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.

Factors Influencing the Maturation and Developmental Competence of Yak (Bos grunniens) Oocytes In Vitro

“…Specifically, high-dose (10 −6 mol/L) RFRP-3 inhibited yak oocyte proliferation, induced oocyte apoptosis, and decreased 17β-estradiol and progesterone (P4) concentrations. RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3b-HSD) were downregulated in a dose-dependent manner [143].…”

Dynamic Changes in Proteome during Yak Oocyte Maturation Analyzed Using iTRAQ Technology

“…Then, they were transferred to a well-balanced maturation medium (M199 1 μg/mL LH + 1 μg/mL FSH + 1 μg/mL E2 + 1 μg/mL EGF + 0.2 μg/mL sodium pyruvate + 0.03 μg/mL NaOH + 0.03 μg/mL glucose + 10% FBS). The COCs at GV (0 h), MI (12 h), and MII (24 h) stages were collected, and the granulosa cells were removed using 0.1% hyaluronidase and placed in RNase-free tubes [ 21 ].…”

Impact of stress on male fertility: role of gonadotropin inhibitory hormone

“…GnIH is also referred to as RF amide-related peptides (RFRPs) in mammals, while it is known as LP X RF-amide in teleosts because it has LP X RF ( X = L or Q) amide motif at the C-termini ( 14 , 17 ). The two forms of GnIH found in mammals are RFRP-1 and RFRP-3, and have been identified to regulate the hypothalamic-pituitary gonadal (HPG) axis in men ( 13 , 14 , 18 ). GnIH directly inhibits GnRH since its axon is in contact with GnRH neurons ( 19 , 20 ).…”