Study Of Arginine On Folliculogenesis In Nutrient Restriction Ewes Through Nitric Oxide Pathway

Hu sheep is a local breeding in China and is characterized by precociousness and high-prolificacy.At present,the feeding and management of Hu sheep has expanded,however,some improper feed management may lead to chronic malnutrition,leading to delays in puberty and/or disruption of the estrous cycle.Short-term nutritional supplementation with high-energy substrates increases the number of large follicles,the ovulation rate and decreases follicular atresia.Therefore,short-term supplementation(i.e.,focus feeding)has been developed to improve the reproductive performance of Hu sheep.Arginine(Arg),a conditionally essential amino acid of ruminants,is the main precursor of nitric oxide in vivo.It hasfunctions such as regulating follicular development,atresia,ovulation,oocyte quality,sexual behavior,pregnancy,infertility and luteal function in mammals.Until now,the effects of nutrition and/or Arg supplementation on endocrine and ovarian function in non-pregnant animals have not been well studied.Considering the above situation,this study added exogenous Arg to the diet to observe whether it can alleviate the delayed ovulation of Hu sheep and explore the possible mechanism of Arg-NO signaling pathway regulating the development of follicles.This study consists of the following four parts:1.Effects of nutrient restriction and arginine treatment on follicular development and NO-cGMP signaling in Hu sheep during the luteal phaseThis study was aimed to determine whether arginine(Arg)supplementation in malnourished ewes affects the expression of NO-cGMP signaling pathway genes in the ovary.On days 6-15 of the estrous cycle,24 multiparous Hu sheep(BW=43.56±1.53 kg)were randomly assigned to three groups:control group(CG;n=6),restriction group(RG;n=9)and L-Arginine group(AG;n=9)and the L-Arginine group was administered Arg treatment(or vehicle)three times per day.The ewes were slaughtered at the end of treatment and blood samples and ovaries were collected for analysis.The results showed that short-term feed-restriction and/or supplementation with L-Arg-HCl affected the number of different size follicles observed in the ovary,and the relative date at which estrus behavior begins.Specifically,the relative date of estrus behavior initiation was significantly advanced in AG compared with that in RG ewes(P<0.05).Compared with CG ewes,the number of-ovarian follicles≤2 mm(P<0.05)and the total number of ovarian follicles(P<0.05)were significantly increased in the RG and AG ewes.The proportion of follicles ≤ 2 mm in RG ewes was higher(P<0.05),but the proportion of follicles>5 mm was lower than that in CG ewes(P<0.05).Compared with CG or RG ewes,the mean number of corpus lutea>5 mm in AG ewes was significantly increased.In addition,the expression of eNOS,nNOS,iNOS,PDE5A,PDE9A and PRKG2 varied significantly among the treatment groups(P<0.05).Compared with CG ewes,GUCY1A3 mRNA levels were significantly increased in RG and AG ewes(P<0.05),whereas conversely,GUCY1B3 mRNA levels were significantly decreased in CG and RG compared to AG ewes(P<0.05).In conclusion,the results of the present study indicate that feed-restriction has a negative impact on follicular development,and that Arg-supplementation may modulate the expression of NO-cGMP signaling pathway genes in the ovary,thereby accelerating ovulation and the estrous rate.To elucidate the mechanism of these effects of Arg on gene expression in the ovaries of ewes requires further investigation.2.Effect of nutrient restriction and arginine supplementation on the expression of reproductive hormone related genes in Hu sheep during the luteal phaseIn this study,the effects of Arg-NO signaling pathway on reproductive hormone-related genes and proteins in the hypothalamic-pituitary-ovarian axis of sheep were investigated by limulus-term feeding and arginine injection.A total of 24 purebred Hu sheep of similar ages(2-3 years old)were selected in this experiment,and the experiment began in the next emotional period after estrus.On the 6th day after the trial,they were divided into 3 groups(control group,CG;restriction group,RG;Arginine group,AG).After 9 days of processing,they were slaughtered during estrus,separating ≤2.5 mm and>2.5 mm follicles,and collecting serum and hypothalamus,pituitary and ovary for further study.nutrient restriction significantly increased GnIH concentration in the hypothalamus and GnIH gene expression in the hypothalamus-pituitary and the expression of 3BHSD and CYP19A1 genes in the ovary(P<0.05),and significantly decreased the expression of GnRH in the hypothalamus and FSHR gene in pituitary-ovarian tissue(P<0.05).The gene expression in the AG group showed an opposite trend to the RG group.The concentration of E2 in follicles increased significantly,and the expression of 3BHSD and CYP19A1 genes in follicles>2.5 mm was significantly higher in the feed restriction group than that in the control group(P<0.05).The concentration of P4 in follicles of RG group was significantly lower than that of the control group(P<0.05).The Aeg treatment group significantly increased the concentration of E2 in the follicles≤2.5 mm,significantly decreased the concentration of E2 in the follicles>2.5 mm,and significantly increased the concentration of P4 in the follicles>2.5 mm(P<0.05).In summary,short-term nutrient restriction of the corpus luteum prior to dissolution significantly up-regulated the expression of GnIH in the hypothalamus-pituitary,affecting E2 and P4 concentrations in the late follicles,and might inhibit follicular development by affecting the expression of 3BHSD,CYP19A1 in follicles.Arg may down-regulate the expression of GnIH in the hypothalamus-pituitary via the NO pathway and maintain the balance of E2 and P4 in follicles by affecting the expression levels of StAR、3BHSD and CYP19A1 in follicles,thereby promoting the follicular development of nutrient restriction ovarian follicles.3.Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of Hu sheep during the luteal phaseThe aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase.On days 6-15 of the estrous cycle,24 multiparous Hu sheep(BW=43.56±1.53 kg)were randomly assigned to three groups:control group(CG;n=6),restriction group(RG;n=9),and L-arginine group(AG;n=9),the AG group received Arg treatment(or vehicle)three times per day.The ewes were slaughtered at the end of treatment,and blood samples and ovaries were collected for analysis.In this study,the expression levels of antioxidase enzymes(SOD2,CAT and GPX1)and mitochondrial biogenesis-related genes(ESRRA and TFAM),as well as antioxidase activity and mitochondrial function were examined in ovarian tissue.Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number,while arginine treatment led to a significant recovery of ovarian tissue.In addition,we observed an increase in AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes,and these effects decreased with arginine treatment.In conclusion,the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress.These changes disrupted the redox balance,thus leading to apoptosis through the mitochondria-dependent apoptosis pathway.Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress.The results presented here suggest that reducing the temporary undernutrition of ewes by using dietary supplementation with arginine can increase the agricultural productivity and economic benefits of the sheep industry.4.Effects of nitric oxide on proliferation and apoptosis of goat granulosa cellsThe granulosa cells from healthy follicles were isolated and cultured in vitro.L-NAME was used to inhibit NO product and SNP treatment to study the regulation of NO on granulosa cells cultured in vitro.The results showed that compared with the control group and the L-NAME group,the SNP group significantly increased the NO metabolite content(NO3-/NO2-)in the medium and the cGMP concentration in the granulosa cells(P<0.05).SNP increased the expression of StAR,3BHSD and CYP19A1 mRNA in granulosa cells,and significantly increased the concentrations of E2 and P4 in culture medium(P<0.05).Inhibition of NO significantly increased the apoptotic rate of granulosa cells,the expression of BAX and the ratio of BAX/BBCL-2 and decreased the expression of BCL-2(P<0.05).SNP treatment had an opposite effect Inhibition of NO production led to a significant increase in oxidative damage-related indicators(eg,8-OhdG and GSH/GSSG,P<0.05),decreased antioxidant enzyme activity and gene and protein expression(SOD,CAT and GSH-Px activities and SOD2,CAT and GPX1 gene and protein levels,P<0.05),SNP treatment maintained the balance of cellular oxidation and antioxidant system.Inhibition of NO significantly reduced the copy number of mtDNA(P<0.05)and decreased the expression levels of mitochondrial biogenesis related genes and proteins(PPARGC1A,NRF1 and TFAM),In the comparison between the SNP+L-NAME group and the L-NAME group,SNP significantly increased mtDNA copy number(P<0.05)and the expression of PPARGC1A,NRF1 and TFAM genes and proteins(P<0.05).The above results indicate that NO may up-regulate the expression of PPARGC1A and its downstream factors through the cGMP pathway,thereby reducing granulosa cell apoptosis,and participating in the regulation of granulocyte steroid production through the mitochondrial-dependent pathway.However,the specific molecular mechanisms require further research and verification.