RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence

Abstract

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10^-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E₂ and P₄ were significantly lower with 10^-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10^-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3β-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Keywords: RFRP-3; apoptosis; cumulus cell; development; steroidogenesis.

Figure 1

The expression profiles of GnIH/RFRP-3 and GPR147 in growing follicles and corpus luteum. Immunohistochemical (IHC) analysis showed the expression patterns of GnIH/RFRP-3 and GPR147 in the primordial follicle (A,A’), the primary follicle (B,B’), the preantral follicle (C,C’), the antral follicle (D,D’), the corpus luteum (E,E’), and the negative control (F,F’). Sections of yak ovary were incubated with GnIH and GPR147 antibodies and then stained with hematoxylin. The scale bar is 50 μm. GC: granulosa cell, TC: theca cell, OO: oocyte, CC: cumulus cell, CL: corpus luteum, PF: preantral follicle.

Figure 2

Subcellular localization and expression patterns of GnIH/RFRP-3 and GPR147 in isolated CCs of yak. The expression of FSHR, GnIH/RFRP-3, and GPR147 in CCs were detected by Immunofluorescence. The nuclei of CCs were counterstained with DAPI and observed with a fluorescence microscope. The scale bar is 20 μm (white).

Figure 3

Effects of RFRP-3 and RF9 on proliferation and proliferation-related factors in yak CCs. (A) Various dosages (0, 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ mol/L) of RFRP-3 that influenced the cellular viability of yak CCs were detected with CCK-8 assay. (B) The proliferation activity of CCs was evaluated with EdU analysis after combining treatment with RFRP-3 and RF9. The scale (white) is 20 μm. (C) The statistical analysis of proliferation rates in cumulus cells. (D–F) Relative mRNA expression abundances Cdc42, Ccnd and Pcna after yak CCs incubated with 10⁻⁶ mol/L RFRP-3 and/or 10 μM RF9 for 24 h. Error bars represent mean ± SEM. * indicates a remarkable difference (p < 0.05), and ** indicates a considerable difference (p < 0.01) from that of the control counterparts. n.s. represents no significant difference.

Figure 4

Effects of RFRP-3 and RF9 on apoptosis of yak CCs. (A) The statistical analysis of apoptotic rates after CCs were incubated with different doses of RFRP-3 with or without 10 μM RF9. (B) Apoptosis of CCs was assessed with TUNEL analysis, and the apoptotic cell of yak CCs were marked in green, and nuclei stained with DAPI (blue). The scale bar (white) is 20 μm. Relative mRNA expression abundances of Caspase-3 (C), Bax (D), and Bcl-2 (E) were detected by RT-qPCR. Error bars represent mean ± SEM. Different letters (a–c) suggested significantly different (p < 0.05) compared to the controls.

Figure 5

Effects of RFRP-3 and RF9 on the secretion of P₄ and E₂ in yak CCs. The concentrations of E₂ (A) and P₄ (B) in the supernatant of yak CCs after treatment with RFRP-3 and/or RF9. (C–E) Relative expression levels of the steroidogenic pathway genes. Error bars represent mean ± SEM. Different letters (a–d) indicate a remarkable difference (p < 0.05) from that of the controls.

Figure 6

Dose-related effects of RFRP-3 and RF9 on oocyte meiosis progression and subsequent developmental competence. (A) Subcellular localization and expression of GnIH and GPR147 in yak oocytes. (B) Treatment of RFRP-3 and RF9 impairs the CCs expansion of yak COCs after 24 h IVM. (C) The progression of oocyte maturation was evaluated with the nuclear morphology using DAPI staining. (D) Statistical analysis of the oocyte meiosis progression after RFRP-3 and/or RF9 treatment. IVM medium was added with different doses of RFRP-3 for 6, 12, or 24 h. GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II. (E) Statistical analysis of the PA embryonic development after oocytes treated with RFRP-3 and RF9. Results are presented as means ± SEM. The scale bar is 50 μm. Different letters (a–c) indicate a significant difference (p < 0.05) from that of the controls.