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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.

H2S plays an important role in various inflammatory diseases. However, the role of H2S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-β-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H2S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H2S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H2S in Holstein cows.

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