There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome. We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis. The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway. Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

The corpus luteum number in sows is a key reproductive indicator for measuring ovulation rate and reproductive efficiency. Its formation is precisely regulated by a complex gene regulatory network composed of multi-level molecular interactions. To systematically elucidate the molecular basis of this trait, this study comprehensively analyzed genes located within QTL for corpus luteum number. This approach identified a series of key regulatory molecules specifically expressed in the ovary, including transcription factors (TFs), long non-coding RNAs (lncRNAs), and microRNAs (miRNAs). Using bioinformatics methods to predict the target genes of candidate miRNAs, combined with functional enrichment analysis, revealed that these target genes were significantly enriched in multiple core reproductive pathways closely related to cell proliferation, differentiation, and hormone regulation, including the ErbB signaling pathway, PI3K-Akt signaling pathway, and TGF-beta signaling pathway. Based on the above findings, this study ultimately constructed a TF-lncRNA-miRNA-mRNA network, which is associated with QTL for corpus luteum number. Furthermore, key genes were validated via quantitative real-time PCR (qRT-PCR). Significant positive correlations were identified between the transcription factor NEUROG2 and lncRNA LOC102167554, along with its potential target gene ESRP1, as well as between transcription factor SNAI2 and lncRNAs (LOC102167554, LOC102167796) and their potential target genes (FXR, ERBB4). In addition, the functional validation results showed that the interference of LOC102167554 significantly reduced the proliferation ability of sGCs. These key genes represent potential targets for genetic improvement of sow reproduction.