Transcriptomic Changes of Photoperiodic Response in the Hypothalamus Were Identified in Ovariectomized and Estradiol-Treated Sheep

Abstract

Accurate timing of seasonal changes is an essential ability for an animal's survival, and the change in the photoperiod is the key factor affecting reproductive seasonality in mammals. Emerging evidence has suggested that multiple hypothalamic genes participate in the photoperiod-induced regulation of reproductive activities in sheep, but the mechanism is still unclear. In this study, we initially examined the plasma level of two major reproductive hormones, namely, follicle-stimulating hormone (FSH) and prolactin (PRL), under different photoperiods in ovariectomized and estradiol-treated (OVX + E₂) sheep using radioimmunoassay (RIA). Of the two hormones, the concentration of PRL significantly increased with the extension of the photoperiod, while FSH showed the opposite trend. Subsequently, an examination of the transcriptomic variation between the short photoperiod (SP) and long photoperiod (LP) was conducted. Differential expression analyses and functional annotation showed that several key genes in the insulin secretion (VAMP2, PRKACB, PRKCG, and PLCB1), GnRH (MAPK13, CGA, CDC42, ATF4, and LHB) pathways, and circadian entrainment (KCNJ5, PER1, GNB2, MTNR1A, and RASD1), as well as numerous lncRNAs, including XR_173257.3, XR_173415.3, XR_001435315.1, XR_001024596.2, and XR_001023464.2, were shown potentially vital for the hypothalamic photoperiodic response. Four of the differentially expressed mRNAs and lncRNAs were validated by qPCR. The constructed mRNA-mRNA interaction networks further revealed that transcripts potentially participated in hypothalamic thyroid hormone synthesis, endocrine resistance, and neuroactive ligand-receptor interactions. The interactome analysis of lncRNAs and their targets implied that XR_173257.3 and its target arylalkylamine N-acetyltransferase (AANAT) and XR_173415.3 and its target TH might participate in the regulation of seasonal reproduction. Together, the changes in reproductive hormones and transcriptome will help to determine the important photoperiod-induced lncRNAs and mRNAs and provide a valuable resource for further research on reproductive seasonality in sheep.

Keywords: hormone; hypothalamus; lncRNAs; mRNAs; pathways; photoperiodic response.

FIGURE 1

Hormone preparation and sample collection timeline. All OVX + E₂ ewes were assigned to three experimental groups in three rooms: short photoperiod (SP), long photoperiod (LP), and short photoperiod transfer to long photoperiod (SP-LP). A total of 24 pink triangles represent the day of blood sample collection in SP-LP groups, and seven green circles were the time of hypothalamus sample collection. In addition, the tissue and blood samples were collected at 8 a.m. and 2 p.m., respectively.

FIGURE 2

Hormonal profiling of the response to the different photoperiodic treatments. (A) Mean follicle-stimulating hormone (FSH) levels in blood serum of ewes sampled twice weekly throughout the experiment. (B) Mean of FSH for the SP and LP (12 time points each treatment). (C,D) Prolactin (PRL) profiles, the picture interpretation identical to those for FSH. Results were expressed as mean ± SE, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

FIGURE 3

Screening of the candidate lncRNAs in the ovine hypothalamus transcriptome. (A) 522,061 transcripts were assembled and 11,965 putative non-coding transcripts were retained for the next analysis after five steps filtering. (B) lncRNAs identification was also used in CPC, PFAM, and CNCI.

FIGURE 4

Characterization of candidate lncRNA and mRNA. (A–C) Length, exon number, open reading frame (ORF) length distribution of mRNA, annotated lncRNA, and novel lncRNA. (D) violin plot of expression level about mRNAs, annotated-lncRNAs, and novel-lncRNAs.

FIGURE 5

Top enriched Gene Ontology (GO) terms of differentially expressed mRNA between the short photoperiod and long photoperiod in the hypothalamus of Sunite sheep. The GO terms in (A–F) are from SP42 vs. LP42, SP42 vs. SP-LP3, SP42 vs. SP-LP7, SP42 vs. SP-LP15, SP42 vs. SP-LP21, and SP42 vs. SP-LP42, respectively.

FIGURE 6

Top enriched Gene Ontology (GO) terms of differentially expressed lncRNAs between the short photoperiod and long photoperiod in the hypothalamus of Sunite sheep. The GO terms in (A–F) are from SP42 vs. LP42, SP42 vs. SP-LP3, SP42 vs. SP-LP7, SP42 vs. SP-LP15, SP42 vs. SP-LP21, and SP42 vs. SP-LP42, respectively.

FIGURE 7

KEGG analysis of differentially expressed mRNAs and lncRNAs targets in hypothalamus about photoperiod responsiveness. (A) Five of the top KEGG enrichment pathway for differentially expressed mRNAs; (B) three to five of the top KEGG enrichment pathway for differentially expressed lncRNA targets. The longitudinal axis represents the enrichment pathways in different compared groups, and the horizontal axis represents the rich factor of these pathways. The spot size and color represent the number of differentially expressed genes and statistical significance of each pathway, respectively.

FIGURE 8

Interaction network in the hypothalamus about ovine photoperiod responsiveness and estrous seasonality. (A) mRNA–mRNA interaction network involved in photoperiodic responsiveness, the arrow direction represents the targeting direction; (B) lncRNA–mRNA interaction network related to ovine estrus regulation with the photoperiodic change. Circles and “V” represent mRNAs and lncRNAs, red and green represent upregulated and downregulated transcripts, respectively, and the arrow indicates the direction of the target relationship.