Biphasic Maturation Culture Of Sheep Oocytes And Expression And Regulation Of P66Shc In Sheep Oocytes And Early Embryos

The low efficiency of in vitro production of mammalian embryos,is largely due to poor oocyte maturation in vitro and oxidative stress caused by exposure of oocytes and early embryos to static,suboptimal in vitro culture conditions that are chemically synthesized.The maintenance of cytoplasmic redox homeostasis is one of the key factors that influence the quality of oocytes maturation in vitro and embryo developmental potential.Scientific studies have shown that the physiological paracrine factor C-type natriuretic peptide(CNP)produced by mural granulosa cells in ovarian follicles has the property of maintaining meiotic arrest in mammalian oocytes.In addition,recent studies showed that the 66 KDa protein isoform encoded by proto-oncogenes SHC(src homology 2 domain-containing transforming protein C,SHC)and its signal characteristics played an important role in the regulation of cellular oxidative stress,apoptosis,and the process of organism aging.The aim of this study was to investigate the effects of pre-incubation with C-type natriuretic peptide on nuclear meiotic process and maturation quality of sheep oocytes.C-type natriuretic peptide was used to temporarily inhibit nuclear meiosis process of sheep oocytes to prolong the maturation time of cytoplasm and promote the synchronous maturation of nuclear and cytoplasmic,in order to improve the oocyte developmental competence and subsequent development ability of fertilized embryos.Simultaneously,the expression characteristics of oxidative stress protein p66 Shc in sheep oocytes and embryos and the molecular mechanism of its regulation on cytoplasmic redox homeostasis were studied in order to provide theoretical basis for improving the in vitro culture system and enhancing the efficiency of in vitro embryo production.In this study,we established a "biphasic maturation" culture system of sheep oocytes in vitro and analyzed the expression patterns of m RNA and protein of oxidative stress gene p66 Shc in oocytes and early embryos,as well as its relationship with mitochondrial distribution and activity,ROS level and redox homeostasis,with the aim of providing a theoretical basis for further study the molecular mechanism network of p66 Shc on the redox regulatory of oocytes and early embryos and the functional role of p66 Shc.In summary,the results obtained are as follows:1.C-type natriuretic peptide was used to construct "biphasic maturation" culture system of sheep oocytes in vitro.Cumulus Oocyte Complexes(COCs)were incubated with physiological meiosis inhibitor C-type natriuretic peptide,and then transferred into a maturation solution for the second phase of in vitro maturation.The results showed that 200 n M/L CNP could effectively maintain the meiotic arrest of sheep COCs within 4 h.Moreover,CNP receptor NPR2 m RNA is mainly expressed in mural granulosa cells(MGCs).Compared with the conventional standard 24 h IVM and other experimental groups,COCs pretreated with 200 n M/L CNP for 4 h followed by 24 h IVM showed significantly(P < 0.05)higher blastocyst rate and blastocyst quality than other experimental groups.Furthermore,mitochondria in the germinal vesicle(GV)oocytes mainly showed brilliant circumferential and fine diffuse distribution of mitochondria throughout the cytoplasm.By comparison,200 n M CNP pre-treated COCs for 4 h led to cytoplasmic mitochondrial granule localization to the peripheral and perinuclear regions.Moreover,oocytes pre-treated with 200 n M CNP for 4 h followed by 24 h IVM,showed mitochondrial organization pattern were similar to those of conventional 24 h matured oocytes in which mitochondria were aggregated more toward the cortical regions of the oocytes,but with more and larger clumps of stained mitochondria.These results indicate that CNP pre-treatment improves the quality and developmental competence of sheep oocytes and has great potential for facilitating in vitro embryo production.2.The expression characteristics of p66 Shc in sheep oocytes and its relationship with cytoplasmic redox homeostasis.The expression abundance of p66 Shc m RNA in different quality of sheep oocytes before and after maturation was analyzed by real-time quantitative PCR(RT-q PCR).The p66 Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with Mito Tracker Red probe.Simultaneously,the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte autofluorescence,respectively.In addition,exogenous H2O2-induced oxidative stress was used to treat high quality immature oocytes.The results showed that the transcripts and protein of p66 Shc in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes(P < 0.05).Compared with high quality immature oocytes and conventional 24 h matured oocytes,poor quality immature oocytes and aged oocytes showed disordered mitochondrial distribution and decreased activity,increased ROS levels and imbalance redox homeostasis.In addition,exogenous H2O2-induced oxidative stress significantly upregulated the expression of p66 Shc protein(P < 0.05)and induced p66 Shc from the cytoplasm to the nuclei.These results indicate that the increased expression of p66 Shc in poor quality immature oocytes and aged oocyte is associated with imbalance of cytoplasmic redox homeostasis in sheep oocytes.3.The expression and spatial location pattern of p66 Shc during preimplantation embryo development in sheep.The expression abundance of p66 Shc m RNA,protein level and spatial localization pattern were detected by RT-q PCR and immunofluorescence in early embryonic development,respectively.The results showed that p66 Shc m RNA and protein expressed in all stages of sheep early embryos.p66 Shc m RNA was significantly down-regulated in 4-8 cell stage(P < 0.05),and significantly up-regulated in morula and blastocyst stage after embryonic genome activation(EGA)(P < 0.05).Immunofluorescent staining showed that the p66 Shc protein was mainly located in the peripheral region of the blastomeres cytoplasm at different stages of preimplantation embryonic development.It was noteworthy that the serine(Ser-36)phosphorylated p66 Shc only localized in the cytoplasm in 2-8 cell stage prior to EGA,while the phosphorylated(Ser-36)p66Shc localized not only in the cytoplasm area but predominantly located in the nuclei after EGA.Therefore,it is speculated that the expression and localization of p66 Shc gene is closely related to the regulation of early embryonic development in sheep.4.p66 Shc is involved in hydrogen peroxide-induced cytoplasmic oxidative stress during preimplantation embryo development in sheep.The results showed that the blastocyst rate(66.67%)of early cleavage embryos(? 28 h)was significantly higher than that of blastocyst rate(22.93%)of late cleavage embryos(> 28 h)(P < 0.05).Moreover,the increased expression of p66 Shc and decreased mitochondrial activity were correlated with poor quality of sheep embryos.In addition,the exogenous hydrogen peroxide(H2O2)induced oxidative stress significantly reduced embryo developmental capacity(P < 0.05)and increased the expression of p66 Shc protein(P < 0.05).However,antioxidants(1 U/ul catalase and 10-6 mol/L melatonin)treatment ameliorated H2O2-induced the recession of developmental capacity,ROS accumulation and p66 Shc expression.Importantly,exogenous H2O2-induced oxidative stress induced the localization of serine 36(Ser-36)phosphorylation of p66 Shc from the cytoplasm to the nuclei.Collectively,these observations suggest p66 Shc and its serine phosphorylation(Ser-36)are involved in cytoplasmic oxidative stress regulation during sheep preimplantation embryo development.5.Knockdown of p66 Shc by si RNA microinjection reduces the risk of oxidative damage and improves embryo developmental potential in sheep.Three specific si RNA molecular targeted the specific CH2 domain of p66 Shc were microinjected into the cytoplasm at zygote stage.Simultaneously,three control groups(non-injected,RNase-free water-injected and negative control si RNA-injected)were also designed to test potential effects of the microinjection technique and specificity of the interfering molecules.The results showed that microinjection of specific p66 Shc si RNA into sheep zygotes resulted in a markedly decrease in both m RNA and protein levels of p66Shc(P < 0.05).Knockdown of p66 Shc exhibited reduced intracellular ROS(P < 0.05),decreased DNA damage marker 8-hydroxy-2'-deoxyguanosine(8-OHd G)(P < 0.05)and a greater propensity for development to the morula stage.These findings indicate that knockdown of p66 Shc may increase the resistance of embryos to oxidative stress during embryonic genome activation.