Mechanism Study On The Role Of Autophagy During Primordial Follicle Formation In Perinatal Mouse Ovary

The reproductive life of female mammalian is decided by the number of follicles in the ovary,which is correspondingly decided by the establishment of primordial follicle during the perinatal stage.Because there are about two thirds of oocytes died during the formation of primordial follicle.The reason that caused germ cell loss and the machanism underlying the disintegration of died cells in perinatal ovaries are becoming the key research contents in reproductive and developmental biology.The loss of germ cell in perinatal mammalin ovaries involves many biological process,such as cellular signaling pathway and epigenetic regulation.Autophagy is an evolutionarily conserved process in eukaryocyte,which disintegrats and transports cellular elements.During which,the damaged proteins and organelles are encircled by a double layer autophagic vacuole to form the autophagosome,and then the autophagosome combines with lysosome to disintegrate the contents for the nutrient cycle.Research in recent years found that autophagy plays an improtant role during the development of germ cell in mammals.But the detailed mechanism remains unkown whether autophagy participates in the establishment of primordial follicle during the perinatal stage.Then,this resrach focused on the mechanism of germ cell loss and the role of autophagy during formation of primordial follicle,using perinatal mouse ovaries.By adding the inducer or inhibitor of autophagy and RNAi,as well as technology of RT-PCR,Westen Blot,IHC and TEM,we denonstrated that autophagy plays a role in the formation of primordial follicle,reducing the death of oocytes.Which provides theoretical basis for the research on female mammalin germ cell development,and helps us to investigate the function of autophagy during primordial follicle formation.And our main results are as follows:1.Great loss of oocyte in perinatal mouse ovary couples with autophagyThere is a great loss of oocyte in mammalian perinatal ovary.For example,in human,20 th weekfemale fetus has 6 – 7 million germ cells,whereas only 2 – 3 million left after birth.We checked the development of oocyte in 17.5 dpc,0 dpp,3 dpp and 5 dpp ovaries and found that there was a about two-thirds oocyte loss in 3 and 5 dpp ovaries,compaired to 17.5 dpc ovaries,proving the great loss of oocytes.Through TEM ovservation,we found that a great number of oocytes undergo degradation and ipidation in their nucleus,furthermore,oil red O staining and detection of CPT1 A in 17.5 dpc – 4 dpp mouse ovaries indicating active fatty acid metabolism.We also found the existence of autophagosome and autolysosome in 1 dpp –3 dpp mouse ovaries,as well as the expression of BECLIN1 and LC3 B.All of these results demonstrated that there is a great germ cell loss during the formation of primordial follicle in mouse perinatal ovary,and autophagy may play a role in this process.2.Autophagy influences the formation of primordial follicle in mouse perinatal ovaryTo investigate the role of autophagy during the development of primordial follicle in mouse perinatal ovary,we isolated 17.5 dpc mouse ovaries and cultured them for 2 days,then added rapamycin(a inducer of autophagy)or 3-MA(an inhibitor of autophagy)for the further culture.We found that ovaries that under rapamycin treatment for 3 days showed a remarkable delay in germ cell cyst breakdown compared to control group.After 5 days treatment,rapamycin adding group formed more primordial follicles with higher expressions of follicle-development-relevant genes compared to control group,whereas 3-MA adding group showed an opposite result.We also performed starvation on newborn mouse and found that starvation treated ovaries have smaller size in diameter and weight.What’s more,starvation treated ovaries showed a higher level of autophagy and a delay in germ cell cyst breakdown,as well as a similar number of oocytes in 7 dpp and 21 dpp ovaries compared to the control group.These results proved that autophagy depressed the germ cell cyst breakdown during the formation of primordial follicle,resulting more oocytes survived.3.Mechanism analysis of the role of autophagy during primordial follicle formation in mice ovaries.To investigate the mechanism that autophagy influence the formation of primordial follicle,we detected the expression of LC3 B and found that there was a migration for LC3 B expression.In germ cell cyst,LC3 B was expressed in the oocyts,whereas in follicles,LC3 B was only expressed in granular cells,but not in the oocytes.This migration is in accordance with the nutrition supply for the survived oocytes.We cultured mouse perinatal ovary in vitro adding rapamycin or 3-MA and found that ovaries that under rapamycin treatment showed a remarkable decrease in oocyte death,whereas 3-MA adding group showed an opposite result.We also added EX527(an inhibitor of Sirt1)or performed RNAi for Sirt1 in cultured mouse ovaries and found Sirt1 suppressive group show a decreased level of autophagy and less primordial follicles survived after 5 days treatment compared to control group.These results demonstrated that autophagy activity is n accordance with the nutrition supply for the survived oocytes,and an increased level of autophagy decreased oocyte death during primordial follicle formation,this process maybe regulated by Sirt1-mediated epigenetic regulation.Our research provides new insights for revealing the mechanism of autophagy influence on primordial follicle formation in mammalian perinatal ovary,meanwhile supplies theoretical and technical support in promoting the efficiency of livestock production,as well as postponing woman menopause.