Abstract The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-β-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

Abstract This study aimed to investigate the effects of bacterial endotoxin lipopolysaccharide (LPS) on hormone production and gene expression in duck Leydig cells and its underlying mechanisms. Leydig cells were collected from 200-day-old mallard ducks and divided into five treatment groups (0, 50, 100, 200, and 400 ng/mL LPS). After treatment with LPS for 6, 12, 24, and 48 h, testosterone, activin, and inhibin levels in the cell supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression levels of testosterone synthesis-related genes, including steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom), and reproductive-related genes, including gonadotropin-inhibitory hormone receptor (GnIHR), follicle stimulating hormone receptor (FSHR), and luteinizing hormone receptor (LHR) were detected using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully isolated and cultured duck Leydig cells with cell purity above 90%. Compared with the control group, the levels of testosterone, activin, and inhibin secreted in Leydig cells decreased gradually with increasing LPS concentration. After treatment with LPS, the expression of StAR and 3β-HSD genes in Leydig cells was upregulated at 12 h, and that of GnIHR was upregulated at 24 h; whereas the expression of FSHR and LHR was reduced at 24 h. This study indicates that LPS can inhibit the secretion of hormones and regulate the expression of related genes in duck Leydig cells.