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Published on: January 10, 2025
EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis
Lingling Zhang1, Lijun Ding2, Yifan Li3
1NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Pharmacy, Fudan University, Shanghai 200032, China; NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Fudan University, Shanghai 200032, China; Center for Reproductive Medicine, Drum Tower Clinic Medical College of Nanjing Medical University, Nanjing 210008, China; Center for Molecular Reproductive Medicine, Nanjing University, Nanjing 210008, China.
Aims:
Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis.
Main Methods:
Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology.
Key Findings:
EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1f/f mice.
Significance:
This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.
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