Mutations in the forkhead transcription factor gene FOXL2 are involved in ovarian failure, which occurs in human BPES syndrome. This syndrome presents a sexually dimorphic expression, specific to the ovary in several vertebrates. We cloned the open reading frame of chicken FOXL2 (cFoxL2) and studied cFoxL2 expression in developing gonads and during adulthood to examine the role of FOXL2 in ovarian differentiation and function in birds. The spatial and temporal dynamics of cFoxL2 and aromatase expression were analyzed in parallel by using real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in attempt to investigate the possible role of cFoxL2 in the regulation of aromatase. The expression patterns of cFoxL2 and aromatase transcripts were highly correlated during the sex-differentiation period (4.7-12.7 days of incubation). Aromatase and cFoxL2 proteins were colocalized in the medullar part of female gonads on embryonic day 14. Fourteen days after hatching, cFoxL2 protein was mainly detected in granulosa cells of developing follicles. In adult ovary follicular envelopes, apart from granulosa cells, cFoxL2 transcript and protein were detected at lower levels in theca cells where aromatase was present. A high level of cFoxL2 transcription was also observed in maturing and ovulated oocytes. Our results confirm that FoxL2 is an early regulator of ovarian development in birds and may be involved in aromatase transcription regulation. Developmental Dynamics 231:859 -870, 2004.

The bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9) genes are two members of the transforming growth factor-b superfamily. In mammals, these genes are known to be specifically expressed in oocytes and to be essential for female fertility. However, potential ovarian roles of BMPs remain unexplored in birds. The aim of the present work was to study for the first time the expression of Bmp15 in the hen ovary, to compare its expression pattern with that of Gdf9, and then to investigate the effects of BMP15 on granulosa cell (GC) proliferation and steroidogenesis. We found that chicken Bmp15 and Gdf9 genes were preferentially expressed in the ovary. We showed using in situ hybridization that Bmp15 and Gdf9 mRNAs were specifically localized in oocytes of all ovarian follicles examined. We also demonstrated using real-time quantitative RT-PCR that Bmp15 and Gdf9 expression was maintained during hierarchical follicular maturation in the gerrminal disc region and then progressively declined after ovulation. BMP15 was able to activate Smad1 (mothers against decapentaplegichomolog1) signaling pathway in hen GCs. Moreover, we showed a strong inhibitory effect of BMP15 on gonadotropin-induced progesterone production in hen GCs. This inhibitory effect was associated with a decrease in steroidogenic acute regulatory protein (STAR) level. Taken together, our results suggest that BMP15 may have a key role in the female fertility of birds.

BackgroundIn birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s) involved in gonad differentiation is still incomplete.Methodology/Principal FindingsWith the aim of improving characterization of the molecular pathway(s) involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein) and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor β family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry.Conclusion/SignificanceThis study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors of chicken gonad differentiation and provides evidence of the preferential expression of BMPs in the developing ovary and Inhibin/Activin subunits in the developing testis.

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Background: The initial stages of development depend on mRNA and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. The aim of this study was first to search for avian oocyte-specific genes using an in silico and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis.

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Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryo cryopreservation allows preservation of genetics from both male and female and is the fastest method to restore a breed. Embryo production in vivo is limited in equids. We recently established a technique of ovum pick up (OPU) in the donkey. Conditions of in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture of zygotes have been evaluated. Equine abattoirederived oocytes were used as controls. Donkey cumulus-oocyte complexes (COCs) collected by OPU were matured in vitro in TCM199 with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. Forty-four percent were in metaphase 2 after 34 hours. In our conditions, IVM of donkey oocytes was slower than that of equine oocytes and the optimal duration for donkey oocytes IVM may be 34 hours. Oocytes we co-incubated with frozen-thawed donkey semen treated with procaine for 18 hours and cultured for 30 hours in a Dulbecco Modified Eagle Medium-F12based medium. Only 15% of jennies oocytes contained 2 pronuclei after co-incubation, and none of them developed further after 48 hours after IVF. Treatment of donkey sperm with procaine may not be efficient for IVF. Some parthenogenetic activation occurred. In conclusion, we confirm that our conditions for OPU in jennies yielded high recovery rates that improved with operator experience. Maturation rates of 44% can be achieved using the IVM medium routinely used for equine oocytes in our lab. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.