| Mice and rats have been the comprehensively used in animal models,and a considerable number of synchronously developed oocytes of mice and rats are indispensable materials for reproductive and developmental basic research.To meet this demand,in the present study,we have tried to establish the adequate technique of synchronous follicular development induced by administration of diethylstilbestrol(DES),an artificial nonsteroidal compound possesses strong estrogen bio-activity,as well as the in vitro maturation(IVM) technique of pre-mature oocytes. The results showed that,the peritoneal administration of DES at an appropriate dose in the prepubertal mice and rats could result in the significant increase in the amount of tertiary follicles,owing to the synchronous development of ovarian follicles.The oocytes prepared from the tertiary follicles by manual puncturation were in vitro cultured to mature,and subsequently used for in vitro fertilization.It was found that,no significant difference in maturation rate or fertilization rate was observed between DES-treated and control groups.Furthermore,for the active patterns of DES and E2 played both on the follicle development and IVM of premature oocytes were extremely similar,we deduced that the influence of DES on the synchronous development of ovarian follicles was mediated by the estrogen bio-activity of DES.It has been previously demonstated that,ubiquitin carboxy terminal hydrolases L1(UCH-L1) plays important roles in the development of sperm and fertilization by binding with c-Jun activation domain binding protein 1(Jab1) to regulate the ubiquination dependent degradation of cyclin dependent kinase inhibitive factor(p27Kip1) in cell apoptosis.Seeing that cell proliferation and apoptosis are widely involved in the process of follicle development,we herewith detected the influence of DES on the expression patterns of UCH-L1,Jab1 and p27Kip1 in the mouse ovary by immunohistochemical(IHC) and Western blot analyses,with a view to explore the molecular mechanism of the stimulative effects of DES on follicle development in mice.The results of IHC showed that,no obvious alteration in the distribution of these three molecules in the mouse ovary was detected between the DES-treated and control samples.Both UCH-L1 and Jab1 were localized in oocytes,whereas p27Kip1 was extensively expressed in various ovarian cells.The results of Western blot analyses showed that,the protein levels of UCH-L1,Jab1 and p27Kip1 in the ovarian tissues were slightly increased by the treatment of DES in prepubertal mice,however, no difference in protein levels of these 3 proteins with statistical significance was found between DES-treated and control animals.Interestingly,the ovarian exression levels of UCH-L1 and p27Kip1 proteins continuously increased along with the age during 1-4 weeks after the birth of mouse,although no significant change in protein level of Jab1 was detected during the same period of postnatal development.Accordingly,it could be presumed that,UCH-L1 might be involved in the regulation of ovarian follicle development.In order to further reveal the molecular mechanism underlying the DES-induced synchronous development of ovarian follicles,the cDNA microarray technique was applied to elucidate the global gene changes in mouse ovary induced by the treatment of DES.A total of 38 genes related to cell proliferation(24 genes) or apoptosis(14 genes) were screened out to be upor down-regulated(≥2-fold) in DES group compared with control group.Among these genes,14 genes were validated by real-time quantitative RT-PCR,and the ovarian expression level changes of 4 genes,including Insl3,Il1b,Cuzd1 and Elf3 were finally confirmed,indiating the participation of these 4 genes in the regulation of ovary follicle development.In summary,a large number of oocytes with normal abilities of in vitro maturation and fertilization could be obtained by the treatment of DES in prepubertal mice and rats.The stimulative effects of DES on the synchronous development of ovarian follicles might be caused by the regulation of UCH-L1,Jab1 and p27Kip1.Furthermore,the ovarian expression levels of Insl3,Il1b,Cuzd1 and Elf3 were affected by the treatment of DES,presenting new targets for future studies on the follicular development. |
