大鼠胚胎Leydig细胞提早凋亡致成年细胞异常发育机理与邻苯二甲酸酯的影响

Leydig cell development and the impacts by environmental pollutants are critical area in medicine. The fetal Leydig cell (FLC) develops during embryogenesis and produces androgens and INSL3. After birth, FLC undergoes apoptosis and disappears at postnatal day 21, accompanied by the development of new generation of the adult Leydig cell (ALC). It is still unclear that the influence of FLC apoptosis on the development (from stem-progenitor-immature-adult) of ALC. Different from the effectiveness to ALC, Leydig cell can be specifically eliminated by the ethane-1, 2-dimethyl sulfonate within 24 hours, thus leading to establish a novel development model with earlier disappearance of FLC induced androgen-free environment in the neonatal testis. Using this novel model, we ask a key scientific question: how does the earlier disappearance of FLC affect the postnatal development of ALC and what is the mechanism? The objectives of the present proposal are to investigate: (1) the developmental changes of ALC after earlier disappearance of FLC and the effects of in utero (2) and lactational (3) exposures of diisononyl phthalate on development of ALC in this androgen-free model. After this project, we expect to better understand the mechanism of the earlier disappearance of FLC on the postnatal development of ALC and use this novel model to study the developmental biology and to study environmental toxicology.

Leydig细胞发育及环境污染物对其的影响是重要的医学命题。胚胎Leydig细胞（FLC）在胎儿期发育并分泌雄激素和INSL3。在大鼠出生后21天FLC凋亡并伴随着新一代的成年Leydig细胞（ALC）发育。FLC的自动凋亡对ALC发育（干-祖-幼-成年Leydig细胞）过程的影响尚不清楚。与大鼠正常发育过程不同，乙烷二甲烷磺酸酯可在1天时间内完全杀灭FLC。基于此，我们建立了崭新FLC提前凋亡致无雄激素发育模型。利用这一模型，提出关键科学问题“FLC过早凋亡对ALC的发育及功能所造成的影响及机制是什么？”拟利用初生大鼠FLC凋亡模型研究：（1）ALC在FLC凋亡条件下的发育过程变化；（2）宫内及（3）哺乳期暴露邻苯二甲酸二异壬酯对无睾酮新生动物ALC发育的影响。力争揭示ALC发育过程受FLC凋亡和环境污染物暴露的影响和调控机制，为将此动物模型应用于发育生物学和环境毒理学研究建立理论基础。

初生动物Leydig干细胞发育过程是随着胚胎Leydig细胞的凋亡开始的，在大鼠出生后21天FLC凋亡并伴随着新一代的成年Leydig细胞发育。本项目的研究重点着眼于胚胎Leydig细胞的凋亡对Leydig干细胞发育的影响。为了造成一种胚胎Leydig细胞提前凋亡的模型，采用乙烷二甲烷磺酸酯在处理3天后完全杀灭胚胎Leydig细胞，并且对胚胎Leydig 细胞人工诱导凋亡对成年Leydig 细胞的发育及功能所造成的影响以及在无睾酮的成年Leydig细胞发育过程中邻苯二甲酸二异壬酯（DINP）对成年动物生殖功能的影响进行一系列的研究。探讨了有关胚胎Leydig细胞被乙烷二甲烷磺酸盐诱导凋亡后成年Leydig细胞发发育过程中Leydig细胞的不同发育阶段及与正常生理状态下发育的Leydig细胞在形态、功能等方面的差异及对机体生殖内分泌和生育功能的影响及作用机制的研究工作。并且，应用胚胎Leydig细胞提早凋亡-成年Leydig细胞发发育异常动物模型，探讨了DINP通过母体子宫内暴露后初生动物的睾丸Leydig细胞发育过程、功能变化及可能的作用机制。在采用乙烷二甲烷磺酸盐杀灭初生大鼠胚胎Leydig细胞模型后，初生大鼠的成年Leydig细胞提前发育，其发育过程与正常的生长发育过程具有显著差异，其中多个类固醇合成代谢酶参与这一过程，而在成年期后动物的睾丸类固醇合成能力受到影响，睾酮生成下降明显。宫内暴露DINP对乙烷二甲烷磺酸盐消除胚胎Leydig细胞后的无睾酮发育环境中的新生大鼠睾丸Leydig细胞发育及其功能以及其调控机制。乙烷二甲烷磺酸盐处理后消除了胚胎Leydig细胞的初生大鼠，在宫内暴露DINP后，诱导胚胎Leydig细胞聚集，反而在一定程度上抵抗了单次剂量的乙烷二甲烷磺酸盐的杀灭，保留了一定的胚胎Leydig细胞功能。这些保留的功能是通过HSD3B1等关键类固醇合成酶类所实现的。胚胎Leydig细胞被EDS诱导凋亡后成熟Leydig细胞发育过程中Leydig细胞的不同发育阶段及与正常生理状态下发育的Leydig细胞在形态、功能等方面发生了多层次的变化。这些变化主要集中在睾丸睾酮合成能力的差异、Leydig细胞发育过程的改变、睾丸功能性基因的表达差异以及睾酮合成代谢相关基因的表达差异。