| Follicle is the basic structure and function unit of ovary,which main function is ovulation and hormone secretion.The excessive apoptosis of granulosa cells would induce follicular atresia and reduce the frequency of estrus of female animals.Thus,excessive apoptosis of granulosa cells could affect female animal productivity.Histone methylation is an important epigenetic regulation,which regulates the activity of gene transcriptional by altering the binding ability between histone and DNA.In this research,to explore how H3K4me3 affect the mechanism of porcine follicular development,porcine granulosa cells(p GCs)were first treated with H3K4me3 inhibitor(BCl-121)and agonist(PBIT).Firstly,setting p GCs as cell model,Ed U assay,CCK-8 kit detection,Annexin V-FITC flow test,Caspase3/ 7 kit detection,Western Blot,and other experimental techniques were applied to investigate effects of H3K4me3 on cell proliferation,apoptosis and cycle.Subsequently,Ch IP-Seq was performed on 3～5 and 7～10 mm follicles.RBP1 and RSPO3 genes were screened for which promoter regions held significant difference enrichment of H3K4me3.to explore the effect of RBP1/RSPO3 gene on p GC function,overexpression vectors of RBP1/RSPO3 gene were constructed and interference fragments were designed.Finally,to explore the effect of H3K4me3 on concentration of gonadal axis in mice serum,H3K4me3 inhibitor and agonist were intraperitoneal injected.The main results of this study are as follows:(1)The content of H3K4me3 in large,medium and small follicles is different.Increasing the level of H3K4me3 in p GCs could promote the proliferation of p GCs,inhibit the apoptosis of p GCs,reduce the proportion of p GCs blocked in G0/G1 phase,and promote the secretion of E2 of p GCs.(2)RBP1/ RSPO3 could significantly promote the p GCs proliferation and inhibit the p GCs apoptosis.(3)Compared with the control group,inhibiting the H3K4me3 level could decrease serum Gn RH of 42 days old mice and serum LH and FSH of 35 and 42 days old mice.Promoting the level of H3K4me3 could increase serum Gn RH of 42 days old mice and serum E2 of 35,42 and 49 days old mice.When the mice reach body maturity(49 days old),inhibiting or promoting H3K4me3 level would not significantly affect serum Gn RH,LH and FSH.These results revealed that H3K4me3 level could affect the proliferation,apoptosis and cycle of p GCs.H3K4me3 could promote the transcription of RBP1/ RSPO3,while RBP1/ RSPO3 could significantly promote the proliferation and inhibit apoptosis of p GCs.H3K4me3 could affect the concentration of gonadal axis in mice serum.This study laid a foundation for understanding the mechanism of H3K4me3 and H3K4me3-RBP1/RSPO3 regulating the development of mammalian ovarian follicles. |
