Function Of Ufmylation Involved In Apoptosis Of Goat Follicular Granulosa Cells

There are a large number of follicles on the ovary of mammals,but in the process of follicular development,a very small part?about 1%?can develop to be mature and then ovulate,and the rest of the more than 99%of follicles will be blocked.Follicular atresia greatly limits the reproductive utilization of elite breeding stocks.With the continuous improvement of animal-assisted reproductive technology,especially nuclear transfer and transgenic technology,how to obtain a large number of high-quality oocytes has become a bottleneck restricting the promotion and application of new technologies.Apoptosis of granulosa cells is the main cause of follicular atresia,revealing its regulatory mechanism is the core of studying follicular atresia.UFM1 is a newly discovered small molecule ubiquitin protein with a modification function similar to ubiquitin which is involved in the cell regulation process by covalently linking to the substrate protein through a three-step enzymatic reaction.This phenomenon is called Ufmylation modification.Recently,the Ufmylation modification system can participate in various biological regulation processes such as cell division,DNA damage and repair,as well as osteoblast development.When related enzymes and the modified substrates of Ufmylation suffer from damage of the structure or function,the cells will cause abnormal self-differentiation and proliferation,and eventually bring about apoptosis through the pathways such as ERS.However,relative to other tissues,there have been few reports on the functional studies of Ufmylation in the ovary.Here,it is used to clarify the Ufmylation pathway by these technologies such as immunohistochemistry,Real-time PCR,Western Blot,cellular immunofluorescence and FCM.To elucidate the role of UFM1,based on the definition of the Ufmylation pathway involved in the regulation of goat follicular atresia,further through gene overexpression and RNA interference,it is explained that the role of UFM1 in apoptosis of goat follicular granulosa cells and its correlation with oxidative stress and ERS pathway,which provides new ideas and theoretical basis for comprehensive understanding of the regulation mechanism of follicular development and atresia.The results were as follows:?1?In goat ovarian tissue,Ufmylation key protein UFM1 and substrate protein DDRGK1 were mainly localized in granulosa cells,oocytes and luteal cells,and expressed in follicles at different developmental stages.With luminal follicle atresia,the expression levels of UFM1 mRNA and protein were obviously increased,while those of DDRGK1were significantly decreased.When granulosa cells underwent early apoptosis,UFM1 and DDRGK1 proteins distribution were transferred from cytoplasm to nucleus,but UFM1 and DDRGK1 were not positively expressed in apoptotic bodies.These results indicate that the Ufmylation modification system is involved in the development and atresia of goat follicles and plays a role in regulating granulosa cell apoptosis.?2?ERS specific inducers Tg and Tm induced granulosa cell apoptosis,and the expression levels of UFM1,DDRGK1,UBA5,UFC1 and UFL1 mRNA in Ufmylation pathway were significantly increased,indicating that Ufmylation is involved in ERS-mediated granulosa cell apoptosis.H₂O₂-induced apoptosis had no significant effect on Ufmylation-related gene expression.?3?Overexpression of UFM1 had no obvious effect on proliferation and apoptosis of goat follicular granulosa cells,while interference with UFM1 inhibited granulosa cell proliferation and significantly induced granulosa cell apoptosis.After interference with UFM1,treatment with the addition of Tg and Tm showed that interference with UFM1reduced the tolerance of cells to Tg and Tm treatment,and cells showed apoptosis more rapidly.At the same time,after interfering with UFM1 expression,the expression levels of key genes IRE1?,XBP1,ATF4,ATF6,eIF2?,and PERK in ERS three pathways were significantly increased.Moreover,the expression level of apoptosis-related gene Bax/Bcl-2mRNA was also significantly up-regulated.In summary,Ufmylation is closely related to goat follicular development and atresia,and UFM1 plays an important regulatory role in inhibiting ERS-induced apoptosis of goat follicular granulosa cells..