| Both the intracellular and extracellular calcium plays important roles in female reproductive process.The molecular identification of the extracellular calcium sensing receptor-CASR has opened the possibility that extracellular calcium could serve as the first messenger outside cells.The present study was designed to investigate the role of CASR in porcine oocyte maturation,activation and early embryo development.Firstly,we explored the function of CASR in porcine oocytes in vitro maturation.The western blot and immunofluorescence results showed that CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium.Cortical distribution of CASR was enhanced with gonadotropins but not EGF(Epidermal Growth Factor).Supplementation of a CASR agonist(NPS R-568)in the gonadotropin(FSH(Follicle stimulating hormone)and/or LH(Luteinizing hormone))-containing maturation medium significantly enhanced oocyte nuclear maturation(73.80±1.55 vs 65.20±1.15;67.57±2.73 vs 59.00±1.00;79.05±0.76 vs 66.15±0.71;p<0.05).Addition of NPS2390,a CASR antagonist,compromised oocyte nuclear maturation.Furthermore,a higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist,NPS R-568.MAPK3/1 phosphorylation was increased during in vitro maturation.Taken together,our data showed that the CASR,as a gonadotropin regulated factor,which promotes porcine oocyte maturation through MAPK3/1 signaling pathway.The second study focused on the function of CASR in porcine oocyte activation process.Western blot analysis showed that CASR expression was markedly upregulated following oocyte activation.The addition of a CASR agonist to the medium significantly enhanced pronuclear formation rates,while addition of a CASR antagonist compromised pronuclear formation.To investigate whether these effects were Ca2+-mediated,activation was performed in intracellular Ca2+chelator BAPTA-AM and NPS R568 containing medium.BAPTA-AM abolished the action of the CASR mediated response in oocyte activation.The presence of KN-93,an inhibitor of CaMKII,significantly reduced CASR expression during oocyte activation.Furthermore,CASR activation increased the levels of the active p-CaMK?,supporting an interaction between CaMK? activation and CASR upregulation.Altogether,these findings indicating the interaction between CASR and CaMK? during oocyte activation,which is involved in porcine oocyte activation.The aim of the third study was the extracellular calcium effector called CASR with its expression in porcine gametes and embryos,and with its function during fertilization and early embryo development.By using reversed transcription polymerase chain reaction(RT-PCR),CASR was found to be expressed in porcine gametes and embryos at different developmental stages.Functionally,supplementation of a CASR agonist or an antagonist during in vitro fertilization(IVF)and in vitro culture(IVC)medium was tested.During fertilization,the presence of CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilization rate.During embryo development,agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group.It was concluded that CASR,as the effector of extracellular calcium,modulates porcine fertilization and early embryo development.Taken together,CASR is a gonadotropin regulated factor that promotes porcine oocyte maturation in a MAPK3/1-dependent manner.There is an interaction between CASR and CaMK? during oocyte activation,and the cooperation between them regulated oocyte activation.CASR as the effector of extracellular calcium modulates porcine fertilization and early embryo development. |
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