Effect Of Dietary Methyl Donors Supplementation To Sows Diet On The Follicular Development And Its Underlying Mechanism

Adequate management of follicular development and fertility by nutritional interventions is supposed to be one of the main targets in modern pig production.Existing studies have found that DNA methylation changes drastically during follicular development,which may be a potential mechanism for regulating the expression of key genes in follicular development.From the existing literature,methyl donors?MET?are the most effective substances for regulating DNA methylation.A burgeoning literature have found that MET(eg,folic acid,VB₁₂,etc.)supplementation to female animals or women diets could improve follicular development and the quality of oocytes,but the exact mechanism remains unclear.The primordial follicle pools determine the reproductive potential of female animals.However,it is unclear whether MET can affect the primordial follicle growth and development in female mammals.Therefore,the purpose of this study was to examine the effect of MET supplementation to gestating and lactating sows,as well as gilts diets on follicualr development,and the regulatory effect of DNA methylation.The main research contents and results are as follows:Exp.1 Effects of MET supplementation to sows diets during gestation and lactation on sows'performance and follicular development of offspring and its mechanismThe study aimed to investigate the effects of maternal MET supplementation to sows diets during gestation and lactation?key period of forming primodial follicles?on the performance of sows and follicular development of offspring and its mechanism.A total of48 Landrace×Yorkshire crossbred sows equally balanced by parity?5 to 7?were artificially inseminated upon observation of oestrus with the pooled semen obtained from two littermate boars.After artificial insemination,sows were randomly allocated to the four dietary group,receiving basal diet?CON,n=12?;basal diet supplemented with BPA?BPA,n=12?;basal diet supplemented with MET?MET,n=12?;and basal diet supplemented with BPA and MET?BPA+MET,n=12?.Sows fed the BPA gestational diet were supplemented with 50 mg/kg diet of BPA?Sigma-Aldrich,St.Louis,MO,USA?.A MET supplement premix was added to the gestational basal diet containing 3 g/kg diet of betaine?98%betaine hydrochlorides?,400mg/kg diet of choline?50%choline chloride?,15mg/kg diet of folate?Sigma Aldrich?and 150?g/kg diet of vitamin B12(VB₁₂)?Sigma Aldrich?.During gestation,sows were fed 2.28 kg/d during 1-90,and 2.72 kg/d of diet from day 91 until farrowing.Within 24 h of post-farrowing,litters were equalized to 10–11per sow by cross-fostering within treatment.After parturition,sows received 2,2.5,3.5,4.5,5.5 kg/d followed by ad libitum on the 5th day of lactation.In order to ensure the sows receiving enough amounts of MET or BPA,a special treatment protocol was used as follows.A MET supplement premix pellet containing 29.66 mg/kg.BW of betaine?98%betaine hydrochlorides?,3.95 mg/kg.BW of choline?50%choline chloride?,0.15mg/kg.BW of folate?98%,Sigma Aldrich?and 0.0014 mg/kg.BW of VB12?99%,Sigma Aldrich?,or a BPA pellet containing 0.49 mg/kg.BW of BPA?99%,Sigma Aldrich?was given to the corresponding treatment once per day?8:00?.The lactation period lasted 4weeks.After weaning,12 gilts from each trreatment?average BW±10%?were selected to continue being fed with the same diet until the 19^th day of third estrus.Two gilts within treatment were housed in an individual pen,with 6 pens per group at the weaning,growing,and sexual maturation stages.Gilts were offered free access to feed?three times per day,i.e.0800,1200 and 1500 h?from 28 to 150 d.From 150 d until slaughter,feed intake was controlled at 2.5 kg/d?two times per day,i.e.0800 and 1600 h?,and gilts were exposed?with fence?to mature boars to detect estrous twice?i.e.0900 and 1600 h?per day.Blood samples and ovaries were sampled on the weaning day?weaning?and on the 19^th day?gilts?of the third estrus.The results were presented as following:1.Maternal MET supplementation did not affect 110 d body weight?BW?,total born,total born alive,birth weights and litter weights?P>0.05?.Maternal MET supplementation significantly reduced the mummification and malformation rate?P<0.05?,and increased the number of weaned piglets and weaned litter weights?P<0.05?.Maternal BPA supplementation significantly reduced the birth weights?P<0.05?,and BW and litter weights of piglets on the 21d and 28 d?P<0.05?.2.Maternal MET or BPA supplementation did not affect puberty performance?age,BW and backfat thickness?,estrus interval and ovrian index of offspring?P>0.05?.For follicular development of weaning offspring,maternal MET supplementation reduced the apoptotic follicles rate?P<0.05?,while no differences were observed in primodial,primary and secondary follicles rate of offspring?P>0.05?.Maternal BPA supplementation reduced the primodial follicles rate?P<0.05?,but increased primary and apoptotic follicels rate of offspring?P<0.05?.For follicular development of gilts offspring,maternal MET supplementation increased the primodial follicles rate and numbers of laege follicles?diameter?5 mm?and corpus luteums?diameter?3 mm??P<0.05?,while reduced the apoptotic and primary follicles rates of offspring?P<0.05?.Maternal BPA supplementation reduced the primodial follicles rate?P<0.05?,but increased apoptotic follicels rate of offspring?P<0.05?.3.Maternal MET supplementation reduced the serum HCY contents in farrowing and weaning sows,and weaning offspring?P<0.05?,and increased serum SAM level and SAM/SAH ratio in weaning offspring?P<0.05?.Maternal BPA supplementation did not affect the serum HCY contents in farrowing and weaning sows,and weaning offspring,as well as serum SAM level and SAM/SAH ratio in weaning offspring?P>0.05?.Maternal MET or BPA supplementation did not affect the estradiol and progesterone concentrations of offspring?P>0.05?.4.For ovarian folliculogenesis,one caborn metabolism and DNA methylation related genes and proteins of weanling offsprings,maternal MET supplementation upregulated BMP15?P<0.05?,GDF-9?P=0.08?,MTHFR?P<0.05?,MS?P=0.08?,BHMT?P=0.08?and GNMT?P=0.05?mRNA levels,and increased DNMT3a gene and protein expressions?P<0.05?,while reduced BAX gene and protein expressions of offsprings?P<0.05?.Maternal BPA supplementation downregulated DNMT3a gene and protein expressions?P<0.05?,while upregulated BAX gene and protein expressions?P<0.05?,and no differences were observed in one caborn metabolism related genes expressions of offsprings?P>0.05?.For ovarian folliculogenesis,one caborn metabolism and DNA methylation related genes and proteins of gilts offsprings,maternal MET supplementation upregulated Bcl-2,GDF-9?P<0.05?mRNA levels,and reduced Bim gene and BAX gene and protein expressions?P<0.05?,while no differences were observed in one caborn metabolism related genes expressions of offsprings?P>0.05?.Maternal BPA supplementation upregulated BAX gene?P<0.05?and protein?P=0.05?expressions,and no differences were observed in one caborn metabolism related genes expressions of offsprings?P>0.05?.5.Maternal MET supplementation up-regulated the ovarian DNA methylation of global and the promoter of the BAX gene of offsprings?P<0.01?,while maternal BPA supplementation down-regulated the ovarian DNA methylation of global?P<0.05?and the promoter of the BAX gene?P<0.01?of offsprings.For ovarian CpG sites of BAX gene promotor of weanling offsprings,maternal MET supplementation increased the DNA methylation at CpG 3,CpG 18-19,CpG 44-45?P<0.05?and CpG 9?P=0.07?sites,while maternal BPA supplementation reduced the DNA methylation at CpG 3,CpG 18-19,CpG 44-45,CpG 8,CpG 24-25?P<0.05?sites of offsprings.For ovarian CpG sites of BAX gene promotor of gilts offsprings,maternal MET supplementation increased the DNA methylation at CpG 1-2,CpG 33-34,CpG 5-6,CpG18-19 and CpG 48-49sites?P<0.05?,while maternal BPA supplementation reduced the DNA methylation at CpG 1-2,CpG 3,CpG 33-34,CpG 5-6 and CpG 48-49 sites of offsprings?P<0.05?.Collectively,maternal MET supplementation reduced the mummification and malformation rate,and increased the number of weaned piglets and weaned litter weights,which maybe related to lower HCY levels.Moreover,maternal MET supplementation increased the primordial follicles rate and large follicles numbers in gilts,as well as decreased the apoptotic follicles rates of offspring,associated with a reduced HCY levels,which in turn mediated DNA hypermethylation of ovarian BAX gene promotor and persistent dowregulated ovarian BAX gene and protein expressions of offspirngs.Exp.2 Effects of MET supplementation to gilts diets on follicular development and and its mechanismThe results of Exp.1 had showed that maternal MET supplementation improve follicular development of offsprings,associated with an increased ovarian DNA methylation.A growing body of literature suggested that the MET supplementation to diets during follicular growth can improve follicular development,but the mechanism remains unclear.A total of 48 Landrace×Yorkshire gilts?28 d?with similar BW?7.43±0.38 kg?were randomly allocated to the four dietary group,receiving basal diet?CON,n=6 repetition?;basal diet supplemented with BPA?BPA,n=6 repetition?;basal diet supplemented with MET?MET,n=6 repetition?;and basal diet supplemented with BPA and MET?BPA+MET,n=6 repetition?.A MET or BPA supplement premix pellet was given to the corresponding treatment once per day?8:00?.The doses of MET and BPA were the same as the gilts of Exp.1.Two gilts were housed in an individual pen,with 6 pens per group at the weaning,growing,and sexual maturation stages.The feed management,sampling procedures and experimental duration were the same as the gilts of Exp.1.The results were presented as following:1.Dietary MET supplementation increased the average BW on 90d and 180 d,average daily gain?ADG??20-180 d?and average daily feed intake?ADFI??28-33 d??P<0.05?.Dietary BPA supplementation decreased the BW of 90 d and 150 d,and ADG?28-90 d;28-180 d?,as well as feed:gain ratio?90-150 d and 28-180 d??P<0.05?.2.Dietary MET or BPA supplementation delayed puberty?P<0.05?,while addition of MET increased puberty BW?P<0.05?.Dietary MET or BPA supplementation did not affet estrus interval?P>0.05?.Dietary MET supplementation had more numbers of large follicles?diameter?5 mm??P<0.05?and corpus luteum?diameter?3 mm??P=0.06?,as well as primordial?P=0.09?and secondary?P=0.08?follicles rate.Dietary BPA supplementation decreased the number of large follicles?diameter?5mm??P<0.05?and primordial follicles?P=0.08?,and increased the apoptotic follicles rates?P=0.05?.3.Dietary MET supplementation increased the SAM level,SAM/SAH ratios and estradiol concentrations in serum and follicular fluids,and decreased the HCY concentration in serum and follicular fluid of the gilts?P<0.05?.Dietary BPA supplementation decreased the SAM/SAH ratio in serum?P=0.09?,and estradiol concentrations in serum?P=0.07?and follicular fluids?P<0.05?.Dietary MET or BPA supplementation had no effect on serum progesterone levels of gilts?P>0.05?.4.Dietary MET supplementation up-regulated the ovarian CYP19A1,ER?mRNA and protein expressions,and there existed interaction between MET and BPA in CYP19A1mRNA expression?P<0.05?.The greater mRNA expressions of ovarian CYP17A1,MS,MTHFR,DNMT1 and DNMT3a genes were observed in MET group?P<0.05?.Dietary BPA supplementation decreased the expressions of CYP19A1mRNA and protein,and ER?protein,as well as DNMT1 and DNMT3a mRNA?P<0.05?.5.Dietary MET supplementation up-regulated the ovarian DNA methylation of global and the promoter of the Cyp19A1 gene?P<0.05?,and CpG 1,CpG 2 and CpG 4 sites of the Cyp19A1 promoter in gilts?P<0.05?.Dietary BPA supplementation down-regulated the ovarian DNA methylation of global?P<0.05?and the promoter of the Cyp19A1 gene?P=0.058?,and the CpG 1,CpG 2 and CpG 4 sites in gilts?P<0.05?.Collectively,dietary MET supplementation increased puberty BW and the numbers of large follicles?diameter?5 mm?,associated with a reduced HCY levels,which upregulated ovarian Cyp19A1 gene expression through DNA hypermethylation,and then increased estradiol synthesis.The activated E₂-ER?pathway improved follicular development.Exp.3 Effects of homocysteine on ovarian development of neonatal mice in vitro and its mechanismThe results of Exp.1 and Exp.2 had showed that addition of MET to sow diets could improve the follicualr developement,which may be related to the lower HCY levels,and mediating DNA hypermethylation of ovarian folliculogenesis and steroidogenesis genes.Whether HCY could regulate follicular development through ovarian DNA hypomethylation,and it was still unclear.First,we used 100?M HCY to treat neonatal mouse ovaries in vitro.We found that the ovary of HCY group had more secondary and apoptotic follicles rate,with less primordial folliclesrate?P<0.05?.Moreover,HCY decreased the concentration of estradiol in culture medium?P<0.05?.The results showed that HCY maybe induced premature primordial follicle initiation and accelerated ovarian development.Using real-time quantitative PCR and western blot to measure the expressions of folliculogenesis and steroidogenesis genes and proteins,HCY up-regulated the ovarian BAX mRNA and protein expressions,and down-regulated ovarian Cyp19A1 and ER?mRNA and protein expressions of neonatal mice?P<0.05?.In addition,HCY increased the levels of ovarian BMP15 mRNA?P<0.05?.The results indicated that HCY accelerated ovarian development through regulating the key genes of follicular apoptosis and estradiol synthesis.Excessive accumulation of HCY could change the levels of other metabolites in one-carbon metabolism.Our results showed that HCY increased the SAH concentration and decreased the SAM:SAH ratio in the medium?P<0.05?.It suggested that HCY may induce ovarian hypomethylation of neonatal mice in vitro.Furthermore,we measured the DNA methylation in the promoter regions of Bax and Cyp19A1 genes.The results showed that HCY induced the promotor of BAX hypomethylation,and reduced the DNA methylation levels of CpG2 and CpG11 sites?P<0.05?,while no difference was observed in DNA methylation of ovarian Cyp19A1 promotor?P>0.05?.To further test this hypothesis,we used a DNA methyltransferase inhibitor?5-Aza?and HCY to treat neonatal mice ovaries.The results showed that both HCY or 5-Aza or co-administered groups reduced the global ovarian DNA methylation,DNMT3a mRNA and protein expressions in neonatal mice in vitro?P<0.05?.It demonstrated that high HCY impaird follicular development through DNA hypomethylation of folliculogenesis key gene.Collectively,HCY increased the SAH level and mediated the CpG2 and CpG11 sites in ovarian BAX gene promoter hypomethylation,and then up-regulated BAX gene and protein expressions,which cause preantral follicle apoptosis and impaird primodial follicles development.Moreover,HCY reduced estradiol synthesis,associated with reduced mRNA and protein expressions of ovarian Cyp19A1 and ER?,which inhibited the E₂-ER?pathway,and induced preantral follicle apoptosis.In conclusion:?1?maternal MET supplementation reduced the mummification and malformation rate,and increased the number of weaned piglets and weaned litter weights,which maybe related to lower HCY levels.Moreover,maternal MET supplementation increased the primordial follicles rate and large follicles numbers in gilts,as well as decreased the apoptotic follicles rates of offspring,associated with a reduced HCY levels,which in turn mediated DNA hypermethylation of ovarian BAX gene promotor and persistent dowregulated ovarian BAX gene and protein expressions of offspirngs.?2?Dietary MET supplementation increased puberty BW and the numbers of large follicles?diameter?5 mm?,associated with a reduced HCY levels,which upregulated ovarian Cyp19A1 gene expression through DNA hypermethylation,and then increased estradiol synthesis.The activated E₂-ER?pathway improved follicular development.?3?HCY increased the SAH level and mediated the CpG2 and CpG11 sites in ovarian BAX gene promoter hypomethylation,and then up-regulated BAX gene and protein expressions,which cause preantral follicle apoptosis and impaird primodial follicles development.