TGF-β、Dhh信号轴对睾丸器官重构的作用及机制研究

Although mechanisms regulating testis cord formation during sex-determination have been well-studied, the genes, signaling pathways and cellular interactions are not well characterized. How TGFβ1/2/3-TGFBR3-ALK4/5/7 and Dhh/Pch1 signaling modulates the formation of testis. In our previous study, we have been achieved to reconstruct the seminiferous tubules using in 3-dimensional (D) re-aggregated culture of testicular cells, which were dissociated from 6-dpp neonatal mice inside a collagen matrix. And we found that the formation of testis cord are regarded the regulation using inhibitors of TGF-β, DHH and endothelial cells in 3 dimensional (D) re-aggregated culture of testicular cells. Therefore, we aim to study the mechanisms of regulating testis development underlying TGF-β, Dhh signaling pathway and interaction between the Sertoli cell and endothelial cells . We (1) select the unknown factors associated with testicular development; (2) investigate the change for mRNA and protein of down-regulated of TGF-β and Dhh signaling pathways; (3) observe the behavior of Sertoli cells using SOX-9 GFP transgenic mice using time-lapse technique;(4) explore the mechanism of endothelial cells of the seminiferous tubules reconstruction after the removal of CD34+ cells by MACS. We demonstrate that molecular mechanism underlying TGF-β , Dhh signaling pathway and endothelial cell regulated testis development. This study thus provides a novel avenue and methods for further investigation of the improvement of testicular development, spermatogenesis and male infertility .

TGF-β、Dhh信号通路及细胞间相互作用在睾丸器官形成中有重要作用。TGFβ1/2/3-TGFBR3-ALK4/5/7及Dhh/Pch信号轴的下游信号通路调控睾丸形成的具体机制尚不清楚。我们前期研究显示，采用三维立体培养，通过添加KSR，成功的重构了睾丸生精小管，并通过添加抑制剂发现TGF-β、Dhh信号通路及血管内皮细胞对生精小管重构有调控作用。本申请项目进一步研究上述信号轴及细胞间相互作用对睾丸器官重构的分子调控机制：筛选出与睾丸发育相关的未知因子；采用PCR、WB探讨TGF-β及Dhh信号轴相关基因、蛋白的变化；利用SOX9-GFP转基因小鼠，采用live-imaging技术动态的观察支持细胞的运动；采用MACS去除CD34+细胞后，探讨内皮细胞对生精小管重构的作用机制。项目将初步明确TGF-β、Dhh信号轴及细胞间分子调节机制，为后续的睾丸发育、精子发生、男性不育等提供理论依据。

TGF-β、Dhh信号通路及细胞间相互作用在睾丸器官形成中有重要作用。TGFβ1/2/3-TGFBR3-ALK4/5/7及Dhh/Pch信号轴的下游信号通路调控睾丸形成的具体机制尚不清楚。我们前期研究显示，采用三维立体培养，通过添加KSR，成功的重构了睾丸生精小管，并通过添加抑制剂发现TGF-β、Dhh信号通路及血管内皮细胞对生精小管重构有调控作用。本申请项目进一步研究上述信号轴及细胞间相互作用对睾丸器官重构的分子调控机制：通过分别添加DHH信号通路抑制剂（Cyclopamine），激动剂（SAG）及TGF-β信号通路抑制剂（SB431542）和重组蛋白（rTGF-β1）后，我们发现，与对照组相比，抑制剂组支持细胞失去极性，管周肌样细胞不连续，睾丸间质细胞随机分布，Laminin分泌受抑，生精小管样结构被破坏。而激动剂和重组蛋白组支持细胞具有极性，管周肌样细胞呈线性排列，睾丸间质细胞分布于两个相邻生精小管样结构之间，Laminin分泌呈线性，有大量生精小管样结构形成。通过WB，q-PCR技术检测DHH，TGF-β信号通路下游分子变化情况，发现Dhh通过调控下游分子Gli1 的表达参与生精小管样结构的重构。TGF-β信号通路通过调控下游分子p-Smad2、p-Smad3的表达，影响管周肌样细胞、睾丸间质细胞、生殖细胞的增殖；影响管周肌样细胞变形、迁移, 极性蛋白的分泌，以及生精小管样结构的形成。项目初步明确了TGF-β、Dhh信号轴及细胞间分子调节机制，为后续的睾丸发育、精子发生、男性不育等提供理论依据。