Follicular atresia is closely related to both apoptosis and autophagy of granulosa cells (GCs) in ovarian follicles. In the present study, GCs were isolated from pig ovaries in small, medium and large antral follicles, and the current results showed that the proliferation of GCs was higher in medium follicles, and lower in large follicles compared to small follicles. The Bax and Caspase 3 mRNA levels were significantly higher, but the ratio of Bcl-2/Bax was lower in GCs of large follicles. The marker genes of autophagy, Atg3, Atg7 and LC3 mRNA levels were higher in GCs from medium follicles. Apoptosis- and autophagy-related proteins had a similar expression pattern to the mRNA level. Our results showed that phosphorylated ERK (p-ERK) was activated in GCs of large follicles, while phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) were inhibited in GCs of medium follicles. Labeling of autophagic vesicles with 4’,6-diamidino-2-phenylindole (DAPI) and monodansylcadaverine (MDC) confirmed the results of gene transcription and protein expression in GCs of different size follicles. We conclude that autophagy and apoptosis of GCs occurred in different size follicles during follicular development, and autophagy was mainly found in GCs of medium follicles, while apoptosis was mainly found in GCs of large follicles.

It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death; however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. miR-21-3p regulates many fundamental biological processes and is pivotal in the autophagy of tumor cells; nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. miR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while it increased with transfection of miR-21-3p inhibitor. In addition, small interference-mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling; however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and its phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.

In the present study, AA was used to challenge bovine ovarian granulosa cells in vitro and the related parameters of cellular and molecular biology were measured. The results indicated that lower doses of AA increased survival of bovine granulosa cells whereas higher doses of AA suppressed survival. While lower doses of AA induced accumulation of lipid droplet in granulosa cells, the higher dose of AA inhibited lipid accumulation, and AA increased abundance of FABP3, CD36 and SLC27A1 mRNA. Higher doses of AA decreased the secretion of E2 and increased the secretion of P4 accompanied by down-regulation of the mRNA abundance of CYP19A1, FSHR, HSD3B1 and STAR in granulosa cells. The signaling pathways employed by AA in the stimulation of genes expression included both ERK1/2 and Akt. Together, AA specifically affects physiological features, gene expression levels and steroid hormone secretion, and thus altering the functionality of granulosa cells of cattle.

Background: Zinc oxide nanoparticle (ZnO NP) is one of the metal nanomaterials with extensive use in many fields such as feed additive and textile, which is an emerging threat to human health due to widely distributed in the environment. Thus, there is an urgent need to understand the toxic effects associated with ZnO NPs. Although previous studies have found accumulation of ZnO NPs in testis, the molecular mechanism of ZnO NPs dominated a decline in male fertility have not been elucidated. Results: We reported that ZnO NPs exposure caused testicular dysfunction and identified spermatocytes as the primary damaged site induced by ZnO NPs. ZnO NPs led to the dysfunction of spermatocytes, including impaired cell proliferation and mitochondrial damage. In addition, we found that ZnO NPs induced ferroptosis of spermatocytes through the increase of intracellular chelatable iron content and lipid peroxidation level. Moreover, the transcriptome analysis of testis indicated that ZnO NPs weakened the expression of miR-342-5p, which can target Erc1 to block the NF-κB pathway. Eventually, ferroptosis of spermatocytes was ameliorated by suppressing the expression of Erc1. Conclusions: The present study reveals a novel mechanism in that miR-342-5p targeted Erc1 to activate NF-κB signaling pathway is required for ZnO NPs-induced ferroptosis, and provide potential targets for further research on the prevention and treatment of male reproductive disorders related to ZnO NPs. Graphical