Changes And Roles Of Histone Acetylation During Fetal Oocytes Attrition In Mouse

The quantity and quality of germ cells is critical to the reproductive capacity of mammals.The number of female oocytes is very limited.More than half of the oocytes are eliminated before birth,and only a few oocytes are activated and mature at the time of animal estrus.When the oocyte is activated or lost,the ovary cannot replenish the new oocyte,so the reproductive efficiency and reproductive age of the female are greatly limited.However,the causes and mechanisms leading to a continuous and rapid decline in the number of oocytes have not been clear so far,and there is less research work about it.Epigenetic modification plays a crucial role in embryonic development.Histone acetylation is involved in the process of germ development,migration and differentiation as an important modification in epigenetics.This study sought to establish a method for in vitro culture of fetal gonads of mouse.By adding histone deacetylase inhibitor to the culture medium,the whole tissue immunofluorescence 3D imaging technique was used to study the oocyte regression phenomenon and the changes and roles of histone acetylation during fetal ovary development,and reveals its possible mechanism of action.1.Establishment of in vitro culture method for fetal gonads of mouseSince the germ cells in both male and female gonads are migrating and differentiated from primordial genn cells,in order to study the process of oocyte regression,the whole tissue immunofluorescence 3D imaging technique was used to observe the gonadal development and analyze the distribution and quantity of germ cells in vivo.Establishing an in vitro culture method for fetal gonads to study the optimal system.Testicle as a control,The gonads of mice of different gestational ages were collected,the germ cells were labeled with the specific antibody Tra98,and the distribution of germ cells in the gonads was determined by laser confocal microscopy.The 3D model was used to generate the number of germ cells by Imaris software On this basis,fetal mouse gonad kidney complex at 13.5 dpc was collected and cultured in four different in vitro condition,which are ovarian adherent culture,ovarian mid-coastal adherent culture,ovarian mid-semi-suspension culture and ovarian semi-suspension culture.The development of gonads in vitro culture was observed,and the distribution and number of Tra98-labeled germ cells were observed and counted by whole tissue immunofluorescence after different time of culture.The results showed that the germ cells in the fetus ovary were evenly distributed and the number decreased significantly with the increase of gestational age,while the germ cells in the fetus testis were tubular distribution,but the number could not be accurately counted.In vitro culture,ovarian semi-suspension culture is the best culture condition.The morphology and structure of ovary and the distribution and quantity of germ cells in vitro culture are basically consistent with the ovary in vivo.However,the morphology of testis in vitro is inconsistent with the testis in vivo,and germ cells are not tubular distribution.This suggests that the semi-suspension culture is suitable for the ovary,but may not be suitable for the testis,or that other conditions are required for in vitro culture of the testes.2.Changes and roles of histone acetylation during fetal oocytes attritionHistone modification plays an important role in embryo and germ cells development.The levels of histone acetylation(H4K8ac and H4K12ac)and histone deacetylase(HDAC1)in gonads of different gestational ages were analyzed by immunoblotting.On this basis,by added into the culture medium,the effect of histone deacetylase inhibitor(TSA)on oocyte regression was studied by whole tissue immunofluorescence 3D imaging technique,and the change of histone acetylation was analyzed by immunoblotting.The results showed that the levels of H4K8ac and H4K12ac in the male and female gonads decreased significantly with increasing gestational age,but the levels of HDAC1 remained constant.Under in vitro culture conditions,TSA has a significant effect on the ovary by a dose-dependent.High concentrations of TSA(50-1000 ng/ml)produce significant toxicity,and fetal ovary stops developing and shrinks.While the morphology of the fetal mouse ovaries treated with low concentration of TSA(10 ng/ml)was not significantly different from that of the control group.Further analysis showed that the number of germ cells in the ovary after treatment with low concentration of TSA was significantly lower than that of the control group,while H4K8ac and H4K12ac were significantly increased,but there was no significant difference in HDAC1.This suggests that TSA inhibits the decline of H4K8ac and H4K12ac in the gonads,which in turn accelerates the trend of germ cell decline.In summary,ovarian alone in semi-suspended culture can have higher replication of the gonads in vivo,and the decrease in histone acetylation is associated with regression of oocytes.while TSA inhibits the decrease in histone acetylation and accelerate the process of fetal oocytes attrition.