Both hypothalamic neurotransmitters and serum steroid hormones are impacted by photoperiod and have effects on physiology and seasonal reproductive. However, the relationship between circulating gonadal steroids and hypothalamic neurotransmitters underlying different photoperiod is still unclear. To further understand the crosstalk of neurotransmitters and steroids in seasonal reproduction, metabolic changes of 27 neurotransmitters concentrated in hypothalamus tissues and 42 steroids hormones in serum were assessed during two artificial photoperiodic programs. The results showed that photoperiod induce testicular atrophy and recrudescence. In L-to-S groups, significantly decreased levels of testosterone concentration were found in serum (P < 0.001) and increased 11-Dehydrocorticosterone (P < 0.05); Testosterone were almost undetectable at SD_14d. In addition, the hypothalamus exhibited significantly increased arginine and 4-aminobutyric acid (GABA) concentration and decreased serotonin and epinephrine content (P < 0.01 or P < 0.05). Accordingly, serum testosterone and androstenedione became detectable at LD_3d in the S-to-L group and were markedly increase at LD_7d. Furthermore, Serum androstenedione showed a significant increase with long light expose (P < 0.01). Additionally, the hypothalamus exhibited both significantly increased L.Tryptophan and phenylalanine concentration, as well as decreased L-glutamine and L-glutamine.acid content (P < 0.01 or P < 0.05). Serotonin metabolism showed significant differences between L-to-S group and S-to-L group. Furthermore, in the correlation analysis, serum testosterone had a positive correlation with 5-Hydroxyindole-3-acetic acid (5-HIAA), while Androstenedione was significantly negative with L.Tryptophan in L-to-S (P < 0.05). However, in S-to-L group, serum testosterone showed strong negative correlation with both serotonin and 5-HIAA (P < 0.05), but positive correlation with L.Tryptophan (P < 0.01), while Androstenedione was significantly negative correlation with both serotonin (P < 0.05) and L-Glutamine (P < 0.01). Photoperiod also had significant effects on the mRNA expression. We found significant differences in gene expression patterns of both serotonin signaling and steroid biosynthesis, while MAOB, NR5A1, and 3β-HSD showed an opposite tendency between two groups. Taken together, our results revealed that circulating gonadal steroids and hypothalamic neurotransmitters were significantly impact quail’s seasonal reproduction. Circulating gonadal steroids have different effects on neurotransmitter at different photoperiodism, which may coordinately influence the seasonal reproduction of quails.

The incubation behavior of geese seriously affects their egg production performance. Studies on incubation behavior have identified functional genes, but the regulatory architecture relationship between functional genes and chromatin accessibility remains poorly understood. Here, we present an integrated analysis of open chromatin profiles and transcriptome to identify the cis-regulatory element and their potential transcription factors involved in regulating incubation behavior in goose pituitary. Assay for transposase-accessible chromatin sequencing (ATAC-seq) revealed that open chromatin regions increased in the pituitary during the transition from incubation behavior to laying. We identified 920 significant differential accessible regions (DARs) in the pituitary. Compared to the laying stage, most DARs had higher chromatin accessibility in the brooding stage. Motif analysis of open DARs showed that the most significant transcription factor (TF) occupied sites predominantly enriched in motifs binding to the RFX family (RFX5, RFX2, and RFX1). While the majority of TF motifs enriched under sites of the nuclear receptor (NR) family (ARE, GRE, and PGR) in closed DARs at the incubation behavior stage. Footprint analysis indicated that the transcription factor RFX family exhibited higher binding on chromatin at the brooding stage. To further elucidate the effect of changes in chromatin accessibility on gene expression levels, a comparison of the transcriptome revealed 279 differentially expressed genes (DEGs). The transcriptome changes were associated with processes of steroid biosynthesis. By integrating ATAC-seq and RNA-seq, few DARs directly affect incubation behavior by regulating the transcription levels of genes. Five DAR-related DEGs were found to be closely related to maintaining the incubation behavior in geese. Footprinting analysis revealed a set of transcription factors (RFX1, RFX2, RFX3, RFX5, BHLHA15, SIX1, and DUX) which displayed the highest activity at the brooding stage. SREBF2 was predicted to be the unique differentially expressed transcription factor whose mRNA level was down-regulated and enriched in hyper-accessible regions of PRL in the broody stage. In the present study, we comprehensively profiled the transcriptome and chromatin accessibility in the pituitary related to incubation behavior. Our findings provided insight into the identification and analysis of regulatory elements in goose incubation behavior. The epigenetic alterations profiled here can help decipher the epigenetic mechanisms that contribute to the regulation of incubation behavior in birds.

Background Domestic geese are seasonal breeders and have the lowest reproductive capacity among all poultry species. Magang geese is a topical short-day breeder, short photoperiod exposure stimulates its reproductive activity while long photoperiod inhibits. To explore epigenetic change that could influence reproductive activity, we performed whole genome bisulfite sequencing and transcriptome sequencing in the hypothalamus at three reproductive stages during long-light exposure in male Magang geese. Results A total number of 10,602 differentially methylated regions (DMRs) were identified among three comparison groups. We observed that the vast majority of DMRs were enriched in intron regions. By integrating the BS-sequencing and RNA-seq data, the correlation between methylation changes of CG DMRs and expression changes of their associated genes was significant only for genes containing CG DMRs in their intron. A total of 278 DMR-associated DEGs were obtained among the three stages. KEGG analysis revealed that the DMR-associated DEGs were mainly involved in 11 pathways. Among them, the neuroactive ligand-receptor interaction pathway was significantly enriched in both two comparisons (RA vs.RD and RD vs.RI); the Wnt signaling pathway, apelin signaling pathway, melanogenesis, calcium signaling pathway, focal adhesion, and adherens junction were significantly enriched in the RA vs. RI comparison. In addition, the expression level of two serotonin-metabolic genes was significantly altered during reproductive axis inactivation by the methylation status of their promoter region (TPH2) and intron region (SLC18A2), respectively. These results were confirmed by Bisulfite sequencing PCR (BSP), pyrosequencing, and real-time qPCR, indicating that serotonin metabolic signaling may play a key role in decreasing the reproductive activity of Magang geese induced by long-light exposure. Furthermore, we performed a metabolomics approach to investigate the concentration of neurotransmitters among the three stages, and found that 5-HIAA, the last product of the serotonin metabolic pathway, was significantly decreased in the hypothalamus during RI. Conclusions Our study reveals that the methylation status of the serotonin metabolic pathway in the hypothalamus is associated with reproductive inactivation, and provided new insight into the effect of DNA methylation on the reproductive regulation of the hypothalamus in Magang geese.