Ovarian gene expression in the absence of FIGLA, an oocyte-specific transcription factor
© Joshi et al; licensee BioMed Central Ltd. 2007
Received: 11 December 2006
Accepted: 13 June 2007
Published: 13 June 2007
Ovarian folliculogenesis in mammals is a complex process involving interactions between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific, basic helix-loop-helix transcription factor, FIGLA (Factor In the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes.
Taking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream target genes. Using high stringent cutoffs, we find that FIGLA functions as a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. FIGLA also inhibits expression of male germ cell specific genes that might otherwise disrupt normal oogenesis.
These data implicate FIGLA as a central regulator of oocyte-specific genes that play roles in folliculogenesis, fertilization and early development.
Primordial germ cells migrate to and colonize the mouse gonad, completing the process during embryonic day 10.5 (E10.5) to E12.5 . Subsequent phenotypic sexual dimorphism is defined by the gonad and mice lacking Sry located on the Y chromosome follow a constitutive female pathway , presumably instructed by a unique set of genes . Entrance of female germ cells into meiosis at E13.5 is a defining event mediated by retinoid responsive genes [4, 5]. A second major transition occurs perinatally when flattened granulosa cells surround individual germ cells to form primordial follicles , forming a reservoir of eggs available for subsequent fertilization . Germ cells that fail to interact with the gonadal somatic cells, either by ectopic location  or in the absence of a critical oocyte-specific transcription factor, FIGLA (previously known as FIGα), do not survive.
FIGLA (Factor In the GermLine, Alpha), a basic helix-loop-helix transcription factor, was first defined in the coordinate regulation of three genes (Zp1, Zp2, Zp3) encoding proteins that form the zona pellucida surrounding ovulated eggs . FIGLA transcripts are detected as early as E13.5 in the embryonic gonad and persist in the adult ovary . FIGLA protein, however, is not detected until E19 based on sensitive gel mobility shift assays . Mice lacking FIGLA have normal embryonic gonad development, but primordial follicles do not form at birth and germ cells are lost within days. Female, but not male, mice are sterile . These data suggested that FIGLA plays critical roles in female germline and follicle development, but the full complement of downstream gene targets involved in these processes and when in development they become activated have not been defined
Using cDNA microarrays, we have compared the transcriptomes of normal and Figla null ovaries at four developmental time points (E12.5 to newborn). These results have been complemented with SAGE (Serial Analysis of Gene Expression) libraries derived from newborn ovaries to identify potential direct and indirect gene targets of FIGLA in female gonad development.
Microarray data analysis
To identify potential downstream gene targets of FIGLA, total RNA was obtained from normal and Figla null gonads isolated from E12.5, E14.5, E17.5 and newborn female mice. Three independent biological samples obtained from each embryonic time point and four from newborn gonads were linearly amplified, labeled with Cy3 and Cy5 and hybridized to the National Institute of Aging (NIA) cDNA microarray consists of ~22K features enriched for transcripts from newborn ovaries, pre- and peri-implantation embryos [12, 13]. After washing, the average statistically significant intensities for each element were analyzed with Gene Spring GX software. Features that varied more than 2-fold (with a coefficient of variance less than 30%) between normal and Figla null gonads were selected for further analysis.
Developmental hierarchical cluster analysis of FIGLA regulated genes
Differentially regulated genes
Nucleic acid binding proteins and transcription factors
Pou5f1, E2f5, Stat3, Lcp1, Zfp313, Og2X, Rex, Helic1, Cpeb3, Elav2, Cpeb1, Dhx40Top3b, Ccrn41, E430034L04b, 5830484A20
Pou5f1, Sp3, Dtx2, Peg3, Tcf21, Eef1a1, Rps2, Hdac2
Phtf1, Taf7la, Mrg1, Ncam2, Tia1, 1110031M08
Mxd3, Pbx3, MkI1, Sox17, Jarid1c, Sp8, Mjd, Mkrn1, FhI4, Sdpr, Miz1, BC029103, Papolb, Rnaseh2a, Sox17, Tdrd6, Sf3a1, 5, Tnp2 , Mgmt, Hils1, AV34037
Kit, Grid2, Drd3, Trpm7
Gabra2, Pvrl3, Tcp10b, Plxnb3, Mass1
Extracellular matrix proteins
Zp2, Zp3, Col9a3
Zp2, Leprel2, Col1a1, Mfap2,
Cell adhesion molecules
Clgn Dnajb1, CPN60, Osp94, Dnajb3, Hsbp9
Kif14, Rdx, Lmo7, Lcp1, AI427122, 2900002G04
Kif2b, Dncl2b, Actl7a, Tuba3, Tekt1, Fscn3 , Tuba5, Rsn, Dnahc8 , Tns
Defense and immunity
And, H2-Bf, Pvrl3
Thedc1, Helic1, Exo1, D7Ertd445e, 2610020H15
Rnaseh2a, Ephx2, Asrgl1, Dnahc8, Dpysl3, AV340375,
Paox, Prdx1, Akr1a4
Ldhal6b, Sdh1, Ldhc , Cyp11a1, Txndc2, Gpd2, Sqrdl, prdx6-rs1, Cyp17a1, Akr1c19, Mcsp, AL024210
Stk33, Dyrk3, Plxnb3, Cdc2l6,
Herc3, Slc27a2, Rnf35, 2610020H15
Herc4, Fbox36, Rnf133, Acsl1, Miz1, Glul, Rnf139,, Senp7, 630025C22
And, Tpp2, Ctsd, 3, Asrgl1, Klk6, Adam3, 1700074P1
Chst10, Fdft1, Bcat1, Nmna3, Ipmk, Ak44930403J22, 4930487N19
Tcp10b, Sia5, B4galt1, Mgmt, Sas
Cell junction proteins
Membrane traffic proteins
Gabra2, Kcne3, Kcnk4
Select regulatory molecules
Chn1, Argap20, Top3b, Serpinb6C
Oaz3, Akap3 , Ppp1r3a, Dusp18, 4933414G08
Ovarian genes affected by Figlaexpression
Total RNA was isolated from 9 normal newborn organs and assayed for gene expression which was normalized to HPRT and standardized to 100% in the ovary (Fig. 3A). As previously reported, all five Nalp genes were expressed in the ovary with low levels of transcripts (< 5% of ovarian expression) observed in the uterus for Nalp14 and in the liver for Nalp4a of newborn mice. The developmental profiles of transcript abundance from E12.5 to newborn were consistent with FIGLA regulating expression of Nalp4b, Nalp5, Nalp4f and Nalp14. No expression was detected in E12.5, E14.5, E17.5 or newborn gonads isolated from Figla null mice, but all five genes were expressed in normal newborn ovaries. However, the absence of FIGLA did not preclude expression of Nalp4a in newborn ovaries, although expression was ~60% of normal. Although the members of the Nalp gene family share structural motifs and are co-expressed in oocytes, they are not functionally redundant and the inactivation of one (Nalp5) is sufficient to arrest pre-implantation development .
Among the other annotated genes, each was preferentially expressed in the ovary with only low levels (< 5% of ovarian expression) of Oas1d, Serpinb6C and Arhgap20 transcripts in other tissues in newborn mice (Fig. 4A). Expression of Pdzk1 and Elavl2 was less tightly controlled with transcripts detected in kidney (~25% of ovarian expression) and brain (~40%), respectively. Each of these five genes was first detected in the newborn ovary where their expression was dependent on FIGLA (Fig. 4B). These observations are consistent with each gene being a direct downstream target of FIGLA, but transcripts of each are detected in adult tissues, suggesting that other transcription factors play a role in developmental and organ specific expression. Oas1d encodes an oocyte-specific dsRNA, 2'5'-oligoadenylate synthetase that is ~60% identical to OAS1A that has been implicated in the interferon defense response against viral infections. Genetically altered mice, lacking OAS1D, are fertile, but have reduced fecundity associated with lowered ovulatory capacity and defects in early embryonic development . Mice lacking PDZK1, implicated in controlling ion transport and cholesterol metabolism , had normal fertility and produced off-spring with the expected Mendelian inheritance of the Pdzk1 null allele .
Tissue and developmental expression of unannotated genes
Five additional genes that were more abundant in normal than in Figla null newborn ovaries and functionally un-annotated were also selected for further evaluation (Fig. 5). Three [E330009P21Rik, E3300017A01Rik, Genbank:BC052883] were detected only in the ovary by qRT-PCR and the remaining two [C330003B14Rik, E330034G19Rik] had only low levels of expression in other tissues. All five genes had similar developmental profiles as the aforementioned genes with little expression prior to birth and virtual absence of expression in Figla null ovaries.
SAGE analysis of normal and Figlanull newborn ovaries
Using the Audic and Claverie algorithm that incorporates Bayesian and false alarm analyses , 977 genes more highly expressed in the normal library and 1308 genes more highly expressed in the Figla null library were identified (Fig. 7B). The data also were analyzed with other statistical tests commonly used for SAGE  and Fisher's exact test provided similar results (> 90% overlap) with 810 genes more highly expressed in normal and 1318 more highly expressed in null ovaries. However, only Pou5f1 (Oct4) and Zp2 were observed as up-regulated in both newborn microarrays and SAGE libraries and no commonly down regulated genes were observed among those identified by SAGE and by microarray analyses. Although the microarray and SAGE screens were selected to complement one another in identifying potential downstream targets of FIGLA, the lack of overlap in targets was unanticipated. The data from the newborn normal and Figla null microarray data were reexamined by False Discovery Analysis . Of 2,233 identified features (FDR threshold of 5%) from the microarray data, 1,550 had single unigene annotations , 202 mapped to multiple clusters and 248 were not mapped. Of the annotated genes, 768 were up-regulated (see Additional file 5) and 782 were down-regulated (see Additional file 6). Eleven genes (marked in Additional files 5, 6), including Pou5fl and Zp2 from our initial analysis, were also present in our SAGE analysis. Eight were up-regulated (Pou5f1, Zp2, Ivns1abp, Vbp1, Padi6, Rbpms2, Genbank:EG626058, 6430591E23Rik) and three were down-regulated (Sp3, Hdac2, Ogt) in newborn normal ovaries. Because alternative statistical analyses [24–26] may be useful for the comparison of these data sets, we have made the original microarray and SAGE data available at Gene Expression Omnibus  under accession GSE5558 and GSE5802, respectively.
Of the genes identified in the SAGE screen, 64 (see Additional file 7) of the potentially up-regulated genes had ≥ 10 tags in normal and none in the null and 312 (see Additional file 8) of the potentially down-regulated genes had ≥ 10 tags in the null and none in normal ovaries. These 376 tags were reliably mapped to genes using the NCBI SAGE database  and ontology of 112 was determined by PANTHER (Table 1). The greatest percentage (26%) of genes were nucleic-acid binding proteins (including transcription factors) followed by oxidoreductases (13%), cytoskeletonal proteins (10%) and ligases (8%). Ten of the down-regulated genes (Tnp2, Hils1, Clgn [Calmegin], Tekt1, Fscn3, Dnahc8, Ldh3, Adam3 [Cyritestin], Oaz3, Akap3), scattered among the categories, have sperm-associated patterns of expression (Table 1).
Expression of eight up-regulated genes from the SAGE analysis
Pou5f1 (Oct4) is expressed in pluripotent cells during mouse development before becoming restricted to germ cells  and regulates a significant number of downstream target genes by itself and in tandem with other transcription factors . The complex pattern of POU5F1 transcripts in normal and Figla null mice (Fig 8B) indicates participation of other transcription factors in controlling its expression. Expression of the other seven genes was not detected in Figla null mice (except for a very modest accumulation of [Genbank:BG084789] at E15.5) and was first observed in normal ovaries at E15.5 (Dppa3, [Genbank:AK139812] or E19.5 (Oash1, Padi6, [Genbank:AK087784, 2410146L05Rik, Genbank:AK139812])(Fig. 8B).
Dppa3 (Stella), originally implicated in germ cell lineage specification , is a maternal effect gene required for pre-implantation development [32, 33]. Oas1h encodes a 2'5'-oligoadenylate synthetase and clusters with Oas1d (detected by microarray analysis) along with 6 other closely related synthetases on mouse Chr5:119,938,130-120,073,460 . Padi6 (Padi5) is one of 4 similar genes (Padi1–4) clustered on mouse Chr4:139,787,689-139,623,878 that encodes a peptidylarginine deiminase which converts arginine residues into citrulline . It is expressed during oogenesis where it is associate with cytoplasmic sheets and the protein persists in the early embryo up to the blastocyst stage .
Each of the remaining four SAGE tags matched either cDNA or spliced EST that was expressed in ovarian tissue and/or the early mouse embryo. [Genbank:AK087784] is a full-length cDNA from a 2 days pregnant adult female ovary [E330020D12Rik] that maps to mouse Chr1:153,290,975-153,292,514 and [2410146L05Rik] is a female germline specific cDNA that encodes a hypothetical 166 amino acid protein mapping to mouse Chr9:78,577,285-78,578,468. [Genbank:BG074389], a 532 bp spliced EST (Chr11:7,077,950-7,079,635), is part of the NIA15K cDNA clone set and was up-regulated in the microarray screen, albeit not to a statistically significant extent. [Genbank:AK139812] was reliably matched by a SAGE tag that also matched two other sequences, neither of which was ovary specific. [Genbank:AK139812] corresponds to a full-length cDNA [B020018122Rik] obtained from a 2-cell embryonic library and maps as a spliced EST on mouse Chr12:107,907,672-107,914,726.
During ovarian development, oocytes accumulate maternal products necessary for successful folliculogenesis, fertilization and pre-implantation development. FIGLA is an oocyte-specific, basic helix-loop-helix transcription factor required for perinatal formation of primordial follicles and the establishment of the extracellular zona pellucida matrix that surrounds eggs to mediate fertilization and an embryonic block to polyspermy. Transcripts encoding FIGLA are first detected at E13.5 in female gonads and persist in adult ovaries [9, 10]. The involvement of FIGLA in two, independent, oocyte-specific genetic pathways and its persistence during critical periods of ovarian development suggest that FIGLA may regulate other genes critical for successful gonadogenesis and development. Two complementary approaches using spotted-glass microarrays and Serial Analysis of Gene Expression (SAGE) have been combined to identify additional potential downstream targets of FIGLA.
Taking advantage of Figla null mice and the NIA ~22K microarray that contains elements enriched for expression in oocyte and early development [12, 13], ovarian gene expression was profiled at four embryonic time points from E12.5 to newborn. No statistically significant differences (≥ 2X, ρ ≤ 0.05) in transcript abundance between normal and Figla null mice was observed at E12.5 and E14.5 and only two transcripts differed at E17.5. However, in newborn ovaries, 165 genes were up-regulated (i.e., less abundant in Figla null) and 38 were down-regulated (i.e., more abundant in Figla null). This developmental time point is just after FIGLA protein is detected in a sensitive gel mobility shift assay  and coincident with the first phenotypic manifestation of Figla null mice . Thus, although Figla transcripts are detected as early as E13.5, the major affect on ovarian gene expression occurs perinatally.
Microarrays are limited by the elements spotted on glass during their fabrication. Therefore, to broaden the search for potential direct or indirect downstream gene targets, SAGE libraries were constructed from poly(A)+ RNA isolated from newborn normal and Figla null ovaries. A 10 base tag immediately adjacent to the 3' most Sau3A1 restriction enzyme cleavage site was used to identify 7,715 transcripts in normal and 9,455 transcripts in Figla null mice. Of the genes encoding these transcripts, 977 were up-regulated and 1,308 were down-regulated when analyzed with statistics that incorporate Bayesian and false alarm analyses . Unigene designations were available for 838 of the up-regulated and 648 of the down-regulated genes and 31% (334 up-regulated; 131 down-regulated) were represented among the elements on the NIA microarray. Initially only two (Pou5f1 and Zp2) were identified as up-regulated on both platforms. However, reanalysis of the newborn microarray data with an FDR threshold of 5%  identified 11 genes in common, eight of which were up-regulated (Pou5f1, Zp2, Ivns1abp, Vbp1, Padi6, Rbpms2, Genbank:EG626058, 6430591E23Rik) and three of which were down-regulated (Sp3, Hdac2, Ogt) in newborn normal ovaries. A similar lack of concordance among different platforms analyzing the same biological samples, particularly for genes with low abundance, has been observed previously [38–41].
'Guilt by association' has been invoked to identify genetic hierarchies, members of which are co-regulated by specific transcription factors (for review, see ). In examining the ontology of the differentially regulated genes detected by microarray and SAGE analysis, nucleic acid binding proteins and transcription factors formed the largest group. One gene, Pou5f1 (Oct4) in this grouping was up-regulated by FIGLA on both platforms and is initially present in primordial oogonia prior to down-regulation as female germ cells enter the prophase of meiosis I (~13.5E). Perinatally, Pou5f1 transcripts and POU5F1 protein once again become abundant in growing oocytes  and persist in the early embryo prior to zygotic expression which begins in 4-cell embryos. Although initially present in all blastomeres, Pou5f1 transcripts becomes restricted to the inner cell mass, the epiblast and finally primordial germ cells by E8.5 . The expression of Pou5f1 is regulated by a TATA-less promoter with distal and proximal enhancers implicated in regulating transcript levels during embryogenesis . However, the molecular control of Pou5f1 expression during gametogenesis has not been determined. A canonical E-box (CANNTG) is present -261 bp upstream of the Pou5f1 transcription start site and represents a well-defined binding sites for basic helix-loop-helix transcription factors . FIGLA protein is first detected at E19  just prior to the post-natal up-regulation of Pou5f1  and in both microarray and SAGE analyses, Pou5f1 expression is down-regulated in Figla null mice. These data are consistent with FIGLA acting as a regulator of Pou5f1 expression in the female germline, although other factors must play a role as well.
Structural proteins, including those involved with extracellular matrices and intracellular cytoskeleton were also well represented in the gene ontology analysis and one, Zp2, was up-regulated by FIGLA on both platforms (microarray and SAGE). Zp2 encodes a major component of the mouse zona pellucida that surrounds growing oocytes, ovulated eggs and pre-implantation embryos . Mice lacking ZP2 initially form a thin zona matrix composed of ZP1 and ZP3 that does not persist and no zona pellucida is observed in ovulated eggs which renders female mice sterile . FIGLA was initially defined as transcription activating activity that bound to a conserved E-box, within the first 200 bp of Zp1, Zp2 and Zp3 promoters and was subsequently isolated by expression cloning [10, 49]. ZP2 transcripts can be detected as early as E19, although the zona matrix does not form until oocytes begin to grow after birth; no ZP2 transcripts are detected in Figla null mice . Thus, Zp2 expression appears to reflect direct targeting of FIGLA during the onset of oogenesis.
All twenty-three of the genes selected from the microarray and SAGE screens were preferentially expressed in newborn ovarian tissue, although significant levels of Pdzk1 and Elavl2 transcripts were detected in kidney and brain, respectively. There was considerable variation in levels of expression in normal newborn ovaries, ranging from 14–400% of HPRT levels which may indicate the importance of additional co-factors in regulating gene expression, although differing efficiencies of PCR amplification may affect these comparisons. All genes (except Nalp4a) were virtually absent in newborn Figla null ovaries. Those genes expressed uniquely in oocytes, may be of particular importance in ovarian gonadogenesis.
Although well appreciated in simple model organisms, only in recent years have maternal effect genes have become well documented in mice. The protein products of these genes accumulate during oocyte growth and are required for successful embryogenesis. While it remains controversial whether such maternal products play a role in establishing embryonic polarity [50, 51], there is increasingly ample molecular evidence that maternal effect genes are critical in pre- [16, 32, 33, 52–59] and post-implantation [57, 60–63] development. Mater (M aternal a ntigen T hat E mbryos R equire) was one of the earliest maternal effect genes molecularly characterized in mice. Mice lacking this 125 kDa cytoplasmic protein have normal gonadogenesis and ovulate eggs that can be fertilized, but do not progress beyond the two-cell stage . Mater (Nalp5) is a member of the Nalp family and genetic studies are underway to determine if other members regulated by FIGLA (Nalp4b, Nalp4f, Nalp14) affect pre-implantation in mice. A second maternal effect gene Dppa3 (Stella), also required for pre-implantation development [32, 33], was detected by the SAGE analysis and there may be others as well.
Of the 350 genes that were significantly less abundant in normal ovaries (see Additional files 2 and 6), 30 were testis-associated and 12 were classified by the Panther gene ontology software (Table 1). Two were transcription factors, preferentially expressed in the testis, Taf7l and Phtf1. TAF7L is a TATA box binding protein involved in differentiation of spermatogonia to spermatocytes  and PHTF1 is a putative homeodomain transcription factor with male germ cell specific expression that binds feminizing factor FEM1B [65, 66]. Another two, Tnp2 and Hils1, encode transition protein 2 and a spermatid-specific linker histone H1-like proteins, respectively, which are involved with chromatin remodeling during spermatogenesis [67, 68]. The remaining 8 genes (Clgn [Calmegin], Tekt1, Fscn3, Dnahc8, Ldhc, Adam3 [Cyritestin], Oaz3, Akap3) have well characterized functions in spermatogenesis [69–76]. Thus, FIGLA appears to play a role in preventing expression of male germ cell associated genes during oogenesis. If true, additional co-factors must interact with FIGLA in determining its affect on downstream gene targets.
Taken together, these data indicate that FIGLA, an oocyte-specific, basic helix-loop-helix transcription factor, plays a pivotal role in modulating multiple genetic hierarchies involved in folliculogenesis, fertilization and pre-implantation development. Although transcripts accumulate earlier in embryogenesis, FIGLA protein is first detected ~E19 and affects female perinatal gonadogenesis. While some of the effects on expression may be as direct regulator of downstream target genes, others may be indirect through the activation (or suppression) of other transcription regulator(s). In addition to involvement in the activation of gene expression, it seems likely that FIGLA will also down-regulate genes, the expression of which would be inappropriate during post-natal oogenesis. The further characterization of the genes that are differentially regulated by FIGLA should prove useful in defining developmental pathways that affect the postnatal female germ cell. These targets represent not only genes that affect folliculogenesis and fertilization, but also maternal effect genes required for successful completion of early mouse development.
Experimental animals and tissue collection
CF1 mice were obtained commercially and Figla homozygous null mice  were identified by genotyping tail DNA using three primers (see Additional file 9) in a PCR reaction (95°C 5 min, 94°C 30 sec, 60°C 30 sec, 72°C 30 sec for 28 cycles). RNA was extracted from gonads isolated at embryonic day 12.5 (E12.5), E13.5, E14.5, E15.5, E17.5, E19.5, newborn (NB), 2 days post-partum (2dpp) and 7dpp females; all other tissues were obtained from newborn mice. All experiments were conducted in compliance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health under a Division of Intramural Research, NIDDK approved animal study protocol.
RNA extraction and labeling
RNA was extracted from tissue using Absolutely RNA RT PCR Miniprep Kit (Stratagene, La Jolla, CA) and its integrity was determined with RNA 6000 Nano Lab-on-Chip (Agilent Technologies, Waldbronn, Germany). For hybridization to microarrays, 50 μg of total RNA (20 ovaries) was linearly amplified with the Aminoallyl RNA Amplification and Labeling System (NuGen Technologies, San Carlos, CA) and labeled with ester-linked Cy3 and Cy5 dyes (Amersham Biosciences, Piscataway, NJ).
Labeled cDNA probes were hybridized to NIA cDNA microarrays on glass slides containing 20,996 features [12, 13] according to Vanderbilt Microarray Shared Resources protocols . In brief, arrays were washed (0.2% SDS) at RT and incubated in a pre-hybridization solution (5 × SSC, 0.1% SDS, 1% BSA) at 55°C for 45 min . After 5 rinses in Milli Q water and 1 in iso-propanol, arrays were air dried and overlaid with coverslips. Utilizing a Maui Hyb Station (BioMicro Systems, Salt Lake City, UT), the arrays were hybridized (25% formamide, 5 × SSC, 0.1% SDS, 1 μg poly(A)+ RNA) at 42°C for 16 h and then washed (5 min, 55°C) sequentially with 2 × SSC, 1 × SSC and 0.1 × SSC, each with 0.1% SDS After drying by centrifugation (20 × g), the arrays were scanned using the Axon 4000 B (Axon Instruments, Union City, CA).
Microarray data analysis
Hybridizations were performed in triplicate (E12.5, E14.5, E17.5) or quadruplicate (newborn) with dye-swapped pairs of cDNA. The raw data were analyzed with GeneSpring GX 7.2 (Agilent Technologies, Waldbronn, Germany) to remove data that did not meet the following criteria: 1) Cy5 signal to background intensity ratio less than 1.5; 2) Cy3 signal to background intensity ratio less than 1.5; 3) Cy5 signal less than 200; and 4) Cy3 signal less than 200. Data were then normalized by the LOWESS sub-grid method and features with a coefficient of variance of less than 30% (across replicate arrays) and ≥ 2-fold changes between normal and Figla null gonads were identified. Analysis of Variance (ANOVA) was performed on the set of genes identified in newborn ovaries. Hierarchical clustering of transcripts during development was determined by GeneSpring GX 7.2.
Alternatively, all eight replicates of newborn ovaries were used in a one-sample Student's t-test to determine whether the mean normalized expression value for each element was statistically different from 1. False Discovery Rate  at a threshold of 5% was used as a multiple testing correction to decrease the number of false positives.
SAGE library construction
Total RNA (~50 μg) was obtained from newborn normal and Figla null ovaries with an RNeasy mini kit (Qiagen, Valencia, CA). The presence of MSY2 and the absence of ZP2 transcripts was assayed by one-step qRT-PCR according to the manufacturer's protocol (Qiagen, Valencia, CA) using oligonucleotide primers (see Additional file 3) to confirm the identity of Figla null ovaries. Poly(A)+ RNA was isolated with oligo d(T)-conjugated magnetic beads (Dynal-Invitrogen, Carlsbad, CA) and 5 μg was used to construct libraries using a modification  of the original SAGE protocol . The final concatenated ditags were cloned into Bluescript pKS (Stratagene, La Jolla, CA) using the BamHI site for blue-white screening. Plating, picking, DNA preparation and sequencing were performed at the NIH Intramural Sequencing Center (NISC). Low quality sequence data were eliminated by Phred analysis.
eSAGE  was used to extract tags and remove duplicate ditags from the raw sequencing data. 79,095 tags from the normal were compared to 77,851 tags from the Figla null libraries to determine statistically significant differences either by the Audic and Claverie test  using eSAGE  or Fisher's exact test  using IDEG6 . Tags with ρ ≤ 0.05 were extracted from both lists and further analysis was performed on tags present at least 10 times in normal and absent from Figla null ovaries. These lists of tags were then mapped to Unigene clusters using reliable mapping from NCBI Unigne build #138. A group of 13 genes from the microarray data and 8 from the SAGE analysis that were up-regulated in normal newborn ovaries were selected for more detailed analysis based on their expression profile in the SOURCE data base at the Genetics Department at Stanford University .
Analysis of gene expression
Transcript abundance was determined by quantitative real-time polymerase chain reaction (qRT-PCR) on an ABI 7700 HT Sequence Detection System (Applied Biosystems, Foster City, CA) using QuantiTect SyBR green PCR kit (Qiagen, Valencia, CA) and primers (see Additional file 3) designed by Primer Express 2.0 (Applied Biosystems, Foster City, CA). Independently obtained biological samples (2–6), analyzed in triplicate were normalized to HPRT (averaged for tissue or developmental time point) and expressed as an average (± s.e.m). PCR products from the SAGE analysis were separated by agarose gel electrophoresis and stained with ethidium bromide (0.1 ug/ml). Tissue-specific expression for the in situ hybridization was performed on ovarian sections obtained from normal, 6–8 week old mice. Ovaries were fixed (4% paraformaldehyde, PBS, pH 7.4), and processed for paraffin sections (5 μm) which were then re-hydrated stepwise through alcohol washes (100%, 70%, 50%, 30%) and distilled water prior to PBS. Sections were hybridized with sense or antisense probes (200 ng/ml) (see Additional file 4) labeled with digoxigenin (DIG) according to the manufacturer's instructions (GenPoint, Dako, Carpenteria, CA) except for the use of rabbit anti-DIG-HRP antibodies (1:200, Dako, Carpenteria, CA). Sections were then counter-stained with hematoxylin and de-hydrated stepwise in ethanol (30%, 50%, 70%, 100%) prior to permount mounting (Fisher Scientific, Fair Lawn, NJ). Images were acquired with an Axioplan microscope equipped with a CCD camera using AxioVision software (Carl Zeiss, Gottingen, Germany).
We appreciate the contribution of Braden Boone to the FDR analysis of the newborn microarray data. This research was supported by the Intramural Research Program of the National Institutes of Health, NIDDK.
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