Effects of BAMBI on luteinized follicular granulosa cell proliferation and steroid hormone production in sheep.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) regulates mammalian ovarian follicle growth and maturation; however, its effect on luteinized granulosa cells (LGCs) in sheep ovarian follicles remains unknown. Here we explored the regulatory role of LGC functions and steroid hormone synthesis by BAMBI. Multiple sequence alignment revealed that the sheep BAMBI gene sequence was relatively conserved. Sheep LGCs were strongly positive for BAMBI. LGC proliferation increased when BAMBI was silenced and decreased when BAMBI was overexpressed. After BAMBI overexpression, the expression of CASP3, CASP8, CASP9, and BAX significantly increased, whereas that of BCL2 and the ratio of BCL2/BAX expression decreased. The opposite was observed after BAMBI silencing. CDKN1A, CCND1, and CCND2 were downregulated with BAMBI overexpression and upregulated with BAMBI silencing. Expression of steroid hormone-related genes (CYP11A1, STAR, and 3BHSD), except CYP19A1, significantly increased after BAMBI overexpression. Moreover, estrogen and progesterone secretion increased after BAMBI overexpression and decreased after BAMBI interference. The effect of the exogenous addition of bone morphogenetic protein 2 (BMP2) on GCs was similar to that of BAMBI overexpression. In conclusion, BAMBI can regulate the proliferation and steroid hormone synthesis of sheep LGCs, and BMP2 can affect LGCs as an activator of BAMBI. These findings provide a basis for further research on the physiological role of BAMBI.

Protective effects of MNQ against Lipopolysaccharide-induced inflammatory damage in bovine ovarian follicular granulosa cells in Vitro.

Inflammation of the reproductive tract in dairy cows lead to functional disorders of follicular granulosa cells (GCs) in mammalian ovaries resulting in infertility and serious losses to the livestock industry. Lipopolysaccharide (LPS) can induce an inflammatory response in follicular granulosa cells in vitro. The aim of this study was to investigate the cellular regulatory mechanism of MNQ (2-methoxy-1,4-naphthoquinone) on eliminating the inflammatory response and restoring normal functions for bovine ovarian follicular GCs cultured in vitro exposed to LPS. The cytotoxicity of MNQ and LPS on GCs were detected by MTT method to determine the safe concentration. The relative expression of inflammatory factors and steroid synthesis-related genes were detected by qRT-PCR. The concentration of steroid hormones in the culture broth were detected by ELISA. Differential gene expressions were analyzed by RNA-seq. There were no toxic effects on GCs at MNQ and LPS concentrations of less than 3 µM and 10 µg/mL, respectively and treated in 12 h. The relative expressions of IL-6, IL-1ß and TNF-α were significantly higher in the LPS group compared with the CK group when GCs cultured in vitro were treated with the above concentrations and times (P < 0.05), but significantly lower in the MNQ+LPS group compared with the LPS group (P < 0.05). The levels of E2 and P4 in the culture solution were significantly reduced in the LPS group compared to the CK group (P < 0.05), and restored in the MNQ+LPS group. The relative expressions of CYP19A1, CYP11A1, 3ß-HSD, and STAR were significantly decreased in the LPS group compared with the CK group (P < 0.05), while the MNQ+LPS group also recovered to some extent. There were 407 differential genes shared by LPS vs CK and MNQ+LPS vs LPS by RNA-seq analysis, which were mainly enriched in steroid biosynthesis and TNF signaling pathway. We screened 10 genes for analysis and found consistent results for RNA-seq and qRT-PCR. In this study, we confirmed the protective effect of MNQ, an extract from Impatiens balsamina L, on LPS-induced inflammatory responses in bovine follicular granulosa cells in vitro as well as functional damage, and acted through steroid biosynthesis and TNF signaling pathways.