RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat

Abstract

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

Gonadotropin Inhibitory Hormone and Its Receptor: Potential Key to the Integration and Coordination of Metabolic Status and Reproduction

“…Non-seasonal breeders include spontaneous ovulating species, such as humans, rats, mice, and naked mole rats, as well as induced ovulating species, such as the domestic cat. Unlike short-day and long-day breeders, the literature on non-seasonal breeders is more consistent, and GnIH and its receptor were shown to be present in all three levels of the HPG axis ( 13 , 15 , 30 , 35 , 105 ). Interestingly, puberty in humans has been hypothesized to be better anticipated by measuring the accumulation of fat rather than using age or environmental stimuli as a predictor ( 106 ).…”

“…Although it is clear that GnIH and its receptor are key to the hypothalamic control of reproduction, the presence of the GnIH-R at the lower level of the HPG axis (gonads) has also been reported in avian species, including the Japanese quail, the European starling, the white-crowned sparrow ( 10 ), and the domestic chicken ( 7 , 230 ), as well as various fish species including clownfish ( 57 ), Nile tilapia ( 56 ), Indian carp ( 61 ), common carp ( 54 ), zebrafish ( 51 ), and goldfish ( 49 ), and the rat ( 13 , 15 ). Additionally, the GnIH-R has been identified in the ovary of humans ( 30 ), swine ( 33 ), felines ( 35 ), tongue sole ( 60 ), and in the testis of Syrian hamsters ( 26 ) and house sparrows ( 11 ) ( Table 1 ). Furthermore, treatment with GnIH was shown to effectively shut down reproduction in the ovary and testis not only by indirectly reducing gonadotropins release from the pituitary as previously discussed, but also directly by decreasing cell viability in the ovary and reducing the levels of testosterone in the testis ( 5 , 8 , 10 , 11 , 46 , 51 ).…”

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

“…Previous studies confirmed that the GnIH receptor GPR147, also named NPFF receptor 1, is considered a novel member of the G-protein-coupled receptors [ 50 ]. The interaction between GnIH/RFRP-3 and GPR147 is known from studies of their mRNA and protein expression profiles in cat and rat ovarian cells [ 45 , 51 ]. Biochemical examination demonstrated that RFRP-3 has a greater affinity with GPR147, so GPR147 is one of the major receptors for RFRP-3 [ 50 , 52 ].…”

Chronic Stress Detrimentally Affects In Vivo Maturation in Rat Oocytes and Oocyte Viability at All Phases of the Estrous Cycle

“…It was reported that Kisspeptin at a concentration of 100 nM decreases granulosa cell viability [ 28 ]. In addition, RFRP-3 has been reported to decrease the number of estrous cycles in cold-stressed rats [ 29 ], and to decrease follicular viability in cats [ 30 ]. Follicles’ exposure to 1 μM of RFRP-3 increased the proportion of follicles with cell death.…”

“…Follicles’ exposure to 1 μM of RFRP-3 increased the proportion of follicles with cell death. One of the mechanisms proposed by the authors is that RFRP-3 can promote follicular degradation through paracrine signaling [ 30 ]. Additionally, RFRP-3 has been reported to decrease porcine granulosa cell viability at different concentrations [ 31 ].…”