Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.     

Incorporation of [1-14C]-acetate into testosterone,androstenediol, dehydroepiandrosterone and progestogens by ram testis following human chorionic gonadotropin administration

Testicular steroidogenesis in rams was examined by constant infusion (3 hr) of [1-¹⁴C]-acetate into the testicular artery of four conscious standing animals.The following steroids (in order of decreasing levels of [¹⁴C] labeling) were secreted by the testis and found in testicular tissue: testosterone, dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one, androstenediol, 5-androsten-3β,17β-diol and 17-hydroxy-4-pregnene-3,20-dione. In addition, [¹⁴C] labeling of 17,20α-dihydroxy-4-pregnen-3-one occurred in testicular tissue but not in blood. This $\text{in vivo}$ system with the conscious standing ram demonstrated an operative Δ⁵ steroidal pathway to testosterone. The physiological significance of 17,20α-dihydroxy-4-pregnen-3-one is not yet explained in this species.     

The adrenal gland and progesterone stimulates testicular steroidogenesis in the rat in vivo

Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.     

Effect of ethyl alcohol on plasma testosterone level in mice

The effect of ethanol ingestion on testicular steroidogenesis in mice was evaluated by measuring plasma testosterone level. Four groups of CBA/J male mice were treated with one of the following doses of ethanol: 1.240, 0.620, 0.310 or 0.155 g ethanol/Kg body weight. A control group was given water. The data showed no effect of the treatment on testicular weight. The concentration of testosterone in the plasma was significantly reduced in animals treated with alcohol. There was also a significant relationship between the dose of alcohol and the plasma testosterone level, with the decrease in testosterone being from 2 to 18 fold in various groups.     

Local Effect of PACAP and VIP on Testicular Function in Immature and Adult Rats

Csaba, Zs., V. Csernus, and I. Gerendai. Local effect of PACAP and VIP on testicular function in immature and adult rats. Peptides 18(10) 1561–1567, 1997.—PACAP, VIP, anti-PACAP and anti-VIP antisera were injected intratesticularly. In 9-day-old hemicastrated rats PACAP or VIP decreased basal testosterone secretion. In 22-day-old hemicastrates VIP but not PACAP reduced compensatory testicular hypertrophy, however, neither PACAP nor VIP altered steroidogenesis. Anti-VIP antiserum to this age group increased testosterone production and enhanced compensatory testicular hypertrophy. In adult hemicastrates neither the peptides nor the antisera influenced steroidogenesis. Neither in immatures nor in adults treatment of both testes with PACAP or VIP had any effect. Data indicate that both PACAP and VIP might exert a local action on testicular steroidogenesis, on compensatory testicular hypertrophy, and these effects are age-dependent.     

Adrenalectomy does not influence basal secretion of testosterone in rat in vivo

We have studied the effect of adrenalectomy on the testicular secretion of testosterone in the rat. In the acute period following adrenalectomy plasma testosterone levels were reduced but this was no different from those levels in appropriate sham-operated controls. This reduction in plasma testosterone levels is probably a result of direct effects of anaesthesia and surgical stress. Whilst studies on the late effect of adrenalectomy avoided this problem, plasma testosterone levels were normal in both adrenalectomised and sham-operated animals. Resetting of anterior pituitary-gonadal relationships may mask the absence of any contribution made by the adrenal gland to testicular steroidogenesis. In contrast to previous data we were unable to demonstrate that adrenalectomy influenced the secretion of testosterone in the male rat.     

Simvastatin Treatment Ameliorates Injury of Rat Testes Induced by Cadmium Toxicity

Cadmium-induced testicular toxicity is mediated through oxidative stress and inflammation which eventually lead to cell death. Simvastatin, the antihyperlipidemic agent, exhibits additional antioxidant and anti-inflammatory activities. The aim of the present work was to investigate the protective effect of simvastatin against cadmium-induced testicular toxicity in rats. The rats received a single intraperitoneal (i.p.) injection of cadmium chloride (2 mg/kg). Simvastatin treatment (5 mg/kg/day, i.p.) was applied for three consecutive days, starting 1 day before cadmium administration. Cadmium significantly decreased serum testosterone, and testicular reduced glutathione and catalase activity, and significantly increased testicular malondialdehyde, nitric oxide, and cadmium ion levels. Simvastatin significantly ameliorated the biochemical changes induced by cadmium. Cadmium-induced testicular tissue injury observed by histopathological examination was attenuated by simvastatin. In addition, simvastatin significantly decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, nuclear factor-κB, and caspase-3, and increased heme oxygenase-1 expression in testicular tissue of rats exposed to cadmium toxicity. It was concluded that simvastatin, through its antioxidant and anti-inflammatory activities, provided a significant protective effect against cadmium-induced testicular toxicity in rats. However, starting treatment with simvastatin before cadmium exposure, as done in the present work, is not clinically applicable. Therefore, other investigations are needed to assess the protective effect of simvastatin treatment following induction of cadmium testicular toxicity.     

Vitamin C and vitamin E protect the rat testes from cadmium-induced reactive oxygen species

Cadmium is an environmental and industrial pollutant that affects the male reproductive system of humans and animals. However, the mechanism of its adverse effect on Leydig cell steroidogenesis remains unknown. The present study points to the possible involvement of oxidative stress in the suppression of steroidogenesis. Cadmium administration caused an increase in reactive oxygen species (ROS) by elevating testicular malondialdehyde (MDA) and decreasing the activities of testicular antioxidant enzymes such as glutathione peroxidase and superoxide dismutase. The mRNA of Steroid Acute Regulatory (StAR) protein was substantially reduced. The activities of testicular delta5-3beta and 17-beta-hydroxysteroid dehydrogenases (HSD) as well as serum testosterone level were also lowered, suggesting that cadmium-induced ROS inhibit testicular steroidogenesis. Supplementation with vitamin C (VC) and or vitamin E (VE) reduced testicular ROS and restored normal testicular function in Cd-exposed rats. We conclude that VC and VE prevent oxidative stress and play vital roles in co-regulating StAR gene expression and steroid production in cadmium-exposed rats.     

Insulin augmentation of testosterone production in a primary culture of rat testicular cells

The direct effects of insulin on basal and human chorionic gonadotropin (hCG)-stimulated accumulation of testosterone were investigated in vitro using a primary culture system of rat testicular cells from adult hypophysectomized male rats. The basal accumulation of testosterone was low throughout the 10-day incubation period. Treatment of testicular cells with insulin (10 micrograms/ml) by itself was without effect on the basal accumulation of testosterone, while treatment with increasing concentrations (0.1--10 ng/ml) of hCG resulted in dose-dependent increases in the accumulation of testosterone. Furthermore, concomitant treatment with increasing concentrations (0.01--10 micrograms/ml) of insulin led to a dose-dependent augmentation (up to 116% on Day 10) in the hCG-stimulated accumulation of testosterone, as well as a 1.6-fold increase in the testicular responsiveness to hCG. In contrast, treatment with desoctapeptide insulin (10 micrograms/ml), a trypsin degraded insulin, was without effect on the hCG-stimulated accumulation of testosterone. Increasing duration (12--72 h) of treatment with insulin resulted in time-dependent increases in the hCG-stimulated accumulation of testosterone achieving statistical significance (P less than 0.05) by 36 h. In addition, pretreatment with insulin (10 micrograms/ml) brought about significant (P less than 0.01) increases in the choleragen and Bt2cAMP-stimulated accumulation of testosterone. The augmenting effect of insulin was equally effective upon culturing in a glucose-free medium and was not associated with significant alterations in testicular cell number or cellular DNA or protein content. It is concluded that diminished testicular steroidogenesis in the diabetic rats may represent, at least in part, a direct consequence of insulin deficiency at the testicular level and that insulin may play an important role in the augmentation of testicular androgen production.     

Profiles of ligands of sex hormone binding globulin in human serum

Ligands of the sex hormone-binding globulin (SHBG) in samples of human serum were extracted into diethyl ether and the dried extracts chromatographed using Sephadex LH-20 chromatography. The resulting fractions were assayed by competitive binding to SHBG against a testosterone standard. Values for dihydrotestosterone and testosterone were similar to those obtained using radioimmunoassay. While the bulk of the material in male and non-pregnant female serum corresponded to other known ligands (5-androstane-3 alpha,17 beta-diol and 5-androstene-3 beta,17 beta-diol), the quantities of material in the androstanediol and androstenediol regions exceeded the known values for these steroids in hirsute women and in late pregnancy, suggesting the presence of other steroids as well. In addition, there was a large amount of material of low polarity present in pregnancy which was not accounted for by recognized circulating ligands. A normal pattern was found in a man with Addison's disease, suggesting that the bulk of SHBG ligands in men are derived from the testis. This was also indicated by the 60-fold higher levels of testosterone and androstenediol seen in normal testicular vein serum. High values of testosterone, androstanediol and androstenediol in a woman with untreated 21-hydroxylase deficiency suggested that large amounts of these compounds (or their precursors) can be produced by the adrenal and that their production by the adrenal is regulated at least in part by ACTH.     

Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-L-arginine and comparisons to alcohol.

Recent studies suggest that nitric oxide (NO) may regulate hormone biosynthesis and secretion. This was tested by treating male rats with NG-nitro-L-arginine methyl ester (NAME), a NO synthase inhibitor, and measuring serum and testicular interstitial fluid testosterone and serum corticosterone, luteinizing hormone (LH), and prolactin (PRL). The effect of NG-nitro-L-arginine (NA), a less-soluble form of the same NO synthase inhibitor, on the reproductive suppressant actions of alcohol was also examined. NAME increased testosterone and corticosterone secretion dose-dependently without affecting LH and PRL secretion. The alcohol-induced suppression of testosterone or LH secretion was not altered by treatment with NA. Although effects of NAME and NA on other systems may be involved, these results indicate that testicular and adrenal steroidogenesis are negatively regulated by endogenous NO and that NO does not regulate LH and PRL secretion or inhibit the testicular steroidogenic pathway in the same way as alcohol.     

Alteration in expression of estrogen receptor isoforms alpha and beta, and aromatase in the testis and its relation with changes in nitric oxide during aging in mice

The aim of present study was to investigate the changes in the testicular expression of aromatase, ER alpha, ER beta and iNOS protein and correlate these with serum testosterone and nitric oxide levels, to elucidate the role of estrogen and nitric oxide in the testis during aging. This study showed localization of aromatase and ER alpha mainly in the Leydig cell and showed close correlation of testicular aromatase level with circulating testosterone level suggesting that estrogen may be modulating testicular steroidogenesis. Localization ER alpha mainly in the mitotically active germ cell suggest possible role of estrogen in germ cell proliferation. This study showed basal level of nitric oxide during reproductively active period, whereas increased serum nitric oxide coincides with decreased testicular activity in old age. This study showed inverse correlation between aromatase and NO level. Treatment with either SNP or L-NAME on testicular steroidogenic factor (3-beta HSD/ StAR) or germ cell survival factor (Bcl2) showed that increased NO causes decreased steroidogenesis and increased germ cell apoptosis. In conclusion this study suggest that estrogen modulate steroidogenesis and germ cell survival in reproductively active period whereas in old age decreased estrogen concentration causes increased nitric oxide which in turn decreases testicular steroidogenesis and germ cell apoptosis.     

Absence of direct effects of GnRH on testicular steroid secretion in the ram

Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0.5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.     

Effects of exogenous and endogenous opiates on the hypothalamic--pituitary--gonadal axis in the male

Narcotics acutely depress serum testosterone levels in the male. Three mechanisms could be involved: an enhancement of the degradation of testosterone; a direct inhibition of testicular steroidogenesis; or, finally, an inhibition of the hypothalamic-pituitary-luteinizing hormone (LH) axis resulting in a reduction in LH-dependent testicular steroidogenesis. The currently available evidence indicates that narcotics do not affect the catabolism of testosterone by the liver or testicular steroidogenesis. Rather, the data favor a direct action on the hypothalamic--pituitary--LH axis, probably by inhibiting the secretion of LH-releasing hormone (LH-RH) from the hypothalamus. The effects of narcotics on serum LH appear to be mediated via specific opioid receptors, suggesting that a naturally occurring opioid-like substance exists that normally inhibits LH. In support of this conclusion, opiate receptor blockers markedly increase serum LH levels shortly after their subcutaneous administration. In addition, endogenous opioids also seem to participate in testosterone's negative feedback control of the hypothalamic--pituitary--LH axis. Thus, it appears that opiate drugs inhibit the function of the hypothalamic-pituitary-gonadal axis by occupying opiate receptors in the hypothalamus and, moreover, that endogenous opioids exist that normally bind to these receptors and regulate activity in this axis.     

Reduced testicular steroidogenesis in tumor necrosis factor-alpha knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.     

Role of ethanol metabolism in the inhibition of testosterone biosynthesis in rats in vivo: importance of gonadotropin stimulation

The mechanisms by which ethanol (EtOH, 1.5 g/kg) inhibits testicular testosterone synthesis were studied in nonstimulated and human chorionic gonadotropin (hCG, 50 IU/kg)-treated male rats. To dissociate the effects caused by ethanol metabolism, the alcohol dehydrogenase inhibitor 4-methylpyrazole (4MP, 10 mg/kg) was given to half of the rats 30 min before EtOH. The 4MP had little or no effect in the nonstimulated rats on the EtOH-induced decreases in the concentrations of serum testosterone and of the intratesticular steroids of the testosterone biosynthetic pathway measured, but reduced the EtOH-induced elevation in the intratesticular pregnenolone-to-progesterone ratio. In contrast, 4MP pretreatment markedly reversed the EtOH-induced decrease in serum and intratesticular testosterone and increase in intratesticular pregnenolone concentrations in the hCG-stimulated rats. Simultaneously, the EtOH-induced elevations in the intratesticular pregnenolone/progesterone and androstenedione/testosterone ratios were abolished. In the EtOH-treated rats whose EtOH metabolism was blocked by 4MP pretreatment, the intratesticular testosterone concentrations were negatively correlated with the elevated serum corticosterone levels. It is concluded that: (1) EtOH metabolism is involved in the inhibition of testicular steroidogenesis in vivo. This effect is pronounced during gonadotropin-stimulated conditions. Thus, previously reported "discrepancies" between the in vivo and in vitro results are clarified; (2) corticosterone seems also to be involved in the EtOH-induced inhibition of steroidogenesis. This effect is also pronounced during gonadotropin-stimulated conditions; and (3) without external gonadotropin stimulation other inhibitory mechanisms, such as decreased stimulation by luteinizing hormone, are prevalent.     

Effect of tamoxifen on the activity of enzymes of testicular steroidogenesis

Testicular homogenates of tamoxifen-treated rats were incubated with labeled steroid precursors (progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione or testosterone) in order to study the effect of tamoxifen on testicular steroidogenesis. The results indicate that a 9 day treatment with a daily dose of 1 mg tamoxifen produces a reduction of the synthesis of testosterone. Inhibition of the 17 alpha-hydroxylase and C17,20-desmolase enzyme systems was observed together with an increased 20 alpha-hydroxysteroid dehydrogenase activity.     

Effect of experimental infection with Trypanosoma congolense and scrotal insulation on Leydig cell steroidogenesis in the ram

Testicular steroid content and Leydig cell steroidogenesis in vitro were investigated in rams on Days 28 and 58 after Trypanosoma congolense infection and were compared with those of rams in which testicular temperature had been raised artificially by insulation of the scrotum for 58 d. Testicular testosterone content increased significantly on Day 28 after infection but was lower than that of controls on Day 58 while it increased in scrotal-insulated rams compared with that of controls by Day 58. Testicular progesterone was undetectable in the control and trypanosome-infected groups throughout the experiment, but it increased in the insulated rams by day 58. Basal (unstimulated) Leydig cell testosterone production in the infected rams was similar to that of control rams on Day 28 but was significantly lower on Day 58. Stimulation of Leydig cell testosterone production with hCG or 22R-hydroxycholesterol (22ROHC) significantly reduced in infected rams at both 28 and 58 d after infection as well as in scrotal-insulated rams on Day 58. It is concluded that the increase in testicular testosterone content in the infected and scrotal-insulated rams on Days 28 and 58, respectively, was induced by elevation of testicular temperature by trypanosome infection, perhaps through an effect on testicular blood flow. Reduced testosterone production by Leydig cells from infected and scrotal-insulated rams in response to hCG and 22ROHC suggests that trypanosome-induced pyrexia might be involved in reducing Leydig cell steroidogenesis and subsequent plasma testosterone levels, possibly by affecting enzymes involved in steroid biosynthesis.     

Potentiation by epidermal growth factor of the in vitro HCG stimulation of testicular steroidogenesis in hamsters.

Epidermal Growth Factor (EGF) has been reported to stimulate or inhibit steroidogenesis in murid Leydig cells depending on the experimental conditions used. In the present study, testicular fragments from an adult cricetid rodent, the Syrian hamster, were incubated with various doses of mouse EGF (0-2.0 micrograms/ml media), in the presence or absence of HCG (0-12.5 mlU/ml media). Although EGF alone did not affect in vitro testicular steroidogenesis, it significantly potentiated the HCG-induced elevation of the accumulation of testosterone and 17-hydroxyprogesterone in the media. In contrast, the effect of HCG on media progesterone concentration was not affected by EGF. Since in the Syrian hamster intracellular calcium loading functions as a gonadotropic stimulus, the present results could be a consequence of the EGF-induced increase in cellular calcium levels.     

Effects of ethanol and acetaldehyde on the biosynthesis of testosterone in the rodent testes

The effects of ethanol and acetaldehyde on testicular steroidogenesis were examined in enzymatically dispersed cells of the rodent testes. Both drugs significantly inhibited gonadotropin-stimulated steroidogenesis, but acetaldehyde was considerably more potent (>1000 times) than ethanol. To determine the step in testosterone's biosynthetic pathway which was inhibited by the two drugs, cells were incubated in the presence of [³H]pregnenolone and [³H]progesterone, and the amount of label incorporated into testosterone and its precursors was determined. Ethanol and acetaldehyde inhibited only the conversion of androstenedione to testosterone; none of the other precursors of testosterone was affected.