| Glucose plays an important role in oocyte maturation.Oocyte maturation consists of nuclear maturation(the process of resumption of the first meiosis to the extrusion of the first polar body)and cytoplasmic maturation(the ability to obtain fertilization and preimplantation embryo development).At present,although many studies reported effects of glucose metabolism on oocyte nuclear maturation,few reported on cytoplasmic maturation.And most of them are studied in the form of adding inhibitors of glucose metabolic pathways in vitro,but the inhibitors have unavoidable drawbacks such as cytotoxicity and non-specificity.In addition,most of the culture objects are cumulus-oocyte complexes(COCs),it could not differentiate whether glucose metabolism of cumulus cells(CCs)or that of denuded oocytes(DOs)supported oocyte maturation,so it is necessary to use more specific way,such as RNA interference(RNAi)approach to study the effects of glucose metabolic pathways of cumulus cells and DOs on oocyte nuclear maturation and cytoplasmic maturation in detail.In the study,the effects of glucose metabolism on cytoplasmic maturation of pig oocytes were studied using a special maturation medium that could support nuclear maturation but could not support cytoplasmic maturation when glucose metabolism was inhibited.Whether glucose metabolism in pig CCs or DOs supported oocyte maturation was differentiated by RNAi gene silencing.Finally,the capacity to utilize glucose,pyruvate,and lactate was compared between pig and mouse DOs,so that we can more fully understand the mechanisms by which glucose metabolism affects oocyte maturation.The experimental results demonstrated that:1.Glucose could support about 25% of pig DOs to complete nuclear maturation,lactate could support about 40% of pig DOs to complete nuclear maturation,and pyruvate could support about 55% of pig DOs to complete nuclear maturation.2.After small interfering RNA(siRNA)transfection downregulated glucose-6-phosphate dehydrogenase(G6PD)or glyceraldehyde-3-phosphate dehydrogenase(GAPDH)protein expression in CCs,the nuclear maturation rate of co-cultured pig DOs,the rate of morula or blastocysts after parthenogenetic activation or the content of adenosine triphosphate(ATP)in the conditioned culture medium was significantly reduced,and the morula and blastocyst rates after downregulated G6 PD were lower.3.Transfection of siRNA downregulated mitochondrial pyruvate carrier 1(MPC1),NADH dehydrogenase flavoprotein 1(NDUFV1)or lactate dehydrogenase B(LDHB)protein expression,the nuclear maturation rate of co-cultured pig DOs was significantly reduced.4.After α-Cyano-4-hydroxy-cinnamate(4-CIN),rotenone and sodium oxalate inhibited monocarboxylate transporter(MCT),mitochondrial respiratory chain and lactate dehydrogenase(LDH)activity in oocytes,respectively,the nuclear maturation rate of pig DOs was significantly reduced.5.After siRNA microinjection downregulated the expression of MPC1,NDUFV1 or LDHB protein in oocytes,the nuclear maturation rate of pig DOs was significantly reduced.6.After the addition of rotenone to the lactate-contained medium to inhibit the mitochondrial respiratory chain activity of pig or mouse COCs,the rates of morula and blastocysts after parthenogenetic activation of oocytes were significantly reduced,and the nuclear maturation rate of pig DOs was significantly reduced after the addition of rotenone to the lactate-contained medium to inhibit the mitochondrial respiratory chain activity of pig DOs.Conclusions of this study are: 1)Pig DOs had limited ability to utilize glucose,and could use pyruvate and lactate to support partial nuclear maturation;2)Glucose metabolism in CCs promoted pig oocyte maturation by releasing metabolites from both pentose phosphate pathway(PPP)and glycolysis;3)Pyruvate and lactate were transferred into pig DOs by monocarboxylate transporter and pyruvate was further delivered into mitochondria by mitochondrial pyruvate carrier in both pig DOs and CCs;4)In both pig DOs and CCs,pyruvate and lactate were utilized through mitochondrial electron transport and LDHcatalyzed oxidation to pyruvate,respectively;5)Pig and mouse DOs differed in their CC dependency for glucose,pyruvate and lactate utilization;6)Pig DOs could use the lactatederived pyruvate contrary to mouse DOs.Glucose also plays an important role in early embryo development.In vitro studies have found that high concentrations of glucose are detrimental to early embryo development.In vivo experiments have also found that maternal hyperglycemia could impair early embryo development,but most studies have not distinguished between damage to oocytes and damage to early embryos.In addition,glucose is beneficial for early embryo development,especially at the later developmental stages.Therefore,we studied the effects of different concentrations of glucose on early embryo development by adding different concentrations of glucose in vitro,and the effects of in vivo maternal hyperglycemia on early embryo development were studied by drug induction.The experimental results demonstrated that:1.Different concentrations of glucose had no effect on the embryo development rates of mouse preimplantation embryos at various stages,but the blastocyst cell numbers of lowconcentration glucose or high-concentration glucose were fewer.2.Injection of 200 mg/Kg streptozocin(STZ)could induce maternal hyperglycemia and significantly reduced the rate of blastocyst development in vivo.Conclusions of this study are: 1)Optimal concentration of glucose could improve mouse early embryo development,but high concentrations of glucose impaired embryo development;2)Maternal hyperglycemia impaired early embryo development.At present,the epidemic trend of diabetes is becoming increasingly severe,and studies have shown that maternal hyperglycemia could increase the risk of diabetes in offspring,we do not know whether maternal hyperglycemia before embryo implantation has had an impact on embryos,thereby increasing the risk of diabetes in offspring.And this should also be an important research content of the genetic mechanisms of diabetes.On the other hand,glucose is currently added to routine in vitro culture systems in humans and animals,and the effects of this on glucose metabolism in offspring have not yet been studied.We used in vivo and in vitro models to study the effects of glucose exposure on glucose metabolism in offspring in mice.We cultured mouse zygotes by adding different concentrations of glucose in vitro,and transferred the blastocysts into recipients,and detected glucose metabolism of the offspring produced.We established an in vivo model of maternal hyperglycemia by drug induction and transferred the blastocysts into recipients,then detected the glucose metabolism of the offspring.The experimental results demonstrated that:1.F1 male offspring following embryo culture with 12,14 or 20 m M glucose developed glucose intolerance and insulin resistance,but no reduction of insulin secretion.2.F2 male offspring from F1 fathers following embryo culture with 12,14 or 20 m M glucose developed glucose intolerance,and no reduction of insulin secretion.3.Injection of 200 mg/Kg STZ caused glucose intolerance and insulin resistance in F1 male mice,but no reduction of insulin secretion.4.F2 male offspring from F1 fathers from mothers that had been injected with 200mg/Kg STZ developed glucose intolerance and insulin resistance,but no reduction of insulin secretion.5.STZ injection caused a significant increase in glucose concentrations in oviductal fluids.6.High glucose exposure of preimplantation embryos caused abnormal expression of glucose and lipid metabolism genes in liver of male offspring.Conclusions of this study are: 1)High glucose exposure of mouse preimplantation embryos caused male offspring diabetic-like phenotype,and this phenotype could be passed from father to son;9)High glucose exposure of mouse preimplantation embryos caused abnormal expression of glucose and lipid metabolism genes in liver of male offspring.This research is of great importance for understanding the maturation mechanisms of oocytes,pathogenesis of diabetes and improving in vitro maturation systems. |
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