Inhibition Of Testosterone Secretion Research Articles

Effects of sugar cane extract on steroidogenesis in testicular interstitial cells of male Japanese quail (Coturnix japonica)

Sugar cane extract (SCE) is the end product of glucose, fructose, and sucrose elimination in molasses. SCE has various biological effects, such as anti-inflammation and antioxidation, and it is commonly found in animal feed. The present research is aimed at investigating the reproductive endocrine influence of SCE in male Japanese quails (Coturnix japonica) by feeding SCE containing food. In addition, in vitro Leydig cell culture was conducted to clarify the mechanism of SCE's influence. Our results showed that SCE feed extended the latency to the first neck grab, decreased male quail testis and epididymis weights, cloaca gland size, and reduced serum testosterone concentrations. Steroidogenic enzymes 3βHSD, 17βHSD, P450c17, and P450scc gene expression in the testis were decreased in the SCE groups. Western blot analysis showed decreased 3βHSD in the testis after feeding SCE. Isolated testicular interstitial cells cultured with SCE and ovine-LH suppressed testosterone secretion and 3βHSD gene expression. In conclusion, SCE as a feed additive has an impact on the sexual behavior and reproductive function of male Japanese quail, with the suppression of steroidogenesis in the Leydig cell. Our results may provide beneficial information to the livestock management and the poultry industry.

Leydig cell function in adult male rats is disrupted by perfluorotetradecanoic acid through increasing oxidative stress and apoptosis.

Perfluorotetradecanoic acid (PFTeDA) is a long-chain perfluoroalkyl compound with increased applications. Its effect on Leydig cell function and its underlying mechanism remain unclear. Male Sprague-Dawley rats (60 days old) were gavaged with PFTeDA at doses of 0, 1, 5, and 10 mg/kg/day from 60 to 87 days after birth. PFTeDA significantly reduced serum testosterone levels at 1 mg/kg and higher doses, while markedly increasing serum luteinizing hormone level at 10 mg/kg and follicle-stimulating hormone at ≥1 mg/kg. PFTeDA significantly reduced the sperm number at the cauda of epididymis at ≥1 mg/kg. PFTeDA also reduced the number of CYP11A1-positive Leydig cells due to increased apoptosis shown by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. PFTeDA significantly repressed the expression of Cyp17a1 and Star and their proteins at 1-10 mg/kg, while it increased the expression of Srd5a1 and its protein (an immature Leydig cell biomarker) at 10 mg/kg. PFTeDA markedly increased testicular malondialdehyde level, while inhibiting antioxidants (SOD1, SOD2, and CAT), triggering oxidative stress, thereby further inducing BAX and CASP3 while inhibiting BCL2, which led to cell apoptosis. PFTeDA also reduced DHH level secreted by Sertoli cells, which indirectly affected Leydig cell function. PFTeDA inhibited testosterone secretion in primary Leydig cells in vitro by increasing reactive oxygen species and inducing apoptosis at 50 μM. In conclusion, PFTeDA inhibits the function of Leydig cells by inducing oxidative stress and subsequently stimulating cell apoptosis.

Differential Endocrine and Metabolic Effects of Testosterone Suppressive Agents in Transgender Women

Suppression of testosterone secretion and/or action in transgender women using cyproterone acetate (CPA), spironolactone, or gonadotropin-releasing hormone analogues (GA) is achieved through various mechanisms. Our objective was to characterize possible differential effects of these compounds on metabolic and endocrine variables. We conducted a historic cohort study of transgender patients treated in a tertiary referral center. A longitudinal analysis of treatment naïve patients and a cross-sectional analysis of the whole cohort at the last visit was carried out. Among 126 transgender women (75 treatment-naïve), CPA was the predominant androgen suppressive therapy (70%), followed by spironolactone (17.6%), and GA (10.2%). Among those who were treatment-naïve, the increase in serum prolactin levels over baseline was greater at 3 months following CPA initiation (mean change 397 ± 335 mIU/L) than following spironolactone (20.1 ± 87 mIU/L) or GA initiation (64.6 ± 268 mIU/L; P = .0002). Prolactin levels remained higher in the CPA-treated group throughout follow-up, irrespective of estradiol levels, which were similar between the groups. A worse metabolic profile was associated with treatment with CPA than with spironolactone or GA. In the CPA compared to the spironolactone and GA groups, high-density lipoprotein-cholesterol levels were lower (47.1 ± 10.4, 54.4 ± 12.2, and 60.3 ± 13, respectively; P = .0076), while body mass index levels (24.3 ± 5, 21.7 ± 2.3, and 20.7±3.1 kg/m2; P = .03), and systolic (117 ± 12.1, 109 ± 12.2, and 105 ± 13.3mm Hg; P = .01) and diastolic (74 ± 9, 65.6 ± 5.5, and 65.4 ± 11 mm Hg; P = .0008) blood pressure levels were higher at the last visit. Treatment of transgender women with CPA was associated with hyperprolactinemia and a worse cardiovascular risk profile than treatment with spironolactone or GA. BMI = body mass index; CPA = cyproterone acetate; E2 = estradiol; FSH = follicle-stimulating hormone; GA = gonadotropin-releasing hormone analogues; LH = luteinizing hormone.

The endoplasmic reticulum stress and related signal pathway mediated the glyphosate-induced testosterone synthesis inhibition in TM3 cells

Glyphosate is the most widely used herbicide in the world. In recent years, many studies have demonstrated that exposure to glyphosate-based herbicides (GHBs) was related to the decrease of serum testosterone and the decline in semen quality. However, the molecular mechanism of glyphosate-induced testosterone synthesis disorders is still unclear. In the present study, the effects of glyphosate on testosterone secretion and the role of endoplasmic reticulum (ER) stress in the process were investigated in TM3 cells. The effects of glyphosate at different concentrations on the viability of TM3 cells were detected by CCK8 method. The effect of glyphosate exposure on testosterone secretion was determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of testosterone synthases and ER stress-related proteins were detected by Western blot and Immunofluorescence stain. Results showed that exposure to glyphosate at concentrations below 200 mg/L had no effect on cell viability, while the glyphosate above 0.5 mg/L could inhibit the testosterone secretion in TM3 cells. Treatment TM3 cells with glyphosate at 5 mg/L not only reduced the protein levels of testosterone synthase StAR and CYP17A1, inhibited testosterone secretion, but also increased the protein level of ER stress molecule Bip and promoted the phosphorylation of PERK and eIF2α. Pretreatment cells with PBA, an inhibitor of ER stress, alleviated glyphosate-induced increase in Bip, p-PERK and p-eIF2α protein levels, meanwhile rescuing glyphosate-induced testosterone synthesis disorders. When pretreatment with GSK2606414, a PERK inhibitor, the glyphosate-induced phosphorylation of PERK and eIF2α was blocked, and the glyphosate-inhibited testosterone synthesis and secretion was also restored. Overall, our findings suggest that glyphosate can interfere with the expression of StAR and CYP17A1 and inhibit testosterone synthesis and secretion via ER stress-mediated the activation of PERK/eIF2α signaling pathway in Leydig cells.

PEGylated leuprolide with improved pharmacokinetic properties

Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, suffers from a short circulating half-life like other peptide therapeutics. As an attempt to improve its pharmacokinetic properties, two PEGylated leuprolides with different molecular weight were synthesized utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. The reaction conditions, including reaction temperature, reaction time and feed ratio of the reactants, were optimized to obtain a higher yield. Reverse-phase high performance liquid chromatography (RP-HPLC) characterization indicates a high purity of the resulting conjugates. Matrix-assisted laser desorption mass spectrometry (MALDI-MS) characterization suggests a 1:1 PEGylation. 1H NMR study reveals that the reaction occurs on the imidazolyl group on the histidine residue and the conjugates are stable in pH7.4 aqueous solutions. The in vitro bioactivity of the conjugates was evaluated using both hormone-sensitive and hormone-insensitive cell lines. It was found that the PEGylated peptides can still counteract the stimulatory action of androgens and the mitogenic action of epidermal growth factor on cell proliferation. The in vivo bioactivity of the conjugates was also tested. Like the unmodified peptide, administration of the conjugates to male rats leads to an initial testosterone surge, followed by a suppression of testosterone secretion. Pharmacokinetics of the drugs after i.v. and s.c. administrations were determined. In both cases, a prolonged circulating half-life, an increased AUC, and a decreased Cl_F were observed for the PEGylated drugs.

ERO1α Knockdown Attenuates Palmitic Acid-Inhibited Testosterone Secretion by Endoplasmic Reticulum Stress in Testicular Leydig Cells

Background and objectivePalmitic acid (PA), the most common saturated free fatty acid (FFA) in food, is related to obesity-related male infertility. The possible mechanism is PA-mediated inhibition of testosterone secretion. Endoplasmic reticulum (ER) oxidoreductin-1alpha (ERO1α), an oxidase that is localized in the ER, plays an essential role in maintaining ER homeostasis and is related to hormone secretion. However, the role and underlying mechanisms of ERO1α in PA-mediated inhibition of testosterone secretion have not been reported. Material and methodsMurine Leydig tumor cell line 1 (MLTC-1) cells were treated with different doses of PA. Cell viability, testosterone secretion, and ERO1α expression were measured by the Cell Counting Kit 8 (CCK-8) assay, enzyme-linked immunesorbent assay (ELISA), and Western blotting, respectively. Moreover, the expres-sion of ER stress marker proteins (glucose-regulated protein 78 [GRP78] and CCAAT/enhancer-binding protein homologous protein [CHOP]) was also measured after treatment. Subsequently, the expression ofERO1α was knocked down, and cell viability, testosterone secretion, and ER stress were measured after treatment with the PA or the ER stress agonist thapsigargin (TG, an ER stress inducer). Also, testosterone secretion was measured by ELISA when ER stress was inhibited by 4-phenylbutyric acid (4-PBA, an ER stress inhibitor). ResultsPA treatment reduced cell viability, induced ERO1α expression, and enhanced the expression of the ER stress marker GRP78 and CHOP, while ERO1α knockdown inhibits ER stress marker expression, promotes testosterone secretion, and enhances cell viability in PA-treated MLTC-1 cells. In addition, ERO1α knock-down rescued the TG-induced the decrease in testosterone secretion and cell viability. ConclusionsThese findings suggest that PA inhibits testosterone secretion via ER stress and that ERO1α knockdown ameliorates PA-induced decreases in testosterone via ER stress in testicular Leydig cells. Our results indi-cate the necessity of exploring the potential applications of ERO1α as a target gene for restoring fertility in obese men.

SB223412, a neurokinin-3 receptor-selective antagonist, suppresses testosterone secretion in male guinea pigs

Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species.

RFamide-related peptide 3 and gonadotropin-releasing hormone-II are autocrine-paracrine regulators of testicular function in the boar.

Widespread use of artificial insemination in swine requires millions of doses of boar semen each year. Subfertility of boars remains a major constraint, which can impact the reproductive efficiency of thousands of sows, so a better understanding of testicular function is needed in order to develop methods to improve semen production. With this in mind, the effects of RFamide-related peptide 3 (RFRP3) and Gonadotropin-releasing hormone-II (GnRH-II) on gonadotropin secretion and testicular function of pigs are reviewed here. Receptors for RFRP3 are present in the pig hypothalamus, adenohypophysis, and testis. Evidence from in vitro studies indicates that RFRP3 could be a hypophysiotropic hormone in the pig by suppressing secretion of GnRH-I from the hypothalamus and luteinizing hormone (LH) from the pituitary gland; however, effects of RFRP3 on in vivo secretion of LH in pigs are minimal. Within the pig testis, RFRP3 suppresses testosterone secretion by inhibiting steroidogenic enzymes. GnRH-II and its receptor (GnRHR-II) are abundant in pig testes. Interstitial cells express GnRHR-II, and exogenous GnRH-II robustly stimulates secretion of testosterone in boars, despite minimal secretion of LH. Data illustrate that GnRH-II directly stimulates secretion of testosterone from the testes of mature boars. Thus, the primary function of RFRP3 and GnRH-II in the boar appears to be autocrine-paracrine inhibition and stimulation, respectively, of testosterone secretion within the testis. A better understanding of changes in the RFRP3 and GnRH-II systems within the testis during development will provide important clues about how to improve the testicular function of boars.

A high-fat diet impairs reproduction by decreasing the IL1\u03b2 level in mice treated at immature stage

Obesity causes low-grade inflammation that is involved in male infertility. Interleukin 1 beta (IL1β) plays an important role in this process. A high-fat diet (HFD) is the most common cause of obesity. However, the effect of a HFD on IL1β and its consequence in reproduction remain unclear. We established a HFD model in mice treated at immature stage (mice-TIS) and mice treated at mature stage (mice-TMS). Surprisingly, we found that a HFD decreased IL1β levels and was accompanied by an increase in testosterone in mice-TIS, while the reverse results were observed in mice-TMS. In addition, a HFD caused a reduction in testis macrophages and in the expression of inflammasome-related genes and proteins in mice-TIS. Furthermore, we found that IL1β inhibited testosterone secretion through down-regulating the gene expression of P450SCC and P450c17. However, the influence on mice-TIS that were induced by a HFD was recovered by stopping the HFD. In this study, we are the first to report that a HFD impairs the reproductive system by decreasing IL1β and enhancing testosterone levels in mice-TIS, which are different from the effects in mice-TMS. This provides new ideas for the treatment of obesity-induced infertility.

Advanced glycation end products inhibit testosterone secretion by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products(AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs(25, 50, 100 and200µg/ml). Testosterone production induced by human chorionic gonadotropin(hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein(StAR), cholesterol side-chain cleavage enzyme(P450scc) and 3β-hydroxysteroid dehydrogenase(3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitativePCR and western blot analyssi, respectively. Reactive oxygen species(ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate(DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins[C/EBP homologous protein(CHOP)andglucose-regulated protein78(GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control(P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control(P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine(NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid(TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Male gonadal axis function in patients with type 2 diabetes.

Patients with type 2 diabetes have lower serum testosterone levels and a higher prevalence of hypogonadism than non-diabetic patients, independently of the metabolic control of disease. The mechanisms underlying a decrease in testosterone might be related to age, obesity and insulin resistance, often present in patients with type 2 diabetes. The increase in estrogens due to higher aromatase enzyme activity in increased adipose tissue might exert negative-feedback inhibition centrally. Insulin stimulates gonadal axis activity at all three levels and therefore insulin resistance might account for the lower testosterone production. Leptin exerts a central stimulatory effect but inhibits testicular testosterone secretion. Thus, resistance to leptin in obese subjects with type 2 diabetes determines lower central effects of leptin with lower gonadotropin-releasing hormone (GnRH) secretion and, on the other hand, hyperleptinemia secondary to leptin resistance inhibits testosterone secretion at the testicular level. However, lower testosterone levels in patients with diabetes are observed independently of age, weight and body mass index, which leads to the assumption that hyperglycemia per se might play a role in the decrease in testosterone. Several studies have shown that an overload of glucose results in decreased serum testosterone levels. The aim of this review is to assess changes in the male gonadal axis that occur in patients with type 2 diabetes.

Immunisation against gonadotrophin-releasing hormone (GnRH) reduces agonistic behaviours in male rangeland goats

Rangeland goat bucks were used to evaluate the efficacy of a commercially available anti-gonadotrophin-releasing hormone vaccine, Improvac (Zoetis Australia, West Ryde, NSW, Australia). The hypothesis tested was that immunisation would suppress testosterone secretion by the testis and agonistic behaviour between male goats. We also compared intervals of 2 and 4 weeks between primary and booster immunisations and monitored responses over a 2-month period. The 45 goats were split into three groups (n = 15): one group receiving the vaccination booster on Day 14, one group receiving the vaccination booster on Day 28, and the Control group receiving sterile saline injections. Body mass, body condition score and scrotal circumference were measured fortnightly, and blood was collected at 2-week intervals and analysed for testosterone concentration. Behavioural interaction tests of 2-min duration were also conducted fortnightly. There was a significant decrease in paired testicular circumference (P &lt; 0.05) and testosterone concentration (P &lt; 0.01) in both vaccination groups by the end of the experiment at Day 60, compared with the Control group. Agonistic interactions measured at Day 60 were significantly reduced in both vaccination groups (P &lt; 0.05) compared with the Control group. These results support the efficacy of Improvac in reducing agonistic behaviours in rangeland goat bucks and suggest that the use of the vaccine may assist in reducing social stress and possible injury in groups of confined male goats.

Intratesticular hypertonic sodium chloride solution treatment as a method of chemical castration in cattle

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control—surgically castrated immediately after birth; positive control —intact males; G1—ITI from 1- to 5-day old; G2—ITI from 15- to 20-day old; and G3—ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P < 0.05), in which no testicular structure and sperm cells were observed during breeding soundness evaluation. Rectal and scrotal temperatures were not affected by different procedures. In conclusion, ITI of hypertonic NaCl solution induces sterility and completely suppresses testosterone secretion when performed during the first 20 days of life.

Hyperleptinemia directly affects testicular maturation at different sexual stages in mice, and suppressor of cytokine signaling 3 is involved in this process

BackgroundLeptin plays an important role in reproductive function, and the mechanism of this phenomenon primarily focuses on the hypothalamic–pituitary–gonadal axis. However, until now, the direct effects of leptin on the testes during development from infancy to adulthood remained unclear. The aim of the present study was to explore the effects and molecular mechanisms that underlie leptin’s action in the testes during sexual maturation.MethodsWe used a monosodium glutamate (MSG)-treated mouse model to assess the effects of endogenous hyperleptinemia on the development of the testes from infancy to adulthood. Then, a variety of reproductive parameters were measured, including the concentration of testosterone, the weight and volume of the testicles, the diameter of the seminiferous tubules, and numbers of spermatogonia, spermatocytes, sperm, Leydig cells and offspring. In addition, we assessed the direct role of leptin and suppressor of cytokine signalling 3 (SOCS3)/phosphorylated signal transducer and activator of transcription 3 (pSTAT3) on the testes in vitro.ResultsTestosterone secretion exhibited a diverse response: a low concentration of leptin induced testosterone secretion, and a high concentration inhibited testosterone secretion both in vivo and in vitro. A variety of reproductive parameters decreased in hyperleptinemic mice, including the weight and volume of the testicles, the diameter of the seminiferous tubules, and the numbers of spermatocytes, sperm, Leydig cells and offspring. The amount of spermatogonia was also elevated. The development of the testes was partially recovered after hyperleptinemia withdrawal. A high concentration of leptin induced SOCS3 expression and inhibited pSTAT3 expression in the testes.ConclusionsThe results indicated that MSG-induced hyperleptinemia directly affects testicular structure and function and that SOCS3/pSTAT3 played an important role in this process. These results also indicated the importance of monitoring and controlling leptin levels in obese male children. SOCS3 is a potential therapeutic target for leptin-induced dysgenesis.

Central and Direct Regulation of Testicular Activity by Gonadotropin-Inhibitory Hormone and Its Receptor

Gonadotropin-inhibitory hormone (GnIH) was first identified in Japanese quail to be an inhibitor of gonadotropin synthesis and release. GnIH peptides have since been identified in all vertebrates, and all share an LPXRFamide (X = L or Q) motif at their C-termini. The receptor for GnIH is the G protein-coupled receptor 147 (GPR147), which inhibits cAMP signaling. Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in most mammals. GnIH neurons in the PVN or DMH project to the median eminence to control anterior pituitary function via GPR147 expressed in gonadotropes. Further, GnIH inhibits gonadotropin-releasing hormone (GnRH)-induced gonadotropin subunit gene transcription by inhibiting the adenylate cyclase/cAMP/PKA-dependent ERK pathway in an immortalized mouse gonadotrope cell line (LβT2 cells). GnIH neurons also project to GnRH neurons that express GPR147 in the preoptic area (POA) in birds and mammals. Accordingly, GnIH can inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as by directly inhibiting pituitary gonadotrope activity. GnIH and GPR147 can thus centrally suppress testosterone secretion and spermatogenesis by acting in the hypothalamic–pituitary–gonadal axis. GnIH and GPR147 are also expressed in the testis of birds and mammals, possibly acting in an autocrine/paracrine manner to suppress testosterone secretion and spermatogenesis. GnIH expression is also regulated by melatonin, stress, and social environment in birds and mammals. Accordingly, the GnIH–GPR147 system may play a role in transducing physical and social environmental information to regulate optimal testicular activity in birds and mammals. This review discusses central and direct inhibitory effects of GnIH and GPR147 on testosterone secretion and spermatogenesis in birds and mammals.

Stress-Induced Changes in Testosterone Secretion in Male Rats: Role of Oxidative Stress and Modulation by Antioxidants

Seventy adult male albino rats were randomly allotted into 3 main groups: control group (n = 10), acute stress-exposed group (n = 30) and chronic stress-exposed group (n = 30). Each of the stressed groups was subdivided into 3 equal subgroups (n = 10/subgroup, SG): subgroup 1 animals were exposed to immobilization stress, SG2 animals, were given immobilization stress and supplemented with α-tocopherol (vitamin E), SG3 animals were exposed to immobilization stress and supplemented with ascorbic acid (vitamin C). Immobilization stress exposure was applied once for 6 continuous hours in the acute stressed group and was 6 hours daily for 10 consecutive days in the chronic stressed group. In all vitamin supplemented groups, both vitamin E and C were administered orally mixed with the diet in a similar dose of 500 mg/kg diet. This supplementation started 6 weeks prior to the stress exposure and continued throughout the experimental period. At the end of the last immobilization session, sera were harvested from all animals thereafter, animals were sacrificed and the testes were immediately excised and processed for further biochemical investigations. Serum testosterone and luteinizing hormone levels were measured and the activities of antioxidant enzymes [catalase (CAT) & glutathione-s-transferase (GST)] as well as malondialdehyde (MDA) concentrations were determined in sera and testes. Compared to control, the results revealed that acute and chronic immobilization stress caused significant decrease in levels of serum testosterone and luteinizing hormone (LH). Also, significant reductions (P < 0.01) were found in the activities of CAT and GST in sera and testes. Contrariwise, there existed a significant (P < 0.05) increase in MDA concentrations in serum and testis. Co-administration of vitamin E or C relatively restored (P < 0.01) the above parameters. Thus, this study draws a conclusion that immobilization stress of male rats significantly inhibited testosterone secretion and induced oxidative stress which partially mediated this inhibition. It also proved a protective role of vitamin E and C against the oxidative stress-induced down-regulation of testosterone secretion with a better efficacy of vitamin E.

Aflatoxin B1 disrupts the androgen biosynthetic pathway in rat Leydig cells

The present study investigated if Aflatoxin B1 (AFB1), a potent and naturally occurring mycotoxin, interferes with the steroidogenic pathway in rat Leydig cells. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone (T) that maintains the male phenotype and support fertility. Leydig cells, isolated from 35-day-old male Long-Evans rats (Rattus norvegicus), were incubated with AFB1 at 0, 0.01, 0.1, 1, 10μM followed by measurement of T secretion by radioimmunoassay and analysis of protein expression in western blots. Results demonstrated that AFB1 suppressed testosterone secretion in a dose-dependent manner and inhibited expression of cholesterol transporter steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes [(3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase enzyme (HSD17B3)]. Protein expression analysis showed that AFB1 treatment increased ERK phosphorylation but suppressed p38 MAPK and JNK activation in Leydig cells. AFB1-induced inhibition of Leydig cells was alleviated by co-treatment with the ERK inhibitor UO 126, implying that ERK mediates, at least in part, the inhibitory effects of AFB1 in Leydig cells. The findings highlight potential extra-hepatic effects of aflatoxin exposure and indicate that exposure to AFB1 has significant reproductive health implications for consumers of contaminated products even under conditions of low dietary toxin levels.

Melatonin and diet-induced metabolic syndrome in rats: impact on the hypophysial-testicular axis.

Abstract Combinations of fructose- and fat-rich diets in experimental animals can model the human metabolic syndrome (MS). In rats, the increase in blood pressure (BP) after diet manipulation is sex related and highly dependent on testosterone secretion. However, the extent of the impact of diet on rodent hypophysial-testicular axis remains undefined. In the present study, rats drinking a 10% fructose solution or fed a high-fat (35%) diet for 10 weeks had higher plasma levels of luteinizing hormone (LH) and lower plasma levels of testosterone, without significant changes in circulating follicle-stimulating hormone or the weight of most reproductive organs. Diet manipulation brought about a significant increase in body weight, systolic BP, area under the curve (AUC) of glycemia after an intraperitoneal glucose tolerance test (IPGTT), and plasma low-density lipoprotein cholesterol, cholesterol, triglycerides, and uric acid levels. The concomitant administration of melatonin (25 μg/mL of drinking water) normalized the abnormally high LH levels but did not affect the inhibited testosterone secretion found in fructose- or high-fat-fed rats. Rather, melatonin per se inhibited testosterone secretion. Melatonin significantly blunted the body weight and systolic BP increase, the increase in the AUC of glycemia after an IPGTT, and the changes in circulating lipid profile and uric acid found in both MS models. The results are compatible with a primary inhibition of testicular function in diet-induced MS in rats and with the partial effectiveness of melatonin to counteract the metabolic but not the testicular sequelae of rodent MS.

Effects of capsaicin on testis ghrelin expression in mice

Capsaicin (CAP), the active substance of red hot peppers, has been reported to stimulate development of the gonad. Ghrelin is an acylated polypeptide hormone that is secreted predominantly by endocrine cells of the stomach. There is evidence that ghrelin is involved in reproductive function. Ghrelin significantly inhibits testosterone secretion in a dose-dependent manner. We investigated the effect of CAP on ghrelin expression in testes of mice and on testosterone levels during pubertal and adult periods. We used a variety of morphometric, immunohistochemical and biochemical methods, and western blot analysis. The animals were divided into two age groups: puberty and adult. Control groups for both age groups were fed with standard diet and experimental groups were fed with a diet containing 0.02% CAP. Testes were collected quickly after sacrifice. After dehydration, the specimens were embedded in paraffin and 5 μm sections were cut, and Crossman's triple staining and immunohistochemical staining for ghrelin were applied. Immunohistochemical staining with ghrelin antibody for both age groups demonstrated immunoreaction especially in Leydig and Sertoli cells, but no reaction was observed in spermatogenic cells. Ghrelin immunoreaction was less intense in the experimental groups. Serum testosterone levels were increased in both experimental groups, especially in adults. More spermatocytes were observed in the experimental group compared to the control group. In both pubertal and adult experimental groups, the seminiferous epithelium was thick. CAP appears to enhance testicular cell proliferation and can affect the release of ghrelin and testosterone directly or indirectly.

Mechanism of phytoestrogen action in Leydig cells of ganders (Anser anser domesticus): Interaction with estrogen receptors and steroidogenic enzymes

Phytoestrogens (PE) are plant-derived compounds that have an estrogen-like activity and they can influence male and female reproduction. The possible mechanisms of PE action may be including: the binding to estrogen receptors (ER) and the interaction with the key steroidogenic enzymes. The aim of this study was to investigate if PE has effect on steroidogenesis of gander testicular cells by above-described pathways. The Leydig cells were isolated from testes of White Kołuda® ganders at the peak of their reproductive activity (March). These Leydig cells (1×105per mL) were pre-incubated with the ER inhibitor – ICI 182, 780 (100 nM) for 3 h and then these cells were incubated with PE (5 and 50 μM): genistein, daidzein, equol and coumestrol during next 20 h or untreated control and the Leydig cells that were previously treated (20 h) with genistein (5 and 50 μM) were incubated for next 6 h with steroid intermediates (20 μM) as testosterone (T) precursors: hydroxycholesterol, pregnenolone, progesterone and androstenedione. Concentrations of T in the samples of incubation medium were measured using radioimmunoassay. Genistein, daidzein, and equol (5 and 50 μM) decreased (P < 0.05) T secretion by incubated gander Leydig cells and ICI 182, 780 did not eliminate the inhibitory effect of these PE. After genistein (50 μM) treatment, basal and stimulated with 22R-hydroxycholesterol, pregnenolone, progesterone and androstenedione, T production by testicular cells was decreased (P < 0.05). In contrast, genistein at lower dose (5 μM) did not affect the stimulatory effects of testosterone precursors. In conclusion, the inhibition of testosterone secretion by the phytoestrogens in gander Leydig cells did not depend on estrogen receptors. The suppression of steroidogenesis in these cells may be in part conducted by interaction of phytoestrogens with key steroidogenic enzymes. However, further studies are required to elucidate the phytoestrogen mechanism of action in gander testicular cells.