Global Long Noncoding RNA and mRNA Expression Changes between Ovarian Granulosa Cell Treatment with GnRH agonist(Alarelin) and antagonist(Cetrorelix) in goose

Gonadotropin-releasing hormone (GnRH) might combine with other reproductive hormones to affect follicular development by regulation of the cell cycle in granulosa cells (GCs).To investigate the function of GnRH in GCs, we examined the effects of a GnRH agonist (alarelin acetate) and an antagonist (cetrorelix acetate) on the proliferation of primary goose (Anser cygnoides) ovarian GCs by colorimetry and 5-ethynyl-2¢-deoxyuridine (EdU) cell proliferation assays. Differently expressed genes (DEGs) affected by these GnRH analogues in goose GCs were detected by RNA-seq and the results were verified using quantitative reverse transcription polymerase chain reaction (RT–qPCR) and western blotting. The optical densities in the colorimetry assays and the S phase cell cycle ratio of GCs in the EdU cell proliferation assays gradually increased and decreased with increasing concentrations in the alarelin-treated and cetrorelix-treated cells in dose-dependent manners, respectively. Statistically significances were achieved at concentrations of 10−5 mol/L alarelin and 1 mg/L cetrorelix acetate RNAseq analysis showed that 134 DEGs and 226 DEGs were detected following GC treatment with 10−5M GnRH alarelin (76 downregulated and 58 upregulated) and 1 mg/L cetrorelix (90 downregulated and 136 upregulated). Many of these DEGs are involved in the actions of genes encoding G protein-coupled hormone receptors, such as estrogen receptor 2 (ESR2), oxytocin receptor (OXTR), and prolactin receptor (PRLR), which are essential for folliculogenesis. The mRNA and protein expression levels of ESR2 in GCs were decreased and increased by the GnRH agonist and antagonist, respectively. Interestingly, ESR2 was increasingly expressed in pre-hierarchical follicles, most strongly expressed in GCs of F5 stage follicles and reduced in preovulatory follicles, which implicates a potential role in follicle selection. Thus, GnRH might act as an autocrine or paracrine factor to regulate GC proliferation by interacting with some key steroid hormone G protein-coupled receptors genes participating in follicular development. Ovaries were collected 2 hr before laying for cell culture and isolation of ovarian GCs from preovulatory follicles and pre-hierarchical follicles (4–12 mm diameter) in 40W Sichuan white goose. The cell were treated with different concentrations of GnRH agonist (alarelin acetate) and an antagonist (cetrorelix acetate). The proliferation assay were performance by the colorimetry and 5-ethynyl-2¢-deoxyuridine (EdU) cell proliferation assays. The cell treatment with optimal concentration of GnRH analogues were collected and frozen immediately in liquid nitrogen and stored at -80°C until required for RNA extraction, sequencing and transcriptome analysis.