Transcriptome and DNA Methylation Analysis of Goat Pineal Gland During Puberty [RNA-seq]

Epigenetic regulation of gene transcription may play a critical role in the onset of puberty. Previous studies have confirmed that methylation regulates gene transcription in the hypothalamus-pituitary-gonadal axis during puberty initiation, but little is known about the regulation of DNA methylation on gene expression in the pineal gland. This study aims to screen pineal gland candidate genes related to the onset of goat puberty and regulated by genome methylation, to clarify the initiation mechanism of goat puberty and lay the foundation for improving goat reproductive performance. Pineal gland was collected from three female Anhui white goats during prepuberty (2.5 to 3.0 mo) and three during puberty (4.0 to 4.5 mo). Genome-wide hydrogen sulfite sequencing determined the DNA methylation profile, while RNA sequencing determined the transcriptome. The relationship between gene transcription and DNA methylation was determined by joint analysis. We found that there was no significant change in the whole genome methylation level of goat pineal gland before and after puberty, but the methylation pattern changed significantly, indicating that genomic DNA methylation of the pineal gland may be involved in regulating goat puberty initiation. Changes in DNA methylation patterns affected some pineal gland transcriptomes, while the transcriptional level of most genes was not affected by DNA methylation differences. Genes regulated by DNA methylation were mainly involved in metabolic processes, oxidative phosphorylation (OXPHOS), and signaling pathways related to thermogenesis. The differential genes ATP5F1D, CACNB2, and PTEN were significantly regulated by methylation, and the difference multiples of LIN28B, GIP, OPN1SW, and DCC were the most significant, possibly involved in puberty initiation. Overall design: The cunnus of pubertal goats was inflamed, with histological observation of some mature follicles in the ovaries. The goats were euthanized and their pineal gland were quickly collected. All the pineal glands sample were quickly frozen in liquid nitrogen for RNA and DNA methylation sequencing analysis.

基因转录的表观遗传调控（epigenetic regulation）在青春期启动过程中可能发挥关键作用。既往研究已证实，青春期启动阶段下丘脑-垂体-性腺轴（hypothalamus-pituitary-gonadal axis）中的甲基化可调控基因转录，但目前对松果体中DNA甲基化对基因表达的调控机制仍知之甚少。本研究旨在筛选与山羊青春期启动相关且受基因组甲基化调控的松果体候选基因，以阐明山羊青春期启动机制，为提升山羊繁殖性能奠定基础。本研究采集了青春期前（2.5~3.0月龄）和青春期（4.0~4.5月龄）的雌性安徽白山羊各3头的松果体组织。通过全基因组亚硫酸氢盐测序（genome-wide hydrogen sulfite sequencing）获取DNA甲基化谱，同时通过RNA测序获取转录组数据。通过联合分析明确基因转录与DNA甲基化之间的关联。研究结果显示，山羊松果体全基因组甲基化水平在青春期前后无显著变化，但甲基化模式发生了显著改变，提示松果体基因组DNA甲基化可能参与调控山羊青春期启动。DNA甲基化模式的改变会影响部分松果体转录组，但绝大多数基因的转录水平并未受DNA甲基化差异的调控。受DNA甲基化调控的基因主要参与代谢过程、氧化磷酸化（OXPHOS）以及产热相关信号通路。差异基因ATP5F1D、CACNB2及PTEN受甲基化调控显著，而LIN28B、GIP、OPN1SW与DCC的表达差异倍数最为显著，可能参与青春期启动过程。实验设计：青春期山羊的阴户存在炎症，且卵巢组织学观察可见部分成熟卵泡。随后对山羊实施安乐死，快速采集其松果体组织，所有样本均迅速置于液氮中冷冻，用于RNA测序及DNA甲基化测序分析。