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BOR - Papers in Press, published online ahead of print November 13, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008557
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BIOLOGY OF REPRODUCTION 68, 985–995 (2003)
DOI: 10.1095/biolreprod.102.008557
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Expression Profiles and Chromosomal Localization of Genes Controlling Meiosis and Follicular Development in the Sheep Ovary1

Béatrice Mandon-Pépin2,a, Anne Oustry-Vaimanb, Bernard Vigiera, François Piumic, Edmond Cribiub, and Corinne Cotinota

a Unité Biologie du développement et Biotechnologies, INRA, 78350 Jouy en Josas, France b Laboratoire de Génétique Biochimique et Cytogénétique, INRA, 78350 Jouy en Josas, France c Laboratoire Radiobiologie et Etude des Génomes, INRA, 78350 Jouy en Josas, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In female sheep fetuses, two of the most crucial stages of ovarian development are prophase of meiosis I and follicle formation. In the present study, sheep ovaries collected on Days 25, 38, 49, 56, 67, 75, 94, and 120 of gestation, at birth, and in adulthood were tested by reverse transcription-polymerase chain reaction (RT-PCR) for the expression of 14 genes known to be involved in the ovarian differentiation in diverse organisms. The aim of this study was to determine 1) the expression pattern of six genes involved in germ cell development or meiosis (DMC1, SPO11, MSH4, MSH5, DAZL, and Boule) and five ovary-derived factors (OVOL1, SIAH2, DIAPH2, FOXL2, and FGF9), 2) the onset of gene expression for several members of the bone morphogenetic protein (BMP) pathway involved in follicular development (GDF9, BMP15, BMPR-IB), and 3) the chromosomal localization of seven of these genes in the sheep genome. The RT-PCR analysis revealed that the two germline-specific genes, DAZL and Boule, were expressed between 49 and 94 days postcoitum (dpc) with a similar pattern to typical meiosis genes (DMC1, MSH4, and MSH5), suggesting their possible participation in prophase of meiosis I. GDF9 and OVOL1 gene transcription started at 56 dpc and extended until birth, while BMP15 presented a more restricted window of expression between 94 dpc and birth, corresponding to the formation of first growing follicles. The homologous ovine genes for SPO11, DMC1, MSH5, DAZL, FGF9, DIAPH2, and SIAH2 were located on OAR 13q21–22, 3q35, 20q22, 19q13, 10q15, Xq44, and 1q41–42, respectively. In sheep, quantitative trait loci affecting female reproductive capacities are currently being detected. The ontology and precise mapping of ovarian genes will be useful to identify potential candidate genes that might underlie these effects.

follicular development, meiosis, ovary, sheep


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sheep play a major role in modern agriculture, and their size and physiology provide an appropriate model to study a variety of mammalian biological functions, including reproduction, embryology, and fetal development. Additionally, extensive efforts have been devoted to studying the sheep genome, and a comprehensive sheep linkage map comprising more than 1000 loci has recently been developed [1]. Sheep have several heritable traits of economic interest, such as those affecting fertility. These genetic variations occur in different breeds and provide interesting models to study the mechanisms regulating the formation and development of ovarian follicles. Among these variations, the Booroola trait (FecB), an autosomal mutation that affects ovulation rate, has recently been identified by several teams [2–4]. A nonconservative substitution in the bone morphogenetic protein receptor type IB (BMPR-IB) coding sequence was found to be fully associated with the hyperprolificacy phenotype of Booroola ewes. Similarly, the Inverdale (FecXI) sheep carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+) but primary ovarian failure in homozygotes (FecXI/FecXI)[5, 6]. The FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2–11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B, GDF9B) [7]. These findings establish that some members of the BMP pathway are essential to female fertility. Natural mutations in these factors can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner [7]. Most of these ovary-derived factors have been studied during adulthood, and little data exist concerning their expression during fetal life [8]. However, two of the most crucial stages of ovarian differentiation occur during the fetal period: initiation of prophase I of meiosis and follicle formation.

Among the genes involved in meiosis, some of them have been cloned in humans and mice, but very little data exist concerning the initiation of their transcription and their expression pattern during female gametogenesis in mammals. Among these genes, Dmc1 (disrupted meiotic cDNA1, yeast homolog of), Spo11 (SPO11 Saccharomyces cerevisiae homolog of), and Msh4 and Msh5 (Mut S, Escherichia coli homolog of) are specifically involved in different stages of meiosis: synapsis, DNA double-strand break, DNA strand exchange, and postreplicative DNA mismatch repair, respectively [9–15]. Members of the DAZ (deleted in azoospermia) gene family such as DAZL (deleted in azoospermia-like) and Boule are also reported to be expressed by germ cells [16, 17]. The DAZ genes encode potential RNA-binding proteins that are expressed in prenatal and postnatal germ cells and are serious candidates for human fertility factors [18]. Homologs of DAZ have been identified in diverse organisms [16, 19, 20]. Homologs in these organisms are required for germ cells to develop but differ in null phenotypes and expression patterns. In flies, disruption of the DAZ homolog, Boule, causes male meiotic arrest [21]. In Caenorhabditis elegans, disruption of the DAZ homolog causes meiotic arrest in oogenesis only [22], and in mice, it leads to the loss of germ cells and the complete absence of gamete production in both males and females [16]. Recently, the identification of an additional member of the DAZ gene family, called BOULE, has been reported in humans [17]. The time course of expression of these genes during early ovarian development in mammals remains little documented.

Moreover, several ovary-derived factors first isolated in Drosophila melanogaster have been characterized in mammals. Among them are Ovo, a female germ-line-specific nuclear protein known to play a critical role in Drosophila oogenesis and germ-line differentiation [23, 24], Siah-2 (seven-in-absentia Drosophila homolog of 2), which is one of three murine homologs of the Drosophila gene seven in absentia (sina) expressed in germ cells within both the mouse ovary and testis [25], and the DIAPH2 gene (diaphanous Drosophila homolog of 2), the human homolog of the Drosophila diaphanous gene [26]. Members of the latter protein family affect cytokinesis and other actin-mediated morphogenetic processes required in the early steps of development. It has been suggested that the human DIAPH2 gene was one of the genes responsible for premature ovarian failure and that it affected the cell divisions that lead to ovarian follicular development [26].

The aim of this study was to determine 1) the profile of gene expression for multiple ovary-derived factors such as growth/differentiation factor 9 (GDF9), BMP15, BMPR-IB, OVO Drosophila homolog-like 1 (OVOL1), DIAPH2, SIAH2, fibroblast growth factor 9 (FGF9), forkhead transcription factor (FOXL2), and germ-cell-specific genes such as Boule, DAZL, DMC1, SPO11, MSH4, and MSH5 by reverse transcription-polymerase chain reaction (RT-PCR) during ovarian development and 2) the chromosomal localization of seven of them in the sheep genome by screening a sheep bacterial artificial chromosome library [27] to find one bacterial artificial chromosome (BAC) corresponding to each nonassigned gene by fluorescence in situ hybridization (FISH).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Procedures for handling animals in this study were conducted in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Cyclic Ile-de-France ewes were synchronized with an intravaginal progestogen sponge. Animals were inseminated at Day 0. Pregnant female tracts at different developmental stages were collected at slaughter and rapidly dissected to extract fetuses. Fetuses were collected on Days 25, 38, 43, 49, 56, 67, 75, 82, 94, and 120 of gestation. The gonads of newborn and adult animals were collected at slaughter. The neonate ovaries were used in full, while the testes were cut up into two pieces. After puberty, gonads of both sexes were cut up into small pieces before freezing (about 1/4 for the ovary and 1/20 for the testis). The adult animals were slaughtered during reproductive season but without regard to the stage of the estrous cycle. All gonads were first detached and immediately frozen in liquid nitrogen, then different organs, such as the liver, were taken. At 25 days postcoitum (dpc), mesonephros and gonad were not separated. The number of animals used and the number of independent RT experiments performed at each stage are summarized in Table 1.


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  		TABLE 1. Number of animals (before and after birth) used in RT-PCR analysis

Sexing of Fetuses

All fetuses were sexed by PCR. Part of the liver was used to obtain genomic DNA. SRY and ZFX, markers of the Y and X chromosome, respectively, were used to amplify liver genomic DNA and obtain the chromosomal sex of each fetus. The PCR conditions for the SRY gene were 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec with 0.5 U of Taq polymerase (Takara; Verviers, Belgium) and M11 and M13 primers (described in [28]). The PCR conditions and primers for ZFX gene were described by Aasen and Medrano [29].

RNA Extraction, DNase Treatment, and Reverse Transcription

Total RNA was extracted from frozen gonads using RNA-plus solution (Bioprobe Systems; Montreuil-sous-Bois, France) and was quantified with a spectrophotometer at 260 nm. Twenty micrograms of each sample were treated with 40 U of DNaseI, RNase-free (Roche, Saclay, France), for 2 h at 37°C to avoid genomic DNA contamination. After phenol/chloroform extraction and ethanol precipitation, the DNase-treated RNA was resuspended in RNase-free water, and 1 µl was electrophoresed on a 0.8% agarose gel to check the quality and quantity of each sample. For each independent series (from 25 dpc until adulthood), only samples yielding a comparable signal were used for reverse transcription. Five micrograms of DNase-treated RNA were reverse transcribed in 20 µl at 42°C for 50 min using 200 U of Superscript II RNase H-reverse transcriptase (Gibco BRL; Cergy-Pontoise, France), 1 mM of each dNTP, and 7.5 µM random hexamers (Roche) in the presence of 20 U of RNase inhibitor (Roche). Reverse transcription was followed by a 5-min denaturation at 95°C.

Polymerase Chain Reaction

Two microliters of each RT reaction were amplified in a 50-µl PCR reaction using 0.5 U of Taq polymerase (Takara) for 29–35 cycles, depending on primers, with 200 µM of each dNTPs and 150 nM of each primer. PCR conditions and primer sequences for each gene studied are reported in Table 2. As for RT reactions, PCR reactions were performed for a series of all gestational stages for a given gene. After amplification, 12 µl of RT-PCR products were electrophoresed in agarose-TBE gels, then Southern blotted on Nylon N+ membrane (Amersham Biosciences, Saclay, France) and probed with a [{gamma}-32P]ATP-radiolabeled internal oligonucleotide. The sequences of probes used for each gene are shown in Table 3. Autoradiograms were quantified on the films by densitometry using an image analysis system (Advanced Image Data Analyzer software; Fujifilm, Düsseldorf, Germany).


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  		TABLE 2. Sequences of PCR primers and conditions


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  		TABLE 3. Sequences of internal primers (probes)

Histology

Freshly dissected gonads were fixed in Bouin fluid for 24 h at room temperature, dehydrated, embedded in paraffin, and serially sectioned at 7 µm. One section out of 10 was mounted and stained according to the trichrome technique of Tuchmann-Duplessis [30].

BAC Screening, Isolation, and FISH Mapping

Metaphase chromosome spreads were obtained by standard procedures from a primary diploid fibroblast cell culture derived from a 56, XY sheep [31]. Clones containing genes were isolated from a sheep BAC library [27] using PCR and were miniprepped. BACs (400 ng) were labeled by Nick-translation with the Kit Bionick (Gibco BRL). Probes were then mapped by FISH as described by Godard et al. [32].

Slides were stained with iodide propidium and observed under an epifluorescence microscope (Zeiss). Images were analyzed with an Applied Imaging system. The R-banded chromosomes were identified according to the recommendation of the ISCNDB (2000) [33].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Germ-Cell-Derived Factors

By histologic analysis of the ovaries at different stages (Days 43, 49, 56, and 82 of gestation, at birth, and during adulthood), we observed that germ cells entered into prophase of meiosis I between 49 and 56 dpc (Fig. 1, B and C). From 56 dpc, the leptotene and zygotene stages were clearly identifiable in the ovogonia located within the region of ovigerous cords (Fig. 1C). At the same age (56 dpc), the initiation of DMC1, SPO11, and MSH4 transcription occurred (Fig. 2, A and B). Only the MSH5 gene was expressed earlier, at 49 dpc (Fig. 2B). A strong signal was observed until 75 dpc for the four genes. After that, the expression decreased more or less rapidly and was present at a low level at 94 dpc. On Day 82, histologic analysis showed that few nuclei of ovocytes were again in meiosis (Fig. 1D). At birth, all ovocytes were at the dictyate stage of meiosis (Fig. 1E).



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  		FIG. 1. Histological aspect of the ovary during critical stages of the sheep development. A, B) Sections through fetal ovaries at 43 and 49 dpc, respectively. Ovarian cortex development at the periphery of the gonad containing an increasing number of oogonia (arrows), some of them reaching the preleptotene stage, at around 49 dpc (arrowhead). C) Ovary from a 56-dpc fetus. Germ cells located in the deeper part of the ovarian cortex started the prophase of meiosis I (leptotene and zygotene stages, arrow and arrowhead, respectively). D, E) Ovary from a fetus on Day 82 of gestation (D) and at birth (E): initiation and development of the follicles toward midgestation; primordial follicles (arrow), primary follicles (arrowhead), and secondary follicles in development (asterisk). F) Section of adult ovary. Mature antral follicle of "De Graaf", characterized by a well-developed ovocyte (o) and a large antrum surrounded by several layers of granulosa cells (G) and thecal cells (T). Bar = 50 µm (A, B), 40 µm (C, D), 30 µm (E), and 100 µm (F)



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  		FIG. 2. RT-PCR analysis of four genes involved in meiosis during sheep ovarian development. The left part of the figure shows the autoradiograms and the right part the autoradiogram intensity quantifications. They are expressed as the intensity signal in arbitrary units after normalization by ß-actin gene expression on the same samples (Fig. 6). Expression profiles of the DMC1, SPO11, MSH4, and MSH5 genes in the gonads of females (F) or males (M) at different stages of fetal life (numbered in days postcoitum), at birth, and adulthood. Each PCR product was Southern blotted, hybridized with an internal probe, then autoradiographed. The results for the four genes were similar in male gonads, and only DMC1 is present in both sexes (A). For SPO11, MSH4, and MSH5, only female profiles are shown (B)

Two other genes (Boule and DAZL) also known to be expressed in germ cells but not directly involved in meiosis were studied by RT-PCR. Both presented profiles very similar to meiosis genes, i.e., initiation of active transcription at 49 dpc, maximal expression between 56 and 75 dpc, progressive decrease at 94 dpc, then extinction at 120 dpc (Fig. 3). In males, a basal expression of these six genes was detected during fetal life, with a slight rise around 70 dpc. Furthermore, they were highly expressed during adulthood (DMC1: Fig. 2A; Boule: Fig. 3; SPO11, MSH4, and MSH5: not shown).



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  		FIG. 3. Expression patterns of Boule and DAZL genes from 25 dpc until adulthood (autoradiograms on the left part and quantifications after ß-actin normalization on the right part). For the Boule gene, RT-PCR results from male (M) and female (F) gonads are illustrated, whereas for DAZL, only females are represented. The same procedure as described in Figure 2 was used for these genes. All stages during fetal life are numbered in days postcoitum.

A BAC corresponding to four of these six genes was isolated by PCR from a sheep BAC library. Indeed, the MSH4 and Boule genes were not detected in this BAC library. Then each BAC was located on the metaphase spreads of primary fibroblast cultures by FISH.

The homologous sheep genes for SPO11, DMC1, MSH5, and DAZL were located in the chromosomal regions OAR 13q21–22, 3q35, 20q22, and 19q13, respectively (Fig. 4, A–D). These regions are syntenic with HSA 20q13, 22q12–13, 6p21.3, and 3p24, respectively (Table 4).



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  		FIG. 4. FISH mapping of six gene-containing BAC on sheep metaphase: (A) SPO11 to ovine chromosome 13q21–22, (B) DMC1 to ovine chromosome 3q35, (C) MSH5 to ovine chromosome 20q22, (D) DAZL to 19q13, (E) DIAPH2 to Xq44, and (F) SIAH2 to ovine chromosome 1q41–42. The green-yellow dots (arrowhead) indicate the specific spots on the chromosomes.


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  		TABLE 4. Chromosomal location

Factors Specific to Follicular Development

During fetal ovarian development, initiation of GDF9 transcription started at 56 dpc and progressively increased to reach a maximum at 94 dpc, then decreased after birth (Fig. 5). BMP15 presented a more restricted expression window, with a high level of transcription at the end of gestation between 94 dpc and birth (Fig. 5). Expression of both genes was limited to female gonads during fetal life (no signal was observed from the testes; Fig. 5 and data not shown), and only a weak signal was detected in adult testis. In contrast, expression of BMPR-IB was high from 56 dpc and remained high at all fetal stages and during adulthood (Fig. 5). A low expression was also detected in male gonads, whatever the stage (data not shown). This could be due either to a basal transcription of the gene not sufficient to induce protein production or to a weak amplification of another member of the family of BMP receptors.



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  		FIG. 5. Developmental expression of GDF9, BMP15, BMPR-IB, and OVOL1 during male (M) and female (F) gonadal development in sheep. Separate PCR reactions were performed using the same RT products with different specific primer pairs. RT-PCR products were then Southern blotted and hybridized with a {gamma}-32P-labeled internal oligonucleotide corresponding to each investigated gene. Autoradiography was performed over 3 h. The left part shows the autoradiograms and the right the histograms corresponding to intensity quantifications after ß-actin normalization.

Another gene, OVOL1, presented a profile similar to GDF9: onset of expression at 56 dpc and higher transcription between 94 dpc and birth (Fig. 5). For OVOL1, no expression was detected in male gonads during fetal life (data not shown). The adult gonads of both sexes were also negative.

Ovary-Derived Factors

FOXL2 was expressed as early as 25 dpc in urogenital ridges (mesonephros + gonad) and within gonads of all the developmental stages tested, including the perinatal period and adulthood. Expression was always higher in females than in males (Fig. 6).



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  		FIG. 6. Expression patterns of FOXL2, DIAPH2, and ß-actin in developing male (M) and female (F) sheep gonads. All stages during fetal life are numbered in days postcoitum. We used the procedure described in Figure 5.

FGF9 and SIAH2 genes showed similar profiles, with a basic expression of low intensity without differences between males and females (data not shown). Only the DIAPH2 gene presented a dimorphic pattern between male and female gonads at 56 and 67 dpc (Fig. 6). Finally, the ß-actin gene was amplified in all samples to control the equal quantity of cDNA samples (Fig. 6).

Among these genes, only FOXL2 had been previously located in Bovinae, and we decided to map the three others on the sheep genome. FGF9, DIAPH2, and SIAH2 were localized on OAR 10q15, Xq44, and 1q41–42, respectively (Fig. 4, E and F). These regions corresponded to the human chromosomal regions HSA 13q11–12, Xq21–22, and 3q25, respectively (Table 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Knockout mouse technology has been used over the last decade to define the essential roles of ovary-expressed genes, in particular, genes involved in meiosis, such as DMC1, SPO11, MSH4, and MSH5 [9–15]. In female mice lacking the Dmc1 gene, normal differentiation of oogenesis aborts in fetuses with arrest of gametogenesis in the first meiotic prophase, and germ cells disappear in the adult ovary [9, 10]. In Spo11-deficient females, germ cells complete the early stages of prophase I in the fetal gonad and arrest at the dictyate stage a few days after birth. In 8-wk-old adults, ovarian dysgenesis is apparent, with a severe reduction in ovary size [11, 12]. Disruption of the Msh4 or Msh5 genes in mice results in male and female sterility due to meiotic failure [13, 14]. Mice carrying a disruption in the Msh5 meiotic gene have a diminution of testicular size and a complete loss of ovarian structures [14, 15].

The results presented here on sheep fetal ovaries are in accordance with the phenotypes observed in mice carrying a disruption of meiotic genes. All four genes showed a narrow expression window during early ovarian development corresponding to prophase I of meiosis. In sheep, we showed that these early stages of meiosis occurred between 56 and 75 days of gestation. Knowing the precise timing of this crucial step in ovarian development will be useful in identifying new genes inducing and controlling meiosis in sheep by different approaches, such as subtractive hybridization or transcriptomic assays. Moreover, these genes may affect female fertility in sheep and could be responsible for sterility in cases of gene defects or be involved in QTL affecting female reproduction. For this reason, we finely localized these genes in the sheep genome. Their location is in agreement with the comparative mapping data between human and sheep (http://www.ri.bbsrc.ac.uk/sheepmap/).

The time course of the BOULE gene was unknown in mammals, and the results presented here in sheep describe for the first time its expression pattern during gonadal development. As in invertebrates, sheep BOULE was expressed at the onset of prophase I of meiosis, with a peak of expression at 56 dpc. A strong expression was also observed in adult testes corresponding to spermatogenesis. As in humans and mice, the sheep DAZL gene presented a premeiotic expression at 49 dpc. For both genes, expression stopped after 94 dpc in the sheep ovary. These results support the hypothesis that the DAZ gene family is made up of two subfamilies required for different stages of germ cell development: DAZL for early germ cell function and BOULE for meiotic function [17].

In addition to molecular markers for germ cells and meiosis, we studied genes known to be involved in folliculogenesis (GDF9, BMP15, BMPR-IB) as well as several factors, such as OVOL1, FOXL2, FGF9, SIAH2, DIAPH2, the function of which remains unclear in mammalian ovary development. The two oocyte-secreted growth factors BMP15 and GDF9 have been intensively studied in mice, in which simple and double homozygote mutant females have been obtained [40, 41]. Knockout mice lacking GDF9 are infertile due to a block in folliculogenesis at the primary follicle stage [40]. In sheep, the first expression of GDF9 was detected at 56 dpc, with a maximum between 75 dpc and birth. Our results are in agreement with those of Bodensteiner et al. [8], who detected GDF9 mRNA in 135-dpc sheep ovary by in situ hybridization. The authors did not test earlier stages. They observed that specific hybridization was exclusively limited to oocytes in follicles of type 1 and 1a. The formation of these types of follicles occurs around 75 dpc, when a high level of mRNA was observed in our RT-PCR experiments. Nevertheless, transcription of the GDF9 gene began as early as 56 dpc, preceding the period of primordial follicle formation.

As in humans, the expression of ovarian BMP15 in sheep occurs after that of GDF9 [8, 42]. Our results show that the expression of the BMP15 gene started at 94 dpc, when the first growing (primary) follicles were observed, before secondary follicle formation. These results are in agreement with those of Galloway et al. [7], obtained by RNA in situ hybridization, and also with previous works on sheep homozygous for null mutations in the BMP15 gene (FecXII) that described streak ovaries containing no normal follicle beyond the primary stage of development despite a number of primordial follicles similar to that found in the wild type [5, 43–45].

The Booroola (FecB) phenotype is associated with a mutation in the BMP receptor type 1B (BMPR-IB) gene that affects ovulation rate. McNatty et al. [46] have previously observed that, as early as 30 dpc, the FecB ovaries contained less oogonia than the normal genotype. This observation corroborates the detection of BMPR-IB transcripts in our early stages (from 25 dpc). By in situ hybridization of 135-dpc ovarian sections, BMPR-IB expression was observed in oocytes of type 1 follicles [4]. Our RT-PCR results showed a strong expression of BMPR-IB between 56 dpc and birth. The expression appeared before the primordial follicle formation as for GDF9. The intensity of the signal seems to decrease after birth in adult ovary, in accordance with an arrest of expression after the small antral stage. Nevertheless, we can not exclude the possibility that this decrease of expression was due to a dilution of the cell type expressing the transcripts within the gonad. Moreover, we have tested only a part (1/4 of the volume) of the adult ovary.

Homozygous ovo-/- mice were obtained, but unfortunately phenotypes were studied only in males and no data were available on OVOL1 gene expression during ovarian development in mammals [47]. Interestingly, the sheep OVOL1 gene presented an expression profile similar to GDF9. Because OVOL1 protein contains a zinc finger domain, it may be a transcription factor and be involved in regulating BMP protein or receptor transcription.

Finally, we studied FOXL2, FGF9, DIAPH2, and SIAH2 genes. Transcripts of all four of these genes were present throughout ovarian development. The presence of FOXL2 protein from 36 dpc until adulthood was confirmed in precursors and follicular cells in goats by immunohistochemistry [48]. This gene, involved in human blepharophimosis/ptosis/epicanthus inversus syndrome [49] and polled intersex syndrome in goats [50], appeared as a very early sex-dimorphic marker of ovarian differentiation that plays a multiple role during follicle formation but also later, during adult female fertile life, in maintaining follicle viability. In this previous study [48], we showed that few transcripts present in males around 70 dpc were not sufficient to produce protein FOXL2. Indeed, at these stages, no signal was detected by immunohistochemistry with an anti-FOXL2 antibody.

DIAPH2 is the human homolog of the Drosophila diaphanous gene [26], which participates in the establishment of cell polarity and cytokinesis. In the mouse, Dia transcripts are present in the ovary and the testis at early stages of development (E16) as well as at stages during which the ovarian follicles undergo differentiation (P6–P16) [26]. In sheep, DIAPH2 was also expressed in both sexes, with a maximum at around 56–67 dpc in the ovary. This period probably corresponds to a phase of intensive mitotic multiplication. By incorporation of BrdU into proliferating cells, Sawyer et al. [51] have shown that, from Day 55 to Day 90, surface epithelial cells and oogonia were proliferating and that the maximum number of germ cells present in the fetal ovary of sheep is found at Day 75.

The two remaining genes, FGF9 and SIAH2, did not present sex- or stage-specific patterns. They were expressed in both sexes irrespective of the stage of development. These results are in contrast with those obtained with the Siah-2 gene in mouse. Indeed, Siah-2 expression was absent in primordial oocytes but was detected in all growing oocytes, coincident with their recruitment from the pool of quiescent cells [25]. Likewise, the expression pattern described for Fgf9 in mouse [52] was not found in sheep.

In conclusion, this study extends previous works on sheep gonadal differentiation [53–55] and advances our knowledge of early ovarian differentiation by describing 11 new gene expression patterns (DMC1, SPO11, MSH4, MSH5, DAZL, Boule, OVOL1, DIAPH2, SIAH2, FGF9, and FOXL2) in sheep. Moreover, some genes, such as GDF9, BMP15, and BMPR-IB, for which the cellular location has previously been described were not systemically studied during fetal ovarian development in sheep. The molecular data presented here as well as the detailed description of histoarchitecture of the developing ovary described by others [46, 51] contribute to making sheep an interesting model for studying ovarian differentiation in mammals. Finally, the chromosomal localization of seven of these genes will provide indications on their potential involvement in female reproductive traits. This work fits into a global approach developed to identify the gene(s) controlling meiosis and follicle formation in ruminants.


   ACKNOWLEDGMENTS
 
The authors would like to thank Eric Pailhoux for the Boule, FoxL2, and ß-actin primers; Philippe Mulsant for the BMP15 primers; Philippe Monget and Stéphane Fabre for BMPR-IB primers; Cédric Rougheol and Jean François Alkombre for animal care; and Céline Ducroix-Crépy for technical assistance in BAC library screening.


   FOOTNOTES
 
1 This work was supported by INRA, Institut National de la Recherche Agronomique. Back

2 Correspondence. FAX: 33 1 34 65 22 41; e-mail: pepin{at}jouy.inra.fr Back

Received: 18 June 2002.

First decision: 10 July 2002.

Accepted: 3 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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