Stimulated Testosterone Production Research Articles

37-Year-Old Man With Fatigue and Erectile Dysfunction

37-Year-Old Man With Fatigue and Erectile Dysfunction

Decrease in the Basal and Luteinizing Hormone Receptor Agonist–Stimulated Testosterone Production in Aging Male Rats

In the course of aging, the steroidogenic function of the testes weakens and their gonadotropin sensitivity decreases. However, the underlying mechanisms are poorly understood. The goal of this work was to study the stimulating effects of human chorionic gonadotropin (hCG) and TP03, a low molecular weight agonist of luteinizing hormone (LH)/hCG receptor, on testosterone production and the expression of steroidogenic proteins in young (3-month-old) and aging (15-month-old) male rats and to investigate the activity of the adenylyl cyclase system in the membranes isolated from the rat testes. Treatment with hCG (100 IU/rat/day) and TP03 (15 mg/kg/day) was carried out for 3 days. In the testes of aging rats, adenylyl cyclase stimulation by gonadotropin and guanine nucleotide was decreased, indicating a weakening of the coupling of LH/hCG receptor and Gs protein, the main components of the adenylyl cyclase system regulating the steroidogenesis. In elderly rats, the blood testosterone levels and the expression of the Star, Cyp11a1, and Cyp17a1 genes, which encode the StAR protein and the steroidogenic enzymes cytochromes P450scc and P450-17α in the testes, were decreased. The stimulating effect of both hCG and TP03 on testosterone production diminished with age, although their effects on LH/hCG receptor are mediated by different mechanisms. In both young and aging rats, hCG treatment upregulated the expression of the genes encoding StAR, P450scc and dehydrogenase 3β-HSD; at the same time, Hsd17B expression in aging rats increased, and the expression of the genes encoding P450-17α and 17β-HSD decreased in young rats. In young rats, TP03 treatment upregulated Star and Cyp17a1 expression, and it increased Star and Hsd17B expression in aging rats. Thus, in the testes of aging rats, the coupling between the LH/hCG receptor and Gs protein and LH/hCG receptor sensitivity to agonists were weakened, which led to a decrease in hCG- and TP03-induced testosterone production and altered the basal and LH/hCG receptor agonist–stimulated expression levels of some steroidogenic protein genes.

In utero exposure to hexavalent chromium disrupts rat fetal testis development

Hexavalent chromium (Cr6+) acts as an endocrine disruptor. Herein, we investigated effects of Cr6+ on the development of rat fetal Leydig and Sertoli cells, which support differentiation of the male reproductive tract in late gestation. Female pregnant Sprague Dawley rats were gavaged with potassium dichromate (0, 3, 6, and 12 mg/kg) from gestational days (GD) 12 to GD 21. Leydig and Sertoli cell function was evaluated by investigating serum testosterone levels, cell number and distribution, and the expression levels of Leydig and Sertoli cell genes and proteins. Cr6+ increased serum testosterone level at dose of 3 mg/kg (1.170 ± 0.121 ng/ml vs. 0.720 ± 0.082 ng/ml in the control), while lowered it at dose of 12 mg/kg (0.400 ± 0.098 ng/ml). In addition, it showed that Cr6+ dose-dependently reduced Leydig cell size and cytoplasmic size and decreased the percentage of medium fetal Leydig cell cluster at dose of 12 mg/kg. Further study demonstrated that the expression of Leydig cell (Lhcgr, Scarb1, and Hsd3b1) and Sertoli cell (Fshr, Pdgfa, and Lif) genes in the testis was upregulated at dose of 3 mg/kg while the expression of Lhcgr, Hsd17b3 and Igf1 was downregulated by Cr6+ at dose of 12 mg/kg. In conclusion, Cr6+ had biphasic effects on fetal Leydig cell development with low dose to stimulate testosterone production and high dose to inhibit it, possibly via biphasically regulating growth factor gene expression in fetal Sertoli cells.

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction-tandem mass spectrometry to detect hCG use.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free β-subunit (hCGβ) and β-subunit core fragment (hCGβcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGβ and hCGβcf were 0.04IU/L, 0.47pmol/L and 0.16pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGβ and hCGβcf were 0.21IU/L, 0.40pmol/L, and 1.86pmol/L, respectively. Based on these data, we recommend a threshold of 1.0IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

Pyrethroid Insecticide Cypermethrin Accelerates Pubertal Onset in Male Mice via Disrupting Hypothalamic-Pituitary-Gonadal Axis.

Pyrethroids, a class of insecticides that are widely used worldwide, have been identified as endocrine-disrupting chemicals (EDCs). Our recent epidemiological study reported on an association of increased pyrethroids exposure with elevated gonadotropins levels and earlier pubertal development in Chinese boys. In this study, we further investigated the effects of cypermethrin (CP), one of the most ubiquitous pyrethroid insecticides, on hypothalamic-pituitary-gonadal (HPG) axis and pubertal onset in male animal models. Early postnatal exposure to CP at environmentally relevant doses (0.5, 5, and 50 μg/kg CP) significantly accelerated the age of puberty onset in male mice. Administration of CP induced a dose-dependent increase in serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone in male mice. CP did not affect gonadotropin-releasing hormone (GnRH) gene expression in the hypothalamus, but CP at higher concentrations stimulated GnRH pulse frequency. CP could induce the secretion of LH and FSH, as well as the expression of gonadotropin subunit genes [chorionic gonadotropin α (CGα), LHβ, and FSHβ] in pituitary gonadotropes. CP stimulated testosterone production and the expression of steroidogenesis-related genes [steroidogenic acute regulatory (StAR) and Cytochrome p 450, family 11, subfamily A, polypeptide 1 (CYP11A1)] in testicular Leydig cells. The interference with hypothalamic sodium channels as well as calcium channels in pituitary gonadotropes and testicular Leydig cells was responsible for CP-induced HPG axis maturation. Our findings established in animal models provide further evidence for the biological plausibility of pyrethroid exposure as a potentially environmental contributor to earlier puberty in males.

Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10-9 -10-4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc.

5 TESTICULAR GnRH-II RECEPTOR KNOCKDOWN IMPAIRS DIURNAL TESTOSTERONE SECRETION IN THE BOAR

The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are ubiquitously expressed, with elevated levels in the testis. Gene coding errors prevent their production in many species, but both genes are functional in swine. We demonstrated that GnRHR-II localizes to porcine Leydig cells, and exogenous GnRH-II robustly stimulates testosterone production in vivo, despite minimal luteinizing hormone (LH) secretion. These data suggest that GnRH-II directly effects steroidogenesis in the boar testis. To explore this hypothesis, we produced a GnRHR-II knockdown (KD) swine line. Upon characterisation of this line, serum testosterone concentrations were reduced in GnRHR-II KD compared with littermate control males during pubertal development. However, concentrations of LH were unaffected, indicating that GnRHR-II KD impairs steroidogenesis directly at the testis rather than inhibiting gonadotropin secretion from the anterior pituitary gland. Based on these results, the objective of this study was to compare diurnal secretory patterns of testosterone in mature GnRHR-II KD (n = 5) and littermate control (n = 5) males. Boars were fit with indwelling jugular cannulae and blood was collected every 15 min for 8 h. Serum was assayed for testosterone concentration via radioimmunoassay. Next, GnRHR-II KD (n = 5) and littermate control (n = 4) boars were killed, testis weight was recorded, and testicular tissue was collected for RNA isolation. To confirm KD in these animals, digital droplet PCR was performed to quantify GnRHR-II mRNA abundance (normalized to β-actin). Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with line (transgenic or control) as the fixed effect and litter as a random effect. For hormone data, time and line × time interaction were included as fixed effects, with time as a repeated measure. Although there was no effect of time or line × time interaction (P &gt; 0.05) on serum testosterone concentrations, we observed a line effect (P &lt; 0.05). Differences between lines were dramatic; testosterone was reduced by 82% in GnRHR-II KD (0.75 ± 0.05 ng mL−1) compared with littermate control (4.09 ± 0.29 ng mL−1; P &lt; 0.05) males. Despite divergent testosterone levels, testis weights were similar between lines (P &gt; 0.05) indicative of altered Leydig cell function as opposed to hypertrophy/hyperplasia. Given that testicular GnRHR-II mRNA levels were reduced by 69% in transgenic animals (P &lt; 0.001), these data demonstrate that GnRH-II and its receptor play a critical role in testosterone biosynthesis within porcine Leydig cells. Thus, this report reveals novel mediators of testicular function in the boar and challenges the central dogma of testosterone regulation. Because testosterone dictates male reproductive success, GnRH-II and its receptor represent unique targets to improve fertility in swine. This study was partially supported by NIFA Hatch (NEB-26-199; BRW) and AFRI (2011-67015; CAL) funds.

Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

Testosterone deficiency is associated with sickle cell disease (SCD), but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle) exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH) levels compared with wild type (WT) mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol)- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR), but not cholesterol side-chain cleavage enzyme (P450scc), in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi) exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

Editorial Commentary

No AccessUrology PracticePatient Care1 Jul 2015Editorial Commentary Ranjith Ramasamy, Jason M. Scovell, and Larry I. Lipshultz Ranjith RamasamyRanjith Ramasamy More articles by this author , Jason M. ScovellJason M. Scovell More articles by this author , and Larry I. LipshultzLarry I. Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.urpr.2014.10.021AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Editorial Commentary." Urology Practice, 2(4), pp. 204–205 References 1 : The use of clomiphene citrate in male infertility. J Urol1984; 131: 425. Link, Google Scholar 2 : Drug therapy for idiopathic male infertility: rationale versus evidence. J Urol2006; 176: 1307. Link, Google Scholar 3 : WITHDRAWN: Clomiphene or tamoxifen for idiopathic oligo/asthenospermia. Cochrane Database Syst Rev1996; : CD000151. Google Scholar 4 : Enclomiphene citrate stimulates testosterone production while preventing oligospermia: a randomized phase II clinical trial comparing topical testosterone. Fertil Steril2014; 102: 720. Google Scholar © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 2Issue 4July 2015Page: 204-205 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ranjith Ramasamy More articles by this author Jason M. Scovell More articles by this author Larry I. Lipshultz More articles by this author Expand All Advertisement PDF downloadLoading ...

Opioid-induced androgen deficiency in males (OPIAD syndrome)

Although many of the effects of opioids are well studied, their activity on the hypogonadal axis is less known. Opioids lead to a decreased level in testosterone by changing the normal excretion of gonadotropins or as a result of changes in the anterior pituitary gland response to these hormones. Long-acting opioids, such as morphine sulfate, methadone, oxycodone and fentanyl, which are used in the treatment of chronic pain, often lead to androgen deficiency induced by opioids (OPIAD-Opioid-Induced Androgen Deficiency). A significant decrease in testosterone levels already occurs during application of 100 to 200 mg of morphine oral formulation. This effect is realized by modifying the normal pulsatile release of the gonadotropins, but also by changing in the response of the anterior pituitary gland up gonadotropin-releasing hormone, which altogether lead to reduced secretion of pituitary hormones, follicle-stimulating hormone and luteinizing hormone. As a result, the reduction in the stimulation of testosterone production in the testes occurs and it leads to hypogonadism. The aim of this review to point out to the occurrence of endocrine disorder caused by chronic use of opioids, as well as to emphasize the need for further research in this area in order to provide a better quality of life for patients with malignant diseases.

Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential immunoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

Differential Response to Abiraterone Acetate and Di-n-butyl Phthalate in an Androgen-Sensitive Human Fetal Testis Xenograft Bioassay

In utero exposure to antiandrogenic xenobiotics such as di-n-butyl phthalate (DBP) has been linked to congenital defects of the male reproductive tract, including cryptorchidism and hypospadias, as well as later life effects such as testicular cancer and decreased sperm counts. Experimental evidence indicates that DBP has in utero antiandrogenic effects in the rat. However, it is unclear whether DBP has similar effects on androgen biosynthesis in human fetal testis. To address this issue, we developed a xenograft bioassay with multiple androgen-sensitive physiological endpoints, similar to the rodent Hershberger assay. Adult male athymic nude mice were castrated, and human fetal testis was xenografted into the renal subcapsular space. Hosts were treated with human chorionic gonadotropin for 4 weeks to stimulate testosterone production. During weeks 3 and 4, hosts were exposed to DBP or abiraterone acetate, a CYP17A1 inhibitor. Although abiraterone acetate (14 d, 75 mg/kg/d po) dramatically reduced testosterone and the weights of androgen-sensitive host organs, DBP (14 d, 500 mg/kg/d po) had no effect on androgenic endpoints. DBP did produce a near-significant trend toward increased multinucleated germ cells in the xenografts. Gene expression analysis showed that abiraterone decreased expression of genes related to transcription and cell differentiation while increasing expression of genes involved in epigenetic control of gene expression. DBP induced expression of oxidative stress response genes and altered expression of actin cytoskeleton genes.

Permanent contact with females increases testosterone and improves fresh semen traits in pampas deer (Ozotoceros bezoarticus) males

Socio-sexual signals can stimulate neuroendocrinal control of reproduction. The aim of this work was to test the hypothesis that permanent and close contact with females stimulates testosterone production and enhances fresh seminal characteristics of pampas deer males. Five adult males (group CF) were established in a paddock in five breeding groups each comprising one stag and between five and ten hinds. Six adult males (group IS) were held in another paddock without any direct contact with females. Fecal testosterone concentrations were determined for each male, weekly, from October to May. Semen was collected by electro-ejaculation and evaluated before and after the addition of diluents [November: Fructose-Tris-Glycine with 20% egg yolk (FTG); April: Andromed]. Males in contact with females from mid-December to February exhibited significantly (P=0.04) greater concentrations of testosterone than those without. In November, CF males ejaculated semen of significantly (P=0.03) higher quality, with a significantly (P=0.02) greater proportion of progressive motile spermatozoa than IS males. After dilution with FTG, semen from the CF males had greater quality (P=0.04), a greater percentage of motile spermatozoa (P=0.04), spermatozoa with progressive motility (P=0.01), and spermatozoa with intact acrosomes (P=0.03). Semen from CF males also had lower fractions of spermatozoa with damaged acrosomes (P=0.04). In April, there were no significant differences in semen properties between the two groups. After the addition of extender, semen from CF males had a greater percentage of spermatozoa with damaged acrosomes (P=0.04). It is concluded that contact with females stimulates reproductive patterns of male pampas deer, resulting in better fresh semen characteristics before rutting and higher testosterone concentrations during the rut.

Paracetamol, Aspirin, and Indomethacin Induce Endocrine Disturbances in the Human Fetal Testis Capable of Interfering With Testicular Descent

Masculinization depends on the fetal testis. Exposure of the human fetus during pregnancy to paracetamol and/or to other mild analgesics is associated with an increased risk of cryptorchidism. We aimed to determine whether mild analgesics disrupted the morphology and endocrine function of the human testis. We used an in vitro system based on the culture of human fetal testes exposed or not to paracetamol, its metabolite N-(4-hydroxyphenyl)-arachidonoylethanolamide (AM404), aspirin, indomethacin, and ketoconazole at 10(-4) to 10(-7) M. The study was conducted at the University of Rennes I. Human fetal testes were from pregnant women after induced abortion, between 7 and 12 weeks of gestation (GW). Testosterone (RIA), anti-Müllerian hormone (ELISA), insulin-like factor 3 (RIA), and prostaglandin (PG) D2 and PGE2 (ELISA) were assayed in the medium. Testicular cells were counted using histology and image analysis. The possible nuclear receptor-mediated activities of the analgesics were investigated using reporter cell lines expressing estrogen, androgen, and peroxisome proliferator-activated γ receptors. Indomethacin and aspirin stimulated testosterone production, particularly by the younger testes (8-9 GW vs 10-12 GW). Paracetamol, AM404, and ketoconazole decreased insulin-like factor 3 levels. Aspirin stimulated whereas ketoconazole inhibited AMH production. PGE2 levels were inhibited by paracetamol and aspirin in the 7 to 12 GW testes and by indomethacin but only in 7 to 9.86 GW testes. The inhibitory trends seen for PGD2 were not statistically significant. Analgesics at concentrations relevant to human exposure cause endocrine disturbances in the fetal testis. We suggest that the fetal human testis displays slight critical age windows for sensitivity to direct exposure to aspirin, indomethacin, and paracetamol. The analgesic-induced inhibition of INSL3 may be the mechanism by which analgesics increase the risk of cryptorchidism. Greater caution is required concerning consumption of analgesics during pregnancy.

SET/PP2A system regulates androgen production in ovarian follicles in vitro

SET has multiple cell functions including nucleosome assembly, histone binding, transcription control, and cell apoptosis. In ovaries SET is predominantly expressed in theca cells and oocytes. In our study, SET overexpression in theca cells stimulated testosterone production whereas SET knockdown decreased testosterone production. Moreover, SET negatively regulated PP2A activity. Treatment with PP2A inhibitor okadaic acid (OA) led to increased testosterone synthesis, while treatment with PP2A activators resulted in the decreased testosterone synthesis. Furthermore, PP2A knockdown confirmed the key role of PP2A in the testosterone synthesis, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition of testosterone production. The central role of PP2A in SET-mediated regulation of testosterone production was confirmed by the finding that SET promoted the lyase activity of P450c17 and that PP2A inhibited its lyase activity. Taken together, these results reveal a specific, SET-initiated, PP2A-mediated, pathway that leads to the increased lyase activity of P450c17 and testosterone biosynthesis.

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Transient Rise of Serum Testosterone Level after Single Sildenafil Treatment of Adult Male Rats

Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short-term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil-induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co-immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time- and dose-dependent manner without effect on cAMP level. Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity.

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.

Inhibitory Effects of Digoxin on Testosterone Production in Rat Leydig Cells

Digoxin has beneficial effects on heart failure due to a direct positive inotropic action as an inhibitor of Na^+- K^+-ATPase and endogenous digoxin-like immunoreactive factors (DLIFs) found in mammalian blood also correlates to the same issue. We have demonstrated that digoxin inhibits rat testosterone secretion but the mechanisms remain unclear. In the present study, the direct effects of digoxin on the expression of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) in rat Leydig cells were evaluated. Digoxin inhibited not only testosterone production after incubation of Leydig cells with or without human chorionic gonadotropin (hCG), forskolin, 8-Br-cAMP, but also pregnenolone production following stimulation of 25-hydroxy-cholesterol, suggesting that digoxin reduced adenylyl cyclase and P450scc activity. Digoxin significantly reduced the expression of StAR protein, but not that of P450scc. The stimulation of testosterone production induced by A23187 was decreased by digoxin indicating that digoxin might influence Ca^2+ utilization. The analog of digoxin, ouabain, did not affect testosterone production in rat Leydig cells, which indicated the drug specificity. These results suggested that, in rat Leydig cells, digoxin directly inhibited testosterone production partly by reducing P450scc activity, StAR protein expression, and Ca^2+ utilization. This report not only demonstrates the mechanism digoxin acts, but also implies a probable interaction between gonadal and adrenal axis linked by DLIFs (i.e. digoxin).

Effects ofEurycoma longifoliaon Testosterone Level and Bone Structure in an Aged Orchidectomised Rat Model

Testosterone replacement is the choice of treatment in androgen-deficient osteoporosis. However, long-term use of testosterone is potentially carcinogenic. Eurycoma longifolia (EL) has been reported to enhance testosterone level and prevent bone calcium loss but there is a paucity of research regarding its effect on the bone structural parameters. This study was conducted to explore the bone structural changes following EL treatment in normal and androgen-deficient osteoporosis rat model. Thirty-six male Sprague-Dawley rats aged 12 months were divided into normal control, normal rat supplemented with EL, sham-operated, orchidectomised-control, orchidectomised with testosterone replacement, and orchidectomised with EL supplementation groups. Testosterone serum was measured both before and after the completion of the treatment. After 6 weeks of the treatment, the femora were processed for bone histomorphometry. Testosterone replacement was able to raise the testosterone level and restore the bone volume of orchidectomised rats. EL supplementation failed to emulate both these testosterone actions. The inability of EL to do so may be related to the absence of testes in the androgen deficient osteoporosis model for EL to stimulate testosterone production.