The Effect Of Upp Inhibited On The Gametes Ubiquitination Level And Porcine Sperm Oocyte Binding Ability

The phenomenon of polyspermy was easy to occur during swine in vitro fertilization(IVF),which resulted in lower embryo development rates and lower birth rates.Studies have shown that the ubiquitin proteasome pathway(UPP)plays an important role in the process of IVF ascidians and mammals,and during sperm and oocyte binding it able to degrade Zona pellucida(ZP)proteins to help sperm penetrate the ZP and make successfully fertilize.UPP is the major protein degradation pathway in living creature,it relys on ATP to provide the energy,it can regulate the homeostasis and degrade misfolded and redundant proteins,and it relate to a variety of the human genetic diseases,for example,cancer,tumour,neurological diseases and other diseases.Its main execution process is known as the ubiquitination(Ub)degradation pathway.The it is regulated by El,E2,E2 and deubiquitinating(DUBs)enzymes.It has also been shown that UPP plays an important part in spermatogenesis and oogenesis.But so far,the origin of the Ub protein during the formation of two gametes in IVF,and the role of Ub in the process of sperm and oocyte binding in swines was not clear.Therefore,in this experiment we have studied the Ub protein content in mature sperm,the seminal plasma of mature sperm,immature sperm and the seminal plasma of immature sperm,and Ub protein content in follicular fluid of different diameters and oocyte maturation.Added Inorganic phosphate(PPi),E2 and DUBs inhibitors to the oocyte maturation medium and IVF medium,than to test the Ub degree in sperm capacitation and acrosome reaction(AR),the effects of oocyte maturation rates,and Ub degree of oocytes and polyspermy.To detect the contents of Ub in the different periods corpus luteum and cumulus cells.In addition,PPi was added in boar sperm preservation solution,to detect the influences of the Sperm biological indexs and the Ub related proteins.The results shown that:(1)The concentration of 50 ?M PPi was added to the boar sperm preservation solution can significantly improve the sperm biological index and the protein content of Ub,acrosin inhibitor(AI)and PSMD4 during sperm storage(P<0.05);(2)The content of Ub protein in mature sperm was significantly higher than that in the seminal plasma of mature sperm and immature sperm(P<0.05),there was no Ub protein in the seminal plasma of immature sperm.The content of Ub in follicular fluid of 3-6 mm in diameter was significantly higher than that in 1?3 and above 6 mm(P<0.05).The content of Ub protein in middle luteal phase was significantly higher than that in early and late luteal phase(P<0.05).There was Ub protein in granulosa cells;(3)Different concentrations of E2 and DUBs inhibitors were added to swine sperm capacitation solution,when the concentration reached 10 ?M can significantly reduce the content of Ub,AI and PSMD4 protein(P<0.05).(4)After culturing the oocytes maturation in follicular fluid of different diameters,the WB results showed 3 distinct bands in maturation oocytes,the oocytes maturation rates and Ub level in diameter 3?6 mm was significantly higher than those in the other two groups(P<0.05).Different concentrations of PPi,E2 and DUBs inhibitors were added into swine oocyte IVM medium,the concentration of 1 ?g/mL PPi could significantly improve the oocytes maturation rates(P<0.05).However,the contents of Ub protein was significantly lower than that in control group and other treatment groups.The addition of 10 ?M E2 inhibitor could significantly reduced the oocyte maturation rates and Ub protein contents(P<0.05).With the increase of concentration,the oocyte maturation rates and the contents of Ub protein decreased gradually.The addition of 10 ?M DUBs inhibitor could significantly reduce the oocyte maturation rates and Ub protein contents(P<0.05),with the increase of concentration,the oocyte maturation rate and the content of Ub protein decreased gradually.(5)Different concentrations of PPi,E2 and DUBs inhibitors were added to the IVF medium,the concentration of 1 ?g/mL PPi could significantly increase the ZP hardening times(P<0.05),and significantly reduce the polyspermy rates(P<0.05).The concentration of 10 ?M E2 inhibitor could significantly increase the ZP hardening times(P<0.05),and significantly reduce the polyspermy rates(P<0.05).The concentration of 10 ?M DUBs inhibitor could significantly increase the ZP hardening times(P<0.05),and significantly reduce the polyspermy ratess(P<0.05)Therefore,addition of 50 ?M PPi to the sperm preservation solution could significantly improve the efficiency of sperm preservation,addition of concentration 10 ?M E2 inhibitor and 10 ?M DUBs inhibitor could significantly reduce the Ub and AI content after sprem capacitation and AR,addition of 1 ?g/mL PPi could significantly increase the oocyte maturation rate,significantly reduced the Ub protein content and the polyspermy rate,addition of 10 ?M E2 inhibitor could significantly increase the oocyte maturation rate,significantly reduced the Ub protein content and the polyspermy rate,addition of 10 ?M DUBs inhibitor can significantly reduce the oocyte maturation rate,the Ub protein content and the polyspermy rate.At the same time,it also shown that UPP plays a very important role in procine spermatogenesis,oocyte maturation and IVF.It is formed in the process of sperm maturation and oocyte maturation.