P-023 Exogenous glial cell-derived neurotrophic factor contributes hepatocyte growth factor-induced in vitro organization of Sertoli cells on matrigel-based three-dimensional culture
Authors: Durkut B , Celik-Ozenci C 
Published: June 2022

Abstract

Study question
Does glial cell-derived neurotrophic factor (GDNF), a crucial factor for spermatogonial stem cell (SSC) self-renewal, have a role in in vitro tubule-formation capacity of Sertoli cells?

Summary answer
Exogenous GDNF treatment together with hepatocyte growth factor (HGF) significantly increased the in vitro tubule-forming capacity of Sertoli cells on matrigel-based three-dimensional (3D) culture system.

What is known already
GDNF increases the tubule-forming capacity of HGF in vitro by regulating c-Met expression during renal tubulogenesis. In vitro testicular organization of Sertoli cells consists of two stages: testicular cord-like structures and tubule-like structures. HGF secreted from Sertoli cells plays a direct role in forming seminiferous tubules in vivo and in vitro by activating c-Met. GDNF maintains SSC numbers by activating GFRα1 and Ret receptors in SSCs and can also directly phosphorylate c-Met in GFRα1-positive cells.

Study design, size, duration
Four groups were established using the 15P-1 Sertoli cell line: Control, HGF-administrated, GDNF-administrated and, HGF and GDNF-administrated. All groups were pursued on a matrigel-based 3D culture system for seven days. Experiments were repeated three times in all groups. Kit ligand (KitL, Sertoli cell marker; positive control) and c-kit (germ cell marker; negative control) were used as the control for Sertoli cell characterization. This work was supported by TUBITAK (SBAG-120S554) and Akdeniz University Research Foundation (TYL-2020-5481).

Participants/materials, setting, methods
The cord-forming and tubule-forming capacity of Sertoli cells on matrigel-based 3D culture was evaluated under an inverted microscope, and c-Met and p-c-Met expression levels were analyzed using western blot. In addition to KitL and c-kit protein expression, zonula occludens-1 (ZO-1) protein expression for polarization of Sertoli cells was evaluated by immunofluorescence staining. Data were analyzed using one-way ANOVA and Tukey post hoc test for multiple comparisons. p &lt; 0.05 was considered statistically significant.

Main results and the role of chance
In the first 24 hours, interaction and aggregation between Sertoli cells began. The highest cellular interaction was found in the group treated with HGF and GDNF compared to the other groups (p &lt; 0.05). At 48 and 72 hours, the formation of cord-like structures was observed by forming connections between the clusters formed in all groups in the first 24 hours. The number of cord-like structures was higher in the groups treated with only HGF and HGF and GDNF at 48th and 72nd hours (p &lt; 0.05). At 96th and 120th hours, structures forming spaces were observed, and these structures were determined to be lumen-like structures. The capacity of 15P-1 Sertoli cells to form tubule-like structures was higher in the HGF and GDNF treated groups than in the other groups (p &lt; 0.05). c-Met and p-c-Met protein expression levels were high in HGF, GDNF, HGF, and GDNF groups compared to control (P &lt; 0.05). Sertoli cells retained their identity until the end of the culture and formed tight junctions in the HGF and GDNF-administrated group as evaluated by ZO-1 expression.

Limitations, reasons for caution
The cord- and tubule-forming capacity of Sertoli cells was evaluated in vitro utilizing exogenous GDNF treatment but the effect of testicular endothelial cell-derived GDNF should be also evaluated.

Wider implications of the findings
Exogenous GDNF treatment enhances HGF-induced in vitro tubule-formation capacity of Sertoli cells by directly phosphorylating c-Met in Sertoli cells. Our findings highlight the importance of designing studies related to GDNF-induced cell signaling pathway for in vitro tubulogenesis of Sertoli cells to support ex vivo spermatogenesis.

Trial registration number
None