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.
Highlights
In spite of their name, neuropeptides are synthesized in and act on many peripheral tissues in addition to their classical actions in the nervous system (McGuire & Bentley, 2010)
Presence of feline RF-amide related peptide 3 (RFRP3) (fRFRP3) and receptors in ovarian tissues fRFRP3 was expressed in ovarian tissue of domestic cats (Fig. 3A)
Both primary (NPFFR1) and secondary (NPFFR2) fRFRP3 receptors were expressed in domestic cat ovaries (Figs. 3B, 3C)
Summary
In spite of their name, neuropeptides are synthesized in and act on many peripheral tissues in addition to their classical actions in the nervous system (McGuire & Bentley, 2010). While some neuropeptides have peripheral effects that serve distinct functions from their role in the central nervous system (e.g., the neuropeptide gonadotropin-releasing hormone (GnRH); Iwakoshi-Ukena et al, 2004; Bentley et al, 2017), other neuropeptides exhibit putatively concordant function in the central nervous system and periphery. One such example is the nonapeptide RF-amide related peptide 3 (RFRP3). There are far fewer studies that have assessed function of RFRP3 in the ovary; of the 17 species in which ovarian RFRP3 has been identified, only seven have had any function evaluated (Table 1)
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