Leptin Secreted in the Testicular Microenvironment Promotes the Endogenous Function of Leydig Cells Through Hedgehog Signaling Pathway

Objective: Testosterone deficiency (TD) is a common health concern, affecting around 1 in 5 men globally. However, the factors responsible for TD remain largely unknown. Leydig cells produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. Leydig stem cells (LSC) have the potential to differentiate into adult Leydig cells, which can increase testosterone levels; however, the factors promoting differentiation are unknown. In the present study we evaluated the paracrine factors released from the testicular microenvironment (TME) (comprised of Sertoli and peritubular myoid cells) that modulate the differentiation of Leydig stem cells to adult Leydig cells. Additionally, we explored the underlying mechanism of action of these paracrine factors. Methods: Tissue samples were obtained from a total of 13 men with testicular failure, who underwent testis biopsies for sperm retrieval. Using an IRB approved protocol, about 10mg of testicular tissue from each sample were processed for LSC isolation, culturing, and characterization. Cytokine antibody array was performed to identify the paracrine factors released by Sertoli and peritubular myoid cells using unsorted and CD146(+ve) sorted cells. The cells were treated with hedgehog signaling agonist and antagonist to validate the specificity of paracrine factors identified. Immunostaining was performed to evaluate changes at the protein level. Flow cytometry was performed to study the shift in the population of cells post leptin treatment. GraphPad Prism (GraphPad Software) was used for statistical analysis. Results: This study revealed that the TME plays an instrumental role in Leydig stem cell differentiation and testosterone production through regulation of the desert hedgehog (DHH) signaling pathway. TME-secreted leptin induces LSC differentiation and increases testosterone production. However, these effects are inversely concentration-dependent: positive at low leptin doses and negative at higher leptin doses. Mechanistically, leptin acts on LSCs upstream of DHH in a unidirectional fashion, as DHH gain or loss of function was shown to have no effects on Leptin levels. Conclusions: These findings identify leptin as a key paracrine factor released by cells within the TME that modulate LSC differentiation and testosterone production from adult Leydig cells, a finding that is key to developing new niche therapies for TD.