Traditional Chinese Medicine Department, Children's Hospital of Fudan University, Shanghai 201102, China.
Department of Integrative Medicine and Neurobiology, Fudan University, Shanghai 200032, China.
Curr Mol Med. 2019;19(10):766-775. doi: 10.2174/1566524019666190906142445.
Pubertal development is a complex physiological process regulated by the neuroendocrine system and hypothalamic-pituitary-gonadal axis. Sexual precocity is a common childhood endocrine disease.The pathogenesis of sexual precocity has not been fully elucidated. RFRP3/GPRl47 signal pathway is able to inhibit the reproductive capability in avians and mammals, probably by acting on the GnRH neuron and pituitary to regulate gonadotrophin synthesis and release. However, little is known about the role of RFRP3 in puberty development and sexual precocity.
To observe the dynamic changes of RFamide related peptide 3/G proteincoupled receptor 147 (RFRP3/GPR147) in hypothalamic during puberty development and explore their role in precocious puberty based on a female rat model.
The Sprague-Dawley female rats were randomly divided into three groups, normal, vehicle, and precocious puberty model. At 5 days old, the rat model with precocious puberty was prepared by subcutaneously injecting a mixture of danazoldissolved ethanol and glycol. At different day-age (15, 25, 30, 35, and 40 days), the levels of estradiol(E2), follicle-stimulating hormone(FSH), and luteinizing hormone (LH) in the peripheral blood were detected by the enzyme-linked immunosorbent assay, the messenger ribonucleic acid (mRNA) expressions of RFRP3, gonadotropin releasing hormone and GPR147 were examined by real-time polymerase chain reaction(R-T PCR). RFRP3 positive cells were observed using Immunofluorescence confocal microscopy.
At 25 and 30 days, the levels of sex hormones and the uterus coefficients were significantly higher in the precocious puberty model group than those in the normal and vehicle groups. The ovarian morphological development in the precocious puberty model rats was significantly earlier than those in the normal and vehicle groups. The mRNA expressions of RFRP3/GPR147 and GnRH in the precocious puberty model group gradually increased and peaked at 25 days. The different day-age and the interaction have significant statistical significance on the expression of RFRP3 mRNA, while the levels of RFRP3 mRNA in the model group and vehicle groups have no significant statistical significance. There was statistical significance between the model group and vehicle groups in different day-age on the expression of GPR147 mRNA.The expression of hypothalamic RFRP3/GPR147 mRNA and RFRP3 positive cells gradually decreased with puberty onset. At 35 days, the levels of RFRP3 mRNA and GPR147 mRNA were significantly lower in the precocious puberty model group than those in the vehicle groups. Meanwhile, the levels of LH in the precocious puberty model rats reached its peak at this age. In the vehicle group, the levels of RFRP3 mRNA and serum LH were gradually increased and LH nearly peaked at 35 day-age. Subsequently, it gradually decreased and reached the lowest level at 35 day-age. The expression of RFRP3 mRNA and LH were positively correlated.
The findings suggested that RFRP3/GPR147 signaling pathway may be involved in the pathogenesis of sexual precocity by regulating puberty development and sexual maturity in rats.
青春期发育是一个受神经内分泌系统和下丘脑-垂体-性腺轴调控的复杂生理过程。性早熟是一种常见的儿童内分泌疾病。性早熟的发病机制尚未完全阐明。RFRP3/GPR147 信号通路能够抑制禽类和哺乳动物的生殖能力，可能通过作用于 GnRH 神经元和垂体来调节促性腺激素的合成和释放。然而，关于 RFRP3 在青春期发育和性早熟中的作用知之甚少。
观察促性腺激素释放激素相关肽 3/G 蛋白偶联受体 147（RFRP3/GPR147）在青春期发育过程中下丘脑的动态变化，并基于雌性大鼠模型探讨其在性早熟中的作用。
将 Sprague-Dawley 雌性大鼠随机分为正常组、溶剂组和性早熟模型组。在 5 天大时，通过皮下注射溶解在乙醇和乙二醇中的丹那唑混合物制备性早熟大鼠模型。在不同的日龄（15、25、30、35 和 40 天），通过酶联免疫吸附试验检测外周血中雌二醇（E2）、卵泡刺激素（FSH）和黄体生成素（LH）的水平，通过实时聚合酶链反应（R-T PCR）检测 RFRP3、促性腺激素释放激素和 GPR147 的信使 RNA（mRNA）表达。使用免疫荧光共聚焦显微镜观察 RFRP3 阳性细胞。
在 25 天和 30 天，性早熟模型组的性激素水平和子宫系数明显高于正常组和溶剂组。性早熟模型大鼠的卵巢形态发育明显早于正常组和溶剂组。性早熟模型组 RFRP3/GPR147 和 GnRH 的 mRNA 表达逐渐增加，并在 25 天达到峰值。不同日龄和交互作用对 RFRP3 mRNA 的表达有显著的统计学意义，而模型组和溶剂组的 RFRP3 mRNA 水平没有显著的统计学意义。在 GPR147 mRNA 的表达上，模型组和溶剂组在不同日龄之间有统计学意义。下丘脑 RFRP3/GPR147 mRNA 和 RFRP3 阳性细胞的表达随着青春期的开始逐渐减少。在 35 天，性早熟模型组的 RFRP3 mRNA 和 GPR147 mRNA 水平明显低于溶剂组。同时，性早熟模型大鼠的 LH 水平在这个年龄达到峰值。在溶剂组中，RFRP3 mRNA 和血清 LH 水平逐渐升高，LH 在 35 天龄时几乎达到峰值。随后逐渐下降，在 35 天龄时达到最低水平。RFRP3 mRNA 和 LH 的表达呈正相关。
这些发现表明，RFRP3/GPR147 信号通路可能通过调节大鼠的青春期发育和性成熟参与性早熟的发病机制。
