Role Of Homocysteine In Mitochondrial Dysfunction Of Oocytes From Polycystic Gilts And The Mechanisms Involved

Polycystic ovarian syndrome(PCOS)is a common reproductive disorder in women of child-bearing age.PCOS is the most common cause of anovulatory infertility,and the incidences was 5%-10%.Follicular cysts disease is also a common infertility diseases in sows.Since follicular disease results in anovulatory,it greatly decreases the reproductive performance of sows and results in severe economic loss to the breeding farm.It is reported that the decreased fertility of female is mainly due to the decreased quality of oocyte.Although polycystic ovarian syndrome patients produce more oocytes and embryos through in vitro fertilization,they still suffer from lower fertilization,cleavage and implantation rate,and higher miscarriage rate.So,the poor oocyte is the key question.Mitochondria is the most abundant organelle in the cytoplasm of oocyte and play an important role in the maturation of oocyte and embryonic development.Elevated serum homocysteine(Hcy)levels are reported in PCOS patients.Moreover,abnormally high level of Hcy is detected in the follicular fluid of polycystic ovaries.However,the mechanisms linking high Hcy level and poor oocyte quality remain unclear.In this study,we use polycystic ovaries of gilts as model to investigate the relationship of mitochondria dysfunction in poor quality oocyte and elevated Hcy.The findings may shed a new light on the pathogenesis of PCOS and provide new hints for identifying novel targets for the prevention and treatment of this disease.1 The relationship between homocysteine and poor oocyte quality from polycystic gilt ovariesHealthy and polycystic ovaries were obtained from the cross-bred prepubertal gilts(Duroc x Landrace x Yorkshire;135-170 days of age;70-120 kg of body weight)slaughtered in a local abattoir located in qixia district,Nanjing.Ovaries in the follicular phase of the ovarian cycle were selected and classified into two groups:healthy ovaries(Control)and polycystic ovaries(PCO).Collected follicular fluid in follicle which diameter is 3-6 mm from healthy ovaries and polycystic ovaries.Cumulus-oocyte complexes(COCs)were aspirated from follicular fluid separately.COCs were used to do in vitro maturation and analysis the development potential of oocyte,or treated with 1%hyaluronidase to remove the cumulus,the remaining oocytes were used to extract DNA,RNA,protein or do staining.Oocytes in the PCO group showed significantly lower survival rate and polar body extrusion rate,as compared with the Control group.No significant difference was observed between two groups in the cleavage rate after parthenogenetic activation at the 2-cell stage,yet the cleavage rate at the 4-cell stage and the blastocyst rate were significantly lower in PCO group compared with the Control group.Cell numbers in blastocysts were also significantly reduced in the PCO group.Mitochondrial distribution pattern in cytoplasm was disturbed in the PCO group.Moreover,mitochondrial membrane potential was decreased in the PCO group.Most of the mitochondria in control group contain clearly visible intact inner membrane,outer membrane,and a well-defined intermembrane space.In contrast,mitochondria in PCO group were predominantly spherical with almost no cristae.Copy number of mtDNA(mitochondria DNA)in the oocyte was significantly lower in PCO group than in Control group.Meanwhile,7 out of 13 mtDNA-encoded genes were down-regulated in PCO group.mRNA abundance of ND1,ND4L,ND5,COX1,and CYTB was significantly lower in the oocytes of PCO group while that of ND2 and COX2 tended to be lower.In addition,the abundance of 12S rRNA and 16S rRNA was also significantly lower in the oocytes of PCO group.The mRNA and protein level of DNA methyltransferase 1(DNMT1)was significantly increased in PCO group.Moreover,the concentration of Hcy in follicular fluid were significantly higher in PCO group compared with the Control group.Furthermore,Betaine-homocysteine S-methyltransferase(BHMT)and Glycine N-methyltransferase(GNMT),which participate in one-carbon metabolic pathway,were significantly up-regulated at the level of both mRNA and protein in PCO group.Methylation status of mtDNA coding for 12S rRNA,16S rRNA,COXl,COX2,COX3 and ND4 was analyzed by bisulfite sequencing in oocytes of Control and PCO group.mtDNA sequences coding for 12S rRNA,16S rRNA and ND4 were significantly hypermethylated in PCO group.Methylation status of D-loop was significantly higher in PCO group compared with the Control group.In summary,this present study provides the first evidence that the abnormal activation of one-carbon metabolism,up-regulated DNMT1 expression and the hypermethylation of mtDNA are associated with mitochondrial malfunction and the poor quality of oocytes derived from polycystic ovaries of gilts.2 Hcy affect one carbon metabolism and mitochondrial function in oocyte from giltsCollected follicular fluid in follicle which diameter is 3-6 mm from healthy ovaries.COCs were aspirated from follicular fluid and randomly divided into two groups(Control group and Hey group).COCs of the Control group were cultured in vitro maturation(IVM)medium.COCs of Hey group were cultured in IVM medium containing 100 ?M or 200 ?M Hey.COCs were used to do in vitro maturation and analysis the development potential of oocyte,or treated with 1%hyaluronidase to remove the cumulus,the remaining oocytes were used to extract DNA,RNA,protein or do staining.Oocytes in the Hey 100 ?M and 200 ?M group showed significantly lower survival rate and polar body extrusion rate,as compared with the Control group.No significant difference was observed between Control group and Hey 100 ?M group in the cleavage rate after parthenogenetic activation at the 2-cell stage and 4-cell stage.No significant difference was observed between Control group and Hey 200 ?M group in the cleavage rate at the 2-cell stage.Cleavage rate at the 4-cell stage were significantly lower in Hey 200 ?M group compared with the control group.Blastocyst rate was significantly lower in both of Hey 100?M and 200 ?M group compared with the Control group.According to data above,both of Hey 100 ?M and 200 ?M treatment can impair oocyte developmental potential.However,Hey 200 ?M treatment have more effect to oocyte developmental potential compare with Hey 100 ?M treatment.Therefore,Hey 200 ?M was choosed as the treatment concentration in the following experiment.Moreover,mitochondrial reactive oxygen species(ROS)in the oocyte was significantly higher in Hey group than in Control group.The copy number of mtDNA in the oocyte was significantly lower in Hey group than in Control group.Furthermore,7 out of 13 mtDNA-encoded genes,including COX2,ND1,ND2,ND3,ND4,ND4L and ND5 were down-regulated in Hey group,as well as 12S rRNA and 16S rRNA.Meanwhile,mRNA level of BHMT,GNMT and DNMT1 was significantly increased in Hcy group as well as the protein level of BHMT and GNMT.Protein level of DNMT1 also increased after Hey treatment,but did not reach a significant level.Taken together,Hey treatment on oocytes activated one carbon metabolism and lead to mitochondrial dysfunction and poor oocyte quality..3 Activated one carbon metabolism and mitochondrial dysfunction in oocytes from gilts was partially rescued by DNA methyltransferase inhibitor 5'AZA treatmentCollected follicular fluid in follicle which diameter is 3-6 mm from healthy ovaries.COCs were aspirated from follicular fluid and randomly divided into four groups.Control group,COCs were cultured in IVM medium;Hcy group,COCs were cultured in IVM medium containing 200 ?M Hcy;Hcy+5'AZA group,COCs were cultured in IVM medium containing 200 ?M Hcy and 10 nM 5-AZA-2'-deoxycytidine(5'AZA);5'AZA group,COCs were cultured in IVM medium containing 10 nM 5'AZA.COCs were used to do in vitro maturation and analysis the development potential of oocyte,or treated with 1%hyaluronidase to remove the cumulus,the remaining oocytes were used to extract DNA,RNA,protein or do staining.When 5'AZA added,the increased protein level of BHMT were rescued as well as the mRNA level of BHMT and GNMT,compared with Hcy group.Protein level of GNMT tender to be rescued after 5'AZA treatment.And mRNA level of DNMT1 decreased after 5'AZA added,although did not reach a significant level.Methylation status of mtDNA coding for 12S rRNA and 16S rRNA was analyzed by bisulfite sequencing in oocytes of Control group,Hey group and Hcy+5'AZA group.No significantly difference were found in methylation status of 12S rRNA.However,mtDNA sequences coding for 16S rRNA were significantly hypermethylated in Hcy group.When 5'AZA added,methylation status of 16S rRNA was significantly decreased compared with Hey group.5'AZA treatment reversed the increased mitochondrial ROS which induced by Hey.Moreover,5'AZA treatment rescued the decreased copy number of mtDNA which induced by Hcy.Furthermore,7 out of 13 mtDNA-encoded genes,12S rRNA and 16S rRNA were down-regulated in Hcy group,compared with Control group.After 5'AZA treatment,there are no significant difference for these genes between Hcy+5'AZA group and Control group.Meanwhile,5'AZA treatment rescued the reduced the survival rate and polar body extrusion rate of oocytes which induced by Hcy.When 5'AZA was added,the decreased cleavage rate after parthenogenetic activation at 4-cell stage and blastocyst rate were also rescued.5'AZA treatment independently had no significantly effect on survival rate,polar body extrusion rate and cleavage rate.Taken together,5'AZA patially resucued activatied one carbon metabolism and mtDNA-encoded genes DNA hypermethylation,mitochondrial dysfunction and poor oocyte quality.4 Hcy affect RNA methylation m6A modification in oocytes from giltsCollected follicular fluid in follicle which diameter is 3-6 mm from healthy ovaries.COCs were aspirated from follicular fluid and randomly divided into two groups(Control group,Hcy group).COCs of the Control group were cultured in IVM medium.COCs of Hcy group were cultured in IVM medium containing 200 ?M Hcy.COCs were treated with 1%hyaluronidase to remove the cumulus,the remaining oocytes were used to extract RNA,protein or do staining.We examined totel N6-methyladenosine(m6A)in oocytes and other tissues.The results showed that m6A modification existed in oocytes from gilts.Moreover,m6A modification was increased in oocytes after Hcy treatment.Meanwhile,both the protein level of m6A methyltransferase METTL3 and demethyltransferase FTO were increased upon Hcy treatment.However,m6A reader YTHDF2 was decreased after Hcy treatment.Hcy treatment increased the fluorescence intensity of m6A in mitochondria.We next analysised m6A-seq data from mouse embryonic fibroblase cell line with or without amino acid starvation.The mRNA of COX3_ND2X ND3?ND4L and ND5 showed m6A peaks.No differential changes of mPA modification were observered upon amino acid starvation.With heat stress,the m6A modification in mRNA of ND5?ND6?CYTB and ATP8 changed,including some m6A peak decreased and other m6A peak increased.12S rRNA and 16S rRNA showed obvious m6A peaks,especially for 16S rRNA.No differential changes of m6A modification of 12S rRNA and 16S rRNA were observered upon amino acid starvation and heat stress.In summary,the transcript of mitochondrial trancript in mice had m6A modification,m6A modification changed differently upon different stress.To summarize,m6A modification existed in oocyte from gilts.Hcy treatment increased total and mitochondrial transcript m6A Uodification,as well as the protein level of METTL3 and FTO.However,YTHDF2 was decreased after Hcy treatment.And the m6A modification of mitochondrial transcript in mice is a potential biological target becaused they changed differently in response to different stress.Taken together,the present study provided the first evidence that Hcy coincided with the abnormal activation of one-carbon metabolism and the hypermethylation of mtDNA which were associated with mitochondrial malfunction and the poor quality of oocytes derived from polycystic ovaries of gilts.Hcy treatment on normal oocyte activated one carbon metabolism and changed mtDNA-encoded gene DNA methylation and RNA methylation m6A pathway,which lead to mitochondrial dysfunction and poor oocyte quality.Whereas,DNA methyltransferase inhibitor 5'AZA treatment partly rescued mitochondrial dysfunction and poor oocyte quality which induced by Hcy.