Effect Of Follicle Stimulating Hormone On In Vitro Culture Of Mouse Preantral Follicles

With the development of modern medical technology,the survival rate after treatment for cancer and other life-threatening diseases is gradually increasing.While saving lives,therapeutic drugs may harm the oocytes in the ovaries,eventually leading to infertility.With the test-tube babies came out,assisted reproductive technology has achieved remarkable progresses in solving infertility.A large number of follicles are stored in the ovaries of female mammals,and their development is regulated by autocrine and paracrine,so there are many factors that affect the development of preantral follicles in vitro.Most follicles will become atresia and eventually degenerate due to internal and external influences during their developmental process.In vitro culture of pre-antral follicles can save these follicles.This study took mouse follicles as the research object,and based on the study of mouse follicle development in vivo,to explore the most suitable method for the isolation and culture of mouse preantral follicles in vitro.(1)The growth and development of mouse ovaries and follicles in vivo.In order to explore the natural development of mouse follicles,this study used mechanical methods to peel off the ovaries and follicles of 1 d-21 d mice to observe the growth and development.By measurement,it was found that the area and diameter of the mouse ovary gradually increased within 21 days.As the age continues to grow,the mouse follicles also continue to grow and develop.The histological analysis of the mouse ovaries within 21 days showed that the mice had only primordial follicles in the ovaries at birth.As the age increased,primordial follicles gradually developed into primary follicles,secondary follicles,antral follicles and mature follicles.There are multiple stages of follicles in the ovaries of mice.It was found that the growth and development of follicles is divided into two stages by analyzing the ratio of the area of mouse follicles to oocytes at different developmental stages.The first stage is the preantral follicles period,and the area of mouse follicles and oocytes increases proportionally;the second stage is the antral follicles period,when the oocytes grow slightly and the follicles increase significantly,and the follicular fluid continues to accumulate at this stage.(2)The effect of FSH on the growth and development of mouse follicles cultured in vitro.In this study,six concentrations of FSH(0 m IU/ml,10 m IU/ml,50 m IU/ml,100 m IU/ml,150 m IU/ml,200 m IU/ml)were added to the culture medium to culture mouse follicles in vitro for 10 days and observed the effect of FSH on the growth and development of mouse follicles.It was found that the follicles without FSH grew slowly and failed to develop the follicular antrum;the follicles with FSH all developed the follicular antrum.The growth area of the follicles cultured with FSH was significantly higher than that of the follicles without FSH(P <0.01).The promoting effect of FSH on the growth of follicles did not show a positive correlation to its concentration.The area of follicles cultured with 50 m IU/ml FSH was the largest,and it was significantly larger than that of 150 m IU/ml FSH and 200 m IU/ml FSH(P <0.05).Follicles cultured with 10 m IU/ml FSH successfully shed the cumulus-oocyte complex,and the oocytes successfully shed the first polar body.This showed that FSH can promote the growth and development of follicles in a certain concentration range.(3)The effect of FSH on steroid hormone synthase in mouse follicles.Cultured in vitro for2 d,4 d,6 d,8 d,10 d,11 d,these six time points were used to explore the effect of FSH on gene expression of follicles and the secretion of estradiol.It was found that FSH can up-regulate the expression of 17β-HSD,CYP19A1 and 3β-HSD in a certain period.The expression level of 3β-HSD is correlated with the growth and development of mouse follicles.In the early stage of culture,the secretion of estradiol is dose-dependent with FSH;in the later stage of culture,there is no strict dose-dependence between FSH and estradiol.Excessive FSH will affect the secretion of estradiol.Studies have shown that FSH can promote the development of follicles in vitro,and that 10 m IU/ml FSH is the most suitable for the development of follicles in vitro.These research results can add theoretical and practical data to the protection of endangered animals and the research of human reproductive medicine.