LncRNA LOC102176306 plays important roles in goat testicular development.

Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.

Estradiol-17ß regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1.

Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells (EEC) as a result of estradiol-17ß (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1 mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17ß treatment the abundance of YAP1 mRNA transcript was similar to that of the control samples. Taken together, estradiol-17ß may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17ß and YAP1 in EEC proliferation and apoptosis of ewes.

MiR-1197-3p regulates testosterone secretion in goat Leydig cells via targeting PPARGC1A.

As a fundamental regulator of mitochondrial function, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) acts as a powerful coactivator of many transcriptional factors that relate to steroidogenesis, while the regulatory mechanism remains unclear. In the present study, testosterone secretion of goat Leydig cells (LCs) mediated by miR-1197-3p via PPARGC1A was investigated. We found PPARGC1A protein was diversely localized in testis, and the expression of PPARGC1A in testis of 9-month-old goat was significantly higher than that in 3-month-old goat. In addition, suppression of PPARGC1A significantly decreased the testosterone secretion in goat LCs, as well as reduced the expressions of key steroidogenesis related genes [steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3 beta-hydroxysteroid dehydrogenase (3BHSD)], and overexpression of PPARGC1A showed the opposite effects. Moreover, we observed suppression of miR-1197-3p increased the synthesis of testosterone and promoted the expressions of PPARGC1A, StAR, CYP11A1, and 3BHSD by directly targeting PPARGC1A in the LCs. Furthermore, overexpression of PPARGC1A could alleviate miR-1197-3p induced aberrant steroidogenesis related gene expressions and testosterone synthesis. Taken together, miR-1197-3p could act as an essential regulator of LC testosterone secretion in goat testis by targeting PPARGC1A. These results provide a novel view of the regulatory mechanisms involved in male sexual maturation and help us to understand the molecular role of PPARGC1A in testosterone synthesis.

Suppression of miR-1197-3p attenuates H2O2-induced apoptosis of goat luteinized granulosa cells via targeting PPARGC1A.

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) acts as a powerful coactivator of many transcriptional factors that relate to granulosa cell (GC) apoptosis. In this study, the miRNAs mediating goat follicular atresia and luteinized granulosa cell (LGC) apoptosis induced by hydrogen peroxide (H2O2) via PPARGC1A were investigated. Our results showed that miR-1197-3p targeted PPARGC1A was predicted by bioinformatics algorithm and verified by luciferase reporter assay. In addition, miR-1197-3p promoted goat LGC apoptosis via PPARGC1A through mitochondrial-dependent apoptosis pathway, and these effects could be restored by PPARGC1A overexpression. Moreover, H2O2-induced LGC apoptosis significantly upregulated miR-1197-3p expression and downregulated PPARGC1A level. Pretreatment of miR-1197-3p inhibitor alleviated LGC apoptosis induced by 400 µM H2O2 for 12 h, and preserved the mitochondrial membrane potential by increasing PPARGC1A expression. In conclusion, miR-1197-3p might act as an essential regulator of goat LGC apoptosis potentially via the mitochondrial-dependent apoptosis pathway by targeting PPARGC1A.

Expression of Hippo signaling pathway components in Hu sheep male reproductive tract and spermatozoa.

Hippo signaling pathway is essential for tissue development and homeostasis, while its specific role in male reproductive tract development is still unclear. The objective of this study is to elucidate the localization and expressions of key Hippo pathway components (MST1/2, LATS1/2 and YAP1) in male reproductive tract (testis, epididymis, and ductus deferens) of prepubertal (3-month-old) and postpubertal (9-month-old) Hu sheep, as well as in the cauda epididymal and ejaculated spermatozoa. Results showed that the Hippo pathway proteins were diversely localized in male reproductive tract portions, and most of their expression levels increased during sheep testicular maturation. In addition, these Hippo components were mainly localized and highly expressed in ejaculated spermatozoa compared with cauda epididymal spermatozoa. In ejaculated spermatozoa, LATS1 was localized in the acrosomal head region, and LATS2 and YAP1 were expressed in the midpiece part. Taken together, the presence of Hippo signaling cascade in the pubertal development of male reproductive tract and spermatogenesis of Hu sheep, provides new insights on the function of these components in the process of male sexual maturation, capacitation and fertilization.