The Regulation Of BMP4/Smad Signaling Pathway On The Apoptosis Of Mouse Primordial Follicle Oocytes

Objective and Significance The resting pool of primordial follicles in human ovaries forms in last of embryonic period or before birth, non-regeneration and non-update once formed, which represents the total number of gametes available to a female throughout her reproductive life. 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. The survival of primordial follicles is a key of follicle growth initially whose mechanism is always a hot topic in reproductive biology. 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 apoptosis of oocytes 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. Atresia is started by the apoptosis of oocytes in primordial follicles and primary follicles. However, little is known concerning the mechanism of primordial follicle oocyte apoptosis, the present study found the apoptosis of oocytes is regulated mainly by the factors secreted from granulosa cells and stroma cells in ovary.Bmp4(bone morphologenetic protein4) is one of the members of transforming growth factor-13(TGF-p) superfamily. Recent years, we found BMP4regulated follicular growth, development, survival and the secretion of estrogen and progesterone via signal transduction system of local in ovary. The present study found BMP4involved in the apoptosis of oocytes, but the mechanism responsible has not yet been clarified. So we explored the effects and mechanisms of BMP4/Smad signaling pathway in mouse primordial follicle oocyte apoptosis.In our study, we cultured mouse primordial follicle oocytes in vitro, TUNEL, Immunohistochemical staining, realtime quantitative PCR, Western Blotting, siRNA interference, sohlh2plasmid transfection and LY294002treatments were performed to explore the effects of BMP4on the survival of the primordial follicle oocyte and its regulatory mechanism. our study provides a new theoretic and experimental evidence for revealing the molecular regulation mechanism of the apoptosis of the primordial follicle oocytes, for making full use of the large follicles resources in ovaries, and curing POF and infertility.Methods1. Examining the effects of BMP4/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes.3-day-old Kunming mouse ovarine tissues were digested by the two-step enzymatic method to extract and purify oocytes. The cultured oocytes were divided into three groups:the normal culture medium (Con group), the medium with BMP4(BMP4group), and the medium with BMP4and BMP4inhibitor (BMP4+inhibitor group), the final concentration of BMP4in each medium was2μmol/L. TUNEL was used to examine the effects of BMP4on the survival of the primordial follicle oocyte; Immunohistochemical staining and realtime quantitative PCR were performed to investigate the expressions of p-Smad1/5/8, sohlh2, c-kit and foxo3a.2. Exploring the mechanism of BMP4/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes.(1) Using siRNA interference, the transfected oocytes were divided into four groups:siCon group, siCon+BMP4group, siSohlh2 group, siSohlh2+BMP4group. After24h transfection, TUNEL was used to examine the difference of oocyte apoptosis in four groups, immunohistochemistry, Western blotting and qRT-PCR were used to examine Sohlh2, c-kit and p-Foxo3a expression at protein and mRNA level among them.(2) Sohlh2plasmid transfection and LY294002treatments The coding regions of mouse sohlh2was inserted between the CAG promoter and the IRES to generate CAG-sohlh2. After transfected, the oocytes were divided into four groups:blank plasmid group, sohlh2plasmid group, blank plasmid+LY294002group, sohlh2plasmid+LY294002group. TUNEL was used to examine the difference of oocyte apoptosis in four groups above, Western blotting was used to examine c-kit and p-Foxo3a expression at protein level among them.Results1、(1) After cultured48h in vitro,TUNEL assay result showed that apoptotic oocytes of BMP4groupwere lower than the Con group and BMP4+inhibitor group(P<0.05), no significant difference in oocyte apoptosis was seen between Con group and BMP4+inhibitor group.(2) Immunohistochemistry result showed that the expression of p-Smad1/5/8of BMP4group was higher than that of Con group and BMP4+inhibitor group in oocyte nucleus(P<0.05). Sohlh2was observed in the cytoplasm and nuclei of the oocytes in primordial follicles, the expression of shlh2in BMP4group was higher than that in Con group and BMP4+inhibitor group(P<0.05). C-kit was localized to the cytoplasm and membrane of oocytes, in the BMP4group, the positive cells and protein levels of c-kit was significantly more than the Con group and the BMP4+inhibitor group(P<0.05). The expression of Foxo3a in nuclei of the oocytes in the BMP4group was significantly more than that of the other two groups(P<0.05).(3) Quantitative real-time PCR results showed that compared with the Con group, the mRNA levels of sohlh2and c-kit were up-regulated in the BMP4group, there was no significant difference for Foxo3a gene expression among the three groups.2、(1) After siRNA transfected24h, the quantitative real-time PCR results showed that sohlh2siRNA1and sohlh2siRNA2were effective, and sohlh2siRNAl is more effective, so we use sohlh2siRNAl doing the next step. TUNEL assay result showed that, compared with the siCon group, the apoptotic oocytes in siCon+BMP4group decreased and in siSohlh2group increased, apoptotic oocytes of siSohlh2+BMP4group were more than the siCon+BMP4group, the difference was significant (P<0.05). Immunohistochemistry result showed that compared with the siCon group, the positive cells and protein levels of sohlh2and c-kit were significantly lower in siSohlh2group but significantly more in siCon+BMP4group(P<0.05), the expression of Foxo3a in nuclei of siSohlh2group was up-regulated(P<0.05), there was no significant difference for total Foxo3a gene expression among the four groups Westem blouing analysis brought into correspondence with the immunohistochemistry result. QRT-PCR results showed that compared with the siCon group, sohlh2and c-kit mRNA levels were significantly lower in siSohlh2group but that of siCon+BMP4group were significantly higher(P<0.05).(2) Sohlh2plasmid transfection and LY294002treatments TUNEL result showed compared with blank plasmid group, apoptotic oocytes of sohlh2plasmid group were significantly little, blank plasmid+LY294002group were significantly more(P<0.05), the ratio of apoptotic oocytes in sohlh2plasmid+LY294002group was higher than sohlh2plasmid group(P<0.05). Western blouing analysis showed the level of c-kit was up-regulated after sohlh2plasmid transfection, LY294002could not effect the expression of c-kit. The expression of Foxo3a was not significantly different among the four groups, but its phosphorylation was diverse, the level of p-Foxo3a is higher in sohlh2plasmid group than blank plasmid group, and it is down-regulated by LY294002.Conclusion1. BMP4/Smad signaling pathway could inhibit oocyte apoptosis in mouse.2. Bmp4/Smad signaling pathway may act by up-regulation of sohlh2and c-kit, then effect PI3k-Akt-Foxo3a signaling pathway.