The Distribution, Expression Patterns and Functional Analysis of NR1D1 and NR4A2 in the Reproductive Axis Tissues of the Male Tianzhu White Yak

SIMPLE SUMMARY: The reproductive function and behaviors of male animals are regulated by HPG tissues that are responsible for hormone synthesis and secretion. The NRs, as a kind of transcription factor, are recruited to some ligands, to convey and translate signals for regulation of hormone-related gene expression. Understanding the distribution, expression and function of NRs in reproductive axis tissues is vital for elucidating the molecular mechanisms of animal reproduction. NR1D1 and NR4A2, members of the NR superfamily, are important receptors of hormones, but lacking in identified target genes and related molecular mechanisms, particularly in yaks. In the present study, we found that NR1D1 and NR4A2 proteins were present in all yak HPG tissues, particularly in the adenohypophysis, Leydig cells, principal cells and cilia of epididymis. Yak testicular tissues from animals of different ages confirmed that the expression levels of NR1D1 and NR4A2 were up-regulated or down-regulated with the maturation or decline of sexual function. These results suggested that NR1D1 and NR4A2, as important mediators, are involved in the regulation of steroid hormones metabolism in male yaks. ABSTRACT: Nuclear hormone receptors NR1D1 and NR4A2 play important roles in the synthesis and metabolism of hormones that are thought to be strictly regulated by the hypothalamus-pituitary-gonad axis (HPG) tissues via gene expression. However, in the yak, the function and regulatory mechanisms of NR1D1 and NR4A2 are not clearly understood. The current study is aimed to investigate the expression patterns, distribution and functions of these two receptors in HPG tissues in male Tianzhu white yaks. Immunohistochemical staining showed NR1D1 and NR4A2 proteins were present in all yak HPG tissues with differential expression patterns and degrees of staining, particularly in Leydig cells that were strongly positive in accordance with the immunofluorescence results. qRT-PCR and Western blot results suggested that the highest expression levels of NR1D1 and NR4A2 mRNA were present in the hypothalamus, while the expression levels of NR1D1 and NR4A2 proteins were higher in the testis and epididymis than in the hypothalamus or pituitary gland. In addition, expression levels of NR1D1 and NR4A2 mRNA and protein in testicular tissues differed by age. Expression levels were significantly higher at 6 years of age. Gene ontology (GO) and pathway analysis enrichment revealed that NR1D1 may directly regulate the synthesis and metabolism of steroid hormones via interaction with different targets, while NR4A2 may indirectly regulate the synthesis and metabolism of steroid hormones. These results showed that NR1D1 and NR4A2, as important mediators, are involved in the regulation of male yak reproduction, and especially of steroid hormones and androgen metabolism. These results will be helpful for the further understanding of the regulatory mechanisms of NR1D1 and NR4A2 in yak reproduction.