Study On The Expression And Functions Of ZO-1/2 In Porcine Follicular Development And Oocyte Maturation

Zonula occludens(ZO)-1 and ZO-2(ZO-1/2),also called tight junction protein(TJP)-1 and TJP2,belong to a family of membrane-associated guanylate kinase homologs(MAGUKs)and are involved in epithelial polarity maintenance,gene transcription,cell proliferation and tumor cell metastasis.Studies have found that downregulating their expression was harmful to the early embryonic development of mouse,indicating that the two genes were involved in regulating embryonic development,but ZO-1/2 has been less studied in livestock,especially the expression and function of them in the follicular development and maturation of oocytes are still unclear.Therefore,in this study,pig was used as model animal,the expression patterns of ZO-1/2were systematacially studied during follicular development and oocyte maturation,in further,their roles and molecular mechanism during follicular development and oocyte maturation were explored by overexpression or sh RNA technology.The study enriches the understanding of the biological functions of ZO-1/2 and provides evidences for elucidating the molecular mechanism of follicular development and oocyte maturation in livestock.1.Expression levels of ZO-1/2 in porcine follicles of different sizes and their actions in the proliferation of granulosa cellsThe Real-time fluoreacent quantitative PCR(qRT-PCR)and Western Blot methods(WB)were applied to analysis the expression patterns of ZO-1/2 in different tissues of pig.Results showed that ZO-1/2 were expressed in pancreas,brain,heart,liver,lung,kidney,bladder,small intestine,ovary and testis tissues,and their expression in testis tissue was significantly higher than that in ovarian tissue(P<0.05).The expression analysis results of ZO-1/2 in porcine follicles of different diameters showed that they were expressed in porcine follicles of different sizes,and the expression trends of RNA and protein levels were consistent.The expression of ZO-1 in granulosa cells(GCs)of small follicles(<2 mm)was significantly higher than that of large follicles(5?8 mm),and the expression of ZO-2 in GCs of small follicles was significantly higher than that of healthy medium(3?5 mm)and large follicles.The expression of ZO-1/2 in GCs of atresia medium follicles was significantly higher than that of healthy medium and large follicles(P<0.05),while there was no significant difference compared with that of small follicles(P>0.05).The expression of ZO-1/2 in cumulus oocyte complexes(COCs)was significantly higher than that of GCs in the same size follicles(P<0.05),their expression in COCs of small follicles was significantly higher than that of medium follicles and large follicles(P<0.05),and the expression of COCs in atretic follicles was significantly higher than that in healthy follicles(P<0.05).The ZO-1/2 lentiviral expression vectors were constructed,and their effects on the proliferation of porcine GCs cultured in vitro(IVC)and the expression of cell cycle and apoptosis-related genes were explored.The results showed that the proliferation of GCs was reduced significantly by overexpression of ZO-1/2(P<0.05).Up-regulating the expression of ZO-1 increased Occludin expression in GCs(P<0.05),decreased the expression of Cyclin A2 and Cyclin B(P<0.05),while had no significant effect on Cyclin D1 expression(P>0.05).The expression of Cyclin A2 and Cyclin D1(P<0.05)in GCs by up-regulating ZO-2expression was significantly decreased,while the expression of Cyclin B and Occludin did not(P>0.05).In addition,overexpression of both ZO-1 and ZO-2did not affect the expression of Bcl-2 and Bax(P>0.05)in GCs,while the expression of Caspase3 and Caspase9 was significantly up-regulated(P<0.05).2.Study on the function of ZO-1/2 in porcine oocytes maturation in vitro and early embryonic developmentThe qRT-PCR and WB methods were applied to analyze the expression patterns of ZO-1/2 during porcine oocyte maturation in vitro(IVM)and early embryonic development.The results showed that ZO-1 and ZO-2 were expressed in cumulus cells(CCs),oocytes and early embryos,while ZO-1?⁺was expressed only in CCs,morulas and blastocysts.The abundance of ZO-1/2 in germinal vesicle(GV)stage oocytes was significantly higher than that in CCs(P<0.01),they had the highest RNA expression level in COCs at GV stage,and the highest protein expression level at 18 h.The effective sh RNA fragments of ZO-1/2 were screened out and lentiviral expression vectors were constructed to study the effects of downregulating ZO-1/2 expression on porcine oocyte IVM and early embryonic development.The qRT-PCR results showed that the addition of ZO-1/2sh RNA lentivirus to the maturation medium significantly reduced the expression of endogenous ZO-1/2in MII stage oocytes and CCs,as well as in the blastocysts(P<0.05).Compared with the untreated groups,cumulus expansion,oocyte nuclear maturation and subsequent cleavage were not influenced by infection of COCs with ZO-1-sh RNA1,ZO-2-sh RNA2 or combined ZO-1-sh RNA1 and ZO-2-sh RNA2lentivirus(P>0.05).The migration of oocytes cortical granules was significantly inhibited by ZO-2sh RNA2 but not ZO-1-sh RNA1,and the blastocyst rate of parthenogenetic embryos was reduced by ZO-1-sh RNA1 but not ZO-2-sh RNA2.The total cell number in blastocysts was decreased by down-regulation of ZO-1/2(P<0.05).Inhibition of ZO-1/2 also decreased(P<0.05)the expression of Cx43,Cx45,PTX3 and PTGS2 in CCs,Cx45,BMP15,ZP3 and C-KIT in oocytes after IVM,as well as Nanog in blastocysts(P<0.05)with the exception of HAS2 expression in CCs and Oct4 expression in blastocysts(P>0.05).3.Study on regulating mechanism of ZO-1/2 expression during porcine follicular development and oocyte maturationFirstly,the effects of PMSG/h CG or MG132 on the expression of ZO-1/2during IVM were analyzed by qRT-PCR or WB methods.The results showed that the PMSG/h CG contained in the maturation medium did not affect the expression of ZO-1/2(P>0.05),and the addition of 5?M MG132 significantly up-regulated the RNA and protein expression levels of ZO-1/2 at 22 h of IVM(P<0.05)Next,the effects of estradiol(E₂)on porcine oocyte IVM,ZO-1/2expression and gap junction were explored.The results showed that low concentrations of E₂ did not affect porcine oocyte nuclear maturation(P>0.05),when the concentration was greater than 100?M,the oocyte nucleus maturation was significantly inhibited(P<0.05).Calcein-Red staining results showed that E₂ treatment promoted gap junctional intercellular communication(GJIC)at 24h of IVM.The qRT-PCR results showed that E₂ treatment significantly up-regulated the expression of Cx43,Cx45,ZO-1 and ZO-2 in COCs at 24 h of IVM(P<0.05),but did not affect that in oocytes(P>0.05).Finally,the effects of E₂ on the proliferation of porcine IVC GCs and ZO-1/2 expression were detected.The results showed that when the concentration of E₂treatment was greater than 50?M,the proliferation rate of GCs decreased significantly(P<0.05),while treatment with estrogen receptor antagonist ICI182780 had no significant effect(P>0.05).Under the culture condition containing 10%FBS,E₂ concentration of 10?M significantly down-regulated the expression of ZO-1/2 in GCs,and concentration of 100?M significantly up-regulated that in GCs(P<0.05),while the combination treatment of 100?M ICI182780 and 100?M E₂ did not affect that in GCs(P>0.05).In conclusion:(1)the expression of ZO-1 and ZO-2 in porcine follicles showed a decreasing trend with increasing of diameter,and the expression of ZO-1/2 in atretic follicles was higher than that in healthy follicles;(2)Overexpression of ZO-1/2 inhibited the proliferation of GCs,down-regulated the expression of cell cycle-related genes and up-regulated the expression of apoptosis-related genes;(3)The expression patterns of ZO-1 and ZO-2 in porcine oocytes during IVM were similar,and inhibiting the expression of ZO-1/2 would reduce the efficiency of porcine oocyte maturation and early embryo development;(4)During the maturation of porcine oocytes,the expression of ZO-1/2 is regulated by the ubiquitin-proteasome pathway and estrogen.