Alterations in mitogenic and steroidogenic activities in rat testicular interstitial fluid after administration of ethane dimethane sulphonate

A single dose of EDS was given to mature male rats and interstitial fluid (IF) was collected to determine the potency of mitogenic and steroidogenic activities therein. The potency of the factor stimulating testosterone secretion in vitro by Percoll-purified Leydig cells was significantly elevated 2 weeks after EDS, whilst the potency of mitogenic activities (stimulation of DNA synthesis by BALB/c 3T3 cells) was not elevated until 4 weeks after EDS treatment. This study suggests that two separate factors, one with mitogenic and the other steroidogenic activity, may be involved in the response of Leydig cells after EDS administration. The mitogenic factor may play a role in Leydig cell regeneration whereas the testosterone-stimulating factor may be involved in testicular testosterone homeostasis.     

Effects of ketoconazole on rat testicular steroidogenic enzymatic activities

Ketoconazole (K) is an antifungal imidazole derivative which has been shown to be a potent inhibitor of testosterone (T) biosynthesis in rodents and humans. To study the effect of K on rat testicular steroidogenesis we measured the activities of five testicular microsomal steroidogenic enzymes in K-treated rats and controls. Thirty male adult rats were given either 2 mg K or water every 12 hours by mouth during 5 days. Mean testicular weight was similar in both groups of animals. The K-treated group had a T serum concentration of 83 +/- 14 ng/dL whereas it was 94 +/- 16 ng/dL in the control group (NS). The K-treated animals had decreased activities of the 3 beta-hydroxysteroid dehydrogenase (830 +/- 48 vs 2,245 +/- 109 pmol/mg protein/min, P less than 0.001), 17-hydroxylase (243 +/- 5 vs 676 +/- 17 pmol/mg protein/min, P less than 0.001), 17-ketosteroid reductase (31 +/- 2 vs 169 +/- 7 pmol/mg protein/min, P less than 0.001), and aromatase enzymes (92 +/- 6 vs 123 +/- 7 pmol/mg protein/min, P less than 0.01). The 17,20-desmolase activity was similar in both groups of animals (210 +/- 4 vs 171 +/- 18 pmol/mg protein/min). We conclude that K given orally to rats inhibits the activity of several testicular steroidogenic enzymes.     

The effects of prolactin on rat testicular steroidogenic enzyme activities

To investigate whether hyperprolactinemia directly affects rat testicular steroidogenesis, we examined the effects of prolactin (PRL) on microsomal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) 17-hydroxylase (17-OH), 17,20-desmolase (17,20-D), 17-ketosteroid reductase (17-KSR) and aromatase enzyme activities. Adult hypophysectomized, gonadotropin-treated Fisher rats were rendered hyperprolactinemic by isografting pituitaries under the kidney capsule. The controls received skeletal muscle. All rats were sacrificed 7 days later and serum PRL was measured in each animal. PRL levels were 198 +/- 14 ng/ml in the hyperprolactinemic rats and 4.3 +/- 0.6 ng/ml in the controls (P less than 0.001). The testes were resected, pooled according to PRL levels, and microsomes were prepared from each pool. The activities of the 3 beta-HSD, 17-OH, 17,20-D, 17-KSR and aromatase were measured using as substrates 14C dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone, respectively. Hyperprolactinemia was associated with significant decreases in 3 beta-HSD, 17-OH, 17,20-D, 17-KSR and aromatase activities when compared to controls (P less than 0.005). We conclude that prolactin may have a direct effect on rat testicular steroidogenesis which appears to be independent of changes in gonadotropin secretion.     

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Rat testicular interstitial fluid contains mediators of vasopermeability

An intradermal injection of testicular interstitial fluid (IF) produced a marked increase in vasopermeability in a dose-dependent manner. Likewise bovine follicular fluid caused a smaller but significant response. The effect of IF was associated with accumulation of polymorphonuclear leucocytes (PMNs) inside the dermal venules and with their adherence to the venular endothelium. A minor but significant response was noticed after injecting anterior chamber fluid, but there was no response after an injection of amniotic fluid or serum intracutaneously. Destroying the Leydig cells with ethane dimethanesulphonate did not change the vasopermeability-increasing effect of IF, but after denaturation of IF proteins the effect was diminished by about 50%. Intravenous administration of hCG did not increase the ability of IF to cause the effect. These results suggest that rat testicular interstitial fluid contains mediators of vasopermeability, probably specific for the testis and also follicular fluid. The vasopermeability effect of IF does not seem to depend on the collecting time or on Leydig cells and is at least partly mediated by PMNs which are seen in the dermal venules shortly after an injection of IF.     

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.     

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.     

Mapping the testicular interstitial fluid proteome from normal rats

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low‐abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI‐MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.     

Altered testicular microsomal steroidogenic enzyme activities in rats with lifetime exposure to soy isoflavones

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c17 (CYP17), 17β-hydroxysteroid dehydrogenase (17β-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5-reducatase was assayed on PND 28. At PND 28, 3β-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0 mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.     

Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats

In order to find out the effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity, male rats fed with ethanol 1.6g/kg body weight per day for four weeks were studied. Besides a drastic reduction in body and testis weight, there was decrease in ascorbic acid, reduced glutathione and activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase in the testicular tissue of the treated animals. Simultaneously, there was increase in lipid peroxidation and glutathione S-transferase activity. Activities of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase were also found decreased in the treated animals. The results indicate that chronic ethanol administration resulted in increase in oxidative stress and decrease in the activities of steroidogenic enzymes in the rat testes.     

Changes in isoproterenol-stimulated adenylate cyclase activity in rat testicular tissue during cryptorchidism and after orchidopexy

Cryptorchidism was associated with increased responsiveness of the isoproterenol-sensitive adenylate cyclase in membrane particles from rat testis. Abdominal testes from uni- and bilaterally cryptorchid rats showed the same activities. The change in isoproterenol-responsive adenylate cyclase was independent of the age at which the animals were made cryptorchid. The isoproterenol response was maximal 3-4 weeks after the rats were made cryptorchid. By 2-3 months after orchidopexy the isoproterenol response in the rat testis had decreased to normal control values.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

Inhibition of testosterone secretion by S-petasin in rat testicular interstitial cells

S-petasin, a kind of sesquiterpene ester, is the anti-inflammatory ann analgesic component of the butterbur (Petasites hybridus). The clinical benefit of S-petasin is the spasmolytic activity, but its side effects on the reproductive endocrinology are not clear yet. The present study was to explore the effects of S-petasin on the secretion of testosterone in vivo and in vitro. We found that single intravenous injection of S-petasin (1 microg/kg) decreased basal plasma testosterone concentration in adult male rats. The enzymatically dispersed rat testicular interstitial cells were incubated with S-petasin (0-4.3 x 10(-5)M) in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (adenylyl cyclase activator, 10(-5) M), and androstenedione (testosterone biosynthesis precursor, 10(-9) M) at 34 degrees C for 1 h. The concentrations of testosterone in the incubation medium were measured by radioimmunoassay. S-petasin at 4.3 x 10(-7) M was effective to reduce the basal and hCG-stimulated release of testosterone in rat testicular interstitial cells. The stimulatory effects of testosterone secretion induced by forskolin and androstenedione were significantly reduced by S-petasin at 4.3 x 10(-5) M and 4.3 x 10(-6) M, respectively. These results suggest that S-petasin inhibits the production of testosterone in rat testicular interstitial cells in part through diminishing the activities of adenylyl cyclase and 17-ketosteroid reductase.     

Stimulation of Leydig cell function by a polypeptide present in testicular interstitial fluid

Rat testicular interstitial fluid (IF) contains a polypeptide factor(s) which when added to Percoll-purified rat Leydig cells in vitro, enhances both basal and hCG-stimulated production of testosterone in a dose-dependent manner. To assess the possible physiological significance of this factor(s), its levels in testicular IF from individual rats were assessed following the suppression of intratesticular levels of testosterone by either (a) treatment with anti-LH, (b) induction of unilateral cryptorchidism, or (c) treatment with ethane-1,2-dimethane sulphonate (EDS). These treatments suppressed testosterone levels in IF by 80 to 99% and in each instance levels of the IF-factor were increased. This increase occurred as early as 5h after anti-LH treatment, but restoration of normal intratesticular levels of testosterone at 20-40 h after anti-LH treatment by testosterone injection failed to normalize levels of the IF-factor. In contrast, injection of LH into normal rats raised IF testosterone to supranormal levels and this was associated with a reduction in levels of the IF-factor, and similar effects of hCG were observed in rats pretreated with anti-LH. Therefore, whilst these studies suggest that the IF-factor(s) may be of physiological importance, its local regulation does not appear to involve a straightforward negative feedback control by testosterone.     

Inhibition of testosterone secretion by digitoxin in rat testicular interstitial cells.

Both in vivo and in vitro experiments were conducted to determined the effects of digitoxin on the secretion of testosterone, and its underlying mechanisms including testicular adenosine 3':5'-cyclic monophosphate (cAMP), and the activities of steroidogenic enzymes. Male rats were injected with digitoxin, human chorionic gonadotropin (hCG), or hCG plus digitoxin via a jugular catheter. Blood samples were collected immediately before and at 30 and 60 min after the challenge, and analyzed for testosterone by radioimmunoassay. In an in vitro study, rat testicular interstitial cells were isolated and incubated with digitoxin, hCG, 8-bromo-cAMP (8-Br-cAMP), digitoxin plus hCG, or digitoxin plus 8-Br-cAMP at 34 degrees C for 1 h. The media were collected and analyzed for testosterone. For studying cAMP accumulation, testicular interstitial cells were incubated for 1 h in the medium containing isobutyl-1-methylxanthine (IBMX) and different doses of digitoxin with the absence or presence of hCG. After incubation, cells were processed for determining cAMP content. Intravenous injection of digitoxin decreased hCG-stimulated, but not basal, plasma testosterone levels. Administration of digitoxin in vitro resulted in an inhibition of both basal and hCG- as well as 8-Br-cAMP-stimulated release of testosterone. In addition, digitoxin diminished hCG-stimulated cAMP accumulation in rat testicular interstitial cells. Furthermore, digitoxin inhibited the activity of cytochrome P450 side chain cleavage enzyme (P450scc) but failed to affect the activities of other steroidogenic enzymes. Taken together, these results suggest that the acute inhibitory effect of digitoxin on the testosterone production in testicular interstitial cells involves, at least partly, an inefficiency of post-cAMP events, and a decrease of P450scc activity.     

The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats

Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function. Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism. Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.