Growth hormone-induced STAT5B regulates Star gene expression through a cooperation with cJUN in mouse MA-10 Leydig cells

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star mRNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (EMSA and supershift) and in vivo (ChIP) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

Bisphenol A decreases expression of insulin-like factor 3 and induces histopathological changes in testes of rat

Bisphenol A (BPA) is a chemical agent known to have detrimental reproductive and developmental effects. The tissue-specific impacts of BPA exposures and target tissues sensitiveness to BPA are still unclear. The aim of this study was to determine the short- and long-term dose-dependent toxic effects of BPA on rat testes. Forty-eight Wistar albino male rats were divided into four groups each containing 12 rats. To induce toxicity, BPA was administered orally at three different dosages (50, 100, and 200 mg/kg) for 14 and 28 days, respectively. Testis tissues were examined using light and electron microscopy, immunohistochemistry, and biochemical methods. Serum testosterone (T) and luteinizing hormone (LH) levels were measured. Additionally, insulin-like factor 3 (INSL3) as a marker of Leydig cell function was evaluated immunohistochemically. Groups administered high doses of BPA showed severe degenerations such as testicular atrophy, spermatogenic arrest, and interstitial edema in testis. Also, a significant decrease in INSL3 immunoreactivity and serum LH and T levels was found. The results indicated that both increased exposure time and dosage of BPA caused more serious detrimental effects on testes in the rat. Decreased INSL3 and T levels was evidence of Leydig cell function impairment due to BPA.

Serum Insulin-Like Factor 3 Levels Are Reduced in Former Users of Anabolic Androgenic Steroids Suggesting Persistent Impaired Leydig Cell Function

Background: Illicit use of anabolic androgenic steroids (AAS) has emerged as a public health concern among men, but the long-term effect on gonadal function is still unresolved. Serum insulin-like factor 3 (INSL3) has emerged as a novel and potentially superior marker of Leydig cell function than serum testosterone per se. INSL3 synthesis and secretion exhibit far less daily variation than testosterone. Further, serum INSL3 levels are not related to body composition. The objective of this study was to investigate INSL3 as a marker of Leydig cell function in former AAS users. Methods: Community-based cross-sectional study including men aged 18 - 50 years, involved in recreational strength training and allocated to one of three groups: current (n = 46) or former AAS users (n = 42) or controls (n = 44). Mean age (SD) of all participants were 32 (7) years and the elapsed duration since AAS cessation, geometric mean (95% CI), was 32 (23; 45) months in former AAS users. All procedures were performed during one visit in the morning hours following overnight fasting. We drew blood through a cannula placed in an antecubital vein following 30 minutes of supine rest. Medical records, testicular size, questionaries, and detailed history of strength training and AAS use were obtained in a structured interview. Serum INSL3 and testosterone were measured using liquid chromatography mass spectrometry. Results: Serum INSL3 was markedly suppressed among current AAS users compared with former AAS users and controls, P < 0.001. Additionally, former AAS users also displayed lower serum INSL3 concentrations than controls, mean (SD), 0.43 (0.31) versus 0.60 (0.22) µg/L, P = 0.006 and the difference remained significant in a multivariate linear regression, (B) (95%CI), -0.17 (-0.28;-0.55) µg/L, P=0.004, adjusted for plasma LH, plasma sexual hormone-binding globulin, age, body fat %, smoking and use of other illicit drugs. Longer accumulated duration of AAS use (log2) was associated with reduced serum INSL3 levels in former AAS users, (B) (95%CI), -0.08 (-0.14;-0.01), P=0.022, suggesting a dose-response relation between AAS use and suppression of serum INSL3. We evaluated the association between INSL3 and total testosterone levels and they were not associated among former users and controls in a multivariate linear regression, P=0.821. We noted recovery of serum inhibin B levels among former AAS users reaching the mean plasma level of controls after elapsed duration since AAS cessation of ≈ 21 months; (B) (95%CI), 2.2 (0.7; 3.7) months, P = 0.006. In contrast, we did not note any recovery of serum INSL3, P = 0.541, or total testosterone, P = 0.861, among former AAS users. Conclusions: Serum INSL3 is decreased years following AAS cessation in former AAS users, independently of plasma testosterone, suggesting persistent impaired Leydig cell function, which should be investigated further.

Androgen and Luteinizing Hormone Stimulate the Function of Rat Immature Leydig Cells Through Different Transcription Signals

The function of immature Leydig cells is regulated by hormones, such as androgen and luteinizing hormone (LH). However, the regulation of this process is still unclear. The objective of this study was to determine whether luteinizing hormone (LH) or androgens contribute to this process. Immature Leydig cells were purified from 35-day-old male Sprague Dawley rats and cultured with LH (1 ng/ml) or androgen (7α-methyl-19- nortestosterone, MENT, 100 nM) for 2 days. LH or MENT treatment significantly increased the androgens produced by immature Leydig cells in rats. Microarray and qPCR and enzymatic tests showed that LH up-regulated the expression of Scarb1, Cyp11a1, Cyp17a1, and Srd5a1 while down-regulated the expression of Sult2a1 and Akr1c14. On the contrary, the expression of Cyp17a1 was up-regulated by MENT. LH and MENT regulate Leydig cell function through different sets of transcription factors. We conclude that LH and androgens participate in the regulation of rat immature Leydig cell function through different transcriptional pathways.

Diabetes Type 1 Negatively Influences Leydig Cell Function in Rats, Which is Partially Reversible By Insulin Treatment

Type 1 diabetes mellitus (T1DM) is associated with impaired spermatogenesis and lower testosterone levels and epididymal weight. However, the underlying processes in the testis are unknown and remain to be elucidated. Therefore, the present study focused on the effects of T1DM on testicular function in a spontaneously diabetic rat model. BB/OKL rats after diabetes manifestation were divided into 3 groups: those without insulin treatment and insulin treatment for a duration of 2 and of 6 weeks. Anthropometrical data, circulating levels of gonadotrophins, testosterone, and inhibin B were measured. Intratesticular testosterone, oxidative stress, inflammation, and apoptosis were analyzed. Key enzymes of steroidogenesis were evaluated in the testis. Untreated diabetic rats had significantly lower serum follicle-stimulating hormone and luteinizing hormone levels. Serum and intratesticular testosterone levels significantly decreased in untreated diabetic rats compared to healthy controls. Key markers of Leydig cell function were significantly downregulated at the RNA level: insulin-like factor 3 (Insl3) by 53% (P = .006), Star by 51% (P = .004), Cyp11A1 by 80% (P = .003), 3Beta-Hsd2 by 61% (P = .005), and Pbr by 52% (P = .002). In the insulin-treated group, only Cyp11A1 and 3Beta-Hsd2 transcripts were significantly lower. Interestingly, the long-term insulin–treated group showed significant upregulation of most steroidogenic enzymes without affecting testosterone levels. Tumor necrosis factor α and apoptosis were significantly increased in the long-term insulin–treated rats. In conclusion T1DM, with a severe lack of insulin, has an adverse action on Leydig cell function. This is partially reversible with well-compensated blood glucose control. Long-term T1DM adversely affects Leydig cell function because of the process of inflammation and apoptosis.

INSL3: A Marker of Leydig Cell Function and Testis-Bone-Skeletal Muscle Network

This article reviews the role of INSL3 as biomarker of Leydig cell function and its systemic action in testis-bone-skeletal muscle crosstalk in adult men. Insulin-like factor 3 (INSL3) is a peptide hormone secreted constitutively in a differentiation-dependent mode by testicular Leydig cells. Besides the role for the testicular descent, this hormone has endocrine anabolic functions on the bone-skeletal muscle unit. INSL3 levels are low in many conditions of undifferentiated or altered Leydig cell status, however the potential clinical utility of INSL3 measurement is not yet well defined. INSL3 levels are modulated by the long-term cytotropic effect of the hypothalamicpituitary- gonadal axis, unlike testosterone that is acutely sensitive to the stimulus by luteinizing hormone (LH). INSL3 directly depends on the number and differentiation state of Leydig cells and therefore it represents the ideal marker of Leydig cell function. This hormone is more sensitive than testosterone to Leydig cell impairment, and the reduction of INSL3 in adult men can precociously detect an endocrine testicular dysfunction. Low INSL3 levels could cause or contribute to some symptoms and signs of male hypogonadism, above all sarcopenia and osteoporosis. The measurement provided suggested that the measurement of INSL3 levels should be considered in the clinical management of male hypogonadism and in the evaluation of testicular endocrine function. The monitoring of INSL3 levels could allow an early detection of Leydig cell damage, even when testosterone levels are still in the normal range.