| In vitro maturation(IVM)of mammalian oocytes is one of basic and key technology in embryo engineering technology,such as somatic cell nuclear transfer and production of transgenic animals.Although IVM of the bovine oocytes has been established and made great progresses,the IVM efficiency of bovine oocytes was still lower than that in vivo due to the environmental difference exist in vivo and vitro.CK1 is a key kinase in cell mitosis and Wnt/?-catenin signaling pathway,which affects cell proliferation and differentiation.In the present study,the effect of CK1 inhibitor D4476 on efficiency of bovine oocytes IVM and its underlying mechanism were investigated.The results we got will provide a theoretical basis for the bovine oocytes IVM system.Firstly,the effects of D4476 on cumulus cells expansion,the efficiency of bovine oocytes IVM and subsequent embryonic development competence were explored.The cumulus oocyte complexes(COCs)were cultured in the maturation medium containing different concentrations of D4476(0,2,5,10,20 ?mol/L)for 24h,and the results showed that 2 ?mol/L and 5 ?mol/L D4476 treated groups could not significantly affect the average scores of cumulus cells expansion compared to the control group(2.57,2.67 vs 2.68,P>0.05),while the average score of the cumulus cells expansion in 10 ?mol/L and 20 ?mol/L D4476 treated groups was significantly lower than that in the control group(1.92,1.26 vs 2.68,P<0.05).We also found that the maturation rate in 5 ?mol/L and 10?mol/L D4476 treated groups were significantly higher than that in the control group(70.15%,73.94%vs 53.65%,P<0.05),while there existed no significant difference in the maturation rate with 2 ?mol/L and 20 ?mol/L D4476 treated groups compared to the control group(62.77%,67.85%vs 53.65%,P>0.05).Then the treated oocytes were fertilized in vitro and the results revealed that there existed no significant difference in the cleavage rate in 2 ?mol/L and 5 ?mol/L D4476 treated groups compared to the control group(70.90%,73.52%vs 69.43%,P>0.05),however,the cleavage rate in 10 ?mol/L and 20?mol/L D4476 treated groups was significantly lower than that in the control group(59.17%,48.57%vs 69.43%,P<0.05).We also found that the blastocyst rates in 2 ?mol/L,5 ?mol/L and 10 ?mol/L D4476 treated groups were significantly higher than that in the control group(20.05%,28%,19.72%vs 15.67%,P<0.05),but there existed few influence in the blastocyst rate after 20 ?mol/L D4476 treated group compared to the control group(14.71%vs 15.67%,P>0.05).Secondly,the under-lying mechanism of D4476 on bovine oocyte during IVM was investigated.The results of quantitative real-time PCR(QPCR)analysis and PI staining revealed that after the oocytes were treated with 5 ?mol/L D4476,the expression of CK1 was decreased significantly(P<0.05),however,the nucleus morphology of oocyte was not significantly affected.The QPCR results also showed that compared to the control group,there was no significant difference in the expression of Wnt signal related genes ?-catenin and Cx43 in oocyte with 5 ?mol/L D4476 treated group(P>0.05),while the expression of TCF-4 in oocyte was significantly up-regulated(P<0.05).After with the COCs were treated with 5 ?mol/L D4476,the expression of the apoptosis gene Bad in cumulus cells was not affected significantly(P>0.05),but the expression of Wnt signal related genes ?-catenin,Cx43 and TCF-4,the proliferation genes CTSB and MAPK,the expansion genes PTGS-2,PTX-3 and TGS-6,apoptosis gene Bax,and anti-apoptosis gene Bcl-2 were all significantly up-regulated in cumulus cells(P<0.05).Western Blot analysis also revealed that the Cx43 protein in cumulus cells was decreased during IVM and increased after the maturation when 5 ?mol/L D4476 was added into the maturation medium.These results showed that addition of 5 ?mol/L D4476 into the maturation medium is beneficial to improve the bovine oocytes maturation as well as its subsequent IVF embryonic development competence.We conclude that D4476 maybe down-regulate the expression of CK1 during the spontaneous meiosis of oocyte and up-regulate the expression of the nuclear transcription factor TCF-4.Moreover,it can promote cumulus cell proliferation and expansion,delay the apoptosis of cumulus cells by simulating the Wnt signal,so that it can improve the oocytes maturation and embryonic development. |
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