Imp2 Follicle Developmental Effects In Rats

Objective:The development of ovarian follicles is a complicated process,mainly containing the oocyte growth,granulosa cell proliferation,differentiation,apoptosis and other important developmental events.During this process,a variety of proteolytic enzymes are involved in the regulation of these important events.Lmp2 proteolytic enzyme is an essential functional subunit of proteasome complexes,which plays a vital role in the hydrolytic activity of proteasome complexes.There had littile research reproductive system about lmp2;particularly in the ovarian development,it's role has not yet been reported.Therefore,we explored lmp2 gene expression and its possible role in the process of follicular development.Methods:In this paper,HE staining,immunohistochemistry,apoptosis detection assay(TUNEL),primary granulosa cell culture in vitro and western blot are used to detect the expression of lmp2 in rat different developmental stage follicles and atresic ones induced by artificial treatment in vitro.Rats(21 days old) were injected with pregnant mare serum(PMSG),and ovaries were collected after PMSG injection for 12,24,36 and 48 hours.Additionally,rats were treated with a 24-hour injection of anti-PMSG antibodies on the basis of PMSG injection for 24 hours,and ovaries were collected for paraffin sections;adjacent serial sections were selected for hematoxylin-eosin(HE) staining to detect the morphology of different developmental stage follicles and atresic ones induced in vitro after PMSG treatment;the expression of lmp2 was determined by immunohistochemistry;TUNEL apoptosis assay was used to clarify the relationship of lmp2 expression and ovarian follicle atresia as well as follicular granulosa cell apoptosis.In addition, follicles containing different developmental stage and atresic ones were punctured with needles, granulose cells were separated with cell mesh sieve 400 under the same condition in vitro. Granulosa cells were split with NP40 lysis buffer to collect whole cell proteins;the expression of lmp2 at different developmental stage and atresic follicle was determined by western blot.The results showed:The expression of lmp2 was variant in the rat ovarian follicle of different development stages Higher expression of lmp2 can be found at the primary follicle stage and lower level of lmp2 was seen in large antral follicles.In according with the immunohistochemitry results,TUNEL assay showed that there were high levels of lmp2 in follicles with death signal;there were a stronger TUNEL signal and a high correspondence between the lmp2 expression location and death signal in the atresic follicles induced in vitro;on the other hand,the western blot results showed that there was a higher lmp2 level in the early follicle,also called primary follicle and a relatively lower expression of lmp2 at antral follicle stage.Conclusion:Therefore,the results of western blot and immunohistochemistry demonstrated that there was a similar expression pattem the lmp2 in different developmental stage follicles.Diseussion:In this paper,we explored lmp2 whether and how to play a role in ovary,and made a depth research the first time.After exploring the theoretical study and experimental validation,results showed that lmp2 had significant different expression in the developing ovarian follicles.This shows that lmp2 may be involved in follicular development, atresia,apoptosis process.In the process of follicular development,lmp2 may be caused by apoptosis of granulosa cells induced follicular atresia and apoptosis.These results suggest that, lmp2 may become a new target in the treatment of ovarian-related diseases area.It may become the provision of the relevant clues in ovarian pathogenesis-related diseases in subsequent studies.