Involvement of KLF14 and egr-1 in the TGF-beta1 action on Leydig cell proliferation.

Transforming growth factor ß1 (TGF-ß1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-ß1 exerts stimulatory and inhibitory effect depending on the type I receptor involved in the signaling pathway. The aim of the present work was to study the signaling mechanisms and the intermediates involved in the action of TGF-ß1 on TM3 Leydig cell proliferation in the presence or absence of progesterone. The MTT assay showed that the presence of progesterone in the culture media lead to a proliferative effect that was blocked by Ru 486, an inhibitor of progesterone receptor; and ALK-5 did not participate in this effect. TGF-ß1 (1 ng/ml) increased the expression of p15 (an inhibitor of cell cycle) in TM3 Leydig cells, and this effect was blocked by progesterone (1µM). The expression of PCNA presented a higher increase in the cell cultured with TGF-ß1 plus progesterone than in cells cultured only with TGF-ß1. Progesterone induced the gene expression of endoglin, a cofactor of TGF-ß1 receptor that leads to a stimulatory signaling pathway, despite of the absence of progesterone response element in endoglin gene. In addition, the presence of progesterone induced the gene expression of egr-1 and also KLF14, indicating that this steroid channels the signaling pathway into a non-canonical mechanism. In conclusion, these findings suggest that the proliferative action of TGF-ß1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone.

Modulation by polyamines of gamma-glutamyl transpeptidase activity and lactate production in cultured Sertoli cells from immature and adult regressed golden hamster.

Polyamines are involved in cellular growth and differentiation. To analyze a possible role of polyamines on the regulation of Sertoli cell function, we studied the effect of putrescine, spermidine, and spermine on gamma-glutamyl transpeptidase (gamma-GTP) activity and lactate production on Sertoli cell cultures obtained from immature and adult-regressed golden hamsters. Sertoli cells were cultured for 7 days. The 72 hour conditioned media obtained on day 6 were used to evaluate lactate levels. Gamma glutamyl transpeptidase activity was determined in the cells harvested on day 7. Cultured Sertoli cells isolated from immature and adult-regressed golden hamsters exhibited a clear morphological response to follicle-stimulating hormone (FSH) and to spermine. Gamma glutamyl transpeptidase activity increased in response to FSH in a dose-dependent manner. Dose-dependent stimulation of lactate production by FSH was also observed. For each functional parameter, a similar ED50 value of FSH stimulation was observed in both groups of animals. Spermine increased basal and FSH-stimulated gamma-GTP activity in immature and adult-regressed Sertoli cell cultures. A stimulatory effect of spermidine and putrescine on gamma-GTP activity was exclusively observed in adult-regressed Sertoli cell cultures. In Sertoli cells obtained from immature hamsters, spermine exerted a stimulatory effect on basal and FSH-stimulated lactate production. These results suggest that, in addition to the known effects of hormones and paracrine factors, polyamines may influence the functionality of Sertoli cells.

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.

Serotonin in golden hamster testes: testicular levels, immunolocalization and role during sexual development and photoperiodic regression-recrudescence transition.

Serotonin (5-HT) is found in the gonads and accessory reproductive organs of several species. The golden (Syrian) hamster is a seasonal breeder. Exposure of male adult hamsters to short days for 14 weeks results in a severe gonadal regression, while after a photoinhibition period of 22 weeks a spontaneous testicular recrudescence occurs. The aim of this study was to investigate the presence of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the gonads of golden hamsters, its immunolocation and its physiological role in the testis. The influence of age and photoperiod was also analyzed. Hamsters of 23, 36, 46, 60 and 90 days of age were kept in long photoperiod (LP: 14:10 h light/dark), and adult animals were exposed either to LP or to short photoperiod (SP: 6:18 h light/dark) for 14 and 22 weeks. Testicular parenchyma and capsule levels of 5-HT and 5-HIAA increased significantly at ages of 36 and 60-90 days, but decreased markedly during the exposure of adult hamsters to SP for 14 and 22 weeks. Mast cells were found exclusively in the testicular capsule. The testicular number of mast cells increased concomitantly with age, but decreased in adult hamsters exposed to SP. Mast and Leydig cells presented 5-HT-positive immunoreactivity. During sexual maturation as well as during the transfer of adult hamsters from LP to SP, the 5-HIAA/5-HT ratio showed the highest values in active adult animals, indicating that the increase in testicular 5-HT levels in adulthood is accompanied by an augment in 5-HT turnover. In vitro basal and hCG-stimulated testosterone production was significantly inhibited in presence of physiological concentrations of 5-HT. In conclusion, the present studies demonstrate the existence of 5-HT in mast cells and Leydig cells of hamster testes, as well as describe an inhibitory action of this neurotransmitter on gonadal testosterone production. Furthermore, the age-dependent and photoperiodic-related changes detected in testicular 5-HT levels suggest that this neurotransmitter might act as an important local modulator of the action of gonadotropins on steroidogenesis during sexual development and during the photoperiodic regression-recrudescence transition in the golden hamster.

Ornithine decarboxylase activity and polyamine levels in the epididymis of prepubertal rat after antiandrogen administration.

The antiandrogens flutamide and casodex have been administered subcutaneously (vehicle, 1, 5 or 10 mg per day) to prepubertal male rats for 10 days. A significant change of epididymal weight has been observed after both treatments, from the lowest dose used. Epididymal dihydrotestosterone concentrations were significantly increased in flutamide- or casodex-treated rats, while epididymal 3 alpha-androstanediol concentrations were affected only after flutamide administration, suggesting a differential effect on androgen metabolism between both antiandrogens. Ornithine decarboxylase (ODC) activity was significantly decreased by flutamide, and to a lesser extent by casodex. Antiandrogen administration resulted in a significant decrease in epididymal content of the polyamines putrescine, spermidine, and spermine. A slight but significant decrease in putrescine and spermidine concentrations, but not in spermine, was observed after flutamide treatment. However, casodex had no effects on polyamines levels. A decrease in putrescine concentration was detected only when ODC activity fell to rather low levels. Interference of the antiandrogens with the biological action of androgens on ODC activity was clearly seen in immature male rats. Therefore, both epididymal growth and differentiation, in correlation to ODC activity, would be severely affected at an early period of sexual development, such as prepuberty.

Polyamine levels in testes and seminal vesicles from adult golden hamsters during gonadal regression-recrudescence.

The exposure of golden hamsters to short days results in early regression of the reproductive organs and subsequent spontaneous recrudescence characterized by active cellular regeneration and differentiation. Thus, adult male hamsters were subjected to short photoperiod (SP, 6L:18D) for 9, 12, 14, 16, 18, and 22 weeks or maintained under long photoperiod (LP, 14L:10D) for 22 weeks, to assess photoperiodic-related changes in testicular and seminal vesicle (SV) levels of polyamines (PA) that are involved in cell growth and differentiation. During the regression phase, the weights of the organs and the circulating levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, testosterone, dihydrotestosterone, and 5 alpha-androstane-3 alpha, 17 beta-diol were significantly diminished and, thereafter, during the recrudescence phase, they recovered total or partially their control values. In both tissues, the exposure to SP for 14-16 weeks resulted in an increase of PA concentrations, followed by a return to control levels in the recrudescence period. At the time of maximal tissue involution, the ornithine decarboxylase (ODC) activity (key regulatory enzyme of PA biosynthesis) showed a significant increase in testis, preceding the sharp peak of PA concentration. However, a marked decrease in ODC activity was detected in SV. The concentration of N-acetyl PA in SV showed an increment at 16 weeks of SP, while no modifications were detected in testicular concentration. When PA, N-acetyl PA, and ODC activity were expressed per testis and per SV, values fell significantly during the involution period, but in the recrudescence phase levels were recovered concomitantly with the restoration of the organ weight and function. In conclusion, the photoperiodic-related changes in PA and their N-acetyl derivatives might play a crucial role in regrowth and differentiation of the male sexual organs during the spontaneous recrudescence phase. Additionally, organ-specific regulation of the PA biosynthesis pathway could also take place.

Testicular gamma-aminobutyric acid and circulating androgens in Syrian and Djungarian hamsters during sexual development.

Several factors, besides luteinizing hormone (LH), participate in the modulation of testicular function. A number of neurotransmitters are reported to be involved in this process, including a stimulatory action of gamma-aminobutyric acid (GABA) on steroidogenesis in the rat testis. The purpose of this study was to investigate the testicular pattern of GABA and glutamic acid, one of its main precursors, during sexual maturation in two seasonally breeding species: Syrian (golden) and Djungarian hamsters. Plasma androgen levels were also measured. The animals were maintained under long-day photoperiod (16:8, L:D) and were killed at 23, 30, 36, 46, 60, and 90 days of age. A different pattern of developmental changes in body and testicular weight was observed in these two species. GABA was present in the testes at all ages studied. GABA concentration and content showed a sharp elevation in the prepubertal period in golden as well as Djungarian hamsters. However, glutamic acid concentrations remained nearly constant during development in both species. Glutamic acid content increased gradually with age in the golden hamster, while a marked peak at 36 days of age was detected in the Djungarian hamster. Plasma testosterone and dihydrotestosterone levels were maximal at pubertal age in both species. The plasma levels of 5 alpha-androstane-3 alpha, 17 beta-diol increased significantly at 30 days of age in the golden hamster while in Djungarian hamsters this steroid remained unchanged. These results suggest that glutamic acid may serve as a precursor for GABA biosynthesis in the testis. In addition, changes in testicular GABA and plasma androgen levels might reflect a modulatory effect of this neurotransmitter in the acquisition of steroidogenic capability during development.

Influence of photoinhibition on GABA and glutamic acid levels, and on glutamate decarboxylase activity in the testis and epididymis of the golden hamster.

Gamma-aminobutyric acid (GABA) is found in the gonads and accessory reproductive organs, and a direct effect on steroidogenesis and sperm viability and motility has been described. The golden (Syrian) hamster is a seasonal breeder, and a pattern of regression-recrudescence in their reproductive organs is observed when adult animals are exposed to less than 12.5 h daylight for an extended period of time. The purpose of this study was to investigate: (1) the presence of GABA in the testis and epididymis of golden hamsters undergoing regression and spontaneous recrudescence; (2) glutamic acid levels and glutamate decarboxylase (GAD) activity in both tissues, and (3) testicular and epididymal testosterone, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol concentrations. Adult golden hamsters were exposed to long (LP 14L:10D) or short (SP 6L:18D) photoperiods for 9, 12, 14, 16, 18 or 22 weeks. When animals were exposed to SP for 14-16 weeks, the testis and epididymis reached maximal involution. Testicular and epididymal androgen levels showed a marked decrease (p < 0.05) during the regression period, and after 18-22 weeks, values began to recover. Between 12 and 18 weeks in SP, the testicular and epididymal content of GABA and glutamic acid was reduced significantly. The concentration of GABA in both tissues showed a sharp rise (p < 0.05), while the concentration of glutamic acid diminished during the period of maximal involution (p < 0.05). In the testis, GAD activity was increased (p < 0.001) after 14 weeks in SP, with no change in the epididymis. In conclusion, glutamic acid via GAD activity could be the main source of GABA in the testis, but not in the epididymis. Furthermore, the presence of GABA in testicular cells and its subsequent photoperiodic variations might act as an important autocrine and/or paracrine modulatory signal in gonadal processes.

Polyamines in the male reproductive system.

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experimental models as well as humans, to their presence in the male gonad, prostate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polyamine biosynthesis, and the relationship of these compounds on cell growth and differentiation, are also discussed. In this regard, an attention is offered to the potential role of polyamines during early spermatogenesis stages and the use of some enzymes involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polyamines, their oxidation products and the relationship with male fertility.

Effects of two non-steroidal antiandrogens on testicular function in prepubertal rats.

The effects of two non-steroidal antiandrogens, flutamide and casodex, were evaluated in prepubertal male rats. Animals (23 days old) were subcutaneously administered vehicle or 1, 2, 5, or 10 mg/day of flutamide or casodex for 10 days. Testis weights were diminished at the 10 mg/day dose of both antiandrogens. A significant increase in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels was detected. Notwithstanding, flutamide influenced LH/FSH levels more severely than casodex. No changes were observed in serum prolactin. Serum testosterone, dihydrotestosterone, and 3 alpha-androstanediol levels were increased in flutamide-treated rats from the 2 mg/day dose, whereas only 3 alpha-androstanediol was modified at 10 mg/day of casodex, suggesting a differential effect on androgen metabolism. An elevation of testicular concentration and basal production of androgens was found, indicating that flutamide and casodex administration are capable of stimulating testicular steroidogenesis, as well as 5 alpha-reduction. However, the in vitro maximal responsiveness of the gonads to human chorionic gonadotropin was preserved. Antiandrogen administration did not modify testicular androgen binding protein concentration. In conclusion, the blockade of androgen action during sexual maturation caused profound changes on the pituitary-gonadal axis in male rats.