Regulation Of Germ Cell Meiotic Initiation In The Embryonic Chicken

At present, people’s consumption and market force the poultry industries to choice high-reproductive performance individuals in the breeding. The reproductive performance depends on the follicular development of poultry. Thus, study of follicular development is the theoretical basis for understanding the regulation of reproductive function and improving the laying performance in poultry. Moreover, the poultry ovary constitutes an ideal model for studies of ovarian biology and follicular development. Here, we evaluated the effects of endocrine, paracrine and autocrine on the development of follicles and their related mechanism.1. Turnover of the germ cell developmental fate in the sex-reversed embryonic chickens by estrogen modulation.2. Gonadotropins regulate ovarian germ cell mitosis/meiosis decision in the embryonic chicken.3. Progesterone (P4) regulates embryonic germ cell meiotic initiation independent of retinoic acid signaling in chicken.4. Basic fibroblast growth factor (bFGF) suppresses meiosis and promotes mitosis of ovarian germ cells in embryonic chickens. From these studies, we expected to provide theoretic guidance for improving the reproductive performance of poultry.1. Turnover of the germ cell developmental fate in the sex-reversed embryonic chickens by estrogen modulationThe hypothesis that sex determination of germ cells depends upon their external environment rather than a cell-autonomous program had been widely accepted, but there exists little direct in vivo evidence to support this notion. Dazl, as an intrinsic factor for meiotic initiation, was strictly localized to the cytoplasm of both male and female embryonic germ cells, which indicates that the intrinsic Dazl may not be involved in sex determination. Then, the sex reversed chicken embryos were used to investigate germ cell development pattern in different microenvironments. Feminization of male (ZZ) gonads was caused by estrogen and masculinization of female (ZW) gonads by aromatase inhibitor. Meiotic germ cells appeared only in the ovarian-like cortex and mitotic germ cells appeared in the testis cord-like structures of both sexes. The abundance of retinoic acid (RA) metabolism enzyme mRNAs indicated that local levels of RA likely represents the signaling factor for germ cell meiotic initiation. Furthermore, the RA-degrading enzyme Cyp26b1was immunolocalised strictly in the testis cord-like structures of both sexes but not in the ovarian-like cortex. We proved through in vivo experiments that the fate of germ cells was predominantly determined by their gonadal environment instead of sex-specific intrinsic factors.2. Gonadotropins regulate ovarian germ cell mitosis/meiosis decision in the embryonic chickenGonadotropins are required for gametogenesis but in embryonic gonads this mechanism is not well understood. Here we use chicken embryos to investigate the mechanism that gonadotropins regulate the ovarian germ cell mitosis/meiosis decision. Treatment with follicle-stimulating hormone (FSH) delayed germ cell meiosis entry and promoted their proliferation. This action was blocked by an aromatase inhibitor. Treatment with luteinizing hormone (LH) accelerated germ cell meiosis entry and promoted transcription of3βHSDII to increase progesterone (P4) production. In the cultured ovaries, P4triggered meiotic initiation in germ cells. MiR181a*, which acts to downregulate the NR6A1transcript to prevent the meiotic initiation, was upregulated by FSH and downregulated by LH. Collectively, gonadotropins regulate germ cells mitosis and meiotic initiation through steroid hormones and a miRl81a*-mediated pathway. In particularly, FSH delays germ cell meiosis entry and promotes cell proliferation via estrogen while LH accelerates the meiotic initiation via elevated P4production.3. P4regulates embryonic germ cell meiotic initiation independent of retinoic acid signaling in chickenMeiosis is a special type of cell division that is restricted to germ cells. Recent studies demonstrate that the signaling molecule RA triggers germ cells to enter meiosis. However, many publications have revealed that RA may not be the only secreted inducer of meiosis. Progestin is an essential factor for the initiation of meiosis in teleosts. Here we used the chicken embryo to investigate whether P4regulates germ cell meiotic initiation in vertebrates which been reported that meiosis entry induced by RA. The female chicken embryos were challenged with P4at embryonic day9.5of incubation that accelerated germ cells meiosis entry. However, treated with P4did no effect on testicular germ cells in vivo but entered meiosis when testis-only culturing. The treatment of a pan-RAR antagonist, AGN193109cannot prevent germ cell meiotic initiation which induced by P4indicating no involvement of RA receptor-mediated signaling. Moreover, no significantly difference in abundances of RA metabolism-related enzymes (Raldh2and Cyp26bl) and RA receptor (RARβ) mRNAs and RA concentration in ovaries by P4treated in vivo. Use siRNA to knockdown both progesterone A and B in germ cell/somatic cell co-culture did not impaired meiosis suggest that PR is not required for P4-stimulated germ cell meiosis. Overall, this study provides new insights into the mechanisms of germ cell meiotic initiation in chicken. We conclude that P4regulates embryonic germ cell meiotic initiation independent of RA signaling.4. The bFGF Suppresses Meiosis and Promotes Mitosis of Ovarian Germ Cells in Embryonic ChickensThe bFGF plays diverse roles in regulating cell proliferation, migration and differentiation during embryo development. In this study, the effect of bFGF on ovarian germ cell development was investigated in the embryonic chicken by in vitro and in vivo experiments. Results showed that a remarkable decrease in bFGF expression in the ovarian cortex was manifested during meiosis progression. With ovary organ culture, we revealed that meiosis was initiated after RA treatment alone but was decreased after combined bFGF treatment that was detected by real time RT-PCR, fluorescence immunohistochemistry and Giemsa staining. Further, no significant difference in mRNA expression of either RA metabolism-related enzymes (Raldh2and Cyp26b1) or RA receptors was displayed after bFGF challenge. This result suggests that the suppression of bFGF on meiosis was unlikely through inhibition of RA signaling. In addition, as a mitogen, bFGF administration increased germ cell proliferation (via BrdU incorporation) in cultured organ or cells in vitro and also in developing embryos in vivo. In conclusion, bFGF suppresses RA-induced entry of germ cells into meiosis to ensure embryonic ovarian germ cells to maintain at undifferentiated status and accelerate germ cell proliferation in the chicken.5. Neo-oogenesis in the adult chicken ovaryIt remains unclear whether neo-oogenesis occurs in postnatal ovaries of avis, based on studies in chicken. We thought to test whether adult chicken ovaries undergo neo-oogenesis.We took the approach of analyzing the expression of meiotic marker genes and genes for germ cell development. First, histological analysis of juvenile and young adult ovaries revealed the presence of large ovoid cells, resembling germ cells of fetal chicken ovaries. Immunohistochemical staining for Dazl, confirmed that these large ovoid cells were of a germline lineage. We show that active meiosis and neo-oogenesis are likely to exist in normal, adult, chicken ovaries. These data establish the existence of germ cells that sustain oocyte and follicle production in the adult chicken ovary.In conclusion, we investigated the developmental changes of chicken germ cells by the methods of morphology and immunohistochemistry and the effects of bFGF on the follicular development by suspension follicle culture and cell culture. The results showed that the loacal growth-promoting factors promoted proliferation of cells via their respective receptors, hence to regulate the development of chicken follicles.These results could be contributed to elucidate the mechanism of follicular development and selection, as well as to represent a reference for improvement of poultry reproductive performance.