The Effects And Mechanism Of Bmp4/Smad Signaling Pathway In Mouse Primordial Follicle Development

Objective and Significance Primordial follicle is the basic unit of the pool of female mammals ovary, non-regeneration and non-update once formed. Although there are millions of the primordial follicles initially,99%of primordial follicles will be atresia and degenerate at last, only very few of which can mature and ovulate. This leads to the huge waste of female reproductive resource.The storage and atresia of primordial follicles directly affect the capacity of reproductive in female mammals. Once the depletion and atresia of primordial follicles is too fast to cause premature ovarian failure,endocrine dyscrasia and infertility. So it is provided with important theoretical and practical significance to observe the activation and survival of primordial follicles and its regulatory mechanism for a better understanding of mammalian reproduction.Primordial follicles keep the state of dormancy until activated. Once started primordial follicles will enter the continuous process of differentiation and development irreversibly till they become dominant follicles which can mature and ovulate or degenerate halfway. The activation of primordial follicles from their resting state into a growing state, which means the transition from primordial follicle to primary follicle.This process is the key step of follicle growth initially whose mechanism is always a hot topic in reproductive biology. The present study found the initiation of primordial follicle development was gonadotmpin-independent, but regulated mainly by paracrine and autocrine factors in ovary.Bmp4(bone morphologenetic protein4) which belongs to a kind of multifuncti-onal glycoproteins is one of the members of transforming growth factor-P (TGF-P) superfamily. It was conformed that Bmp4from ovary involved in the initiation growth of primordial follicles could control and adjust the physiological function of ovary and female mammals reproductive ability, especially play an important role in the regulation of primordial follicle growth. But the specific mechanisms of Bmp4are still under investigation. So we explored the effects and mechanisms of Bmp4/Smad signaling pathway in mouse primordial follicle development.In vitro studies, the object of our experiment was to detect the effects and mechanism of Bmp4/Smad signaling pathway in mouse primordial follicles development by culturing3-day-old Kunming mice.Then we observed the expression of Bmp4, p-Smadl/5/8, Sohlh2and c-Kit in primordial follicles from the neonatal to adult mouse ovaries (0-8weeks), obtained from female mice by immunohistochemis-try in vivo, to further validate the mechanisms of Bmp4-Smad-Sohlh2-c-Kit signa-ling pathway and to aim to provide a new theoretic and experimental evidence for revealing the molecular regulation mechanism of the activation and survival of the primordial follicles, for making full use of the large follicles resources in ovaries, and curing POF and infertility.Methods1. Exploring the effects and mechanism of Bmp4/Smad signaling pathway in mouse primordial follicle development. The ovaries cultured from3-day-old mice in vitro were divided randomly into control group and Bmp4group (medium with recombinant human Bmp4). The size of ovaries cultured8days from two groups were compared by anatomical lens; by HE staining the numbers of follicles at each developmental stage were counted in two serial sections from the largest cross-section through the center of the ovarian slices; TUNEL was used to examine the apoptotic oocytes of two groups. Immunohistochemistry, Western blotting and RT-PCR were used to examine p-Smadl/5/8, Sohlh2and c-Kit expression at protein and mRNA level among them. 2. Observing the expression of Bmp4, p-Smadl/5/8, Sohlh2and c-Kit in primordial follicles from the neonatal to adult mouse ovaries (0-8weeks). Female Kunming mouse ovaries of0,1,3,6and8weeks were obtained.The specimens were sliced into section. Immunohistochemistry method and image analysis were employed to explore the expression of Bmp4, p-Smadl/5/8, Sohlh2and c-Kit protein in primordial follicles.Results1.(DOvarian diameter of Bmp4group was larger than that of control group under anatomical lens after ovaries were cultured8days. HE staining results showed that, ovaries treated with Bmp4were bigger than those of the control group. The number of primary follicles of Bmp4group was significantly more than the control group, yet primordial follicles of Bmp4group was less than the control group(P <0.05), counted in ovarian sections from the largest cross-section through the center of ovaries.②TUNEL assay result showed that apoptotic follicles of Bmp4group were lower than the control group (P<0.05).③Immunohistochemistry result showed that the expression of P-smad1/5/8of Bmp4group was higher than that of control group in oocyte nucleus (P<0.01), Sohlh2and c-Kit levels increased significantly after the treatment of Bmp4for24and48h (P<0.05). Sohlh2was observed in the cytoplasm and nuclei of the oocytes in primordial follicles. c-Kit was localized to the cytoplasm and membrane of oocytes.Western blotting analysis bring into corres-pondence with the immunohistochemistry result. RT-PCR results showed sohlh2and c-kit mRNA level were significantly higher in Bmp4group than that of control group (P<0.05), after the ovaries cultured8h and24h.2. Bmp4, p-Smadl/5/8, Sohlh2and c-Kit protein were determined by immunocy-tochemical in mouse primordial follicles every week. Bmp4and p-Smadl/5/8were presented in cytoplasm and nucleus of oocyte, in addition to pregranulosa cells especially the positive expression were more obvious in nucleus. In the primordial follicles of all stage of mouse ovaries, Sohlh2was presented in the cytoplasm and nucleus of the oocyte, and c-Kit was detected in the cytoplasm and membrane of the oocyte. These proteins exhibited alike expression patterns in primordial follicles were more apparent in the primordial follicles of early-stage ovaries (neonatal to3-week-old), and diminished in the late-stage ovaries (3-week-old to adult). Conclusion1. Bmp4/Smad signaling pathway could promote the primordial-to-primary follicle transition and inhibit oocyte apoptosis in mouse.2. Bmp4/Smad signaling pathway may act by up-regulation of Sohlh2and c-Kit.3. Bmp4, p-Smad1/5/8, Sohlh2and c-Kit protein presented in mouse primordial follicles during the postnatal ovarian development, may be involved in the regulation of the primordial follicle growth in vivo.