Introduction: Several factors like genetic predisposition, lifestyle to environment contribute to male reproductive health. At a cellular level, adult Leydig cells in the testes are essential for reproductive function, as they synthesize and release testosterone. However, the growth and differentiation of Leydig cells could be affected by paracrine factors released by adjacent testicular environment which is constituted of Sertoli and Peritubular myoid cells. In one of our studies, we demonstrated that Leptin, a paracrine factor secreted by Sertoli cells, is critical for Leydig stem cell (LSC) differentiation and subsequent testosterone production via its regulation of desert hedgehog (DHH) signaling. Furthermore, it is well established that obesity plays a role in infertility, and obese men have low testosterone and leptin resistance. Therefore, In the present study, we evaluated the hypothesis that Leptin has specific endogenous effects on Leydig stem cell differentiation that are specific to patient’s BMI.
Methods: A total of 12 men with testicular failure that were subcategorized as obese (BMI >35), normal (BMI 25-30), and lean (BMI <25) underwent testis biopsies for sperm retrieval. Using an IRB approved protocol, about 10mg of testicular tissue from each of these men were processed for Leydig stem cell isolation, culture and characterized. Post characterization via Flow cytometry and Immunostaining, cells were studied for dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) effects of Leptin treatment in the presence or absence of Sertoli and Peritubular Myoid cells. To validate the effects of Leptin over Hedgehog signaling, cells were treated with Leptin in the presence of Hedgehog signaling agonists (SAG) and antagonists (Vismodigib). After treatment, RNA isolation was performed to evaluate the effects of BMI, leptin dose, and leptin treatment time on LSC differentiation. Immunofluorescence staining using 3bHSD, LHR, SOX-9, GLI-1, PDGFR-a, and SMO was performed. Furthermore, flow cytometry analysis for shift in cell population was studied using antibodies against 3bHSD, Nestin, SOX-9, PLZF, PDGFR-a, and Vimentin respectively. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t test.
Results: Results showed BMI specific patterns (p<0.05) of LSC differentiation with varying leptin concentrations. For example, with increasing leptin doses, the expression patterns of Hedgehog signaling markers (GLI and SMO) and LSC differentiation markers (3BHSD) found were positively linear in lean samples, bell-shaped in normal BMI samples, and inverted in obese samples. Furthermore, immunostaining and flow cytometry results showed that in the presence of low doses of leptin in cells from men with normal BMI, 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). However, these patterns were not consistent in cells from lean or obese patients.
Conclusions: Our results demonstrate the critical influence BMI has in affecting leptin signaling and LSC differentiation in the testes. 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. Source of
Funding: Supported by the American Urological Association Research Scholar Award and Stanley Glaser Award to RR. J.M.H. is supported by NIH grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, 5UM1 HL113460 and Soffer Family Foundation.