(083) EMT Induced Transcription Factors Direct Leptin-LEPR-DHH Signaling to Modulate Leydig Stem Cells Differentiation in Testis
Authors: Arora H , Seetharam D , Ramasamy R , Dullea A 
Published: May 2023

Abstract

Introduction
The primary source of testosterone (T) in men are Leydig cells (LCs) and their dysfunction can lead to T deficiency. Testicular microenvironment (TME) which is comparised of cells such as Sertoli and Peritubular myoid cells releases the paracrine factors to influence the growth and differentiation of LCs. Leptin is a 16-kDa protein produced primarily by adipose tissue and exerts a significant influence on hormonal regulation. In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cell (LSCs) differentiation and subsequent T production under the regulation of the desert hedgehog signalling pathway. We also showed that this regulation is inverse concentration dependent such as Leptin increases LSC differentiation at low doses and decrease it at higher doses. However, an in-depth understanding of mechanisms that derive the Leptin secretion and decide the fate of LCs are still unclear and is the focus of the present study.

Objective
To study the mechanisms that derive the Leptin secretion and decide the fate of LCs.

Methods
A total of 18 men with testicular failure underwent testis biopsies. The testicular cells were characterized using Flow cytometry, Immunostaining and RNA sequencing then the cells were also treated with Leptin in a dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) manner. For the functional studies, UCSC genome browser was used to study the transcription factor genes (TFs) binding the leptin receptor (LEPR) promoter. The list of TFs was subjected to enrichment analysis. To evaluate the effects of shortlisted TFs on leptin-LEPR-DHH to induce EMT in LSCs, qPCR and western blotting were performed. For statistical analysis GraphPad Prism was used. For statistical analysis GraphPad Prism was used. All data were presented as the means ± SEM. All data were presented as the means ± SEM

Results
Over 60 TF’s were identified by UCSC Genome browser, which were subjected to enrichment analysis using ENRICHR and Ingenuity pathway analysis. Output revealed TFs; SOX2, SOX8 and SOX9 that were enriched for mechanisms influencing hormonal regulation via hypothalamic pituitary gonadal axis. The role of these TFs has been well established in epithelial to mesenchymal transition in different cancer types, however their impact on LSC differentiation remained underexplored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8 Sox9 respectively. This was confirmed at RNA as well as at protein levels. Consequently, upon inhibition of LEPR, the expression of SOX2, SOX8 and SOX9 was reduced as well. Furthermore we used SOX8, SOX9 and SOX2 antagonists in the presence or absence of Leptin to validate the effects on EMT of LCs.

Conclusions
Our results demonstrate that Leptin-LEPR-DHH induced Leydig stem cells differentiation in testis is modulated by EMT inducing transcription factors which binds to LEPR. Further studies are ongoing to explore the potential therapeutic effects of leptin treatment in improving fertility.

Disclosure
No