Wilsterman Kathryn, Bentley George E, Comizzoli Pierre
Integrative Biology, University of California, Berkeley, Berkeley, CA, United States of America.
Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, United States of America.
PeerJ. 2019 Aug 23;7:e7540. doi: 10.7717/peerj.7540. eCollection 2019.
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 . 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.
下丘脑神经肽RFRP3可抑制下丘脑促性腺激素释放激素（GnRH）神经元的激活，并抑制垂体前叶促性腺激素的释放。RFRP3也在卵巢局部产生，并可抑制许多脊椎动物的类固醇生成和卵泡发育。然而，对于任何食肉动物物种性腺中RFRP3的存在及其调节作用几乎一无所知。这些知识对于制定濒危食肉动物的圈养繁殖计划以及抑制野生种群的繁殖至关重要。以家猫为模型，我们的目标是：（1）证明猫RFRP3（fRFRP3）及其受体在猫卵巢中的表达；（2）评估fRFRP3对卵巢卵泡完整性、存活及类固醇生成的影响。我们首先通过对卵巢RNA的PCR产物进行测序，证实fRFRP3及其受体（NPFFR1和NPFFR2）在猫卵巢中表达。然后，我们在含有10或1 μM fRFRP3 + 促卵泡素（FSH，1 μg/mL）的条件下分离并培养窦前卵泡。我们记录了8天内形态学上存活卵泡（基膜完整性）的百分比，并计算第8天卵泡的存活率（使用细胞存活和死亡的荧光标记物）。最后，我们对培养基中的孕酮积累进行了定量分析。与仅暴露于FSH的卵泡相比，10 μM fRFRP3对卵泡的活力、存活或类固醇生成没有明显影响。然而，第8天，1 μM fRFRP3降低了形态学上存活卵泡的百分比和存活卵泡的百分比。同时，1 μM fRFRP3增加了培养基中孕酮的积累。我们的研究首次表明RFRP3作为一个功能单位对卵泡有直接作用，这也是在食肉动物物种中的首次发现。更广泛地说，我们的结果支持RFRP3对卵巢卵泡发育具有保守的抑制作用，并强调了比较功能研究的重要性。
