The Effects Of Folliculin On Expression Of Vascular Growth Associated Factor In Cultured Mouse Ovaries

Objective Cryopreserved ovarian transplantation is due to resume chemotherapy for cancer patients with premature ovarian failure an important method of fertility, however, ischemia and hypoxia after ovarian transplantation of ovarian transplantation is still the most serious problems. Although studies have shown that gonadotropin intervention given after transplantation can improve the survival rate of follicles, but its long-term intervention may lead to decreased follicle reserve. Therefore, to explore appropriate intervention has become a gonadotropin ovarian transplantation to improve survival and extend the functional life of the issue must be resolved. In this study, the culture medium by adding different concentrations of follicle stimulating hormone (FSH), explore the short-term intervention on the mouse ovary in vitro angiogenesis-related factor expression; and then the best in vitro interference time for ovary transplant FSH effective intervention to provide the time and scientific basis.Method 1. diestrus take 6-8 weeks in vitro short-term cultured mouse ovaries, according to the culture medium in vitro FSH concentrations were divided into four groups (0.00IU/mL, 0.15IU/mL, 0.30IU/mL, 0.60IU/mL), in cultured 0h, 3h, 6h, 12h, 24h to collect specimens, (1) by immunohistochem is try to observe the ovarian tissue VEGF, bFGF, VEGFR, bFGFR, and cellular location of expression of FSHR and the positive intensity changes; (2) the adolescent mouse ovary containing 0.00IU/mL, 0.30IU / mL concentration of FSH in the medium on the culture 3h, 6h, 12h, 24h after the extraction of total RNA, real-time fluorescence quantitative PCR method observed VEGF, bFGF, VEGFR, bFGFR, and FSHR expression at the transcriptional level; (3) to adolescence in the mouse ovary containing 0.00IU/mL, 0.30IU/mL concentration of FSH in the medium on the culture 3h, 6h, 12h, 24h, the extracted total protein by western-blot method of VEGF, bFGF, VEGFR, bFGFR, and quantitative observation of FSHR protein.Results⑴Immunohistochemical staining showed that: intervention group, FSH concentrations increased with VEGF, VEGFR, bFGF and bFGFR expression gradually increased, the concentration of significant differences between groups. 0.15IU/mL FSH and 0.30IU/mL FSH intervention group display VEGF expression in the 12h reached the peak, in addition to the group outside the 24h, with other time groups (P<0.05). 0.6IU/mL FSH intervention group VEGF expression in the 24h reached the peak. And at the same time other groups were different (P<0.05). the control group, VEGF, VEGFR, bFGF, bFGFR with time downward trend. 0.15 IU / mL, 0.30IU/mL and 0.60IU/mL FSH in vitro 3h FSHR expression reached the peak, time of 0.15 IU / mL FSH intervention group were no significant differences (P>0.05), but lower than other groups (P<0.05). 0.6IU/mL FSH 3h and 24h groups did not differ between groups (P> 0.05), but have differences in 6h and 12h groups (P <0.05). Expression of the control group was significantly lower than other groups.⑵Real-time quantitative PCR results showed : the expression of VEGF mRNA with time gradually decreased in the Non-intervention group .0.30IU/mL FSH intervention group between the culture of the time with the expression of VEGF mRNA gradually increased. peaked at 24h group, and other groups were significant (P<0.05). VEGFR mRNA intervention time with the expression of FSH increased, peaked at 24h culture period, with the other groups were significant (P<0.05). bFGF mRNA expression without promoting group peaked at 0h culture group, 0.30IU/mL FSH intervention group peak appears in the 24h group, and other groups were significant (P <0.05). bFGFR mRNA and bFGF similar group of non-peak promote culture in the 0h group, 0.30IU/mL FSH intervention group peak appears in the 24h culture group, 0.30IU/mL FSH intervention group were the other two training groups significance (P<0.05). FSHR mRNA 0.30IU/mL FSH expression peaked at 3h culture group, and other groups were significant (P<0.05).(3) western-blot results of observation: VEGF and its receptor VEGFR expression and immunohistochemical results are similar, showing its expression with the time increased, the peak 12h in intervention group, compared between groups statistically significant (P <0.05). bFGF and its receptors and immunohistochemical results similar expression in the peak 12h group culture among the groups was statistically significant (P<0.05). FSHR and immunohistochemical results was similar.Conclusions 1.FSH in vitro for 24h in mouse ovaries with follicular atresia follicular development and the role of inhibition. 0.60IU/mL group the lowest number of primordial follicles, significant differences between the groups. 2.FSH in vitro intervention can increase the growth factors of mouse ovarian angiogenic associated VEGF, VEGFR, bFGF, bFGFR the mRNA and protein expression within 12h show dose - time-dependent.3.FSH intervention can increase the expression of FSH receptor in development of mouse ovarian.4.The result of FSH in vitro short-term intervention increases the expression of angiogenic factors in mouse ovarian Prompted intervention by FSH before transplantation early in vitro in can start the ovarian blood vessels occur.