Characterization And Comparison Of Early Follicular Growth In Transplanted, In Vitro Cultured And In Vivo Grown Mouse Ovaries

Mouse model has been widely utilized to understand the basic principles ofinitial follicle recruitment (follicle activation) and the role of specific growth anddifferentiation factors. Outside of their natural environment, ovaries must cope withphysical restriction. More importantly, eliminating in vivo regulation system may alterfollicular growth. At present, the methods of ovarian incubation mainly include invitro culture and transplantation under kidney capsule. But there were very feworiginal research articles published on the comparison of different incubation systemsand the differences between incubated and in vivo grown ovaries. In this study, wecompared the numbers of follicles at different stages in cultured, transplanted and invivo grown ovaries. Using immunohistochemical analysis and real-time polymerasechain reaction (PCR), we investigated three key genes expression and their proteinlocation, including zona pellucida3(ZP3), growth and differentiation factor-9(GDF-9) and anti-Müllerian Hormone (AMH). Our results suggest that in vitro cultureaccelerates follicle activation, retards the growth of developing follicles, andinfluences the expression patterns of ZP3, GDF-9and AMH. Although transplantationcauses a small number of primordial follicle loss, transplanted ovaries presented themost similar characters as in vivo grown ovaries, indicates that transplantationprovides the most optimal environment of ovarian incubation. In culture system,alpha-Modified Eagle Medium is the better basal medium. These studies establish thesimilarities and differences between in vivo and incubated ovaries, demonstrate transplantation can mostly imitate the environment of ovary growth in vivo, anddetermine the optimal basal culture medium of the two tested. Objective To investigate the regulatory role of miR-145on granulosa cell proliferation,differentiation and steroidogenesis.Methods Using in situ hybridization (ISH), we detected miR-145expression on ovaries from 3-week-old mice. We measured miR-145expression, differentiation-associated gene andsteroidogenic gene expression in cultured granulosa cells by real-time polymerase chain reaction(RT-PCR). The proliferation of granulosa cells were also examined by EdU assay.Immunohistochemistry were used to evaluate the expression of differentiation-associated protein.Results miR-145was highly expressed in the granulosa cells from antral follicles. FSHsignificantly increased miR-145expression in primary cultured granulosa cells. Down-regulationof miR-145repressed GC proliferation and differentiation. Progestrone synthesis was reduced bydown-regulated miR-145, corresponding with low expression of3β-HSD and cyp11a, whereasno effect on estrogen.Conclusion miR-145is involved in regulation of granulosa cells proliferation, differentiation andsteroidogenesis. The precise mechanism deserves further exploration.