52 Leptin Effects on Leydig Stem Cell Differentiation are Specific to Patients BMI

Introduction Adult Leydig cells in the testes are essential for testosterone production. The growth and differentiation of Leydig cells could be affected by paracrine factors released by testicular microenvironment (TME) (Sertoli and peritubular myoid cells). In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cells (LSCs) differentiation and subsequent testosterone production via its interaction with Leptin receptor on LSCs, followed by downstream regulation of desert hedgehog (DHH) signaling. Although it is well-known that obesity adversely affects male fertility and testosterone production, the endogenous effects of Leptin on Leydig stem cell differentiation and transcription factors genes (TFs) binding to leptin receptor (LEPR) and the mechanism that are specific to patient's BMI (normal, obese and lean men) are understudied. Additionally, the underlying mechanism behind the differential effects of Leptin (if any) is unknown. Therefore, In the present study, we evaluated the impacts and mechanism of Leptin as a paracrine factor on LSC differentiation in patients with different BMI's. Objective To uncover the mechanism of Leptin as a paracrine factor on LSC differentiation in patients with different BMI's. Methods A total of 13 men with testicular failure that were subcategorized as obese (BMI >35), normal (BMI 25-30), and lean (BMI Results Results showed that a strong relationship exists between BMI and leptin levels. Immunostaining, flow cytometry and RNA sequencing results showed that in the men with normal BMI, in the presence of low doses of leptin, there was a shift of the testicular cell population towards adult Leydig cells (increased population of cells staining positive for 3BHSD, LHR) and increased DHH signaling (GLI and SMO). On the contrary, at higher concentrations, the positive effects of leptin on cells from men with normal BMI were neutralized. Furthermore, in cells from lean patients, leptin treatment had a consistent, linear and positive impact on LSC differentiation markers and DHH signaling. Moreover, in cells from obese patients, leptin had a consistent, linear and negative impact on LSC differentiation and markers of DHH signaling. Enrichment analysis identified 50 TFs, of which SOX2, SOX8 and SOX9 were enriched for abnormal pituitary function and therefore for hypothalamic pituitary gonadal axis. The role of these TFs on LSC differentiation remained under-explored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8 Sox9 respectively. Consequently, with increasing leptin doses SOX2, SOX8 Sox9 protein and expression has been upregulated. Mechanistically, Leptin-LEPR-TFs induced DHH signaling regulates LSCs differentiation in a different manner which is specific to patient's BMI. Conclusions Our results demonstrate the influence, BMI has on Leptin induced LSC differentiation. These findings suggest the future potential use of Leptin as a personalized therapy for inducing LSC differentiation and overcoming low Testosterone levels. Further studies are necessary to identify potential therapeutic effects of leptin treatment in improving fertility in the setting of leptin resistance and obesity. Financial Support: Supported by the American Urological Association Research Scholar Award to H.A. J.M.H. is supported by NIH grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, 5UM1 HL113460 and Soffer Family Foundation. Disclosure No