Exploration the role of INHBA in Hu sheep granulosa cells using RNA-Seq.

Activin/inhibin is an important factor for the fecundity of Hu sheep, and it is involved in follicular development in ovaries. Inhibin subunit beta A (INHBA) participates in the synthesis of activin A and inhibin A. In this study, we also noted a positive correlation between INHBA level and the secretion of both activin A and inhibin A in culture medium. Nevertheless, both knockdown and overexpression of INHBA downregulated the expression of Inhibin Subunit Alpha (INHA). Based on RNA-Sequencing, we further examined the effect and molecular mechanism of INHBA knockdown in GCs on mRNA expression. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change ≥ 2; False-discovory-rates (FDR) ≤ 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference group compared with the control groups. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in the regulation of cell cycle, protein serine/threonine kinase activity, and actin cytoskeleton reorganization. Moreover, DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53, progesterone-mediated oocyte maturation, and PI3K-AKT signaling pathways. We also noted a positive correlation between INHBA level and many PI3K/Akt/mTOR pathway-related genes at the gene or/and protein expression. Overall, this study may contribute to a better understanding of the roles of INHBA on GCs of prolific sheep, as well as the molecular effect of low INHBA expression on GCs, clarifying some reproductive failures.

Dietary supplementation with metformin improves testis function and semen quality and increases antioxidants and autophagy capacity in goats.

ATP is essential for mammalian sperm to maintain fertilizing capacity. Metformin (Met) can activate 5'-AMP-activated protein kinase (AMPK) to maintain energy homeostasis. Thus, the aim of the present study was to investigate whether Met can improve testis function, semen quality, antioxidant and autophagy capacity through AMPK mediation of energy metabolism in goats. Twelve adult goats were randomly divided into three dietary treatments. All goats were fed a basal diet for 3 weeks and then assigned to a Met supplementation diet containing 0, 150, or 300 mg/kg for 8 weeks. The results showed that sperm viability, sperm membranal functional integrity, and acrosome integrity increased (P < 0.05) relative to the other treatments in the 300 mg/kg Met group. Growth hormone (GH) and insulin-like growth factor (IGF-1) in the 300 mg/kg Met group significantly decreased (P < 0.05) relative to the control group. Estrogen levels (E2) in the 300 mg/kg Met group remarkably improved (P < 0.05) compared with the control group. The activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GSH-px), and superoxide dismutase (SOD) significantly increased (P < 0.05) in the 300 mg/kg Met group relative to the control group. A significant increase in AMPK and p-AMPK protein expression in the 300 mg/kg Met group was observed relative to the control group (P < 0.05). Belicin-1 and LC3II/I protein expression was significantly increased by adding Met to the diet (P < 0.05) and reached a maximum in the 300 mg/kg Met group. In addition, differentially expressed genes (DEGs) of goat testis were confirmed by RNA-seq. GO enrichment analysis revealed that DEGs were enriched in testicular metabolism and sperm development-related functional pathways. Overall, the results indicate that Met may play an important role in the regulation of testis function, semen quality, antioxidant, and autophagy capacity. These findings will help elucidate the role of Met in goat testis development.

γ-Linolenic Acid Prevents Lipid Metabolism Disorder in Palmitic Acid-Treated Alpha Mouse Liver-12 Cells by Balancing Autophagy and Apoptosis via the LKB1-AMPK-mTOR Pathway.

Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 µM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 µM palmitic acid (PA). GLA reduced lipid content and increased fatty acid ß oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.

Arginine infusion rescues ovarian follicular development in feed-restricted Hu sheep during the luteal phase.

The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 µmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.

Energy restriction affect liver development in Hu sheep ram lambs through Hippo signaling pathway.

This study aimed to evaluate the effects of dietary energy restriction on postnatal liver development in Hu sheep ram lambs. A total of 16 ram lambs were randomly divided into two groups: 100% energy requirements diet and 55% energy requirements diet, which were fed for 75 d. Results showed that the final body and liver weights decreased with energy restriction (p <0.05). Energy restriction caused a significant decrease in the levels of circulating insulin-like growth factor 1 (IGF1) and an increase in growth hormone secretion (p <0.05), which can be explained by the decreased mRNA expression levels of the growth hormone receptor (GHR) and IGF1 (p <0.05). The proliferating cell nuclear antigen (PCNA), Ki-67 and apoptosis-related proteins (BAX and BCL2) were mainly located in the nucleus and the cytoplasm of hepatocytes, respectively. The transcription and protein levels of PCNA and BAX were significantly decreased and increased by energy restriction, respectively (p <0.05). The caspase9 and caspase3 mRNA and activity were increased in energy restriction group (p <0.05). Moreover, Hippo signaling pathway proteins [mammalian sterile 20-like protein kinase 1 (MST1), large tumor suppressor kinase 1 (LATS1), and yes-associated protein 1 (YAP1)] were mainly observed around the hepatic portal area, and the expression levels of their mRNA and proteins were significantly decreased in energy restriction group (p <0.05). In summary, energy restriction in ram lambs impairs liver development by increasing apoptosis, which may occur via the Hippo signaling pathway.