.e., ,100signal) of expression. In addition to the known genes, twogenes of unknown function were dramatically up-regulatedduring the primordial to primary follicle transition. The kid-

248 KEZELE ET AL.

TABLE 4. Primordial to primary up-regulated genes.*

AccessionPrimary

mean signal 6SEMFold

change

X52477AF022147AF022147J02585M14656

Complement component 3Integral membrane–associated protein 1UOTPStearoyl-coenzyme A desaturase 1Secreted phosphoprotein 1 (osteopontin)

428.351262.60375.8957.30

1193.05

187.25184.70104.2622.80289.15

133.8655.2642.9541.0037.58

L24374X98517NM052802U23055M92059

Matrix metalloproteinase 7 (matrilysin)Macrophage metalloproteinaseKAPC-CAM4 geneAdipsin

462.45208.6584.65

254.60231.85

73.5517.7518.2515.6015.34

35.9932.5628.2126.9423.30

X71127AA892775X73371M32062X51529

Complement component c1q b-chainLysozymeImmunoglobin g FC regionFc receptor, IgG, low affinity IIIPlatelet phospholipase A2

282.951611.35117.05334.90

1164.00

42.95655.6550.55

152.70746.60

18.9916.8013.8513.6411.87

U10894AA945737J02962X17053AA946503

Allograft inflammatory factor 1Chemokine receptor (LCR1)Lectin, galactose binding, soluble 3Immediate early serum–responsive JE geneLipocalin 2

207.10142.75736.35456.60951.55

92.9066.1595.3589.10

156.45

10.8710.6510.5610.4410.05

M57276AI639117S53527AI169612M64795

CD53 antigenComplement factor B precursorS-100 b subunitFatty acid binding protein 4MHC class I antigen gene (RT1-u haplotype)

153.10276.10158.40647.80224.55

66.2087.6035.20

135.5066.35

9.789.429.379.169.11

AA800587AA799861M98820L14004U17919

Glutathione peroxidase (GSHPX-1)Interferon regulatory factor 7 (IRF-7)Interleukin 1 bPolymeric immunoglobulin receptorAllograft inflammatory factor 1

198.20103.60152.25122.40140.20

13.4327.0034.5530.9055.00

8.667.857.567.467.38

X17053L03201M23566K00994AI237731

Immediate early serum–responsive JE geneCathepsin Sa-2-macroglobulin geneIntestinal calcium binding proteinLipoprotein lipase

257.00315.10377.20142.55347.25

63.00104.7072.8040.95

138.25

7.267.086.816.806.45

AI072634U90448

Keratin complex 1, acidic, gene 18CXC chemokine LIX

362.90207.60

26.8529.20

6.245.65

U42719L33869AA800750U49062S74141

Complement component, C4 complement proteinCeruloplasminRetrovirus-related POL polyproteinCD24 antigenTyrosine kinase

351.05126.40

1648.30526.45140.10

78.8510.40

116.1538.8823.50

5.054.954.674.644.39

U95368U13275M63656D10354J02612

GABA-A receptor p subunitTransmembrane 4 superfamily member 3Aldolase CGlutamic-pyruvate transaminaseUDP glycosyltransferase 1 family, polypeptide A6

123.70556.30164.60111.45200.70

10.8166.7015.7010.2934.80

4.354.294.234.073.91

U18729M15880S57478AI169104AI170268

Cytochrome b558 a-subunitNeuropeptide YLipocortin IPlatelet factor 4 precursor (PF-4) (CXCL4)Beta-2-microglobulin

658.90194.80423.00215.45

1694.10

160.5034.1078.30

100.35126.12

3.843.763.733.603.58

X07729AA800318AI171962AA894004X13044

Neuron-specific enolaseProtein C inhibitorAnnexin 1PervinCD74 antigen

110.95392.30335.30296.50113.10

10.17138.4027.49

121.5039.20

3.463.403.393.323.27

AF037072S56937L40362M88469AA874848

Carbonic anhydrase 33-methylcholanthrene-inducibleRT1 class Ib genef-spondinThymus cell surface antigen

261.00112.45488.75165.25191.40

22.0010.0763.4556.1548.00

3.263.253.233.223.11

D90404AF065438AB003042AA852004D89730

Cathepsin CPeptidylprolyl isomerase C-associated proteinComplement component 5, receptor 1Glutamine synthetase 1T16 mRNA

325.60461.20134.70132.65159.90

26.4134.4129.6068.0529.80

3.053.043.013.002.95

S76054X62322D90109AI179610X06916

Cytokeratin-8GranulinFatty acid coenzyme A ligase, long chain 2Heme oxygenaseProtein p9Ka homologous to calcium-binding protein

267.451523.60260.80240.15152.95

22.02329.7062.4050.8551.35

2.912.902.882.862.79


TABLE 4. Continued.

AccessionPrimary

mean signal 6SEMFold

change

AA799560AA891690AF083269M10934M15562

N-myc downstream-regulated gene 2Tumor necrosis factor (ligand) member 13Actin-related protein 2/3 complex, subunit 1BRetinol-binding protein (RBP)MHC class II RT1.u-D-a chain

266.10245.25491.80271.05105.60

20.8619.9939.47

108.2520.00

2.712.682.682.642.61

AI639535AI176456X60661

Transporter-like proteinMETALLOTHIONEIN-II (MT-II)Ligand-binding protein

172.70714.40141.45

31.80113.1044.15

2.612.612.59

AI014091AI235585J02722AI008888AI231213

Cbp/p300-interacting transactivatorCathepsin DHeme oxygenaseCystatin BKangai 1

220.80655.55103.95222.05591.15

55.30142.3533.4527.6596.25

2.592.562.552.552.49

AF087943D10729J02791S68135AA850734

CD14 antigenProteasome subunit RC1Acetyl-coenzyme A dehydrogenaseGlucose transporter 1Vascular endothelial growth factor

191.85268.20289.10561.70108.90

16.8622.7724.33

202.2019.60

2.482.422.422.422.41

AF003835X82396AI232783AF025308AF087944

Isopentenyl-diphosphate delta isomeraseCathepsin BGlutamine synthetase 1MHC class Ib antigen (RT1.C1)Monocyte differentiation antigen CD14

183.651046.25384.60475.25156.45

29.15245.55139.7075.2514.91

2.412.382.372.342.31

X96437AI009405X76985U90610AI169327

PRG1 geneInsulin-like growth factor binding protein 3LatexinChemokine receptor (LCR1)Tissue inhibitor of metalloproteinase 1

254.65183.35181.25275.85424.30

23.5878.2567.7597.2538.61

2.292.282.272.272.26

AA892797AA926129M83143U09870J05122

Phosphoglycerate kinase 1Schlafen 4Sialyltransferase 1Major vault proteinBenzodiazepin receptor (peripheral)

1400.80136.30139.55134.25353.40

340.8023.8032.4514.4531.32

2.262.242.242.222.21

J03752M93257AA893235H32189AA925752

Microsomal glutathione S-transferase 1Catechol-O-methyltransferasePutative lymphocyte G0/G1 switch protein 2Glutathione S-transferase, m 1cd36 antigen

1576.20107.15207.60531.95137.75

186.5013.2520.7073.2512.98

2.192.182.182.182.16

M31788D30649L40364C07012X02610U27562

Phosphoglycerate kinase 1Alkaline phosphodiesteraseMHC class I RT1.O type-149Peptidylprolyl isomerase C-associated proteinEnolase, 1, aSPARC-like 1

760.05126.00854.90331.15

1217.10470.35

142.8511.80

109.9028.68

113.05269.95

2.162.132.042.032.032.01


ney-specific androgen-regulated protein (KAP) and theuterus ovary–specific transmembrane protein (UOSTP)were both up-regulated (28- and 42-fold, respectively; Fig.6A).

Of the 50 genes down-regulated during the primordialto primary follicle transition (Tables 1, 5, and 7), threewhere steroidogenic enzymes. The endocrine factors inhib-in and MIS were also down-regulated in a situation recip-rocal to the change between the unassembled and primor-dial follicles (Fig. 6B). Three globin genes were stronglydown-regulated between the primordial and primary folliclestages (Tables 5 and 7).

Further analysis of the data with Pathway Assist soft-ware identified groups of genes in specific cellular path-ways. The relationship of the steroidogenic genes and otherinteracting proteins is shown in Figure 7. Critical steroido-genic genes such as CYP19A1 and STAR have interactionswith a large number of proteins. Some regulate activity(Fig. 7, dashed lines) and some regulate expression (Fig. 7,solid lines). All of the genes identified as changing during

primordial follicle development are linked except Scd1 andDCHR7 (Fig. 7).

DISCUSSION

The current study analyzed the transcriptomes of ovarieswith predominately unassembled follicles, primordial fol-licles, and primary follicles as part of a functional genomicsinvestigation. A similar approach has recently been used tocharacterize the transcriptome of ovarian cell types to as-sess disease states (e.g., polycystic ovarian syndrome) andfolliculogenesis characteristics [23–25]. The genes identi-fied whose transcriptional regulation was correlated withprimordial follicle assembly, and the primordial to primaryfollicle transition are candidate regulators of these devel-opmental processes. A number of candidate genes wereidentified, as well as genes that confirmed and supportedthe experimental approach and methodology. Those genesthat were strongly up-regulated were expressed during onedevelopmental stage exclusively, and each follicle stage be-

250 KEZELE ET AL.

TABLE 5. Primordial to primary down-regulated genes.*

AccessionPrimary

mean signal 6 SEMFold

change

M94918X56325J02773M33986M23721

b-globin gene2-a-1 globin geneFatty acid binding protein 3Cytochrome P450, subfamily 19Carboxypeptidase (CA2)

1298.803661.20369.05109.15194.80

186.60371.40138.5523.4564.50

271.05226.1727.8826.7625.89

S63167D32209AI235576M36453AI178971

3 b-hydroxysteroid dehydrogenase isomerase type IIAcidic nuclear phosphoprotein 32 family, member AHydroxy-d-5-steroid dehydrogenaseInhibin, aHemoglobin, a 1

196.20243.80272.05

1163.05151.35

16.78196.50151.8584.4542.35

25.2924.4024.1924.0323.95

S98336U25264X03347AA875033AI639448

MIS; Mullerian inhibiting substanceSelenoprotein W muscle 1Polyprotein gag-fos-p75; FBR murine osteosarcomaFibulin 5A5D3 protein

487.55326.65116.55169.60896.15

98.1526.5278.8515.0373.50

23.5423.1823.1622.9422.84

J00713M12492AF021936M22400D00575

Carboxypeptidase A1Protein kinase, cAMP dependent regulatory, type II bCdc42-binding protein kinase bGlypican 3Glycoprotein hormones, a subunit

132.15181.30110.70937.40206.25

60.3566.6026.2084.4050.75

22.6622.6122.5822.5022.42

AA891527X07365U16359AI102839J04035

4.5 LIM domains 2Glutathione peroxidaseNitric oxide synthase geneCalbindin 1Elastin

197.401171.95113.40102.85737.35

17.32106.8312.1816.65

180.05

22.3722.3522.2522.2422.21

D14441X06769L22761X74565AA866454

Brain acidic membrane proteinc-fox protein (AA 1-380)GATA-binding protein 4Polypyrimidine tract binding proteinProcollagen, type I, a 2

154.70279.90417.45350.25135.65

15.35181.7066.8546.2528.15

22.1922.1722.1322.1222.07

Z78279L22760AI012275M55017AB011532D49785

Collagen, type 1, a 1GATA-binding protein 6Developmentally regulated protein TPO1Nucleolin geneMEGF6Mitogen-activated protein kinase kinase kinase 12

2479.55349.75983.60128.15125.00250.60

664.5530.24

141.3012.6127.60

111.10

22.0722.0622.0422.0322.0122.01


TABLE 6. Unassembled to primordial follicles genes.

Groups/clustersFold

change

Steroid synthesis 3 b-hydroxysteroid dehydrogenaseAromatase P450, subfamily 19Steroidogenic acute regulatory proteinSteroid 3-alpha-dehydrogenase7-Dehydrocholesterol reductase

13104.894.903.52

Zona pellucida Zona pellucida 2Zona pellucida glycoprotein 3Zona pellucida 1

5.233.383.27

Synaptonemal complex proteins SCP3SCP1

25.7223.99

Hormones and growth factors Mullerian inhibiting substanceInsulin-like growth factor IIinhibin a-subunitndf40 neu differentiation factorNeu development–related proteinNeuregulin

2.4123.46

5.252.534.463.24

Signal transduction Phospholipase DcAMP-protein kinase ANicotinic receptorCDP-diacylglycerol synthase

6.03.52.822.41

Adenosine A2B receptorCadherin 22Mitogen-activated protein kinase 14

3.0624.18

2.05


TABLE 7. Primordial to primary follicles genes.

Groups/clustersFold

change

Immune response Complement component C3Fc G receptorIgE binding proteinOsteopontinComplement protein C1q b chain

101.8813.410.8737.5818.99

C4 complement proteinCXC chemokine LIXMHC class I RT1.Aa a-chainFc receptor, IgG low-affinity IIIComplement factor B

6.465.652.04

13.649.42

Compliment component 4aChemokine receptor (LCR1)Allograft inflammation factor I

5.052.81

10.87Proteases Matrillysin (MMP-7)

Macrophage metalloelastaseSerine proteaseProteosome subunit RC1Cathepsin SCathepsin C

36.0032.605.852.427.083.05

Globin b-globin gene2-a-1 globinEST222653 hemoglobin

271.41226.1724.19

Steroid metabolism Stearyl CoA desaturase3 b-hydroxysteroid dehydrogenase isomerase type II.2Aromatase cytochrome P450Steroid 3-a-dehydrogenase

23.7722.0826.7622.64

Hormones and growth factors Inhibin a-subunit geneMullerian inhibiting substanceVEGFInsulin-like growth factor II

23.6623.54

2.411.89

Interleukin 1 bNeuropeptide YTNF-13

7.553.762.68

Unknown function Kidney-specific androgen-regulated protein (KAP)Uterus-ovary–specific putative transmembrane protein

2842

ing examined displayed a very unique transcriptome. Thisis consistent with the dramatic change in morphology andcell function during primordial follicle assembly and theprimordial to primary follicle transition (Fig. 1).

Changes in gene expression are anticipated as the pri-mordial follicles leave developmental arrest and the somaticcells proliferate. The gene expression changes reflect con-tributions of both the somatic cells and developing oocyte.Of the 148 genes that are up-regulated, 23 are metabolicenzymes suggesting the follicles are generally more meta-bolically active. This increase in metabolic state in the pri-mary stage follicles is anticipated [26]. The increase in ex-pression of the zona pellucida genes during follicular as-sembly validated the experimental methodology and de-sign. The zona pellucida is the protein coat of the oocytethat is produced as the follicle assembles [27]. The rise inzona pellucida gene expression was anticipated as the pri-mordial follicles assembled [27, 28].

The transcriptional profile of MIS is shown in Figure6B. MIS is known to inhibit the primordial to primary fol-licle transition [17]. MIS was up-regulated during primor-dial follicle assembly and down-regulated during the pri-mordial to primary follicle transition, consistent with theinhibitory role of MIS. The accompanying change in in-hibin expression was unexpected (Fig. 6B) [29]. AlthoughMIS and inhibin production are known to be stimulated byestrogen in the antral follicle [30], these early stages offolliculogenesis have negligible steroidogenesis. In addi-tion, the pituitary/gonadal axis is not active at this stage ofdevelopment, and these follicle stages are hormone-inde-

pendent. These data suggest that low levels of inhibin op-erating within the ovary may have a function in maintainingthe primordial follicles in their developmentally arrestedstate. Characterization of this potential unique function forinhibin requires further investigation.

A number of steroidogenic enzymes were up-regulatedin primordial follicles and then down-regulated in primaryfollicles. Although a number of steroidogenic genes haveincreased expression levels (Fig. 7), critical genes in thesteroidogenic pathway (e.g., CYP11A) were not changed.Therefore, steroid production may be minimal. Two im-portant genes, STAR and CYP19A1, were altered (Fig. 7).Previous studies have suggested steroids may be producedand be involved in primordial follicle development [31].This unique role of steroids in early primordial follicle de-velopment requires further investigation.

The transcriptional profile of IGFII and IGFBP2 dis-played a pattern inverse to that of MIS and inhibin (Fig.6C). They were down-regulated during primordial follicleassembly and up-regulated at the primordial to primary fol-licle transition. Although IGFII is known to have importantfunctions in the antral follicle and corpus luteum [32], ithas no known function in the early developing primordialfollicle. The microarray data suggests a potential role forIGFII at this stage of development.

Three neu differentiation factors were up-regulated at thetime of follicular assembly. This large gene family has beenshown to be involved in the differentiation and growth ofmesenchymal and neuronal cells [33]. This may be relatedto the innervation of the ovary by neural cells. Previous

252 KEZELE ET AL.

FIG. 6. A) Signal intensity change of twogenes of unknown function (KAP and theUOSTP) between the primordial and pri-mary follicle stages. B) Representativechange of MIS, inhibin, and aromatase sig-nal between unassembled, primordial, andprimary follicle stages. C) Representativechange of IGFII, IGFBP2, and VEGF signalsbetween unassembled, primordial, and pri-mary follicle stages. Data are displayed asa representative normalized microarraysignal for the different probe sets in unas-sembled, primordial, and primary folliclestages.

studies have suggested ovarian innervation may be criticalfor the normal timing of early folliculogenesis [34]. Be-cause the neu differentiation factor family is such a largeand diverse group of factors, it is possible that these factorsmay act directly on primordial follicles themselves. There-fore, the neu differentiation factors are candidates for po-tential coordinators of primordial follicle assembly.

Interestingly, several growth factors known to promotethe primordial to primary follicle transition such as kit li-gand, leukemia inhibitory factor, and basic fibroblastgrowth factor [10–12] had no change in gene expressionbetween the developmental stages (data not shown). Thiswas in large part due to the low levels of gene expressionof these factors in the whole ovary. These known facilita-


FIG. 7. A Pathway Assist and gene interaction diagram. Open circles represent steroidogenic genes identified in the microarray analysis. Shaded circlesidentify interacting proteins with hatched lines for regulation of activity and solid lines for regulation of gene expression. The interrelationship of thegenes identified is shown with other regulatory factors. Positive (1) and negative (2) regulation of the interaction pathways is also indicated.

tors of the primordial to primary follicle transition may beexpected to be up-regulated during the primordial to pri-mary follicle transition. Alteration of gene expression maynot be required if translational control and/or the cells re-sponsiveness to the growth factors (e.g., receptor expres-sion) are more critical. Although the experimental approachidentifies potential candidate regulatory factors, it is im-portant to note that gene expression changes are not alwaysessential. Further analysis at an individual gene level isrequired to determine the specific function of a given geneproduct.

VEGF (Fig. 6C) gene expression is significantly up-reg-ulated during the primordial to primary follicle transition.This observation was confirmed with a quantitative poly-

merase chain reaction (PCR) procedure that helped validatethe microarray procedure. VEGF effects endothelial cellmigration and proliferation to influence angiogenesis of theovary [35–37]. No function of VEGF that affects early stag-es of folliculogenesis has been identified, but VEGF doesinfluence preantral follicle development [38]. The microar-ray data associates this growth factor with the primordialto primary follicle transition. An obvious function of VEGFin the primordial to primary follicle transition would be inthe angiogenesis of the ovary and follicles. The actual roleof VEGF in the primordial to primary follicle transition willrequire further characterization.

Immune response genes, such as cytokines, were alsofound to be up-regulated during the primordial to primary

254 KEZELE ET AL.

follicle transition. Interleukin 1-b, for example, was up-regulated sevenfold. Interleukins have been shown to haveactivities in the ovary, but not at this early stage of devel-opment [39]. The relationship and functions of these genesin primordial follicle development remains to be elucidated.Microarray observations suggest these factors might alsoplay a role in the coordination of the primordial to primaryfollicle transition.

In addition to genes with a characterized known func-tion, the microarray analysis identified two genes of un-known function that are up-regulated during the primordialto primary follicle transition (Fig. 6A). These are the kid-ney-specific androgen-regulated protein KAP (Genebankreference NM 052802) [40] and the uterus ovary–specifictransmembrane protein (Genebank reference AF022147)[41]. Both of these genes are known only by screening ofcDNA libraries from androgen-treated kidney from the firstcase and from estrogen-treated uterus in the second. Noth-ing can be deduced about the function or localization ofthese proteins except that they are highly expressed at thetime of primordial to primary follicle transition. These twogenes are good candidates for regulators of the primordialto primary follicle transition and require further character-ization of their localization and function.

In conclusion, a gene discovery project was undertakento identify new coordinators of primordial follicle assemblyand the primordial to primary follicle transition. The Af-fymetrix rat U34A 8799 gene chip was used to analyze thetranscriptomes of unassembled, primordial, and primaryfollicles. This gene chip contains 14 000 genes, and there-fore does not reflect the entire genome. The majority ofthese genes did not change expression during primordialfollicle development or were expressed at low levels belowthe cutoff used in the current study. Further investigationsare needed to assess a genome-wide transcriptome of theovary. The unassembled and primordial follicle stages usedin vivo tissue, whereas the primary stage used cultured ova-ries. Although we have shown no effect of culture on organor follicle viability [10–12], changes potentially inducedduring culture need to be considered in any data interpre-tation. A summary of the potential factors involved in thecoordination of primordial follicle development are sum-marized below. The neu differentiation factors are amongthe genes identified as being up-regulated at the time offollicular assembly. The neu factors are candidates for pro-moters of follicular differentiation. MIS was found to behighly expressed at the primordial follicle, consistent withits proposed function as an inhibitor of the primordial toprimary follicle transition. Its site of synthesis and actionremain to be determined. Unexpectedly, the steroidogenicapparatus and inhibin were also highly expressed at theprimordial follicle stage. This suggests that potentially bothinhibin and steroids may have novel functions in maintain-ing the primordial follicle in its developmentally arrestedstate. Estrogen receptor activation has been shown to reg-ulate MIS expression [42]. The speculation is that steroidsmay be performing a similar function in the neonatal ovary.Further studies are needed to characterize the role of MIS,steroids, and inhibin in the coordination of early folliculo-genesis. IGFII was down-regulated in the primordial folliclestage, and therefore may influence follicle assembly. Thegrowth factor VEGF was up-regulated in the primary fol-licle, making the factor a candidate for a facilitator of theprimordial to primary follicle transition. Many cytokinessuch as interleukin 1-b were also up-regulated in the pri-mary follicle. These factors are also candidates for coor-

dinators of the primordial to primary follicle transition. Inaddition, two new genes of unknown function, KAP andthe UOSTP, were up-regulated and are also candidates forcoordinators of the primordial to primary follicle transition.Therefore, the microarray approach identified a number offactors potentially involved in primordial follicle develop-ment.

Primordial follicle assembly and the rate of primordialto primary follicle transition influences the size of the pri-mordial follicle pool [43]. The size of the primordial folliclepool sets the number and availability of follicles in the fe-male. Although it has recently been speculated that newfollicles can be recruited into the primordial follicle poolin adult rodents from germ-line stem cells [2], further in-vestigation is required to confirm this possibility. When theprimordial follicle pool is depleted, reproduction and ste-roidogenesis ends and menopause begins. Dysfunction inprimordial follicle assembly and the primordial to primaryfollicle transition compromises the primordial follicle pooland may lead to an early menopause and/or the conditionof premature ovarian failure. Further analysis of the factorsinvolved in these processes of primordial follicle assemblyand the primordial to primary follicle transition will im-prove our understanding of such pathological conditions aspremature ovarian failure. The genes identified in the cur-rent study provide candidates for further analysis and willhelp elucidate this critical biological process.

ACKNOWLEDGMENTS

We would like to thank Dr. Ingrid Sadler-Riggleman for invaluabletechnical assistance. Thanks also to Jill Griffin and Mary Francis for as-sistance in preparing the manuscript. We acknowledge the use of the Cen-ter for Reproductive Biology, Genomics Core Laboratory, and Bioinfor-matics Core Laboratory, and the technical assistance of Derek Pouchnikand Jim Shima. Electronic access to the microarray database for this studycan be obtained at www.skinner.wsu.edu.

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