The Effect Of Different Months Of Age On The Culture Of Preantal Follicle In Mice

Ovarian follicles with different developmental stages exist in different age mice.In this study,preantral follicles of 10 days old,1 month old and 2 months old mice were used as objects for study,and 10 days old mice were cultured as controls.Using the preantral follicle of control group as a model,the follicular culture conditions were optimized by 4 methods?the method that follicle embedding alginate gel bread,the method that the bottom of dish was covered by the agarose,microdrop culture and microdrop culture?adding 1%PVP??.The optimal culture method was used to culture the early preantral follicles in both the experimental group and the control groups.The morphology of the follicles was observed.The expression level of mitochondrial function related genes and nuclear gene?CytoC,12 sRNA,Tim23,Tom40,Pnpt1,Cox II,Sox2,C-myc?were quantified by qPCR and the E₂ expression was detected by ELISA.1.Development of ovary and follicle in mice The length of the ovary from the 10 days old,1 month old and 2 months old mouse are 1.5mm,1.9 mm and 2.6 mm,respectively.Compared with the control group,the number of preantral follicles in the ovaries of 1 month old and 2 months old mice decreased and the ovarian volume increased with age.2.Optimization of follicle culture system The follicle diameter increased at 1 d culture in which the follicles were embedded in alginate gel bread,but the difference was not significant compared with front day?p>0.05?.At 4 d,follicles diameter significantly decreased comparing with 0 d?p<0.05?,and follicles eventually became atresia.In the other method that the bottle of dish was covered by agarose,the diameter of follicle cultured for 1 d increased significantly?p<0.05?.Compared with the diameter of 0 d,the diameter of follicles at 5 d decreased significantly?p<0.01?.Some follicles extrude naked oocytes during culture.The result of droplet culture was consistent with the agarose method that the bottle of dish was covered by agarose.The MII oocytes normally expelled can be obtained by the combination of droplet method and addition of 1%PVP.3.E₂ levels and P450 gene expression levels in follicles when the follicles were cultured for 3 d,the level of P450 in 2 months old was significantly lower than that of 10 days old and 1 month old mouse?p<0.05?.There was no significant difference in the expression level of E2 between the experimental group and the control group in the early stage of culture?p>0.05?.However,when cultured for 9 days,the level of estrogen in follicles of 2 month old mice decreased significantly,which was significantly lower than that of control mice?p<0.05?.4.Mitochondrial function related genes and nuclear gene expression levels At 3 d of culture,the expression level of 12 s RNA in follicles of 1 month old mice was significantly higher than that of 2 months old mice and control group?p<0.01?;the expression level of Cox II in follicles of 1 month old mice was significantly higher than that of control group?p<0.01?;the expression level of pnpt1 in 2 months old mice was significantly lower than that of 1 month old mice and control group?p<0.05?.When follicles were cultured for 3d and 6 d,the expression levels of Tom40 in 2 months old mice were significantly lower than that of control group and 1 month old mice?p<0.05?and the expression levels of Cox II in the follicles of 2 months old mice were significantly lower than that of control group and 1 month old mice?p<0.01?.At 6 d of culture,the expression level of CytoC in 2 months old mice was significantly higher than that of control group mice?p<0.05?and the expression level of Tim23 and 12 sRNA in 1 month old mice were significantly higher than that of control group?p<0.01?.When cultured for 6 d and 9 d,the levels of 12 sRNA in follicles of 2 month old mice were significantly lower than that of control group and 1 month old mice?p<0.01?.When cultured at 3 d and 9 d,the expression level of Tom40 in 1 month old mice was significantly higher than that in control group?p<0.05?.When the follicle were cultured for 3d,6 d,and 9 d,the expression levels of Tim23 in follicles of 2 month old mice were all significantly lower than that of follicles in control group and 1 month old mice?p<0.01?.At 9d of culture,the expression level of Cox II in follicles of 1 months old mice was significantly higher than that of control group?p<0.01?.For the expression level of nuclear gene,the Sox2 gene of follicle in 1 month old mice was significantly higher than control group and 2 months old mice at 3 d?p<0.01?,while the expression level of C-myc in 2 months old mice follicle was significantly lower than that of 1 month old mice?p<0.05?.This study showed that compared with control mice,the number of preantral follicles in mice decreased with age.The follicle functional activity and cell proliferation ability of the 1month old mice preantral follicles were increased during the culture process,while the follicle development ability and follicular functional activity of 2 months old mice were reduced.The results of this study provide a reference for further research on the mechanism of age affecting the development potential of preantral follicles in mammals.