Effects Of Photoperiod On The Epigenetic Regulation Of Hypothalamus In Sheep Based On OVX+E₂ Model

The seasonal reproduction of sheep is formed by the long-term selection of the external environment such as the photoperiod,and the change of photoperiod is the key factor affecting the reproductive seasonality.Emerging pieces of evidence have indicated that epigenetic modification participated in expression regulation of hypothalamic peptides which controlled reproductive activities,but the mechanism is still unclear.In the present study,Sunite ewes,one of the typical seasonal estrus breeds in China,were selected to establish a photoperiod-controlled model,in which the ewes were ovariectomized and estradiol-implanted?OVX+E₂?.The concentration of serum reproductive hormone and key genes were tested under different photoperiod conditions,RNA-seq and whole-genome bisulfite sequencing?WGBS?were simultaneously used to explore the epigenetic changes of ovine hypothalamus with the photoperiodic change.Experimental ewes were ovariectomized by surgical operation and implanted with estradiol at a concentration of 7.23±2.50 pg/mL under the skin to construct OVX+E₂ model.Then all the OVX+E₂ewes were housed in photoperiod-controlled rooms as 3 groups?short photoperiod,SP;long photoperiod,LP;short photoperiod transfers to long photoperiod,SP-LP?.Compared with SP,the concentration of FSH decreased significantly in LP?P<0.05?,while the concentration of LH and PRL increased significantly?P<0.01?.The secretion rule of FSH and PRL under different photoperiod conditions was in accordance with the physiological characteristics of normal ewes,but the LH is different from previous studies and still needs further verification.The selected 12 genes?KISS1,RFRP-3,DIO2,CLOCK,MTNR1A and PER1 in the hypothalamus;EYA3,TAC1,LHB,LEPR,TSHB and CGA in the pituitary?all reached significant levels under different photoperiod conditions?P<0.05?,and the expression patterns were consistent with previous research results.The results indicate that the OVX+E₂ model could facilitate the study of photoperiod regulation of upstream tissue function related to reproduction in sheep.This study is the first to understand the effect of photoperiod changes on OVX+E₂ ewe on hypothalamic function from the transcriptome level.The main mRNAs?such as VAMP2,PRKACB,PLCB1,RASD1,KCNJ5?and lncRNAs?such as XR?173257.3,XR?173415.3,XR?001023464.2?involving in photoperiod regulation of sheep hypothalamic function were initially selected by comparing and analyzing the SP42,LP42,SP-LP3,SP-LP7,SP-LP15,SP-LP21,and SP-LP42transcriptomes,and these main transcripts mainly regulate the photoperiodic response of the hypothalamus by participating in signal pathways such as circadian rhythm,thyroid hormone synthesis and insulin secretion.Besides,interaction analyses between lncRNAs and mRNAs showed that the above-mentioned lncRNAs have regulatory effects on the expression of their target genes after the photoperiod changes,which in turn affects activities of the photoperiod response and seasonal reproduction pathways in sheep.Through the WGBS analysis of the hypothalamus of 21 ewes,the raw data exceeding the genome coverage of 20×was obtained,in which the C site coverage exceeded 7×.On this basis,a single-base resolution map of ovine hypothalamus genome DNA methylation was drawn.It was initially confirmed that the regulation of the photoperiod on the hypothalamic DNA methylation is dynamic and reversible.Besides,the function analysis showed that many DMRs and DMRs related genes were found and were mainly involved in the hypothalamic neuroendocrine activity and photoperiod regulation through pathways including dopaminergic synapse,insulin secretion and circadian rhythm.This study initially demonstrated that the photoperiod could cause the transformation of the hypothalamic epigenetic regulation pattern,which provides a new idea and theoretical basis for future research on the photoperiod regulation of seasonal estrus in sheep.