| Objective:Primary oocytes,which contain large nuclei,named germinal vesicleand,are stagnant in the prophase of first meiosis after birth in mammals.Studies have shown that cyclic adenosine phosphate(cAMP)can shuttle and flow between the two kinds of cells through the gap connection between the granulosa cells and the oocytes,and plays a role in promoting the maintenance of the meiotic arrest state of oocytes.After Sexually mature,follicle growth is launched.Adenosine monophosphate(cAMP)levels in the inner ring of oocytes are dropped,and oocyte maturation factors expression are increased,resulting in the growth of primary oocyte and the completion of the first meiotic division:the rupture of germinal vesicle and discharge of the first polar body.And after ovulation,oocytes are arrested in the middle of the second meiosis-the MⅡ period.Oocyte maturation includes nuclear maturation and qualitative maturation,both of which are synchronized to optimize oocyte development.Each cell event during oocyte maturation is energy-dependent and the mitochondria are its energy factories.Glucose and/or glucose metabolites,fatty acids and amino acids,may be the metabolic substrates of oocytes,especially glucose product-acetyl-coa,which can be transferred into mitochondria for oxidative respiration and the synthesis of large amounts of ATP.Fatty acid oxidation(FAO)is another important source of energy for oocyte maturation.Oxidation of a fatty acid produces 106 ATP and oxidation of glucose is about 30 ATP,making it an effective source of energy for oocyte maturation and pre-implantation embryo development.Etomos,an inhibitor of beta oxidation,inhibits the nuclear maturation of mouse oocytes,and several other studies have shown that nuclear maturation in mice requires the oxidation of beta fatty acids.However,in addition to mitochondrial productivity,a large number of ROS are also synthesized,and a certain antioxidant system exists in oocytes to remove ROS.In addition to energy supply,these metabolic intermediates regulate intracellular REDOX status,signal transduction,osmotic pressure,and ensure oocyte nuclear and plasma maturation.Once the nuclear maturation and the plasma maturation are not synchronous,the oocyte quality will decline and the embryo development will be poor.Oocytes mature and then wait to be fertilized in a woman’s fallopian tube or in vitro culture,restart and complete a second meiosis.Oocytes are arrested in the middle of the second meiosis after the completion of the first meiosis,and will be discharged from the ovary,reach the oviduct ampulla,waiting for sperm fertilization.Generally,the best insemination period varies from species to species,and the best time for rodents,monkeys and humans is 8-12h,12-14 and<24 hours,respectively.If no fertilization or activation,mature oocytes will gradually aging.Aging after ovulation leads to abnormal mitochondria structure of oocytes,spindle and chromosome arrangement,increase of ROS.And it will also change in apparent heritage,which leads to the decrease of the quality of the oocytes and the poor of embryo development,congenital defects and low birth rate.However,with the application of ART technology,mature oocytes can be cultured for longer in vitro,which will reduce the developmental potential of oocytes.For example,it has been found that prolonged in vitro culture of human and mouse oocytes before IVF and ICSI resulted in decreased oocyte quality.Therefore,it is very important to clarify the changes of cell function and its mechanism during the aging process of post-ovulation oocytes,which is also important for finding the targets to prevent the aging of post-ovulation oocytes.Pyruvate dehydrogenase kinase(PDK)is mainly located in the mitochondrial matrix of cells,but also exists in the cytoplasm.Four PDK isozymes are identified in humans and rodents:PDK1,PDK2,PDK3,and PDK4,with different tissue distributions,specific phosphorylation sites,and structural affinities.Its main role is the phosphorylation of pyruvate dehydrogenase complex(PDC).PDC is a multi-enzyme complex located in the mitochondrial matrix,which can catalyze the oxidation of pyruvate to decarboxylation,thus providing acetyl-coa and NADH for the tricarboxylic acid cycle and lipid biosynthesis,and play a key role in glucose metabolism.PDC is composed of four components:pyruvate dehydrogenase(E1),dihydrothiosyl acetyltransferase(E2),dihydrothiosyl amide dehydrogenase(E3)and E3 binding protein(E3BP).PDC activity is regulated by phosphorylation of E1 component serine residues,phosphorylated by pyruvate dehydrogenase kinase(PDK)and dephosphorylated by pyruvate dehydrogenasephosphatase(PDP).These two regulatory enzymes PDK and PDP have unique tissue expression patterns,kinetic properties and sensitivity to regulatory molecules,and their relative activity determines the degree of phosphorylation of PDC serine residues and hence the activity of PDC complexes.Four PDK isozymes are regulated by different transcription factors,and the expression of PDK1 and PDK3 can be stimulated by HIF1,which has considerable research potential in hypoxia and cancer.The expression of PDK2 and PDK4 is controlled by nutritional factors and hormones,making it a concern in the field of fighting hunger and diabetes.In recent years,some progress has been made in the study of PDK4,including anti-tumor,anti-hiv and anti-osteoporosis.PDK4 inhibitors can improve insulin resistance and reduce blood glucose.Upregulation of PDK4 expression can reduce the release of NEFA in blood and reduce the risk of coronary heart disease.Silencing PDK4 expression can effectively reduce VC and reduce the risk of stroke.Further studies on the mechanism of action revealed that PDK4 expression was regulated by various regulatory factors,such as pgc-1,ERR,hnf-4 and hif-1.Hormone levels also affect PDK4 levels.Therefore,PDK4 expression regulation is affected by a variety of signal regulation mechanisms.We found that PDK4 expression was up-regulated during oocyte maturation,and interference with PDK4 expression increased oocyte oxidative stress and apoptosis.The oocyte apoptosis induced by downregulation of oocytes was inhibited by adding putrescine.At present,the effect and mechanism of PDK4 on oocyte meiosis and post-ovulation oocyte aging are unknown.Putrescine is a small naturally occurring metabolite that is converted from arginine and proline by ornithine decarboxylase(ODC).Putrescine is easily absorbed and diffused throughout the body.High doses of putrescine are not harmful to newborn calves and pigs.T he ovaries of mice showed transient increase of ornithine decarboxylase and putrescine during ovulation.Decarboxylase activity decreased in ovary of elderly mice.Putrescine supplementation during in vitro maturation reduces aneuploidy in older oocytes.Adding 1%putrescine into the drinking water before ovulation can increase the number of mouse blastocyst cells and reduce the abortion rate.putrescine can be quickly absorbed and excreted,and has no toxic effect on the fetus.Male and female offspring can grow normally and have normal fertility.We found that the addition of putrescine to IVM significantly increased the oocyte maturation rate in elderly mice.DFMO was added to IVM to inhibit the generation of endogenous putrescine by inhibiting ODC1,and it was found that the oocyte maturation rate was significantly lower than that of the putrescine group,but there was no statistical difference from the control group,which may be related to the decreased concentration of ODCl and putrescine in the ovaries of elderly mice.Our study also found that the addition of putrescine to IVM significantly reduced ROS in elderly oocytes.Putrescine acts as an antioxidant,protecting bacteria from high oxidative stress in bacterial and cellular experiments.Recent studies have shown that the decrease of mitochondrial membrane potential is an early event of apoptosis.Our results showed that the addition of putrescine to the IVM of elderly mice increased the mitochondrial membrane potential,improved mitochondrial function,and reduced cell apoptosis,indicating that the reduction of putrescine in the oocytes led to a decrease in mitochondrial function,and the reduction of the mitochondrial membrane potential of oocytes.The results showed that putrescine could effectively reduce the ROS and improve mitochondrial function of oocytes in elderly mice.This paper studies the regulation of energy metabolism during oocyte development and maturation,and discusses the role of PDK4 in the regulation of energy metabolism in oocytes;This paper also studies that small molecule-putamine regulates energy metabolism through PDK4 to inhibit intracellular ROS and oxidative stress,thereby delaying the aging of oocytes after ovulation.Materials and methods:(1)Function and mechanism of PDK4 in oocyte meiosis1)We collected GV stage,M II stage oocytes and cumulus cell of 8w young C57/BL6J mice,and M II stage oocytes and cumulus cell of 9 month older mice.Then by western blot,PDK4 expression changes was observated in the these oocytes and cumulus cell;2)Small interference of PDK4 was injected into 8w young C57/6J mouse oocytes at GV phase,and changes in GVBD rate and first body output rate of oocytes were observed under the microscope;3)Small interference of PDK4 was injected into 8w young C57/6J mice oocytes at GV stage,and the distribution of spindle,ROS and mitochondria of oocytes were observed by immunofluorescence;(2)The function and mechanism of PDK4 in the aging process of 8w young C57/6J oocytes after ovulation1)After ovulation,8w young C57/6J oocytes were added with putrescine and morphological changes of oocytes were observed under the microscope.The changes of oocyte apoptosis were observed through TUNEL and immunofluorescence;2)Putrescine was added to 8w young C57/6J oocytes after ovulation,and the changes of ROS in oocytes were observed by immunofluorescence,and the changes of the ROS pathway sirtl-foxo3a-sod2 were observed by western blot;3)Putrescine was added to 8w young C57/6J oocytes after ovulation,and changes in mitochondrial distribution,mitochondrial membrane potential and mitochondrial autophagy of oocytes were observed,as well as the expression of mTOR,an autophagy pathway;4)During the in vitro culture of 8W young C57/6J mice oocytes after ovulation,putamine was added into the culture medium to observe the changes of anti-apoptosis pathway AKT/ERK1/2 protein and the expression changes of Bcl2,BAX and caspase-9 in mitochondrial apoptosis pathway were also observed.5)Putrescine was added to 8w young C57/6J oocytes after ovulation,and changes in the expression of PDK4 in oocytes were observed by immunofluorescence,qPCR and western bolt;6)After ovulation,small interference of PDK4 was injected into the 8w young C57/BL6J oocytes with putrescine,and the expression of PDK4 was observed by western bolt to verify the interference rate.Then,the changes of oocyte ROS,mitochondrial distribution,mitochondrial membrane potential and oocyte apoptosis were observed by immunofluorescence;7)Statistical methods:SPSS 17.0 software was used for statistical processing.All data were expressed as mean standard deviation(Means SD),two groups of data were analyzed by independent sample t test,and three groups of data were analyzed by chi-square test.P<0.05 means statistically significant difference.Results:(1)Function and mechanism of PDK4 in oocyte meiosis1)PDK4 the expression level of MⅡ young mice oocyte was significantly increased compared to the level of the GV stage oocytes(P<0.05);PDK4 expression of MⅡ older mice oocyte was significantly reduced compared to PDK4 expression of MⅡ oocytes in yong mice(P<0.05);the expression level of PDK4 MⅡ cumulus cell in yong mice was significantly higher than the level of GV cumulus cell in young mice(P<0.05).The expression level of PDK4 MⅡ cumulus cell in older was significantly lower mice compared to PDK4 expression of MⅡ oocytes in yong mice(P<0.05);2)After PDK4 was interfered in young mouse oocytes at GV phase,the ratio of GVBD in oocytes was decreased(P<0.05).Exclusion of the first body ratio in oocyte was decreased(P<0.01);3)After PDK4 was interfered by young mouse oocytes in GV phase,oocyte ROS accumulation was increased(p<0.05),oocyte spindle alignment abnormality was increased(P<0.05),and oocyte mitochondria distribution abnormality was increased(P<0.05);(2)The function and mechanism of PDK4 in the aging process of oocytes after ovulation1)24 hours after ovulation,the abnormal morphological changes of oocytes were observed under the microscope,and we found the abnormal morphological changes of oocytes was increased(P<0.05),and the abnormal morphological changes of oocytes was decreased with the addition of putrescine group(P<0.01).Through TUNEL detection,we found the positive rate of cell apoptosis of oocytes increased after ovulation 24 hours(P<0.05),and the rate was decreased after the addition of putrescine(P<0.05).Meanwhile,immunofluorescence was used to observe that the active form of oocyte apoptosis protein caspase3 and it was reported the expression was increased after ovulation 24 hours(P<0.05),and the active form of caspase3 was decreased after the addition of putrescine(P<0.05);2)Immunofluorescence was used to detecte ROS levels of oocytes in 24 h after ovulation.The results were that the ROS level was increased than fresh oocytes(P<0.05),and after adding putrescine,oocytes ROS level was dropped(P<0.01).At the same time,western blot was used to detect Sirtl,Foxo3a,SOD2 expression of oocytes after ovulation 24 h.We found Sirtl,Foxo3a,SOD2 expression of oocytes were decreased after ovulation 24 h(P<0.05,P<0.05,P<0.05),and after adding putrescine,Sirt1,Foxo3a,SOD2 expression of oocytes were increased(P<0.05,P<0.05,P<0.05);3)After ovulation 24h,the abnormal distribution of mitochondria in oocytes was detected by immunofluorescence.Compared with fresh oocytes,the abnormal distribution of mitochondria in oocytes was increased at 24h after ovulation(P<0.05),and was decreased after the addition of putrescine(P<0.05).Compared with fresh oocytes,the mitochondrial membrane potential was decreased 24h after ovulation(P<0.01),and was increased with the addition of putrescine(P<0.05).Compared with fresh oocytes,mitochondrial autophagy was decreased at 24h after ovulation(P<0.05)and was increased after the addition of putrescine(P<0.05).Meanwhile,the expression of p-mTOR in oocytes was detected by western blot at 24h after ovulation.The expression of p-mTOR in oocytes was decreased(P<0.05)after the addition of putrescine compared with that in the group without putrescine(P<0.05).4)p-AKT,p-ERKl/2 protein levels and antiapoptotic BCL2 protein expression were decreased in oocyte after ovulation compared to fresh oocyte protein levels(P<0.05,P<0.05,P<0.05)while protein expression of BAX promoting apoptosis was increased(P<0.01).And After adding the putrescine,p-AKT,p-ERK1/2 protein levels and antiapoptotic BCL2 protein expression were increased in oocyte after ovulation(P<0.05,P<0.05,P<0.05)while protein expression of BAX promoting apoptosis was decreased(P<0.01).Immunofluorescence results showed that the expression of cleaved caspase-9,a protein activated form of mitochondrial apoptosis in oocyte after ovulation,was also increased compared with that of fresh oocytes(P<0.05),while the expression of cleaved caspase-9,in oocytes of putrescine group was decreased(P<0.01).5)After ovulation,the expression of PDK4 in oocytes was detected by immunofluorescence,qPCR and western bolt.After ovulation 24h,the expression of PDK4 in oocytes was lower than that in fresh oocytes(P<0.05,P<0.05,P<0.05).However,the expression of PDK4 in oocytes after ovulation was increased with the addition of putrescine group(P<0.05,P<0.01,P<0.05).6)After ovulation,small interference of PDK4 was injected into oocytes by microinjection while putrescine was added,and the decreased expression of PDK4(P<0.05)was observed by western bolt.Then,immunofluorescence was used and it was observed the increase of ROS level(P<0.05),the increase of abnormal mitochondrial distribution rate(P<0.05),the decrease of mitochondrial membrane potential(P<0.05)and the increase of oocyte apoptosis after intervention in oocyte(P<0.05).Conclusion:(1)Function and mechanism of PDK4 in oocyte meiosis1)PDK4 expression increase during the completion of the first meiosis can promote oocyte maturation by promoting oocyte mitochondrial function;2)PDK4 promotes the completion of meiosis in oocytes,and its specific mechanism may be related to the regulation of energy metabolism,which is of great significance for the understanding of the molecular mechanism of meiosis in oocytes and the search for targets to improve the quality of oocytes in vitro maturation.(2)The function and mechanism of PDK4 in the aging process of oocytes after ovulation1)PDK4 expression is decreased when oocytes aged after ovulation due to overmaturity.PDK4 can inhibit oocyte aging,while small molecule putrescine can inhibit oocyte aging by promoting the expression of PDK4.2)It is of great significance for understanding the specific molecular mechanism of the aging of oocytes after ovulation and finding the target of inhibiting the aging of oocytes after ovulation. |
PDF Full Text Request