MiR-205 Expression Elevated With EDS Treatment and Induced Leydig Cell Apoptosis by Targeting RAP2B via the PI3K/AKT Signaling Pathway.

Yang Cui,Wenxian Zeng,Shuai Yu,Wenjing Yang,Chuanying Pan,Mingyue Chen,Yanghai Zhang,Lin Ma,Rui Chen,Xianyong Lan,Wuzi Dong

doi:10.3389/fcell.2020.00448

Abstract

The adult Leydig cells (ALCs), originated from stem Leydig cells (SLCs), can secrete testosterone which is essential for germ cell development and sexual behavior maintenance. As a synthetic compound, ethane dimethane sulfonate (EDS), a well-known alkylating agent, has been reported to specifically ablate ALCs. In this study, EDS was verified to ablate differentiated pig LCs by experiments. Subsequently, the primary isolated pig LCs (containing SLCs and differentiated LCs) and EDS-treated LCs (almost exclusively SLCs) were collected for RNA-seq 4,904 genes and 15 miRNAs were differently expressed between the two groups. Down-regulated genes in the EDS-treated group were mainly related to steroid hormone biosynthesis. The highest up-regulation miRNAs was miR-205 after EDS treatment. Additionally, miR-205 was expressed more highly in pig SLCs clones compared with differentiated LCs. Through qRT-PCR, western blot (WB), TUNEL, EDU and flow cytometry, miR-205 was found to induce cell apoptosis, but did not affect proliferation or differentiation in both TM3 and GC-1spg mouse cell lines. Through luciferase reporter assays and WB, RAP2B was identified as a target gene of miR-205. Besides, overexpression of miR-205 inhibited the expressions of PI3K, Akt and p-AKT. All these findings were helpful for elucidating the regulation mechanism in pig LCs.

Highlights

The globally increasing male reproductive system abnormality is getting more and more attention
The results showed that miR-205 was highly expressed in porcine stem Leydig cells (SLCs) clones, so miR-205 was selected for further study. miR-205 was verified to have no effect on proliferation of TM3 cells and GC-1spg cells, but it induce apoptosis by targeting RAP2B gene by multiple experimental methods
The Hematoxylin and Eosin (H&E) staining showed that spindle-shaped cells were found in the testicular interstitium of 7-day-old pig testes (Figure 1A), and these cells could be stained with SLCs molecular marker, platelet-derived growth factor receptor α (PDGFRα) (Figure 1B)

Summary

Introduction

The globally increasing male reproductive system abnormality is getting more and more attention. EDS, a well-known alkylating agent, has been reported to ablate ALCs (Ge et al, 2006). In mammals, the main function of ALCs is T synthesis and secretion, which plays an essential role in maintenance of somatic cell function and germ cell development, and maintains male phenotype and sexual behaviors (Smith and Walker, 2014). These function imply that EDS would cause serious damage of male reproduction. The mechanism of EDS ablating Leydig cell remains unclear

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