The Effects Of PLIN3 On Lipids Droplets And The PGC7 Dynamic Pattern On The Maturation Of Pig Oocytes In Vitro

The in vitro maturation(IVM)of pig oocytes is used in many biotechnologies,which is one crucial step for the embryos production in vitro.And oocytes maturation includes nuclear,cytoplasmic and epigenetic maturation.However,many factors can affect the maturation of oocytes,such as the microenvironment,the components of maturation medium,the communication of oocytes and cumulus cells(CC),the accurate regulation of some maternal factors and so on.Enormous lipids droplets(LDs)were noticed in pig oocytes which plays an important role during maturation.PLIN3 is a protein which is related to the metabolism of lipids.This study was designed to detect the dynamic pattern of PLIN3 in pig oocytes and CC during IVM,and to determine the possible relationship between PLIN3 and LDs.Experiments were carried out with different experimental groups that were denuded oocytes(DO)/CC independently(DCC),DO and CC together(which named DTO and DTCC),COC wholly(CEO).In addition,PGC7 as an maternal factors,is related to chromatin condensation and can affect epigenetic modification.Therefore,we observed the dynamic pattern of PGC7 and its related specific epigenetic changes,hence to explore the influence of epigenetic modification on the maturation ability of pig oocytes in vitro.The role of PLIN3 on pig oocytes maturation in vitro(IVM).This study was designed to investigate the possible function of PLEN3 on the oocytes maturation.Results showed that the LDs were larger in the 10 and CEO groups compared to the DO groups.Most of the LDs in the 10 and CEO groups were in the cytoplasm but not around the nucleus,however in the DO group,the LDs were distributed evenly in the cytoplasm.These results indicate that the LDs distribution in oocytes underwent dramatic changes and was sensitive to the CC.In common oocytes,PLIN3 coated the LDs,however in 10 and CEO groups,the PLIN3 protein was also observed around the nucleus over where few LDs could be observed.Although PLIN3 could be detected in the cytoplasm of CCs,but the relationship between PLIN3 and LDs is not clear.It indicated that PLIN3 not only presented on the surface of LDs,but also in the other place of cytoplasm.Our results further confirmed that the pig follicular fluid(PFF)in IVM medium had no effects on PLIN3 mRNA expression of maturated oocytes.The PLIN3 mRNA and protein level in maturated oocytes was significantly lower than immature oocytes(P<0.05),and the triglyceride(TG)content in oocytes was also decreased significantly after IVM(P<0.05).But no significant difference among the maturated oocytes cultured in CEO and DO was observed.The expression of PLIN3 in CC cultured in vitro was increased significantly,while the expression of PLIN3 mRNA in DCC was also at the highest level.It might indicate that the LDs metabolism was different in oocytes and CC.The gap junction affected PLIN3 expression levels in CCs but not in oocytes.The PLIN3 expression acted in parallel with TG accumulation in oocytes.Hence,the PLIN3 seems to play a role in the synthesis of TGs and LDs metabolism.The role of maternal factor PGC7 and epigenetic modification on pig oocytes maturation.Whether the maternal factor PGC7 will affect the maturation of pig oocytes?Our results firstly showed that the expression level of PGC7 and OCT4,DNA methylation,histone H3K4 and H3K9 methylation,H4K12 and H4K16 acetylation in PB1 was higher than those in their nucleus of the maturated oocytes at MII stage.When the CB was added in the IVM medium for 24 h,the mRNA expression of PGC7,OCT4,DNMT1,DNMT3b,TETI,TET2 and TET3 could be inhibited(P<0.01),however,no significant difference of the expression could be observed any more on these genes after 45 h.Most interest to us that with CB treatment,the PGC7 and OCT4 didn't express intensively in chromosome.In the condensed chromosome,the level of H3K9me2 and H4K12ac were higher after the CB treatment.It might indicate that PBI extrusion could be affected by the status of PGC7 and further by the epigenetic changing.With TSA treatment,PB1 extrusion was also inhibited,and the expression of PGC7,OCT4,DNMT1,DNMT3b,TET1,TET2 and TET3 were also accumulated during IVM on the whole as the control group.The dynamic pattern of histone acetylation was disturbed with TSA.Compared to the control group,the acetylation signal of H4K12 was higher in the condensed chromosome after IVM 24h.With TSA treatment,the histone methylation(H3K9me2 and H3K27me3)could be detected all the time and there was no demethylation.In TSA treatment,no difference was observed on the DNA methylation.However,the PGC7 and OCT4 expressed lower in the chromosome after TSA treatment 36h and 45h.It might indicate that the epigenetic modification will affect the expression and distribution pattern of PGC7 and OCT4,and further inhibit the PB1 extrusion.In conclusion,PGC7 will play an important role for the extrusion of PB1 during pig oocytes maturation.