Regulation of testicular function in men: implications for male hormonal contraceptive development

In the adult male, the testes produce both sperm and testosterone. The function of the testicles is directed by the central nervous system and pituitary gland. Precise regulation of testicular function is conferred by an elegant feedback loop in which the secretion of pituitary gonadotropins is stimulated by gonadotropin hormone-releasing hormone (GnRH) from the hypothalamus and modulated by testicular hormones. Testosterone and its metabolites estradiol and dihydrotestosterone (DHT) as well as inhibin B inhibit the secretion of the gonadotropins both directly at the pituitary and centrally at the level of the hypothalamus. In the testes, LH stimulates testosterone synthesis and FSH promotes spermatogenesis, but the exact details of gonadotropin action are incompletely understood. A primary goal of research into understanding the hormonal regulation of testicular function is the development of reversible, safe and effective male hormonal contraceptives. The administration of exogenous testosterone suppresses pituitary gonadotropins and hence spermatogenesis in most, but not all, men. The addition of a second agent such as a progestin or a GnRH antagonist yields more complete gonadotropin suppression; such combination regimens effectively suppress spermatogenesis in almost all men and may soon bring the promise of hormonal male contraception to fruition.     

The evolution of hormonal therapy for prostatic carcinoma

It is well recognized that testosterone has a number of untoward effects on prostatic carcinoma and that castration is associated with significant tumor shrinkage and resolution of symptoms of advanced prostatic carcinoma. Approaches to hormonal therapy have evolved significantly over the last several decades. Initially castration was utilized, which provided effective reduction of testicular androgens, but with adverse psychological factors. The next approach was utilization of diethylstilbestrol, but with significant cardiovascular toxicity in higher doses. The development of the luteinizing hormone-releasing hormone agonists provided an improvement in pharmacologic castration; however, they are associated with a transient testosterone surge and the potential for exacerbation of clinical manifestations of advanced prostate carcinoma (the so-called "testosterone flare"). Recently, gonadotropin-releasing hormone (GnRH) antagonists have been investigated. Abarelix is a pure GnRH antagonist that blocks the anterior pituitary receptor, resulting in prompt and significant reduction not only of luteinizing hormone but also follicle-stimulating hormone. This results in castrate levels of testosterone while avoiding the testosterone surge.     

Seminal parameters in rhesus monkeys under physiological conditions and in studies for male fertility control using FSH antibodies or LH-RH-agonists

To characterize the male rhesus monkey as a nonhuman primate model for human testicular functions, parameters of exocrine and endocrine testicular function were monitored in 16 adult male-rhesus monkeys for 1 and 5 years respectively. Testicular volumes in-season (October–January) were twice as great as in out-of-season animals (March–June). Ejaculations, both spontaneous and electrostimulated, ceased out-of-season. In 37 ejaculates obtained by electrostimulation in-season, sperm counts ranged from 110–1,100 million/ejaculate, 65% of sperm were motile and 60% were normally formed. Testicular histology showed regression of spermatogenesis out-of-season, with the diameter of the tubules being only one third of that in-season. Circannual changes in exocrine testicular function were accompanied by parallel fluctuations in pituitary and endocrine testicular functions, as evidenced by basal hormone levels and the production rate of testosterone, as well as the response to LH-RH throughout the year. As FSH is required for spermatogenesis in rhesus monkeys, we initiated a study on the long-term effects of active immunization against FSH as a possible means of fertility control. After the first 2 years of observation we can conclude that the production of specific antibodies to FSH results in suppression of spermatogenesis (oligospermia and occasional azoospermia) without affecting endocrine function. The lack of adverse side effects may encourage further investigations on this approach to fertility control. LH-RH-agonists exert degenerative effects on testicular function in rats via a down regulation of the pituitary and testis. A 12 week treatment of four adult monkeys in-season with Hoe 766 (Hoechst; 4μg/day for eight weeks, 20μg/day for 4 weeks sc) did not reveal any change in sperm counts or motility, although some pituitary desensitization was evident. It remains to be investigated whether even higher doses may result in a suppression of spermatogenesis.     

Using gonadotropin-releasing hormone (GnRH) and GnRH analogs to modulate testis function and enhance the productivity of domestic animals

Gonadotropin releasing hormone (GnRH) controls the activity of the gonadotrope cells of the pituitary gland and, as a consequence, is a critical component of the endocrine cascade that determines the growth, development, and functional activity of testicular tissue. The use of GnRH and GnRH analogs is common in domestic animal production systems. Although GnRH and GnRH analogs are most commonly used to control the fertility and reproductive events in female animals, GnRH agonists and antagonists are increasingly used to modulate the fertility, behavior, and productivity of male animals as well. This review will focus on recent advances in this use of GnRH agonists and antagonists.     

GnRH analogues--agonists and antagonists

GnRH analogues have achieved widespread clinical use for the control of reproduction in animals. Over 2000 analogues of GnRH have been developed and tested over the last 30 years. Paradoxical anti-fertility effects are seen when the more potent agonists are delivered continuously to animals. The evaluation of agonist potency depends largely on the model used and wide varying potencies are reported for the same agonist. The design of analogues has centered on improving the receptor-binding and subsequent activation for agonists. Antagonists have been produced with strong receptor binding but without activation. Deslorelin is classified as a superagonist, with a potency perhaps 100 times that of GnRH. The interactions between agonist potency, dose and duration of treatment largely determine whether pro- or anti-fertility effects are induced. Due to the peptide nature of the synthetic analogues oral administration and potential gastrointestinal enzymatic degradation poor bioavailability results necessitating a parenteral delivery system. Some GnRH antagonists have been associated with significant histamine release, inhibiting their widespread use. More recently, antagonists have been developed that avoid this side effect without compromising potency. However the GnRH antagonist development has lagged behind that of the agonists, in part related to their high cost of production. In conclusion, GnRH agonists have achieved widespread clinical use in animals for controlling reproduction in either pro- or anti-fertility roles, yet antagonist development has been slower.     

Effects of long-term treatment with the GnrH agonist deslorelin (Suprelorin®) on sexual function in boars

Immunization against GnRH has been proven effective for boar taint removal, and long-term treatment with GnRH analogues has been shown to suppress GnRH dependent reproductive processes in several species. This study was conducted to treat boars (n = 5) with Suprelorin®, i.e., an implant that contains 4.7 mg of the long-acting GnRH analogue deslorelin, and to test the effects on sexual function. Insertion of the implant occurred at the age of 5 weeks and animals were observed until market age at 26-27 weeks. Surgically castrated (n = 4) and intact boars (n = 3) served as controls. Testes growth was markedly reduced and steroidogenesis (testosterone, estrone, estrone sulphate, estradiol 17β) as well as spermatogenesis suppressed in 4 of 5 GnRH treated boars, respectively. The remaining fifth boar resumed testes growth after week 17 of age and had high hormone concentrations when tested at weeks 26 and 27. Restoration of spermatogenesis was observed at 34 weeks of age. There were no effects of treatment on general health, nor were there local inflammatory reactions. Results indicate that suppression of sexual functions in boars due to long-term treatment with the GnRH agonist deslorelin through an implant such as Suprelorin® is possible and can last for several months up to market age; thus it has potential as an alternative to other methods used for boar taint removal. Because the maximum duration of suppression seems to vary between boars, further studies are necessary to refine the treatment.     

Effects of an LH-RH analogue in male rats pretreated with oestradiol benzoate.

Treatment of adult male rats with oestradiol benzoate (OB) for 21 days significantly decreased the body, testicular and accessory sex organ weights but increased anterior pituitary weight. OB treatment also significantly suppressed circulating FSH and LH levels as well as plasma and testicular concentrations of testosterone. The seminiferous tubules and interstitial cells were partly atrophied, and there was some effect on spermatogenesis, with step 14 to 19 spermatids being fewer than normal. Rats treated with OB for 21 days were then treated daily with LH-RH analogue ((D-Leu6, des-Gly-NH2(10))-LH-RH-ethylamide), to see if testicular function could be recovered. Circulating gonadotrophins were significantly elevated, testicular histology was normal and testicular and plasma testosterone concentrations and the accessory sex organ weights remained suppressed. These results suggest possible extra-pituitary effects of the LH-RH analogue, including a direct action on the testes and/or accessory sex organs.     

Effect of an LHRH agonist on pituitary and testicular function in rhesus monkeys

Male rhesus monkeys were given 100 micrograms [(imBzl)-D-His6,Pro9-NEt]-LHRH (LHRH-A), a potent LHRH agonist, s.c. daily for 40 weeks. The first dose of LHRH-A caused acute increases (2-4 h after injection) in serum LH (50-fold), FSH (2 X 5-fold) and testosterone (15-fold) concentrations. Chronic treatment led to a 95% decrease in LH and FSH responses. In spite of a marked decrease in LH response the effect on testosterone response was less evident. Administration of 50 i.u. hCG to control and LHRH-A-treated animals showed that the testicular steroidogenic response was unimpaired by the chronic treatment. Evaluation of the electroejaculated semen at regular intervals showed that there was no consistent reduction in the sperm count of LHRH-A-treated monkeys. Testicular biopsies showed that normal spermatogenesis was occurring in all treated animals, but testicular volume was significantly decreased. These results suggest that, in rhesus monkeys, the pituitary is more susceptible to desensitization by chronic LHRH agonist treatment than are the testes, and that LHRH agonists do not have direct antitesticular effect in rhesus monkeys.     

Effect of continuous infusion of a low dose of GnRH antagonist on serum LH and testosterone concentrations, spermatogenesis and semen quality in the rhesus monkey (Macaca mulatta)

Treatment of 4 adult male rhesus monkeys for 8-12 months with 100-400 micrograms of a GnRH antagonist/day by means of using osmotic minipumps led to suppressed serum concentrations of LH and testosterone followed by various degrees of recovery toward pretreatment values. The serum LH response to a challenge of native GnRH was reduced by 30-75% during antagonist treatment. The serum testosterone response to GnRH was exaggerated above the response in the pretreatment period, suggesting hypersensitivity of the testis to gonadotrophin. Antagonist administration under these conditions did not alter body weight or abolish ejaculatory response. Antagonist infusion caused a 96% decrease in sperm counts. Spermatozoa recovered during the final month of antagonist treatment showed a reduced ability to penetrate denuded hamster ova. Testicular biopsies performed at the end of antagonist treatment revealed persistent spermatogenesis. However, the cellularity of the seminiferous tubules was decreased below that of pretreatment biopsies. The results of this study suggest that the amount of testosterone needed to maintain normal spermatogenesis is greater than that needed to maintain electroejaculatory response in monkeys.     

Negative feedback regulation of the secretion and actions of gonadotropin-releasing hormone in males

This minireview considers the state of knowledge regarding the interactions of testicular hormones to regulate the secretion and actions of GnRH in males, with special focus on research conducted in rams and male rhesus monkeys. In these two species, LH secretion is under the negative feedback regulation of testicular steroids that act predominantly within the central nervous system to suppress GnRH secretion. The extent to which these actions of testicular steroids result from the direct actions of testosterone or its primary metabolites, estradiol or dihydrotestosterone, is unclear. Because GnRH neurons do not contain steroid receptors, the testicular steroids must influence GnRH neurons via afferent neurons, which are largely undefined. The feedback regulation of FSH is controlled by inhibin acting directly at the pituitary gland. In male rhesus monkeys, the feedback regulation of FSH secretion is accounted for totally by the physiologically relevant form of inhibin, which appears to be inhibin B. In rams, the feedback regulation of FSH secretion involves the actions of inhibin and testosterone and interactions between these hormones, but the physiologically relevant form of inhibin has not been determined. The mechanisms of action for inhibin are not known.     

Sustained impairment of pituitary and testicular function in prostatic cancer patients treated with a depot form of a GnRH agonist

Seven patients suffering from prostatic cancer were treated with a slow-release D-Trp-6-LHRH preparation for a period of 24-32 months. LH, FSH, PRL and testosterone levels were evaluated before and at the end of treatment and then 40 days later. Baseline and GnRH-, TRH-, and HCG-stimulated hormonal values decreased after treatment. The possibility that a long-term treatment with GnRH analogues induces a sustained suppression of pituitary and testicular function is suggested.     

Physiological role of putative testicular gonadotrophin releasing hormone (GnRH)

Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non-existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testicular GnRH-like factors: characterization of biologic activity

Observations that gonadotropin releasing hormone and its agonists directly inhibit gonadal function by binding to receptors on the Leydig cells had led to search for testicular GnRH-like peptide(s). This communication presents evidence that GnRH-like factors isolated from rat testis by immunoaffinity chromatography and previously characterized by radioimmunoassay and radioreceptor assay possess biologic activity. The partially purified material led to dose dependent inhibition of oLH stimulated testosterone production in a mixed Sertoli-Leydig cell monolayer culture. Pre-incubation of the cells with a potent GnRH antagonist prevented the inhibitory effects of the partially purified material suggesting that inhibition of oLH stimulated testosterone production may be receptor mediated.     

Pituitary and Leydig cell function in boars actively immunized against gonadotrophin-releasing hormone

Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 micrograms GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 microgram/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0.05 and 0.2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P less than 0.05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.     

Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists

The role of pituitary gonadotropins in the regulation of spermatogenesis has been unequivocally demonstrated, although, the precise mechanism of this regulation is not clearly understood. Previous studies have shown that specific immunoneutralization of LH/testosterone caused apoptotic cell death of meiotic and post-meiotic germ cells while that of FSH resulted in similar death of meiotic cells. In the present study, the death process of germ cells has been characterized by depleting both FSH and testosterone by administering two different potent GnRH antagonists, Cetrorelix and Acyline to both rats and mice. Pro-survival factors like Bcl-2 and Bcl-x/l were unaltered in germ cells due to GnRH antagonist treatment, although a significant increase in several pro-apoptotic markers including Fas and Bax were evident at both protein and RNA levels. This culminated in cytochrome C release from mitochondria and eventually increase in the activity of caspase-8 and caspase-3. These data suggest that both extrinsic and intrinsic apoptotic death pathways are operative in the germ cells death following decrease in FSH and testosterone levels. Multiple injections of GnRH antagonist resulted in complete disappearance of germ cells except the spermatogonial cells and discontinuation of the treatment resulted in full recovery of spermatogenesis. In conclusion our present data suggest that the principal role of FSH and testosterone is to maintain spermatogenic homeostasis by inhibiting death signals for the germ cells.     

Pituitary desensitization and the regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors following chronic administration of a superactive GnRH analog and testosterone

We recently demonstrated that chronic daily administration of a superactive GnRH analog to intact rats resulted in an initial stimulation of serum LH levels with a subsequent return of LH levels to baseline at a time when testosterone levels were marked decreased. These data demonstrated pituatary desensitization following chronic GnRH analog treatment. Administration of GnRH analog with a dose of testosterone which did not markedly lower serum LH levels when administered alone prevented the stimulation of LH secretion by analog. The present studies were undertaken to determine the effects of GnRH analog and testosterone administration on the regulation of pituitary GnRH receptors. Pituitary GnRH receptor binding was increased by analog treatment alone at 20 days and returned to control levels at 40 and 60 days of treatment in parallel to the observed changes in serum LH, demonstrating that one mechanism by which chronic GnRH analog treatment leads to pituitary desensitization is down-regulation of pituitary GnRH receptors. Testosterone administration alone decreased pituitary GnRH receptor binding. Combined GnRH analog and testosterone administration prevented the increase in pituitary GnRH receptors observed with analog administration alone. These studies demonstrate that changes in pituitary GnRH receptor binding correlate with changes in serum LH and that the stimulatory effects of analog administration on LH are sensitive to inhibition by small doses of testosterone.     

Pulsatile gonadotropin-releasing hormone treatment of men with idiopathic hypogonadotropic hypogonadism

OBJECTIVES/METHODS: To induce testicular growth and spermatogenesis, 11 patients with idiopathic hypogonadotropic hypogonadism were treated with long-term subcutaneous pulsatile gonadotropin-releasing hormone (GnRH) administration. Three patients had a history of undescended testes. Patients who did not respond to therapy with a sufficient increase in serum testosterone or spermatogenesis were offered additional injections with hCG or, after discontinuation of GnRH, either combined therapy with hCG and hMG or recombinant FSH. RESULTS: During treatment testicular volume and serum levels of FSH, LH and testosterone increased. Semen analysis revealed the presence of spermatogenesis in 9 of the 11 patients (8 on GnRH alone and in 1 when hCG/hMG was subsequently instituted), and 7 pregnancies have resulted thus far. CONCLUSION: Pulsatile GnRH therapy is a well-tolerated and effective therapy for the induction of spermatogenesis in some men with idiopathic hypogonadotropic hypogonadism. It appears that a significant fraction of them should be treated for a minimum of 1-2 years to maximize testicular growth and achieve spermatogenesis. Cryptorchidism was a negative prognostic factor.     

Efficacy of Pulsatile Gonadotropin-Releasing Hormone Therapy in Male Patients: Comparison between Pituitary Stalk Interruption Syndrome and Congenital Hypogonadotropic Hypogonadism

ObjectivePulsatile gonadotropin-releasing hormone (GnRH), widely used to induce spermatogenesis in congenital hypogonadotropic hypogonadism (CHH) patients, can restore the pituitary-testis axis function in men with pituitary stalk interruption syndrome (PSIS). This retrospective study aimed to compare the differences in the long-term efficacy of pulsatile GnRH therapy on PSIS and CHH.MethodsPatients with PSIS (n = 25) or CHH (n = 64) who received pulsatile GnRH therapy for ≥3 months were included in this retrospective study. The rate of successful spermatogenesis, the median time to achieve spermatogenesis, serum gonadotropins, total testosterone, and testicular size were compared.ResultsBaseline characteristics were comparable except for the lower basal testosterone, triptorelin-stimulated peak luteinizing hormone (LH), and follicle-stimulating hormone in patients with PSIS. With similar duration of treatment and a significantly higher GnRH dose (P < .001), small increments in LH (2.82 [1.4, 4.55] vs 5.89 [3.88, 8.02] IU/L; P < .001), total testosterone (0.38 [0, 1.34] vs 2.34 [1.34, 3.66] ng/mL; P < .001), and testicular volume (5.3 ± 4.5 vs 8.8 ± 4.8 mL, P < .05) were observed. However, spermatogenesis rate (52.0% vs 70.3%, P > .05), median time of sperm appearance (14 vs 11 months, P > .05), sperm concentration, and progressive motility were comparable. Basal testicular volume (hazard ratio, 1.13; 95% CI, 1.01-1.27) and peak LH levels (hazard ratio, 1.11; 95% CI, 1.0-1.23) were predictors for early sperm appearance.ConclusionsPulsatile GnRH therapy can improve gonad function and induce spermatogenesis in men with PSIS. However, its efficacy may be inferior to that in CHH.     

Evaluation of the possible direct effects of gonadotrophin-releasing hormone analogues on the monkey (Macaca mulatta) testis

In Exp. 1, the effect of treatment with a GnRH agonist on basal concentrations of serum testosterone and peak values of serum testosterone after administration of hCG was determined. One group of adult male monkeys was treated with a low dose (5-10 micrograms/day) and a second group with a high dose (25 micrograms/day) of a GnRH agonist for 44 weeks. Basal and peak testosterone concentrations were both significantly reduced by GnRH agonist treatment in all groups compared to untreated control animals, but the % rise in serum testosterone above basal values in response to hCG administration was unchanged by agonist treatment. In Exp. 2, the GnRH agonist (100 or 400 ng) or a GnRH antagonist (4 micrograms) was infused into the testicular arteries of adult monkeys. The agonist did not alter testosterone concentrations in the testicular vein or testosterone and LH values in the femoral vein. In Exp. 3, testicular interstitial cells from monkeys were incubated with three concentrations (10(-9), 10(-7) and 10(-5)M) of the GnRH agonist or a GnRH antagonist with and without hCG. After 24 h, neither basal nor hCG-stimulated testosterone production was affected by the presence of the GnRH agonist or antagonist. The results from all 3 experiments clearly suggest that GnRH agonist treatment does not directly alter steroid production by the monkey testis.     

Gonadotropins, their receptors, and the regulation of testicular functions in fish

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.