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. Here we investigated the effect and molecular mechanism of INHBA on cell cycle, apoptosis, activin A/inhibin A secretion in Hu sheep granulosa cells (GCs) based on RNA-Sequencing. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change = 2 and FDR = 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference groups 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, pathway analysis showed that DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53 pathway, progesterone-mediated oocyte maturation, PI3K-AKT signaling pathway. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) confirmed the differential expression of 7 selected DEGs. In addition, INHBA knockdown promoted GCs to G1 phase and decreased the S phase cell numbers, initiated GC apoptosis through P53 signal pathway, and decreased the concentration of activin A and inhibin A in medium. Above all, our findings may contribute to a better understanding on the law of follicular development and thus improve the reproductive performance of female animals. Overall design: The ovaries of sexually mature Hu sheep were collected from Jiangsu Taicang slaughterhouse (Jincanghu Food Co., Ltd.) during the breeding season (November to January). Ovaries are clipped from connective tissue and adherent fallopian tubes, then immediately immersed in warm (30-37°C) sterile saline (160 IU/mL gentamicin) and transported to the laboratory within 2 hours. After washing with normal saline for 6 times, follicular fluid was aspirated from follicles =3 mm, and GCs were isolated for in vitro culture according to a previous study.Isolated GCs were seeded into a 6-well plate for culture, and when they grow to 70%-80% confluence, mix the lipid complexes containing NC and siRNA-INHBA according to the instructions of Lipofectamine 3000 (Thermo Scientific, MA, USA). After 24h transfection, the RNA of GCs was extracted with Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. The concentration and purity of RNA were detected by the Nanodrop 2000 spectrophotometer (Thermo Scientific, MA, USA). After comfirming the purity of RNA, the RNA sample were sent to sequencing company.