# ATG5 gene regulates testosterone synthesis of testicular Leydig cells in Hezuo pig

## TL;DR

This study shows that the ATG5 gene influences testosterone production in Leydig cells of Hezuo pigs, likely through regulating autophagy.

## Contribution

The study reveals a novel role of ATG5 in regulating testosterone synthesis in Leydig cells of Hezuo pigs.

## Key findings

- ATG5 overexpression increases testosterone levels and autophagy-related gene expression in Leydig cells.
- ATG5 manipulation affects cell proliferation and apoptosis in Leydig cells.
- ATG5 is highly expressed in testicular tissues of 4-month-old Hezuo pigs compared to 1-month-olds.

## Abstract

Autophagy-related gene 5 (ATG5) plays a crucial role in autophagosome formation. Recent studies have investigated the role of autophagy in regulating testosterone production; however, its expression in testicular tissues and Leydig cells of Hezuo pig remains less understood. In this study, we cloned the coding sequence (CDS) region of the ATG5 gene and assessed its expression using qPCR across various tissues and testes at different developmental stages in Hezuo pigs. Subsequently, we constructed silencing and overexpression vectors for ATG5 and transfected them into Leydig cells. Cell proliferation and apoptosis were evaluated using CCK-8 and flow cytometry assays, respectively. Autophagy and testosterone synthesized gene expression were detected by qPCR, while ATG5, StAR and LC3 protein levels were measured by Western blotting. Furthermore, testosterone concentration and the levels of autophagy-related genes BECN1 (Beclin1), NPC1L1 (Niemann-Pick C1-like 1), and TSPO (translocator protein) were quantified via ELISA. The results indicated that the CDS region of the ATG5 gene spans 828 base pairs, encoding 275 amino acids. ATG5 showed high expression in the testis and lung (p < 0.01), with significantly higher expression in the testicular tissues of the 4-month-old group compared to the 1-month-old group (p < 0.01). Compared to the empty vector control (ATG5-PC), the overexpression group (ATG5-OE) exhibited increased cell proliferation (p < 0.05), reduced apoptosis (p < 0.01), elevated autophagy gene expression (BECN1, ATG7, and LC3) (p < 0.01) and testosterone synthesis-related genes (StAR, HSD3B and CYP11A1) (p < 0.01), along with increased ATG5, StAR and LC3protein levels (p < 0.01), as well as significantly increased testosterone levels, BECN1, NPC1L1, and TSPO expression (all p < 0.01). These findings indicate that ATG5 manipulation affects testosterone synthesis in Leydig cells, likely through autophagy regulation. This study offers novel insights into the function of ATG5 in testosterone production within testicular Leydig cells, providing theoretical support for investigating early puberty in Hezuo pigs.

Flowchart illustrating the experimental procedure involving Hezuo pigs. It starts with isolating the ATG5 gene, followed by cloning, sequencing, and bioinformatics analysis. The strategy involves constructing a plasmid vector for cell transfection in primary Leydig cells, isolation, and culture from pig testes. Subsequent analyses include proliferation, apoptosis, qPCR for gene expression (StAR, HSD3B, CYP11A1), Western blot for specific proteins (ATG5, StAR, LC3), and ELISA for testosterone and other markers (BECN1, NPC1L1, TSPO). Images of pigs and testes are included.