. The outcomes of qPCR and Western blot in yak testicular tissues from animals of distinct ages confirmed that the expression levels of NR1D1 and NR4A2 had been up- or down-regulated together with the maturation or decline of sexual function (Figure 5). It was suggested that the expression of NR1D1 and NR4A2 in yak testicular tissues tended to become weak throughout sexual immaturity and strongest inside the most vigorous mating capability (three years old). The outcomes of GO and pathway enrichment analyses showed that NR1D1 and NR4A2 interacted straight or indirectly with proteins associated with steroid hormone biosynthesis, specifically StAR, HSD3B and SRD5A (Figure six), that are rate-limiting enzymes of steroidogenesis. For example, StAR protein translocates cholesterol in the outer to the inner mitochondrial membrane and is utilized further for steroid hormone biosynthesis in steroidogenic cells [39]. Testosterone is converted into 5-dihydrotestosterone by SRD5A in steroidogenic tissues and peripheral tissues [40]. The expression CD40 Antagonist medchemexpress patterns, places, prospective functions and regulatory ETB Activator MedChemExpress networks of NR1D1 and NR4A2 have been elaborated in the present study, but the certain regulation of NR1D1 and NR4A2 in hormone metabolism calls for additional study. These results will aid to improve the understanding of the reproductive physiological traits and to additional boost the reproductive performance of male yaks. five. Conclusions Inside the present study, we described the morphological structures of HPG tissues in male Tianzhu white yaks. The NR1D1 and NR4A2 proteins have been present in all yak HPG tissues, particularly inside the adenohypophysis, Leydig cells, principal cells and cilia on the epididymis. The NR1D1 and NR4A2 proteins have been primarily co-located in Leydig cells plus the cilia within the epididymis. The NR1D1 and NR4A2 mRNAs and proteins have been differentially expressed in all yak reproductive axis tissues and have been particularly hugely expressed in the hypophysis. The expression levels of NR1D1 and NR4A2 mRNAs and proteins inside the distinct testicular tissues were up- or down-regulated with the maturation or decline of sexual function in yaks. NR1D1 and NR4A2 interacted, either straight or indirectly, with proteins related to steroid hormone biosynthesis. These results recommended that NR1D1 and NR4A2 are involved in the regulation of steroid hormones and androgen metabolism in male yaks.Animals 2021, 11,14 ofSupplementary Supplies: The following are available online at mdpi/article/10 .3390/ani11113117/s1, Table S1: qPCR primer sequences, Table S2: The candidate genes or proteins associated with reproductive hormone metabolism, Figure S1: Localization of NR1D1, NR4A2 and 3-HSD in adult yak testis and epididymis tissues employing IF staining. Author Contributions: Conceptualization, Q.Z. and X.Z.; data curation, L.D., J.S., X.B., X.A. and B.Z.; funding acquisition, Q.Z., Y.Z. and X.Z.; investigation, L.D., J.S., X.B. and X.A.; methodology, Q.Z.; project administration, Q.Z. and X.Z.; resources, Q.Z.; Computer software, L.D. and B.Z.; validation, Q.Z. and X.Z.; writing–original draft, L.D. and Q.Z.; writing–review and editing, L.D. and Q.Z. Funding: This study was supported by the Topic Building Funding of Gansu Agricultural University, Gansu Province, China (GAU-XKJS-2018-160), the National Organic Science Foundation of China (No.32060752) along with the Innovation Potential and Improvement Project of Higher Education Department of Gansu Province, China (2019A-049). Institutional Critique Board Statement: The stu
