Temporal relation between leptin and various indices of sexual maturation in the male rat.

Recent studies in humans and rhesus monkeys have suggested the possibility that the adipose tissue hormone leptin has a stimulatory and/or permissive effect on the onset of puberty in the male. We evaluated this hypothesis by measuring leptin in groups of male rats between the ages of 26 days and 96 days. A statistically significant positive correlation was present between serum leptin and age, body weight, prostate, seminal vesicle, and testes weight (both absolute and as a function of body weight). A statistically significant negative correlation was present between leptin and serum FSH and alpha-inhibin. There was not a statistically significant correlation between leptin and testosterone or LH. There was a statistically significant increase in the serum leptin concentrations at day 47. This rise was coincident with the peripubertal growth spurt in the secondary sexual organs and the peripubertal testosterone rise but occurred after the prepubertal rise in testicular weight, the appearance of elongating spermatids in the testes, and the start of the decline in FSH. In animals in which the peripubertal testosterone rise was delayed by the administration of EDS, serum leptin showed statistically significant differences from control. These data do not support the hypothesis that leptin provides a trigger for the onset of puberty in the male rat. They do suggest that leptin may be involved in the secondary sexual organ growth spurt and are consistent with the hypothesis that testosterone stimulates leptin synthesis during puberty.     

The temporal response of rat testes to hCG stimulation during sexual maturation

Serum testosterone responses to a single sc injection of hCG (25 IU/100 g body weight) were monitored for 5 days in rats throughout sexual maturation (22-70 days). Two hours after hCG injection serum testosterone levels rose in 22, 37 and 53 day-old animals and remained elevated for 2 days, returning to control levels on day 3. This response differed markedly from the biphasic secretion of testosterone reported for adult animals. In 70 day-old animals the serum testosterone response approached that seen in adult animals. Testosterone levels were elevated 2 h after hCG injection (25.4 +/- 2.5 ng/ml) and declined significantly at 12 and 24 h to 17.1 +/- 1.0 and 16.1 +/- 3.4 ng/ml, respectively. Testosterone levels tended to increase again on days 2 and 3 (19.9 +/- 2.8 and 21.1 +/- 3.5 ng/ml, respectively) but the increase was not statistically significant. This response differed markedly to the biphasic secretion of testosterone reported for adult animals. In vitro patterns of basal and hCG-stimulated testosterone secretion by decapsulated testes following a single hCG injection also changed during sexual maturation. In 22 day-old animals the testes exhibited refractoriness to in vitro hCG stimulation at 12 h, but testes from 37 day old rats were refractory from 2 to 24 h. In vitro testosterone responses of testes from 53 and 70 day-old rats were similar to that reported for adult rats with a period of refractoriness from 12 h to 2 days. This study demonstrates that during sexual maturation in the rat alterations occur in the temporal patterns of testosterone secretion in vivo and in vitro following hCG stimulation.     

Androgen Dependence of Testicular and Epididymal Angiotensin Converting Enzyme

Treatment with cadmium chloride (CdCl2) and cyproterone acetate (CA) depressed angiotensin converting enzyme (ACE) activity significantly in testes and epididymal regions of the adult rats compared to the corresponding untreated controls. Exogenous testosterone to CA-treated rats significantly increased the enzyme activity both in the testes and epididymis, the effect in the latter being very significant comparable to CA-treated and untreated controls. Testosterone failed to induce ACE activity in the testes and caput epididymis of 30 day-old immature rats, but the enzyme activity was detected in corpus and cauda epididymis. Our findings indicate that ACE activity in the testicular complex is possibly linked with androgen and is concerned with spermatogenesis and sperm maturation.     

Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats

Hypogonadotropism is a common feature of uncontrolled diabetes, for which the ultimate mechanism remains to be elucidated. Kisspeptins, ligands of G protein-coupled receptor 54 (GPR54) encoded by the KiSS-1 gene, have recently emerged as major gatekeepers of the gonadotropic axis. Alteration in the hypothalamic KiSS-1 system has been reported in adverse metabolic conditions linked to suppressed gonadotropins, such as undernutrition. However, its potential contribution to defective gonadotropin secretion in diabetes has not been evaluated. We report herein analyses of luteinizing hormone (LH) responses to kisspeptin and hypothalamic expression of the KiSS-1 gene in streptozotocin (STZ)-induced diabetic male rats. In addition, functional studies involving kisspeptin replacement or continuous administration of leptin and insulin to diabetic male rats are presented. Kisspeptin administration evoked robust LH and testosterone bursts and enhanced postgonadectomy LH concentrations, despite prevailing attenuation of gonadotropic axis in diabetic animals. In addition, hypothalamic KiSS-1 mRNA levels were unambiguously decreased in diabetic male rats, and the postorchidectomy rise in KiSS-1 mRNA was severely blunted. Repeated administration of kisspeptin to diabetic rats evoked persistent LH and testosterone responses and partially rescued prostate and testis weights. In addition, central infusion of leptin, but not insulin, was sufficient to normalize hypothalamic KiSS-1 mRNA levels, as well as LH and testosterone concentrations. In summary, we provide evidence for altered expression of the hypothalamic KiSS-1 system in a model of uncontrolled diabetes. This observation, together with the ability of exogenous kisspeptin to rescue defective LH responses in diabetic rats, unravel the physiopathological implication, and potential therapeutic intervention, of the KiSS-1 system in altered gonadotropin secretion of type 1 diabetes.     

Involvement of inhibin in the regulation of follicle-stimulating hormone secretion in the young adult male Shiba goat

The roles of inhibin and testosterone in the regulation of gonadotropin secretion were investigated in young adult male Shiba goats (8-12 months of age). Plasma levels of inhibin but not testosterone abruptly decreased after hemicastration (75% of the initial level), concomitant with a progressive rise in plasma levels of FSH. Removal of the remaining testis at 33 days after the hemicastration quickly decreased plasma levels of both inhibin and testosterone and induced a progressive increase in plasma FSH and LH. Implantation of testosterone sheets immediately after castration suppressed the increase in plasma FSH in part only, whereas the increase in LH secretion was almost completely suppressed. An i.v. injection of antiserum against [Tyr30] porcine inhibin alpha(1-30) resulted in a significant increase in plasma FSH in a dose-dependent manner, without altering plasma concentrations of LH. These findings clearly indicate that both inhibin and testosterone physiologically regulate FSH secretion and that testosterone is the principal gonadal factor regulating LH secretion in the adult male goat.     

Influence of age and photoperiod on steroidogenic function of the testis in the golden hamster

The golden (Syrian) hamster is a seasonal breeder, and exposure of adult animals to short days results in severe gonadal regression with morphological features that resemble the immature testis. The purpose of this study was to investigate testicular steroidogenic capacity in the golden hamster and to analyse the influence of age and photoperiod on this process. Hamsters aged 36 days were maintained on a long photoperiod (14L:10D), and adult animals were then exposed to a long or a short photoperiod (6L:18D) for 14 weeks (the period of time required to achieve maximal gonadal regression), to assess circulating levels and in vitro production of testosterone, dihydrotestosterone and androstane-3 alpha, 17 beta-diol. In peripubertal hamsters, androstane-3 alpha, 17 beta-diol was the main circulating androgen detected, whereas in active adult animals, testosterone showed the highest serum levels. In hamsters exposed to a short photoperiod, blood testosterone levels were significantly lower than levels in adult hamsters exposed to a long photoperiod. Exposure of adult hamsters to a short photoperiod produced a marked reduction in serum concentrations of dihydrotestosterone and androstane-3 alpha, 17 beta-diol, which was not accompanied by a decrease in testicular 5 alpha-reductase activity. In the in vitro experiments, active adult testes were less sensitive than inactive adult testes to stimulation of androgen production with hCG, but showed similar sensitivity to the gonads from hamsters aged 36 days. In accordance with circulating androgen concentrations, the principal androgens produced in the in vitro assays from peripubertal and normal adult testes were androstane-3 alpha, 17 beta-diol and testosterone, respectively. Unexpectedly, the main androgen produced from regressed testes under in vitro conditions was androstane-3 alpha, 17 beta-diol. Inactive gonads released more androstane-3 alpha, 17 beta-diol than did normal adult testes and total in vitro androgen production (testosterone + dihydrotestosterone + androstane-3 alpha, 17 beta-diol) from adult testes was not diminished by exposure to a short photoperiod. However, in spite of the significant increase detected in production of androstane-3 alpha, 17 beta-diol in vitro from regressed testes, inactive gonads produced less androstane-3 alpha, 17 beta-diol than did peripubertal testes. In summary, our studies suggest that testicular androgen biosynthetic capacity in adult hamsters exposed to short photoperiod is not reduced and these regressed testes represent an intermediate physiological state between peripubertal and active adult testes. The significant decrease detected in serum androgen concentrations during the involution phase could result from the absence of stimulating pituitary factors, together with a negative regulation of steroidogenesis by different non-steroidal signals originating within and/or outside of the testis.     

Experimental Studies on Connective Tissue of the Capsular Ligament: Influences of Aging and Sex Hormones

nfluences of aging and sex hormones on connective tissue metabolism in the hip joint capsule of Wistar rats were analyzed both biochemically and morphologically. As age advanced, collagen content and collagen fibril diameter of the hip joint capsule tended to increase gradually in both sexes until sexual maturation was reached. Collagen content was significantly greater in males than in females after sexual maturation. The collagen content and fibril diameter were considerably increased by ovariectomy and significantly decreased by the administration of estrogen, or estrogen combined with progesterone, whereas they were significantly increased by the administration of testosterone in orchiectomized male rats.     

Pituitary regulation of the expression of the farnesyl pyrophosphate synthetase gene in the testes of the sexually maturing rat

While investigating the coordinate regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and farnesyl pyrophosphate synthetase, the authors observed that rat testes contained high levels of a farnesyl pyrophosphate synthetase mRNA that was larger than that found in most other tissues. This mRNA contains upstream AUG codons that may alter its rate of translation. The developmental and hormonal regulation of this testicular mRNA were investigated. Testicular levels of farnesyl pyrophosphate synthetase mRNA increased in rats between 30 and 40 days of age and remained elevated. Significant increases in serum testosterone concentrations and secondary sexual organ weights first occurred at 50 days of age. Hypophysectomy resulted in nearly undetectable levels of testicular farnesyl pyrophosphate synthetase mRNA. Treatment of hypophysectomized rats with gonadotropins increased the levels of this mRNA toward normal. These data indicate that an increase in farnesyl pyrophosphate synthetase mRNA takes place in testes just before the onset of puberty. This may be induced by the peripubertal rise in follicle-stimulating hormone.     

Effects of testosterone on leptin production in anterior pituitary cells of rats: an immunohistochemical study

The aim of this study was to immunohistochemically examine the effects of orchidectomy and administration of testosterone hormone on leptin production in the rat anterior pituitary. Twenty-one male Wistar rats were divided into three groups. Group I and group II were designated as control (sham-orchidectomized) and orchidectomized rats, respectively. Rats in group III were orchidectomized and injected daily with testosterone propionate for 1 month. At the end of the experimental period, all animals were sacrificed by decapitation. The pituitary glands of all rats were removed and processed for semi-quantitative evaluation of immunohistochemical leptin staining. Intensity of immunostaining was determined on a scale between 0 (no staining) and 5 (heavy staining). Immunostaining of leptin was moderate (3+) in control rats, heavy (5+) in orchidectomized rats, and low (1+) in testosterone-treated orchidectomized rats, respectively. These findings indicate that orchidectomy increases leptin secretion in anterior pituitary cells, and this increase of leptin synthesis can be prevented by administration of testosterone propionate. Thus, testosterone seems to affect leptin production in the anterior pituitary of male rats.     

Effects of inhibitory and stimulatory photoperiods and sexual maturation on the ability of hamster testes to respond to hCG in vitro

The decrease in gonadal weight produced in adult golden hamsters by exposure to short photoperiods was accompanied by a marked reduction in the ability of the testes to produce testosterone from endogenous precursors in vitro, both without and with hCG stimulation. These changes were significant after 4–7 weeks in short photoperiod (5L: 19D) and were even more pronounced after 17–20 weeks. Production of testosterone in vitro by testes of immature hamsters was comparable to values obtained in adult animals with short photoperiod-induced gonadal atrophy. Delay of sexual maturation induced by daily injections of bromocriptine was accompained by a further decrease in testicular testosterone production in vitro. Exposure of gonadally-regressed adult hamsters to a long, stimulatory photoperiod (14L: 10D) produced a rapid and marked increase in testicular testosterone production, which was coincident with the previously demonstrated increase in serum gonadotrophin levels after 1–5 days of photostimulation. Furthermore, testosterone production in vitro by regressed testes of animals exposed to short photoperiod was increased significantly by one large dose of hCG administered 26 h before killing the animals. It is concluded that the suppressive effects of short photoperiods on the ability of the hamster testis to produce testosterone and to respond to hCG stimulation are due to reductions in endogenous LH, FSH and prolactin release, with a consequent loss of testicular LH/hCG receptors and decreased activity of enzymes involved in the biosynthesis of testosterone.     

Modulation of Leydig cell testosterone production by secretory products of macrophages

Summary.  Unstimulated macrophages from testes inhibited the production of testosterone by Leydig cells from adult, but not immature, Sprague-Dawley rats (significant after 48 h). Similar results were observed with unstimulated macrophage-conditioned media, suggesting that the observed effect was mediated by one or more secretory products. None of these substances was interleukin-1, since macrophage supernatants tested negative in an interleukin-1α and interleukin-1β sensitive, thymocyte assay. Interleukin-6 was detected by a B cell proliferation assay.
After stimulation by LPS, testicular macrophages enhanced testosterone production by Leydig cells from adult and immature rats. This enhancement was dose-dependent and required low concentrations (but over 2.5%) of conditioned media. Interleukin-1 and interleukin-6 activities were detected in LPS-stimulated macrophage supernatants. Supernatants of LPS-stimulated, human monocytes had similar effects on Leydig cells. They were rich in interleukin-1, interleukin-1 receptor antagonist and interleukin-6.
The present study suggests that, in adult rats, testicular macrophages modulate Leydig cell steroidogenesis by secretory products whose secretion depends on the physiological state of macrophages. The factor or factors responsible for stimulation are not species-specific. The effect cannot be accounted for by variations in the concentration of the above mentioned interleukins in macrophage supernatants.     

Effects of erythropoietin, bromocryptine and hydralazine on testicular function in rats with chronic renal failure

Summary. We evaluated the effects of chronic renal failure (CRF) on testicular function and semen physiology. A CRF model was created in 48 male rats by performance of five-sixths nephrec-tomies in two-stage procedures, and a control (group A) by two-stage sham operation on six male rats. Seven weeks later, serum urea and creatinine concentrations were assessed, and the nephrectomized rats were then equally divided into four groups, B, C, D and E, and treated with saline, erythropoietin, bromocryptine and hydralazine, respectively. Seventeen weeks after the first surgical procedure, the number of fertile rats, the mean values of epididymal sperm content and motility, the outcome of in vitro fertilization, and peripheral serum testosterone concentrations and responses to human chorionic gonadotropin were significantly higher ( P <0.05) in groups A, G and D than in groups B and E. Serum prolactin concentration was significantly higher ( P <0.05) in all groups of nephrectomized rats than in group A. Our results indicate that bromocryptine and erythropoetin improve Leydig cell function, sper-matogenesis, epididymal sperm maturation, and sperm fertilizing capacity in rats with CRF.     

Effects of follicular fluid administration on serum bioactive and immunoreactive FSH concentrations and compensatory testosterone secretion in hemicastrated adult rats

In adult rats, removal of one testis (hemicastration) results in an elevation of serum follicle-stimulating hormone (FSH) concentrations and a compensation in testosterone secretion by the remaining testis without a corresponding increase in testis size. To determine whether changes in FSH secretion and compensatory androgen production are related, serum testosterone concentrations were measured after inhibin-rich porcine follicular fluid was administered twice daily for 4 days to block the hemicastration-induced rise in FSH. Both serum immunoreactive FSH (immuno-FSH) and bioactive FSH (bio-FSH) concentrations were increased 4 days after hemicastration. The significant increase in serum immuno-FSH in hemicastrated animals was prevented by follicular fluid administration, whereas the serum bio-FSH activity and biologic to immunologic (B/I) ratios were increased in follicular fluid-treated animals. The follicular fluid-induced reduction in serum immuno-FSH had no effect on serum testosterone secretion in hemicastrated rats. Serum inhibin concentrations were reduced 27% in hemicastrated rats compared with intact controls, while administration of exogenous follicular fluid increased serum inhibin concentrations. An elevation in serum immuno-FSH secretion after hemicastration apparently is not required for the compensatory testosterone response. However, the observation of increased bio-FSH in hemicastrated and follicular fluid-treated animals raises questions about the importance of FSH quality (bioactivity), rather than quantity, for controlling testicular steroidogenic activity.     

Carbamazepine damage to rat spermatogenesis in different sexual developmental phases

Carbamazepine (CBZ) is a first-line antiepileptic drug (AED), although it is also utilized for treatment of psychiatric disorders and neuropathic pain. The utilization of CBZ has been associated with damage to male reproduction including hormonal alterations, sexual dysfunction and reduction of sperm quality. Wide and long-term use of CBZ has been a common schedule for children and adolescents, despite the fact it alters the testosterone level in adult rats and humans. In addition, hypothalamic-pituitary-gonadal (HPG) axis during pre-puberty and puberty is more susceptible to toxic agents than in adult phase. The objective of this work was to evaluate the side effects of CBZ on the spermatogenic process of rats from pre-puberty to puberty and sexual maturation. Damage on the seminiferous epithelium, testicular interstitial oedema, reductions of testosterone levels and an increase in oestradiol levels were observed in rats, which were CBZ-treated since the weaning. The results suggest that CBZ, when administered from pre-puberty, provokes specific side effects on rat testes, resulting in more severe damage in the adult phase.     

Primary culture of the rat epididymal epithelial cells as a source of oestrogen

Studies were performed on the rat epithelial cells of the caput and cauda epididymidis cultured in a full medium enriched with foetal calf serum without or with exogenous testosterone. After 3 days of culture, the cells formed a monolayer. The cytoplasm of epididymal epithelial cells cultured with testosterone was rich in lipid droplets, glycogen and PAS-positive substances, while their content was decreased in the cytoplasm of cells cultured without testosterone. The activity of 3beta-hydroxysteroid dehydrogenase was observed both in the cytoplasm of cultured epididymal epithelial cells and epithelial cells of epididymal sections. Hormone assays showed very low levels of dehydroepiandrosterone, androstenedione and testosterone, and the absence of progesterone in the media of cells cultured without testosterone and higher testosterone concentrations when the cells were cultured with exogenous testosterone. However, the concentration of 17beta-oestradiol found in the medium of cells was high, and exceeded many-fold its levels in the control media. Lentaron (Formestan), steroidal inhibitor of cytochrome P450 aromatase added to the culture decreased the secretion of oestradiol. RT-PCR analysis yielded cDNA products of 333 bp in length when primers were chosen to amplify a highly conserved sequence in the 3' region of the cytochrome P450 aromatase gene. This study demonstrates the ability of epididymal epithelial cells in vitro to synthesize androgens and mRNA for cytochrome P450 aromatase in the cultured epididymal epithelial cells of the rat as well as the ability to aromatise the synthesized androgen to 17beta-oestradiol.     

Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of testicular function in immature male rats

We investigated the effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) isolated from diesel exhaust particles (DEP) on the reproductive functions of male rats. Twenty-eight-day-old rats were injected subcutaneously with PNMC (1, 10, or 100 mg/kg) daily for 5 days. The weights of the epididymis, seminal vesicle, and Cowper gland were significantly decreased in rats treated with 10 mg/kg PNMC. The plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly increased by PNMC at 100 mg/kg. However, the plasma concentrations of testosterone and immunoreactive (ir)-inhibin were significantly decreased by PNMC at 100 mg/kg. The testosterone content of the testicles was significantly decreased in the group treated with 100 mg/kg PNMC compared with the control group. Furthermore, testicular concentration of ir-inhibin was significantly decreased by PNMC at 1 mg/kg or 100 mg/kg. To investigate the direct effects of PNMC on the secretion of LH and FSH from the anterior pituitary gland, and on the secretion of testosterone from the testes, we exposed cultured anterior pituitary and interstitial Leydig cells to PNMC (10(-6), 10(-5), 10(-4) M) with or without gonadotropin-releasing hormone (GnRH; 10 nM) (for the LH and FSH tests) and human chorionic gonadotropin (hCG; 0.1 IU/mL) (for the testosterone test) for 24 hours. PNMC did not change either the basal or GnRH-stimulated levels of FSH and LH secretion. However, PNMC significantly inhibited both basal and hCG-stimulated testosterone production. These findings suggest that PNMC has a direct effect on the testes of immature male rats, causing a reduction in testosterone secretion.     

Concentrations of free testosterone, total testosterone, and androgen binding protein in the peripheral serum of male rats during sexual maturation

To investigate the relationship between free testosterone and sexual maturation in the male rat, animals were decapitated every 5 days from 25 through 75 days of life. Serum was assayed for androgen binding protein and total testosterone by radioimmunoassay. Free testosterone concentrations were calculated from the total testosterone concentration and the free testosterone fraction. The free testosterone fraction was determined by ultrafiltration. The pubertal increase in relative prostate and relative seminal vesicle weights began between 45 and 50 days and 40 and 45 days, respectively. Although the over-all trend in the free testosterone fraction was to increase with increasing age (r = 0.46, P less than 0.0001), there was a significant secondary peak at 50 days. The serum concentration of androgen binding protein was highest on day 25, fell rapidly until day 40, and declined slowly thereafter. Despite these variations in both androgen binding protein and the free testosterone fraction during sexual maturation, the calculated serum concentration of free testosterone was remarkably similar in pattern to that of total testosterone (r = 0.99, P less than 0.0001). These data indicate that the serum concentration of total testosterone is an accurate reflection of the serum concentration of free testosterone during the sexual maturation of the male rat.     

Differences in nonhistone protein changes in rat ventral and dorsolateral prostate during sexual maturation

Age-related changes of chromosomal proteins in the dorsolateral and ventral prostates of rats from 6 to 31 weeks of age were studied by SDS-polyacrylamide gel electrophoresis. A nonhistone protein having a molecular weight of about 20,000 (20K-NHP), abundantly localized in the dorsolateral prostate, increased rapidly in content during the early stage of sexual maturation (6-11 weeks of age) in association with increases of serum testosterone concentration and prostatic tissue weight. Serum testosterone concentration decreased after week 11 and then remained constant until week 31. In contrast, the 20K-NHP content continued to increase after 11 weeks of age in the dorsolateral prostate, but not in the ventral prostate. The rapid increase of 20K-NHP in the dorsolateral prostate during the early stage of sexual maturation could not be attained in immature rats (5 weeks of age) by injection of excess amounts of androgens and/or prolactin for a week. But the 20K-NHP content in the ventral prostate of rats treated with testosterone propionate was almost the same as that of mature rats.     

Ablation of the inferior mesenteric plexus in the rat: alteration of sperm storage in the epididymis and vas deferens

The involvement of the sympathetic nervous system in the transport and storage of spermatozoa in the male reproductive tract was examined by surgically ablating the inferior mesenteric plexus (IMP). One to eight weeks after ablation of the IMP, epididymal weight and the total number of spermatozoa present in the cauda epididymidis were significantly greater in IMP-ablated rats than in sham-operated rats. By contrast, the number of spermatozoa present in the initial segment of the vas deferens was significantly greater than in sham operated controls one week after IMP ablation but returned to control levels at two, four, six and eight weeks. Throughout the experiment, no differences were observed between IMP-ablated and control rats in the percentage of motile cauda epididymal spermatozoa, testicular weight, testicular sperm number or serum testosterone. These data demonstrate that the sympathetic nervous system differentially regulates sperm transport and storage in the male reproductive tract and suggest that the IMP may influence the epididymal maturation of spermatozoa.