The direct effects of gonadotropin-releasing hormone on proliferation of granulosa cells and development of follicles in goose

Abstract

ABSTRACT 1. The study objectives were to determine the direct effects of gonadotropin-releasing hormone (GnRH) on the proliferation of ovarian granulosa cells (GCs) and the development of follicles in geese (Anser cygnoides) by colorimetry and ethynyl-2′-deoxyuridine (EdU) cell proliferation assays, in which primary GCs were treated with different concentrations of GnRH agonist (alarelin acetate) and an antagonist (cetrorelix acetate). Differently expressed genes (DEGs) were identified by RNA-sequencing and validated by quantitative reverse transcription polymerase chain reaction (RT–qPCR) and Western blotting. 2. The EdU assays showed that the proliferation of GCs was affected by the GnRH agonist and antagonist in a dose-dependent manner. The effect of treatment on cell proliferation was statistically significant at the concentrations of 10−5 mol/l alarelin and 1 mg/l cetrorelix acetate. A total of 134 DEGs (76 downregulated and 58 upregulated for alarelin treatment) and 226 DEGs (90 downregulated and 136 upregulated for cetrorelix) were identified by RNA-sequencing analysis, respectively. Enrichment analysis indicated that DEGs were enriched in the GO terms of cell–cell signalling and cell junctions. The pathways that regulate the development of follicles were identified, including the biological progress of cAMP accumulation, ovulation cycle and vasculature that are essential to follicular selection. 3. The results suggested that GnRH might directly regulate GC proliferation via autocrine or paracrine pathways related to cell junctions. In particular, it was confirmed that the mRNA and protein expression levels of the oestrogen receptor 2 (ESR2) gene, a negative transcription factor involved in follicular maturation and ovulation, were affected by GnRH agonist or antagonist in GCs. 4. In conclusion, GnRH might play an important role in follicular development by changing the expression of genes that participate in cAMP accumulation, ovulation cycle and cell junctions in ovarian GCs.