出生前邻苯二甲酸酯暴露对雄性大鼠下丘脑生殖神经系统发育的影响及其作用机制

Phthalates (PAEs) are known antiandrogenic EDCs. Prenatal exposure of rats to PAEs inhibited testosterone formation by fetal Leydig cells and disrupted the development of androgen-dependent reproductive tract in male offspring, but very little is known about whether and how PAEs may perturb the hypothalamus, the part of the brain that controls reproduction. This is a major gap in knowledge, because the process of sexual differentiation of the hypothalamus requires normal sex-specific exposures to endogenous steroid hormones. This brain region is also exquisitely sensitive to both exogenous hormonally active EDCs, particularly when exposure occurs during critical developmental windows of gestation. The goal of this study is to test the hypothesis and explore the mechanisms that 'fetal exposure to PAEs can permanently alter hypothalamic development and, by consequence, perturb the hypothalamic control of reproductive function in adulthood of male rats'. We will treat pregnant rat dams with different doses of DEHP. To test our hypothesis, we will: (1) determine the effects on neurodevelopment on the brain regions of sexually dimorphic nuclei (SDN) and anteroventral periventricular nucleus (AVPV) in preoptic area (POA) of hypothalamus, by examining morphological change and neuron apoptosis of neonatal and adult rats; (2) determine the consequences on adult reproductive physiology and behavior by detecting the GnRH content on GnRH neuroterminals, performing serum hormone assays, and determining the mating behavior of male rats. To explore the mechanisms underlying the effects on hypothalamic reproductive neuroendocrine systems caused by prenatal PAEs exposure, (1) we will quantify gene expression using a well-designed 48 genes Taqman low-density arrays (these genes were specifically chosen based on the evidence for their roles in hypothalamic neuroendocrine system development and its control of reproductive functions) to identify candidates genes for protein expression; (2) quantify protein expression levels of those same molecules that are robustly changed at the gene expression level using fluorescence immunohistochemistry method; the gene/protein expression profiles will be compared between different hypothalamic developing periods (neonatal and adulthood) and different sexes (male and female). To further understand the mechanisms of this process, (3) we will move to an in vitro fetal primary hypothalamic culture model. Cultured cells will be treated with DEHP metabolite and steroid hormone (T, E2) and/or AR, ER and P450arom antagonists respectively, to determine the effects of PAEs on the cells' proliferation and apoptosis. The anticipated results will provide theoretical and functional insights into the mechanisms by which the hypothalamus mediates reproductive toxicity effects of phthalates and other EDCs. It can provide information about prevention of EDC's effects on human health.

作为抗雄激素EDCs,出生前PAEs暴露可致胎儿睾丸睾酮合成减少及生殖器官发育异常，但它对下丘脑生殖调控中枢的影响尚不清楚。因雄性下丘脑的性分化和发育依赖雄激素的调节,在其发育关键期PAEs暴露很可能干扰雄性下丘脑生殖中枢的性分化及发育过程，并永久性地影响其对生殖功能的调控作用。我们通过对新生及成年大鼠下丘脑SDN-POA、AVPV核区形态学观察及神经元凋亡检测，成年后下丘脑GnRH表达水平与血清激素测定及性行为观察，评价出生前PAEs暴露对下丘脑生殖神经发育及功能的影响；通过对上述核区与下丘脑发育及生殖内分泌调控相关的多种基因及蛋白表达水平分析，结合胚胎下丘脑原代培养试验，探讨PAEs对生殖神经中枢影响的机制；本研究从生殖神经内分泌干扰的新角度探讨PAEs生殖毒性的靶部位及作用机制，为全面揭示PAEs及其他EDCs的生殖内分泌干扰作用、预防其潜在危害提供新的理论依据、拓展新的研究方向。

近年来，内分泌干扰物（EDCs）对神经系统的干扰作用受到国内外的广泛关注。DEHP是使用量最大的邻苯二甲酸酯类(PAEs)化合物，作为已知的内分泌干扰物，出生前DEHP暴露可致雄性胎儿睾丸睾酮合成减少及生殖器官发育异常，但它对下丘脑生殖内分泌调控中枢发育及功能的影响尚不清楚。本研究于大鼠孕14-19天（G14-G19）分别以0(溶剂对照)、2 mg/kg 、10 mg/kg 及 50 mg/kg DEHP灌胃染毒；通过对子代不同发育阶段（新生及成年后）下丘脑生殖神经内分泌功能调控中枢的神经细胞凋亡分析、成年后大鼠生殖神经内分泌功能相关指标检测及性行为的观察，分别探讨DEHP对雄性及雌性大鼠下丘脑生殖神经系统发育及功能的影响；通过对新生子代大鼠下丘脑视前区（POA），成年后子代大鼠下丘脑生殖神经内分泌调控相关核区内侧视前核（MPN）、前腹侧室旁核（AVPV）及弓状核（ARC）的生殖内分泌调控相关功能基因及蛋白表达水平的分析，结合体外DEHP代谢产物对发育关键期新生大鼠下丘脑神经元增殖、凋亡的影响，探讨DEHP生殖神经内分泌干扰作用的机制。本研究结果发现，出生前DEHP暴露可通过干扰下丘脑生殖神经内分泌功能相关的多种基因/蛋白表达水平（包括促性腺激素释放激素（GnRH）及其受体（GnRHR）系统、类固醇激素信号基因、神经递质信号基因、神经肽基因以及生物节律调节相关基因等）影响下丘脑生殖神经系统的发育及功能。出生前DEHP暴露对大鼠下丘脑生殖神经内分泌中枢发育及功能的影响可从出生后早期持续至成年后，并且表现出性别差异性和发育阶段依赖性。此外，在本研究条件下，大脑发育关键时期DEHP暴露对大鼠下丘脑的生殖神经系统的发育和功能的某些影响具有低剂量效应。通过本研究，可以确认发育中的下丘脑神经内分泌调控中枢为PAEs内分泌干扰作用的重要靶部位，研究结果可为今后全面揭示PAEs及其他内分泌干扰物的生殖内分泌干扰作用提供新的理论依据和科学线索。