Durkut-Kuzu Begum, Celik-Ozenci Ciler
Graduate School of Health Sciences, Reproductive Medicine, Koc University, Istanbul, Turkey.
Department of Histology and Embryology, School of Medicine, Koc University, Istanbul, Turkey.
J Assist Reprod Genet. 2025 May 22. doi: 10.1007/s10815-025-03493-7.
Hepatocyte growth factor (HGF), secreted by Sertoli cells, activates the c-Met receptor, facilitating seminiferous tubule formation. Glial cell-derived neurotrophic factor (GDNF) promotes embryonic Sertoli cell proliferation and cord formation. This study aimed to investigate whether exogenous GDNF contributes to HGF-induced in vitro organization of Sertoli cells in a Matrigel-based three-dimensional (3D) culture system.
Four experimental groups were established using the 15P-1 Sertoli cell line: control, HGF-treated, GDNF-treated, and combined HGF- and GDNF-treated. Tubular structure length and branching were quantified using image analysis software, while the expression of ZO-1 protein was assessed via immunofluorescence staining in 3D Matrigel-based culture for 5 days. Additionally, Gfrα-1, Ret, Ncam, and Met mRNA expression in Sertoli cells were analyzed. The expression levels of ZO-1, c-MET, and p-c-MET were analyzed in two-dimensional (2D) culture after 5 days.
Quantitative analysis revealed a significant increase in the length and branching of tubular-like structures when GDNF was co-administered with HGF, compared to other groups. Additionally, there was a notable increase in ZO-1 protein expression with the combined administration of GDNF and HGF, surpassing levels observed in the control group. Furthermore, co-administration of HGF and GDNF significantly elevated phospho-c-Met levels compared to the control group.
Our study reveals that exogenous GDNF enhances the HGF-induced tubular organization of Sertoli cells in vitro. The concurrent administration of GDNF and HGF markedly augments the formation of tubular structures and the expression of the ZO-1 protein, indicating a potential synergistic influence on the organizational and signaling cascades within Sertoli cells.
支持细胞分泌的肝细胞生长因子（HGF）激活c-Met受体，促进生精小管形成。胶质细胞源性神经营养因子（GDNF）可促进胚胎支持细胞增殖和索状结构形成。本研究旨在探讨在基于基质胶的三维（3D）培养系统中，外源性GDNF是否有助于HGF诱导的支持细胞体外组织化。
使用15P-1支持细胞系建立四个实验组：对照组、HGF处理组、GDNF处理组以及HGF和GDNF联合处理组。使用图像分析软件对管状结构的长度和分支进行定量，同时通过免疫荧光染色评估基于3D基质胶培养5天的支持细胞中ZO-1蛋白的表达。此外，分析支持细胞中Gfrα-1、Ret、Ncam和Met mRNA的表达。5天后在二维（2D）培养中分析ZO-1、c-MET和p-c-MET的表达水平。
定量分析显示，与其他组相比，GDNF与HGF联合使用时，类管状结构的长度和分支显著增加。此外，GDNF和HGF联合使用时，ZO-1蛋白表达显著增加，超过对照组水平。此外，与对照组相比，HGF和GDNF联合使用显著提高了磷酸化c-Met水平。
我们的研究表明，外源性GDNF可增强HGF诱导的支持细胞体外管状组织化。GDNF和HGF同时使用显著增强了管状结构的形成和ZO-1蛋白的表达，表明对支持细胞内的组织化和信号级联可能存在协同影响。
