Effects Of Exogenous ACTH On Estrus,DNA Methylation And Gene Expression Of New Corpus Luteum In Postpartum Weaning Sows

Sows after weaning should get pregnant as soon as possible as this is critical for achieving the best reproductive performance of a herd in the pig production.However,some sows show estrus but without ovulating(pseudo-estrus)or fail to resume the estrous cycle after weaning.Stress is recognized as an environmental contributing factor but the mechanism is still unclear.During the stress response,the hypothalamus-pituitary-adrenal(HPA)axis is activated which involves the sequential release of corticotropin releasing hormone(CRH),adrenocorticotropic hormone(ACTH)and glucocorticoids,such as cortisol.Long term stress or chronic stress prolongs the increases of ACTH and cortisol.The increases of ACTH and cortisol influence the role of the hypothalamus and the pituitary gland,so as to disrupt reproductive processes.As an important stress hormone,ACTH administration is used to study the role of stress hormones in many reproductive processes.Previous studies have revealed the effects of short term repeated ACTH administration on estrus of sows.However,there is a scarcity of information available on the influence of the long term repeated ACTH administration performed in sows after weaning.Especially,the effects of long-term ACTH administration on post-weaning sows during whole period include several ovarian physiological processessuch as follicle development,follicular dominance,ovulation and lutenization is still unclear.In addition,the mechanisms regarding how stress hormones influence the function of CL are poorly understood.The previous investigations were limited to only a small number of genes;a few studies systematically determined the potential mechanism of stress-induced alteration of CL function by using an unbiased genome-wide approach.DNA methylation is an important epigenetic modification and is associated with gene expression and plays critical roles in cellular differentiation,embryogenesis,X-inactivation,genomic imprinting as well as tumorigenesis.However,the DNA methylation pattern in CL and the alterations of DNA methylation pattern in CL formed under chronic stress condition as well as the influence of DNA methylation alteration on gene express have not been reported.In order to clearly reflect the role of long term repeated ACTH administration on the reproductive performance and genomic DNA methylation and gene expression of sows after weaning,9 sows after weaning were chosen and injected with synthetic ACTH at every 8 h intervals for 7 days to construct the model of chronic stress reaction.The blood physiological indexes,serum hormone levels,estrus time statistics,ovarian morphology development were analyzed to examine the effects of long term repeated ACTH administration on reproductive performance of sows after weaning.The alterations of DNA methylome and transcriptome in CL of sows after weaning under ACTH treatment were analyzed by whole-genome bisulfite sequencing(WGBS)and RNA-sequencing(RNA-seq),respectively.The differentially methylated regions and the differentially expressed genes were combined to select the potential genes and pathways that involve in the regulation of development and function of CL under ACTH administration.At last,the luteal cell of sow cultured in-vitro and mouse injected by corticosterone were used to verify the possible role of cortisol during this process.The results showed that:1.Long term repeated ACTH administration significantly upregulated the concentrations of cortisol,glucose,lactate dehydrogenase,creatine kinase,triglyceride and total cholesterol in the plasma of sows after weaned.Results suggested that ACTH administration may influence the glucose metabolism,fat metabolism and energy metabolism of sows.The levels of total protein,albumin as well as the numbers of both white blood cells and neutrophils in the blood of sows were significantly upregulated while the numbers of lymphocyte were significantly decreased by ACTH injection during the experimental phases(P<0.05).These results indicated that ACTH administration induced a reaction similar to stress response in sows.2.All sows displayed normal estrus behavior and standing response to the back-pressure test.There was a significant difference(P<0.05)in the average time of first observed estrus between the ACTH group(99.00 ± 1.22 h)and the control group(84.00±4.95 h).The concentrations of estradiol-17β in ACTH group were significantly lower than the control group at 36 h,42 h and 66 h of experimental period(P<0.05).The concentrations of progesterone in ACTH group were significantly lower than the control group at 132 h,138 h and 147 h of experimental period(P<0.05).ACTH administration significantly decreased LH secretion during the estrus periods(P<0.05).The average concentrations of progesterone in the ACTH group were significantly lower than the control group during the estrus period(P<0.01).All control sows showed the expected LH surgeduring the estrus periods.Three sows in the ACTH group showed LH surge.The LH surge in control group appeared at 108.80± 5.54 h,while the LH surge in the ACTH group appeared at 136.30± 3.75 h,the difference was statistically significant(P<0.05).There was a strong positive correlation between cortisol and progesterone levels(R=0.738,P=0.012)in the ACTH group.No anatomical abnormalities,signs of infection or ovarian cysts were found in the postmortem macroscopic examination of the reproductive organs of the sows.All the ovaries obtained from control sows were ovulated and CLs were observed,however,three sows of the ACTH group had ovulated and formed CLs.The ovulation rate of sows in control group was 100%while it was 60%in the ACTH group.There was no significant difference between the two groups in either the average organ index(ACTH group ovulated sows,8.85 ± 0.88 and control group 7.81 ± 0.57)or the numbers of ovulated follicles(ACTH group ovulated sows,18.50±1.32 and control group 17.33 ± 1.45).What’s more,ACTH administration reduced the mRNA expression of LHR in CL,but didn’t influence the mRNA expression of GR.3.A total of 1.825 billion reads and 228.19 Gb data were generated by performing WGBS.After quality control,there were approximately 1.799 billion clean reads(224.91 Gb),and about 68.55%of the clean reads were aligned to the porcine reference genome(Sscrofa 10.2).The average C site in each sample was around 5172 Mb and each sample contained about 272 Mb GC site.The percent of methylated C sites in the genome of control sample was 4.23%,while it was 4.20%in the ACTH group.Under normal conditions,the methylated cytosine sites were composed of 93.97%mCG,1.72%mCHG,and 4.31%mCHH in CL;After ACTH injection,the methylated cytosine sites consisted of 95.27%mCG,1.29%mCHG,and 3.44%mCHH.The results visually showed the chromosomal distribution of differential regions between control and ACTH group,and the X chromosome exhibited most of the differential regions.By comparing the distribution of gene density and DNA methylation level,it revealed a negative correlation between gene density and methylation level.According to the distribution of gene density in chromosomes,the differences of methylation level between two groups were mainly presented in the regions of chromosomes with low gene density.The CG methylation patterns of two groups in gene functional regions were showed lower CG methylation level in 5’UTRs and hyper-methylation in introns,exons,as well as 3’UTRs.The CG methylation level in regions except promoters were lower in control group than the ACTH group,and the CG methylation density in 5’-UTRs were lower in the control group than the ACTH group.In the context of both CHG and CHH,the patterns of methylation level and density in promoters,5’UTRs,exons,as well as 3’UTRs were largely different between two groups.Furthermore,the methylation level of CG islands(CGIs)located in five genomic regions was analyzed.Similar trends were displayed both in control and ACTH group:the DNA methylation level were lowest in regions of 5 kb upstream,where the transcriptional start sites(TSSs)and highest in introns.According to CG content in the promoters,genes were separated into high CG promoter genes(HCPs)and low CG promoter genes(LCPs).Both in HCPs and LCPs,more than 70%CG sites at region of 5 kb upstream of TSSs were methylated and the gene body was hyper-methylation.Different from LCPs,the methylation pattern of HCPs showed that the methylation level declined abruptly at TSSs while a slight decrease was revealed at TSSs in LCPs.Furthermore,methylation level appeared to drop in introns of HCPs.The curve trends of HCPs and LCPs in the two groups were slightly similar but the differences were presented in the introns of HCPs and 2 kb region downstream of the TES of LCPs.A total of 88557 DMRs were identified;DMRs with higher methylation level were 54530 in the ACTH group compared with the control group while the numbers of DMR with lower methylation level were 34027.The length distribution of DMRs was similar to Gauss distribution.The lengths of DMRs were mainly distributed from 100bp to 500bp in each chromosome.Results showed that 1,899 genes were associated with hyper-methylation DMRs and 118 genes were associated with hypo-methylation DMRs.Most of the genes were significantly gathered in term of metabolic in GO biological processes(P=2.39E-30,GO:0008152).Furthermore,many DMRs related genes were involved in cellular differentiation(P=7.63E-14,GO:0030154).For molecular function analysis,large amount of genes were enriched in the function of binding(GO:0005488)and protein binding(GO:0005515).4.A total of 141.71 million reads were generated and approximately 76.17%clean reads were mapped to the pig genome(Sscrofa 10.2).There were 290 genes expressed in the ACTH group,439 genes expressed in the control group,and 12772 genes were co-expressed between the two groups.9 differentially expressed genes(DEGs)were screened out from co-expressed genes,of which 7 genes were up-regulated and 2 genes were down-regulated in the ACTH group compared with the control group.The GO analysis of DEGs showed that DEGs were strongly associated with oxygen transport both in biological processes and molecular function.The KEGG pathway of DEGs was performed and results showed that two genes enriched in African trypanosomiasis and malaria which may be involved in the process of body resist diseases.The qRT-PCR was used to confirm the differentially expressed genes in CL.Results showed that 7 out of 9 DEGs exhibited significant difference in the expression between the two groups.Four fulfilled genes were detected through condition selection;they were EPB41L3,SLCi5A2,FBX02 and QNS1.Therefore,we predicted that these 4 DMR-related genes were most likely to be involved in the CL function under ACTH administration.5.Both the results of mouse in-vivo experiment and sow’s luteal cell cultured in-vitro demonstrated that EPB41L3,,SLC5A2,FBX02 and QNS1 are involved in the development of the corpus luteum and can be regulated by cortisol/corticosterone.But the effects of cortisol administration were not completely consistent with the ACTH administration.These results indicated that the influences of ACTH administration on DNA methylation and gene expression of CL in sow were not completely or directly dependent on cortisol.In conclusion,our results comprehensively reflected the dynamic changes in gonadotrophins and gonadal hormones during the phase after weaning to ovulation under ACTH administration.Our results suggested that the long term stressful stimuli inducing the hypersecretion of ACTH or cortisol may alter the normal follicle development,suppress the pre-ovulatory surge of LH,block ovulation,and possibly influenced CL function of crossbred sows.Our study delineated the landscape of CL methylome distribution in the genome,analyzed differentially methylated regions,and identified differentially expressed genes that were involved in the development of CL under long term ACTH administration.Our findings might provide novel insights into the pathogenesis mechanism of stress-induced CL functional disorders by identifying the molecular effects of some epigenetic biomarkers in future research.