Abstract
BackgroundThe key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep.ResultsIn order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep.ConclusionWe showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.
Highlights
The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which determine the reproductive lifespan of the ovary
We report on the generation of two sheep fetal ovary cDNA libraries, obtained by applying suppression subtractive hybridization (SSH) using mRNAs isolated from D55 and D82 female gonads In the first subtraction D55 was used as tester and D82 as driver and the forward subtractive library was constructed (M, Meiosis)
The M library, supposed to be enriched in cDNAs representing genes preferentially expressed during prophase I meiosis, was obtained using cDNAs from D55 mRNAs as the 'tester' and cDNAs from D82 mRNAs as the 'driver'
Summary
The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which determine the reproductive lifespan of the ovary In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. A second major event in female gonad differentiation occurs when somatic pregranulosa cells surround individual germ cells to form primordial follicles which constitute a reservoir of oocytes [3], available for subsequent growth and differentiation. This event occurs during fetal life or after birth, depending on the species
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