Hydroxysteroid Dehydrogenase Activity Research Articles

Involvement of porcine and human carbonyl reductases in the metabolism of epiandrosterone, 11-oxygenated steroids, neurosteroids, and corticosteroids

Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C18/C19-steroids,11-keto- and 11β-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) Km values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/β-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3β-HSD and 3α-HSD, respectively). Additionally, 17β-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/β-dihydro-C21-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5β-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/β-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/β-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower Km values (0.3–2.9 μM) for the 3-keto-C21-steroids than pCBR-N1 (Km=10–36 μM). The reduced products of the 3-keto-C21-steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C18/C19/C21-steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/β,17β-HSDs.

A Perspective on Male Contraceptive Therapeutics of Hydro-ethanol Extract of Seeds of <i>Luffa acutangula (L.) Roxb.</i> in Human and Albino Rat: An <i>In-vitro</i> Meta-Analysis Study

Traditionally Luffa acutangula (L.) Roxb. has been utilized as an herbal contraceptive. This in-vitro study has been performed to search out the male contraceptive efficacy of hydro-ethanol (60:40) extract of Luffa acutangula (LAHEE) seeds in a dose-specific manner (1, 2, and 4 mg/ml of in-vitro media). The percentages of motile, viable, hypo-osmotic swelled (HOS), and acrosomal intact of human and rat sperms were declined significantly (p<0.05) at the above-mentioned doses of LAHEE-exposed groups against the placebo group. The inhibitory concentration 50% value (IC50) of LAHEE was 2.5 mg/ml in human and 1.2 mg/ml in rat spermatozoa immobilization. The ∆5,3β and 17β hydroxysteroid dehydrogenase (HSD) activities of rat’s testicular tissues were inhibited significantly at p<0.05 in LAHEE-treated groups than the placebo group. Activities of superoxide dismutase (SOD) and catalase were significantly inhibited (p<0.05) along with the significant increment (p<0.05) in the quantity of thiobarbituric acid reactive substances (TBARS) in rat’s testes and epididymis, sperm pellets of humans and rats in LAHEE-treated groups against the placebo group without any significant difference (p>0.05) in above said sensors in the liver and cardiac tissues of rats. The non-toxic nature of LAHEE was indicated by no significant alterations (p>0.05) in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after LAHEE exposure in said tissues of rats. The LC-MS study of LAHEE identified the presence of oleic acid, dihydroquercetin, luteolin-7-glucoside, formononetin, luteolin 8-C-pentoside-6-C-hexoside, pterosin B, boldine and berberine. These findings interpreted that the LAHEE possesses spermiological inhibitory and hypo-testicular activities, which indicate a promising possibility to develop a potent herbal male contraceptive agent from this plant extract.

Engineering the cofactor binding site of 7α-hydroxysteroid dehydrogenase for improvement of catalytic activity, thermostability, and alteration of substrate preference

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Punica granatum (Pomegranate) Peel Extract Pre-Treatment Alleviates Fenpropathrin-Induced Testicular Injury via Suppression of Oxidative Stress and Inflammation in Adult Male Rats.

Fenpropathrin (FNP) is one of the commonly used insecticides in agriculture and domestically, leading to environmental and health problems. The goal of the current investigation was to determine how well pomegranate peel extract (PGPE) could prevent the testicular toxicity and oxidative stress induced by FNP. Four groups of male Wistar rats were randomly assigned: negative control (corn oil), PGPE (500 mg/kg BW), positive control (FNP; 15 mg/kg BW, 1/15 LD50), and PGPE + FNP. For four weeks, the rats received their doses daily and orally via gavage. The major phytochemical components (total phenolic, flavonoids, and tannins contents) detected in PGPE by GC-MS included ellagic acid, hydroxymethylfurfurole, guanosine, and pyrogallol with high total phenolic, flavonoids, and tannin contents. FNP-treated rats showed a marked elevation in testicular levels of thiobarbituric acid-reactive substances, hydrogen peroxide, and protein carbonyl content, as well as the activity of aminotransferases and phosphatases. Meanwhile. a significant decline in body weight, gonadosomatic index, glutathione, protein contents, enzymatic antioxidants, and hydroxysteroid dehydrogenase (3β HSD, and 17β HSD) activity was observed. In addition, significant alterations in testicular P53, Cas-3, Bcl-2, IL-β, IL-10, testosterone, follicle-stimulating and luteinizing hormones, and sperm quality were detected. Furthermore, biochemical and molecular changes were corroborated testicular histological abnormalities. Moreover, PGPE-pretreated FNP-intoxicated rats demonstrated considerable improvement in the majority of the studied parameters, when compared to FNP-treated groups. Conclusively, PGPE provided a potent protective effect against the testicular toxicity caused by FNP, due to its antioxidant-active components.

Isolation of Environmental Bacteria Able to Degrade Sterols and/or Bile Acids: Determination of Cholesterol Oxidase and Several Hydroxysteroid Dehydrogenase Activities in Rhodococcus, Gordonia, and Pseudomonas putida.

Interest about the isolation and characterization of steroid-catabolizing bacteria has increased over time due to the massive release of these recalcitrant compounds and their deleterious effects or their biotransformation derivatives as endocrine disruptors for wildlife, as well as their potential use in biotechnological approaches for the synthesis of pharmacological compounds. Thus, in this chapter, an isolation protocol to select environmental bacteria able to degrade sterols, bile acids, and androgens is shown. Moreover, procedures for the determination of cholesterol oxidase or different hydroxysteroid dehydrogenase activities in Pseudomonas putida DOC21, Rhodococcus sp. HE24.12, Gordonia sp. HE24.4J and Gordonia sp. HE24.3 are also detailed.

A comparative assessment of artificial and natural energy drinks in the epididymal and testicular milieu

"Artificial and natural energy drinks are both taken for increased energy, physical stamina, and alertness, although they differ in composition. This study investigated the effects of artificial and natural energy drinks on the testicular milieu in male pubertal rats. Eighteen Wistar rats were randomly divided into 3 groups of 6 rats each and all animals had access to food ad libitum. Group 1: (control) received water only; Group 2: (artificial energy drink- AED) received AED; Group 3: (natural energy drink- NED) received NED. A dose of 1.41ml/day/150g animal was administered and this lasted for 28 days. Sperm and testicular variables, biochemical parameters, and hormonal assays were carried out. There were significant decreases in the levels of testosterone, Lactate dehydrogenase, glucose, 3β-Hydroxysteroid dehydrogenase, and 17β- Hydroxysteroid dehydrogenase activities in AED and NED groups when compared to the control group. There was a marked increment in sperm abnormalities in the NED group when compared to AED and control groups. Also, the intake of AED led to an elevated level of Glucose-6-phosphate dehydrogenase compared to the control while a significant reduction was observed in the NED group when compared to the AED group. Artificial and natural energy drinks although consumed for strength and vigor distorted epididymis and testicular integrity via alteration of the testicular metabolism, lowering sperm quality and androgenic hormones in pubertal male Wistar rats. Keywords: energy drink, jaggery, sperm quality, steroidogenic enzymes, lactate dehydrogenase."

Depleting NAD+ pools specifically in the endoplasmic reticulum lumen impairs 11[beta]-hydroxysteroid dehydrogenase activity

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Ovarian and uterine functions in female albino rats fed dietary meal supplemented with Mucuna pruriens (L.) DC. seed powder

Background: While the reproduction-enhancing property of Mucuna pruriens (MP) seed has been widely studied in males, little is known about this property in females despite the rate at which the seed is consumed by both sexes worldwide.
 Objective: To determine the effect of MP seed powder in dietary inclusion on ovarian and uterine functions of adult female albino rats.
 Methods: The rats were randomised into four groups. Group 1 (Control) was given standard rat chow (15g of feed/rat/day only) while groups 2, 3 and 4 were fed diets supplemented with MP seed powder at 0.75 g, 1.5g and 2.25g/day, respectively, for 12 weeks. Serum levels of oestradiol, follicle stimulating hormone, luteinising hormone, ovarian Δ5, 3β- hydroxysteroid dehydrogenase (Δ5, 3β-HSD) and 17 β hydroxysteroid dehydrogenase (17β-HSD) activities, ovarian and uterine peroxidase and tissue cytoarchitectural structures were used as diagnostic markers of reproductive function.
 Results: Significant increases in the serum level of all hormones including ovarian Δ5, 3β-HSD, 17β-HSD activities, ovarian and uterine peroxidase activities, and improvement of the ovarian and uterine cytoarchitectural integrity of the rats fed MP at 0.75g/day compared to other groups were observed. However, MP at 2.25g/day induced reproductive dysfunction characterised by significant reductions in hormones, uterine and ovarian enzyme activities, severe degenerative cytoarchitectural lesions in tissues.
 Conclusions: MP seed improves uterine and ovarian functions at a dose level of 0.75g/day, but a higher dose value may be toxic.

Folic acid protects against fluoride-induced oxidative stress and testicular damage in rats

Objective: To investigate the effects of folic acid on testicular oxidative damage in sodium fluoride-induced male Wistar rats. Methods: A total of 24 male Wistar rats were divided into 4 groups: the control, sodium fluoride (fed with 100 mg/L sodium fluoride through drinking water orally for 21 days), folic acid (36 μg/kg body weight/day, orally), and sodium fluoride plus folic acid (received similar dose orally) groups. At the end of 21 days, epididymal sperm parameters, biochemical analysis of testicular tissue, and serum hormonal levels were performed along with histopathological studies. Results: Sodium fluoride intoxication resulted in marked reduction in gonado somatic index, serum luteinizing hormone, and testosterone level along with 3 β -hydroxysteroid dehydrogenase and 17 β -hydroxysteroid dehydrogenase activities. In addition, reduction in sperm density, as well as loss of sperm motility and sperm viability, were also observed. Besides, increased levels of testicular malondialdehyde, nitrite, interleukin-6 and tumor necrosis factor- α as well as decreased levels of superoxide dismutase and catalase activities and reduced glutathione content were found to be associated with this toxicity. Folic acid co-treatment, on the other hand, could prevent all the sodium fluoride-induced testicular pathophysiology and oxidative stress related parameters. Histological examinations of testicular sections from the experimental rats supported these results. Conclusions: Combining all, this study suggests that being an antioxidant, folic acid plays a beneficial role against fluoride-induced adverse effects on the male reproductive system.

Role of sox30 in regulating testicular steroidogenesis of common carp

Expression of transcription factors is crucial for the regulation of steroidogenesis and gonadal development in fish. SRY-related box (SOX) proteins regulate gene expression of various events related to vertebrate reproduction. This study reports the role of sox30 and its influence on sox9a/b in regulating testicular steroidogenesis of the common carp, Cyprinus carpio. Tissue distribution showed predominant expression of sox30 in gonads, while gonadal ontogeny indicated significant dimorphic expression of sox30 from 120 days post hatch. Higher sox30 transcripts during the spawning season, an elevation of sox30 after human chorionic gonadotropin induction, and 11-ketotestosterone (11-KT) treatment authenticate gonadotropin dependency. Treatment of 17α–methyl-di-hydroxy-testosterone to juvenile common carp for mono-sex induction, vis-à-vis elevated sox30 expression. Sox30 protein was detected abundantly in spermatocytes and spermatid/sperm of carp testis. Transient silencing of sox30 using small interfering RNAs decreased sox9a/b expression, lead to downregulation of certain molecule/factor, transcription factor, germ/stem cell marker, and steroidogenesis-related enzyme genes. Serum testosterone and 11-KT decreased significantly upon transient silencing of sox30, in vivo. Concomitantly, a reduction in testicular microsomal 11-β hydroxysteroid dehydrogenase activity was observed. These results demonstrate the influence of sox30 as well as sox9a/b in the regulation of testicular steroidogenesis in common carp.

Endogenous urinary glucocorticoid metabolites and mortality in prednisolone-treated renal transplant recipients.

BackgroundChronic corticosteroid treatment suppresses HPA‐axis activity and might alter activity of 11β hydroxysteroid dehydrogenases (11β‐HSD). We aimed to investigate whether the endogenous glucocorticoid production and 11β‐HSD activities are altered in prednisolone‐treated renal transplant recipients (RTR) compared with healthy controls and whether this has implications for long‐term survival in RTR.MethodsIn a longitudinal cohort of 693 stable RTR and 275 healthy controls, 24‐hour urinary cortisol, cortisone, tetrahydrocorisol (THF), allotetrahydrocortisol (alloTHF), and tetrahydrocortisone (THE) were measured using liquid chromatography tandem‐mass spectrometry. Twenty‐four‐hour urinary excretion of cortisol and metabolites were used as measures of endogenous glucocorticoid production; (THF + alloTHF)/THE and cortisol/cortisone ratios were used as measures of 11β‐HSD activity.ResultsUrinary cortisol and metabolite excretion were significantly lower in RTR compared with healthy controls (P < .001), whereas (THF + alloTHF)/THE and cortisol/cortisone ratios were significantly higher (P < .001 and P = .002). Lower total urinary metabolite excretion and higher urinary (THF + alloTHF)/THE ratios were associated with increased risk of mortality, independent of age, sex, estimated glomerular filtration rate, C‐reactive protein, body surface area, and daily prednisolone dose, respectively.ConclusionsEndogenous glucocorticoid production and 11β‐HSD activities are altered in prednisolone‐treated RTR. Decreased total urinary endogenous glucocorticoid metabolite excretion and increased urinary (THF + alloTHF)/THE ratios are associated with increased risk of mortality.

Alterations of estrous cycle, 3\u03b2 hydroxysteroid dehydrogenase activity and progesterone synthesis in female rats after exposure to hypobaric hypoxia

The underlying mechanism regulating hypoxia induced alteration in female steroid hormones is first time explored in this study. To understand the mechanistic approach, female Sprague- Dawley rats were exposed to acute and chronic hypobaric hypoxia (282 mm-Hg, ~7620 m, 6 hours, 3 and 7 days). Estrous cycle, body weight, plasma progesterone and estradiol levels, morphology, histology and two key steroidogenic enzymes: 3ß hydroxysteroid dehydrogenase (HSD) and 17ß HSD activity of ovary and adrenal gland were studied. A persistent diestrous phase and a significant decrease in body weight were found in chronic hypoxia groups. Histological study suggested degenerative changes in ovarian corpus luteum of 7 days chronic hypobaric hypoxia (7CHH) group and a declined percentage of adrenocortical cells in 3 days chronic hypobaric hypoxia (3CHH) and 7CHH groups. Plasma estradiol level was unaltered, but progesterone level was decreased significantly in all hypoxic groups. Ovarian 3ß HSD activity was decreased significantly with increasing days of hypoxic treatment along with a significantly low adrenal 3ß HSD activity in 7CHH. In conclusion, hypobaric hypoxia causes a state of low circulatory progesterone level in females likely due to the degenerative changes in the female ovarian and adrenal tissues together with low steroidogenic 3ß HSD enzyme activity.

Antispermatogenic Effect of Piper betel Leaf Stalk Extract with Reference to Kinetic Studies of 17β-hydroxy Steroid Dehydrogenase Enzyme in Testes of Albino Rats

Background: In the development and maintenance of male reproductive function and fertility, steroidogenesis plays a key role. Aims: The aim of the present study was to investigate the effect of the betel leaf stalk extract on 17β- hydroxy steroid dehydrogenase activity levels. Methods: The observed elevation in testicular cholesterol levels may be due to decreased androgen production, which resulted in impaired spermatogenesis. The decreased steroidogenic enzyme 17β- HSD activity represents decreased androgen production by the extract administration. Reduction in enzyme active site density and Km value revealed that there was a reduction in enzyme-substrate affinities and rate of E-S complex breakdown in the administered rat testes. Results &amp; Conclusion: The administration of betel leaf stalk extract resulted in decreased enzyme content probably through impaired synthesis.

Clomiphene citrate ameliorated lead acetate-induced reproductive toxicity in male Wistar rats.

Objective:The current study investigated the effects of clomiphene citrate on the hypothalamic-pituitary-testicular axis, steroidogenesis, sperm parameters, and testicular antioxidant enzyme activity of male Wistar rats submitted to lead acetate (Pb)-induced reproductive toxicity. Methods:Twenty adult male Wistar rats were divided into four groups of equal size as follows: Control; Clomid (0.35 mg/kg); Pb (10 mg/kg); and Clomid + Pb. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, testicular 17-β hydroxysteroid dehydrogenase (17-β HSD) activity, androgen receptors, catalase activity, superoxide dismutase (SOD), malondialdehyde (MDA), sperm motility, viability, counts and morphology were estimated after oral administration of Clomid and/or lead acetate for 35 consecutive days. Data were analyzed using ANOVA at p<0.05.Results:Lead acetate significantly decreased (p<0.05) serum LH and testosterone levels, testicular 17β-HSD activity, androgen receptor expression, sperm motility, viability, counts, catalase activity, and SOD when compared with controls. Abnormal sperm morphology and MDA were significantly increased (p<0.05) in the Pb group compared with controls. Clomid co-administrated with lead acetate significantly increased (p<0.05) serum LH, testosterone levels, testicular 17β-HSD, androgen receptor expression, sperm motility and viability when compared with the group given lead acetate.Conclusions:The present study suggests that clomiphene citrate may stimulate testicular testosterone synthesis, sperm motility and viability via luteinizing hormone in a context of lead acetate-induced reproductive toxicity.

Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase (3β-HSD) in pigs

Recent studies have suggested that apelin has a role in controlling female reproduction. The aims of the present study were, firstly, to investigate the gene expression (mRNA and protein) and immunolocalization of apelin and its receptor APJ in corpora lutea (CL) of pigs collected during the early (CL1), middle (CL2) and late (CL3) luteal phase. Using real time PCR and immunoblotting techniques, it was observed that apelin gene expression was similar in CL1 and CL2, and less in CL3, while relative abundance APJ mRNA and abundance of the protein were similar in CL1 and CL3 and greater in CL2. There was apelin staining in the cytoplasm of both small (SC) and large (LC) luteal cells with the greatest intensity in CL2. In the cytoplasm of CL1, only a few SC cells stained for APJ; in CL2, APJ was located in the cell membrane of LC and in the cytoplasm of SC; and in CL3 was located in the membrane with moderate cytoplasmic APJ staining. Intense APJ staining was noted in epithelium of blood vessels of CL2–3. Secondly, there was an effect of apelin on progesterone (P4) secretion in CL2 and on the molecular mechanisms of these cells. Stimulatory effects of apelin on P4 secretion, 3β–hydroxysteroid dehydrogenase (HSD) activity and protein abundance were observed and this was inhibited in response to APJ and adenosine 5′-monophosphate-activated protein kinase (AMPKα) kinase blockers. In conclusion, the presence of apelin/APJ in the CL of pigs and stimulatory effects of apelin on P4 secretion and 3β-HSD levels suggest potential auto/paracrine regulation by apelin in the luteal phase of the estrous cycle. Moreover, the involvement of APJ and AMPKα kinase in apelin activity in CL was confirmed.

Walnut leaf extract acts as a fertility agent in male Wistar albino rats - A search for herbal male fertility enhancer.

Background Walnut leaf is one of the many medicinal plants used in folklore as male fertility enhancers. The present work was therefore undertaken with an aim to scientifically validate this claim. As such, we evaluated the effect of the aqueous extract from walnut leaves on biomolecules related to fertility in adult male rats and its mode of action as fertility-enhancing agent. Methods Twenty-five rats were randomly divided into five groups of five animals each; Group 1 served as control and received normal (0.9%) saline only; Groups II, III, IV received 50, 500, 1,000 mg/kg body weight (BW) of T. conophorum leaf extract orally, while Group V served as standard and was given suspension of clomiphene citrate orally at the dose of 1.04 mg/kg/ml BW. The extract and drug were given daily and the experiment lasted for 21 consecutive days. Results The testicular biochemical parameters in treated groups showed significant (p<0.05) increase in lactate dehydrogenase activity activity, Glucose-6-phosphate dehydrogenase (G-6PDH) activity, glycogen content, 3β and 17β hydroxysteroid dehydrogenase activities and testicular and epididymal Zn and Se contents with a significant decrease in cholesterol content. A significant increase in testis weight and epididymis weight were also observed. Also, a significant (p<0.05) increase in the level of serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone, sperm count, motility, viability and a decrease in sperm abnormality were observed in the various treated groups when compared with the control group. This increment was concentration dependent, while the extract at the highest concentration showed a more pronounced effect than the standard drug. Also, no sperm DNA fragmentation index was found in all the treatment groups. Photomicrographs from light and scanning electron microscopy showed large fenestrae of interstitial tissue, large fluid space and intact seminiferous epithelium layers fully packed with spermatogenic cells in treated groups than the control group. Conclusions The present study has demonstrated that Tetracarpidium conophorum leaf possesses fertility-enhancing property and have useful effects on spermatogenesis and sperm parameters in rats.

11[beta]-hydroxysteroid dehydrogenase activity, androgen excess, and metabolic outcomes in woman

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Alternations in miRNA Expression in Chronic Stress-Induced Ageing of Leydig Cells

Purpose: Chronic stress represents a critical influence on ageing of Leydig cells in testis. We aimed to investigate expression changes of microRNA (miRNA) associated with stress-induced ageing in Leydig cells.Methods: Brown Norway rats were separated into three groups, young control group, stress group and age group. Physiological changes, including serum corticosterone and testosterone levels, 11β-HSD (11β- hydroxysteroiddehydrogenase) activity, GSH/GSSG (glutathione/glutathione disulfide) ratio and DNA damage after chronic stress treatment were tested by radioimmunoassay and Immunohistochemistry. Differentially expressed miRNAs in Leydig cells between young control group and stress group were identified by miRNA microarray analysis, followed by target prediction of miRNAs. Functional annotation of predicted targets of miRNA was carried out using GeneSpring software and miRNA-target gene interaction network was built by String software.Results: Under chronic stress, increased serum corticosterone levels and reduced testosterone levels were observed. Meanwhile, decline of 11β-HSD activity and GSH/GSSG ratio, increase of DNA damage were also shown in stress group. After performed miRNA microarray analysis, three differentially expressed miRNAs were screened out, including rno-miR-31a-5p, rno-miR-192-5p and rno-miR-191a-3p. Targets of them were mainly involved in apoptosis, cell cycle and transport. In miRNA-target gene interaction network, targets of miR-31a-5p (Tp53, Ywhae and Ndel1), and targets of miR-192-5p (Cdk2, Rac2, Stk3) were with higher degree.Conclusion: These data suggest a central role of miRNA-regulated gene expression in response to chronic stress, especially dys-regulation of rno-miR-31a-5p and rno-miR-192-5p play a vital role in ageing of Leydig cells in testis.

Effects of benzo[a]pyrene as an environmental pollutant and two natural antioxidants on biomarkers of reproductive dysfunction in male rats.

Benzo[a]pyrene (B[a]P) is an environmental toxicant and endocrine disruptor. Therefore, the aim of the present study was to investigate the toxicity of B[a]P in testis of rats and also to study the role of silymarin and thymoquinone (TQ) as natural antioxidants in the alleviation of such toxicity. Data of the present study showed that levels of testosterone, estrogen and progesterone were significantly decreased after treatment of rats with B[a]P. In addition, B[a]P caused downregulation of the expressions of steroidogenic enzymes including CYP17A1 and CP19A1, and decreased the activity of 17-β hydroxysteroid dehydrogenase (17β-HSD). Moreover, B[a]P decreased the activities of antioxidant enzymes including catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD), and significantly increased free radicals levels in testis of male rats. However, pretreatment of rats with silymarin prior to administration of B[a]P was found to restore the level of free radicals, antioxidant status, and activities of steroidogenic enzymes to their normal levels in testicular tissues. Moreover, histopathological finding showed that silymarin recovered the abnormalities occurred in tubules caused by B[a] P in testis of rats. On the other hand, TQ showed pro-oxidant effects and did not ameliorate the toxic effects of B[a] P on the testicular tissue since it decreased antioxidant enzymes activities and inhibited the protein expression of CYP11A1 and CYP21A2 compared to control rats. Moreover, TQ decreased the levels of testosterone, estrogen, and progesterone either in the presence or absence of B[a]P. It is concluded that B[a]P decreased testosterone levels, inhibited antioxidant enzymes activities, caused downregulation of CYP isozymes involved in steroidogenesis, and increased free radical levels in testis. Moreover, silymarin was more effective than TQ in restoring organism health and alleviating the deleterious effects caused by B[a]P in the testis of rats. Due to its negative impact, it is highly recommended to limit the use of TQ as a dietary supplement since millions of people in the Middle East are using it to improve their health.

Biphasic effect of Syzygium aromaticum flower bud on reproductive physiology of male mice.

The flower buds of Syzygium aromaticum (clove) have been used for the treatment of male sexual disorders in indigenous medicines of Indian subcontinent. Therefore to evaluate the efficacy of Syzygium aromaticum on the male reproductive health, chronic oral exposure of aqueous extract of flower buds of Syzygium in three doses (15 mg, 30 mg and 60 mg kg-1 BW) were studied for a single spermatogenic cycle (35 days) in Parkes (P) strain mice. Lower dose (15mg) of Syzygium aromaticum flower buds increased serum testosterone level and testicular hydroxysteroid dehydrogenase (HSD) activities and improved sperm motility, sperm morphology, secretory activity of epididymis and seminal vesicle, and number of litters per female. On the other hand, higher doses (30 and 60mg) of the treatment adversely affected above parameters. Further, higher doses of the extract also had adverse effects on daily sperm production, 1C cell population and on histology of testis. In conclusion, Syzygium aromaticum flower buds extract exhibits biphasic effect on reproductive physiology of male mice. Lower dose of Syzygium aromaticum flower bud extract is androgenic in nature and may have a viable future as an indigenous sexual rejuvenator, while higher doses adversely affected functional physiology of reproductive organs.