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.

Gene expression and functional abnormalities in XX/Sry Leydig cells

The SRY gene induces testis development even in XX individuals. However, XX/Sry testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential for spermatogenesis. XX/Sry Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions through testosterone production. However, whether XX/Sry adult Leydig cells (XX/Sry ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/Sry ALCs demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/Sry ALCs, although downregulated in XX/Sry Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In XX/Sry ALCs, the latter reaction was selectively decreased. The defects in XX/Sry ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/Sry testes.

Expression of Wnt5a defines the major progenitors of fetal and adult Leydig cells

SummaryLeydig cells (LCs) are the major androgen-producing cells in the testes. They arise from steroidogenic progenitors, whose origins, maintenance and differentiation dynamics remain largely unknown. Here, we identified Wnt5a as a specific marker of steroidogenic progenitors, whose expression begins at around E11.5-E12.5 in interstitial cells of the fetal mouse testis. In vivo lineage tracing indicates that Wnt5a-expressing progenitors are initially present in large numbers in the fetal testis and then progressively decrease as development progresses. We provide evidence that Wnt5a-expressing cells are bona fide progenitors of peritubular myoid cells as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis. Additionally, we show in the adult testis that Wnt5a expression is restricted to a subset of LCs exhibiting a slow but noticeable clonal expansion, revealing hitherto unappreciated proliferation of fully differentiated LCs as a contribution to the adult LC pool.

Effects of Osteocalcin on Synthesis of Testosterone and INSL3 during Adult Leydig Cell Differentiation

Proliferation and differentiation of adult Leydig cells are mainly completed in puberty. In many studies, apart from normal postnatal development process, it is widely indicated that, through administrating EDS, Leydig cell population is eliminated and regenerated. It is believed that osteocalcin released from osteoblasts, which is responsible for modulating bone metabolism, induces testosterone production in Leydig cells, independent of the HPG axis. In addition, INSL3 produced by Leydig cells, such as testosterone, plays a critical role in bone metabolism and is known to reflect the development process and functional capacities of Leydig cells. This study is aimed at investigating OC-mediated testosterone regulation and INSL3 synthesis during differentiation of adult Leydig cells that are independent of LH. For this purpose, male rats were divided into 2 groups: prepubertal normal rats and adult EDS-injected rats. Each group was divided into 4 subgroups in which GnRH antagonist or OC was applied. After adult Leydig cells completed their development, testicular tissue samples obtained from the sacrificed rats were examined by light-electron microscopic, immunohistochemical, and biochemical methods. Slight upregulation in 3βHSD, INSL3, and GPRC6A expressions along with the increase in serum testosterone levels was observed in groups treated with osteocalcin against GnRH antagonist. In addition, biochemical and microscopic findings in osteocalcin treated groups were similar to those in control groups. While there was no significant difference in the number of Leydig cells reported, the presence of a significant upregulation in INSL3 and GPRC6A expressions and the increase in serum testosterone and ucOC levels were observed. After evaluation of findings altogether, it is put forward that, for the first time in this study, although osteocalcin treatment made no significant difference in the number of Leydig cells, it increased the level of testosterone through improving the function of existing adult Leydig cells during normal postnatal development process and post-EDS regeneration. This positive correlation between osteocalcin-testosterone and osteocalcin-INSL3 is concluded to be independent of LH at in vivo conditions.

Trimethyltin (TMT) Reduces Testosterone Production in Adult Leydig Cells in Rats

Trimethyltin (TMT) is widely used as a plastic heat stabilizer and can cause severe toxicity. Here, the effects of TMT on testosterone production by adult Leydig cells and the related mechanisms of action were investigated. Eighteen adult male Sprague Dawley rats (56 days old) were randomly divided into 3 groups and given intraperitoneal injection of TMT for 21 consecutive days at the doses of 0 (vehicle control), 5, or 10 mg/kg/d. After treatment, trunk blood was collected for hormonal analysis. In addition, related gene and protein expression in testes was detected. At 10 mg/kg, TMT significantly reduced serum testosterone levels but increased serum luteinizing and follicle-stimulating hormone levels. The messenger RNA and protein levels of luteinizing hormone/chorionic gonadotropin receptor, steroidogenic acute regulatory protein, cytochrome P450 17-hydroxylase/17,20-lyase, follicle-stimulating hormone receptor, and SRY box 9 were significantly lower in the TMT-treated testes than in controls. Immunohistochemical study showed that TMT decreased adult Leydig cell number. In conclusion, these findings indicate that TMT reduced adult Leydig cell testosterone production in vivo by directly downregulating the expression of steroidogenic enzymes and decreasing adult Leydig cell number in the testis.