Intratesticular injection of [D-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.     

Stimulation of leydig cell testoterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone

Injection of a luteinizing hormone-releasing hormone (LHRH) agonist into 55-day-old male rats which had been hypophysectomized 3 days earlier resulted in a 10- to 30-fold increase in the levels of testosterone in serum and testicular interstitial fluid (IF) in the 4h following injection. The levels achieved were within or above the normal range for intact untreated rats of this age. In similar animals, injection of LHRH agonist also enhanced the serum testosterone response to injected hCG at $\text{1}\text{1}\text{2}\text{h}$, but not at later times after injection, and by 24h reduced IF levels of testosterone suggested that LHRH agonist had begun to inhibit stimulation by hCG. $\text{In vitro}$, dispersed Leydig cells from untreated hypophysectomized rats showed a 2-fold increase in testosterone responsiveness to LHRH agonist when compared to cells from intact rats, and this change was associated with an 80% increase in the number of Leydig cell LHRH-receptors.     

Intratesticular distribution of testosterone in rats and the relationship to the concentrations of a peptide that stimulates testosterone secretion

Methods have been established and validated for quantitative assessment of the distribution of testosterone in the testis, by measurement of testosterone concentrations in whole testis, in isolated seminiferous tubules and in testicular interstitial fluid. These measurements were made in individual rats injected 2-40 h previously with saline (0.9% NaCl) or a potent antiserum to ovine LH. Testosterone concentrations in interstitial fluid and seminiferous tubules were closely correlated (r = +0.98; n = 60) and their relationship was log linear over a 200-fold range. However, although the concentrations of testosterone in interstitial fluid and seminiferous tubules decreased progressively with time after LH antiserum injection, this decrease was far more pronounced for interstitial fluid. In association with this change there was a significant increase in the amounts of a locally-produced factor in interstitial fluid which stimulates basal and hCG-stimulated testosterone production by isolated purified Leydig cells. This increase was reversed by injection of hCG but not by peripheral injection of a dose (20 mg) of testosterone propionate which restored normal intratesticular concentrations of testosterone. It is concluded that the tubular 'conservation' of testosterone, which occurs as interstitial fluid levels of this steroid decrease, may be a consequence of restricted diffusion of testosterone out of the tubules, but is also associated with increased amounts of a peptide stimulator of testosterone production.     

Modulation of testicular functions by testicular opioid peptides.

The possible physiological role of testicular opioid peptides in the control of testicular functions has been studied. In neonatal rats intratesticular administration of opiate receptor antagonists (naloxone, nalmefene) stimulates Sertoli cell proliferation and secretion. Both in adult and neonatal rats local injection of the testis with opiate receptor antagonists or with beta-endorphin antiserum results in a decrease in steroidogenesis in long-term studies. Treatment of neonatal testis with an enkephalin analogue induces a short-term suppression of testosterone secretion. Further studies were carried out to investigate whether the above described local effects of opiate agonist or antagonist on testicular function are under the regulatory control of testicular nerves. Partial denervation of the testis was performed by testicular injection of 6-hydroxydopamine (a neurotoxin degenerating sympathetic neural structures) or by vasectomy (cutting the inferior spermatic nerve). If testicular administration of opioid agonist or antagonist was combined with partial denervation of the testis, the effects of pharmacological agents influencing testicular opioid level were not evident. The data indicate that opioid peptides synthesized in the testis are components of the intratesticular regulatory system and that local opioid actions are modulated by testicular nerves.     

Evidence for a major role of inhibin in the feedback control of FSH in the male rat

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1.5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studied in Paraplast-embedded tissue stained with PAS and haematoxylin-eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = -0.70, P less than 0.001) and testicular (r = -0.66, P less than 0.001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0.48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 microliters groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7-14%) of testicular testosterone and unaltered serum FSH concentrations.     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

Prolonged stimulation of adenylate cyclase activity and testosterone production by cholera enterotoxin with suppression of gonadotropin release in rats.

Endocrine effects of cholera enterotoxin (CET) on male gonads were investigated in normal and hypophysectomized rats. After intratesticular injection of 5 micrograms of CET in the bilateral testes of normal rats, serum testosterone concentration remarkably increased after 24 hr, remained significantly elevated for at least 3 days and returned to the control level in 7 days. Serum LH level decreased in the undetectable range after 1--3 days; serum FSH level also significantly decreased after 3 days. Both gonadotropin levels increased 28 days after the injection, when the CET-injected testis decreased in weight and was accompanied by marked loss of germinal cells. When 5 micrograms of CET was injected intratesticularly in the bilateral testes of hypophysectomized rats, adenylate cyclase activity of a CET-injected testis was remarkably stimulated after 6 hr, remained four times elevated for at least 3 days and returned to the control level in 7 days. In relatively good accordance with the increase in adenylate cyclase activity, testosterone content remarkably enhanced in the CET-injected testis. These in vivo data indicate that the intratesticular injection of CET prolongedly stimulates the adenylate cyclase activity of testicular cells including Leydig cells and increases testosterone production, and suggest that the prolonged enzyme stimulation results in the sustained elevation of serum testosterone concentration for at least 3 days, causing the stimulation of the negative feedback mechanism of hypophysealtesticular axis to decrease serum LH levels in the undetectable range.     

Testosterone, androstenedione, progesterone and 17 alpha-hydroxyprogesterone in plasma and testes of immature rats under basal conditions and after hCG stimulation. Effect of bilateral cryptorchidism

Rats were made bilaterally cryptorchid at 21 days of age; sham-operated rats were used as controls. At 35 days, the animals were injected i.m. with saline or with 10 IU hCG. Progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were measured in both testes and plasma under basal conditions and 2, 4, 8, 12, 24 and 72 h respectively after injection. The plasma levels and intratesticular contents of the steroids were generally lower in cryptorchid rats. The patterns of the steroid response to hCG were similar in both groups: in the testes and in the plasma, they increased acutely following hCG injection (except testicular androstenedione), then, after 72 h, returned to normal values in the plasma but remained higher than the basal values in the testes. These results suggest that there are no gross abnormalities in the testicular steroidogenic pathways and that the mechanism of action of hCG on the Leydig cells is unaltered in bilaterally cryptorchid immature rats.     

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

Background

Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO.

Objective

The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion.

Method and Results

EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect.

Conclusions

We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.     

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.     

Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis

The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2x2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N(omega)-nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2x600 microg/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes.     

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.     

Differential effects of follicle-stimulating and luteinizing hormones on testosterone production by mouse testes

In adult mice, direct intratesticular injection of ovine follicle-stimulating hormone (o-FSH-13; AFP 2846-C, from NIAMDD, less than 1% LH contamination) at 10, 100 or 1000 ng significantly elevated concentrations of testosterone (T) within the testis. These effects were rapid, with peak values attained by 15 min, and transient, with return to values comparable to that in the contralateral, saline-injected testis within 90 min. Intratesticular injection of FSH (1 microgram) significantly increased testicular T levels in 15- and 60-day old mice. This contrasted with the effects of intratesticular administration of human chorionic gonadotropin (hCG), which stimulated T production significantly at 30 days of age through adulthood. In adult mice, the equivalent LH to the possible contamination in the FSH preparation (1 ng) had no effect. Intratesticular injection of 10 ng LH produced comparable stimulation to that by 100 ng FSH (approximately 7-fold). Systemic pre-treatment with a charcoal-treated porcine follicular fluid (PFF) extract for 2 days reduced plasma FSH levels [86 +/- 17 (5) vs 700 +/- 8 (6); P less than 0.05], but had no effect on plasma LH. Twenty-four hours after the last treatment, the response to intratesticular injection of hCG (2.5 mIU), FSH (100 ng) or LH (10 ng) was also significantly attenuated in these mice. Intratesticular injection of PFF had no direct effect on testicular T levels. In vitro T production in the presence of hCG, LH or FSH were differentially affected by the concentrations of calcium (Ca2+) or magnesium (Mg2+) in the incubation media. The stimulatory effects of FSH were apparent at significantly lower levels of Ca2+ or Mg2+, than were those of LH or hCG. The results of these studies indicate that FSH is capable of stimulating testicular T production. Furthermore, the responsiveness to FSH is qualitatively different than that to LH/hCG in terms of the age pattern, as well as the dependence on Ca2+ or Mg2+. In addition, plasma FSH levels appear to influence testicular responsiveness to direct exogenous administration of gonadotropins. These studies indicate that FSH stimulation of T production can be differentiated from those of LH, and that these effects of FSH can be observed under physiological conditions.     

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.     

Effects of testosterone on testicular inhibin and fluid production in intact and hypophysectomized adult rats

Rats were given s.c. implants of high (HT) or low (LT) doses of testosterone and 10 days later hypophysectomy or sham-operation was performed. The rats were killed after 50 days. Unilateral efferent duct ligation was performed 16 h before death to measure seminiferous tubule fluid production and the increment in testicular inhibin values (inhibin production). Inhibin levels in testis cytosols were measured by a pituitary cell culture bioassay. The LT implants maintained serum testosterone at control values and decreased testicular weight whereas HT implants raised serum testosterone 3-fold and maintained testicular weight at 75-85% of pretreatment levels. In intact rats, LT implants caused no change in testicular inhibin content but decreased inhibin production; no significant changes occurred with HT implants. After hypophysectomy both values were significantly suppressed and could not be maintained by HT or LT implants. However, the HT implants partly restored inhibin production despite their inability to influence testicular inhibin content. In contrast, tubule fluid production depended mainly on intratesticular testosterone levels and occurred normally in intact or hypophysectomized rats with HT but not LT implants. These results indicate that inhibin and seminiferous tubule fluid production, both functions of the Sertoli cell, are under different hormonal control. The maintenance of inhibin production by the testis requires the support of pituitary hormones, presumably FSH, while seminiferous tubule fluid production requires testosterone, presumably through LH stimulation of Leydig cells. These findings are consistent with the hypothesis that inhibin is produced in response to trophic stimulation by FSH.     

Effects of various doses of testosterone propionate on intratesticular and plasma testosterone levels and maintenance of spermatogenesis in adult hypophysectomized rats.

Adult hypophysectomized rats were maintained on different regimens of testosterone propionate (TP) treatment for 27 days (0.2, 0.4, 0.6 and 1 mg/day) and autopsied 16 hours after the last injection. Blood samples were taken, sex organs were weighed and one testis from each animal was fixed in Bouins fluid for histologic analysis. The other testis and blood were used for testosterone (T) determinations. Both testicular and plasma T were below detectable levels in hypophysectomized control rats. The plasma T level showed a dose response relationship with increasing dose of TP but such was not the case for intratesticular T concentrations. Qualitative and quantitative evaluation of testis sections showed that spermatogenesis was incomplete in rats receiving 0.2 mg TP/day characterized by the absence of step 15 to 19 spermatids, degeneration of some pachytene spermatocytes and a significantly lower yield of B type spermatogonia. Analysis of testis sections from animals treated with 0.4 to 1 mg TP/day showed complete maintenance and maturation of pachytene spermatocytes, meiosis and spermiogenesis. However, even with the highest dose of TP (1 mg/day) the total yield of B type spermatogonia was only about 58% of the intact controls. It is concluded that at least 0.4 mg/day of exogenous TP is essential for qualitative maintenance of spermatogenesis in hypophysectomized rats with an intratesticular T concentration of 17 to 18 ng/gm testis.     

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.     

Alteration of free sulphydryl content of rat sperm heads by suppression of intratesticular testosterone

Subcutaneous injections of testosterone propionate to adult male rats at a dose of 2.5 or 10 mg/kg body weight, 3 times per week for 7 weeks, resulted in a 75% reduction in serum LH and more than 50% reduction in intratesticular testosterone concentration, but serum FSH levels remained unchanged. The free -SH content, measured as iodo[14C]acetamide binding, increased by 70-100% in testicular sperm heads after suppression of testicular testosterone, and by 25-30% in caput epididymal sperm heads but was decreased by 70-80% in cauda epididymal sperm heads. These results demonstrate an alteration in the oxidative state of sperm nuclear basic proteins, suggesting incomplete nuclear maturation. These changes may be specific for the suppression of intratesticular testosterone, thus illustrating the androgen dependency of sperm head maturation. The contrast effects noted between the iodo[14C]iodoacetamide binding by the caput and the cauda epididymal sperm heads indicate that testosterone propionate treatment may affect the mechanisms regulating the oxidation of the sulphydryl residues in sperm heads during epididymal transit. This alteration may not directly relate to the tissue androgen concentrations.     

Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas?

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.     

The effect of testosterone withdrawal and subsequent germ cell depletion on transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the adult rat testis.

We have examined the effects of decreasing intratesticular testosterone concentration and of decreasing germ cell number on levels of transferrin mRNA and sulfated glycoprotein (SGP)-2 mRNA in the adult rat testis. Intact rats received implants of testosterone- and estradiol-filled capsules to suppress LH secretion from the pituitary, thereby suppressing Leydig cell testosterone production. The levels of intratesticular testosterone declined 70% to 20 ng/ml within 3 days, were reduced further to approximately 15 ng/ml by 14 days, and subsequently reached a minimum of about 10 ng/ml. In contrast, the number of elongated spermatids per testis remained unchanged through 14 days, then declined to fewer than 20% of normal between 14 and 28 days, and reached zero by 56 days postimplantation. Likewise, both pachytene spermatocytes and round spermatids declined only after 14 days postimplantation. Northern blots of testicular RNA showed that Sertoli cell transferrin mRNA per testis decreased markedly between 14 and 28 days postimplantation. However, SGP-2 mRNA per testis was unchanged over the time course of the experiment. The decrease in transferrin mRNA, concomitant with germ cell loss, suggests that this mRNA is regulated by the number of germ cells in the testis and not directly by testosterone. In contrast, the constant level of SGP-2 mRNA in the face of reduced intratesticular testosterone and the subsequent loss of germ cells suggests that this mRNA is constitutively maintained in the adult rat testis.