Suppression of spermatogenesis in a nonhuman primate (Macaca fascicularis) by concomitant gonadotropin-releasing hormone antagonist and testosterone treatment

The effects of concomitant testosterone (T)-supplementation on gonadotropin-releasing hormone (GnRH) antagonist-induced testicular regression in cynomolgus monkeys (M. fascicularis) were investigated. Four adult monkeys were infused via osmotic minipumps with daily amounts of 2 mg of a potent GnRH antagonist (N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10)-GnRH (RS-68439) for a period of 104 days. Androgen substitution was provided via T-filled Silastic capsules implanted at initiation of GnRH antagonist treatment. Within 1-4 days of GnRH antagonist administration, serum concentrations of bioactive LH became undetectable. The implants maintained serum T at 50-80% of pre-treatment levels. Sperm production decreased in three out of four monkeys. One animal became azoospermic by the 13th week of treatment and the ejaculates of two other monkeys contained less than 5 X 10(6) sperm. In the fourth monkey, spermatogenesis was less affected. Testicular histology, judging from biopsies at termination of GnRH antagonist treatment, was typical of the hypogonadotropic status in 3 of the 4 monkeys. The most affected tubules contained only spermatogonia and Sertoli cells. Although comparison with GnRH antagonist treatment alone in a previous study indicated a delay of spermatogenic inhibition with testosterone, the present study confirms the potential of GnRH antagonist for male fertility regulation.     

Induction of azoospermia in normal men with combined Nal-Glu gonadotropin-releasing hormone antagonist and testosterone enanthate.

The effects of a combined GnRH antagonist and testosterone (T) replacement regimen on gonadotropins and spermatogenesis were examined to assess its potential as a male contraceptive regimen. The potent Nal-Glu GnRH antagonist ([Ac-D2-Nal1,D4-Cl-Phe2,D3-Pal3,Arg5, D4-p-methoxybenzoyl-2-amino butyric acid6,D-Ala10]GnRH) was administered daily (7.5 mg, sc) to eight normal men for 16 weeks. T enanthate was given im starting at week 2 and every 2 weeks thereafter through week 14 of the treatment phase. Serum LH, FSH, T, and estradiol concentrations were measured frequently during the 5-week control period, the 16-week treatment phase, and the 14-week recovery phase. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. Seven of eight subjects became azoospermic by 6-10 weeks of treatment; the eighth subject, who failed to achieve azoospermia, suppressed his sperm count to 7 million/mL by week 14 (from a mean baseline of 42 million/mL) before treatment was prematurely terminated because of localized swelling at each of his injection sites. Sperm counts returned to baseline 10-14 weeks after the end of Nal-Glu administration. Seven of the eight subjects showed suppression of LH to the limit of assay detection (less than 0.2 U/L), whereas the eighth subject showed incomplete suppression. Serum bioactive and immunoreactive LH concentrations showed concordant responses. Mean serum FSH concentrations were also markedly suppressed. Serum T and estradiol concentrations declined dramatically during the first 2 weeks of Nal-Glu GnRH treatment, but returned to the normal physiological range after T enanthate replacement was initiated. Libido and sexual potency were maintained. No systemic side-effects, other than erythema and induration at injection sites, were observed. These data demonstrate that combined GnRH antagonist plus T treatment can predictably and reversibly induce azoospermia in most men and has potential as a male contraceptive regimen.     

Blockade of neonatal activation of the pituitary-testicular axis: effect on peripubertal luteinizing hormone and testosterone secretion and on testicular development in male monkeys

The objective of this study was to examine the effect of blockade of neonatal activation of the pituitary-testicular axis, using a GnRH agonist, on sexual development in male rhesus monkeys. Monkeys were treated with either a GnRH agonist (10 micrograms/day; n = 8) or vehicle (n = 9) for 112 days using osmotic minipumps beginning at 10-13 days of age. In control monkeys serum LH and testosterone concentrations during the first 3 postnatal months were similar to those in adults; they then declined to very low levels. GnRH agonist administration caused an immediate and precipitous decline in serum LH and testosterone concentrations to very low levels, and both remained low throughout the rest of the agonist administration period. Neither group had any significant elevation in serum LH or testosterone concentrations during the next 2 yr. In the control monkeys serum LH and testosterone began to rise during the third year, with a rapid increase occurring during the fall coincident with the breeding season. This peripubertal rise of LH and testosterone secretion was associated with rapid enlargement of the testes and the appearance of sperm in ejaculates. The monkeys who had received GnRH agonist had subnormal serum LH and testosterone increases, and testicular enlargement was also attenuated compared to that in the control animals during the third year of life. Semen samples were recovered from only 50% of the GnRH agonist-treated monkeys during this period, and the sperm count per ejaculate was suppressed. The serum LH responses of the GnRH agonist-treated monkeys to an iv bolus dose of GnRH (5 micrograms/kg BW) during the third year were normal. These results suggest that the induction of reversible hypogonadotropin-hypogonadism in neonatal male monkeys alters subsequent testicular development and peripubertal endocrine changes. Thus, neonatal activation of the pituitary-testicular axis may be a critical developmental event in the process of sexual development in male primates.     

Combined administration of a gonadotropin-releasing hormone antagonist and testosterone in men induces reversible azoospermia without loss of libido

GnRH antagonists suppress pituitary and gonadal function by competing with endogenous GnRH for binding to receptors on pituitary gonadotrophs. We studied the effects of GnRH antagonist administration to men in a protocol simulating a likely male contraceptive regimen combined with a low dose of testosterone. The GnRH antagonist Nal-Glu was given daily (10 mg, sc) for 20 weeks to eight normal men, and a low dose of testosterone enanthate (25 mg, sc) was given every week. Sperm counts started declining during week 4, and complete azoospermia was reached within 6-12 weeks in six of the eight subjects. Subjects 7 and 8, whose sperm counts and serum gonadotropin levels were not suppressed after 10 weeks, were given 20 mg Nal-Glu starting at week 10. One became azoospermic at week 16, while the other's total sperm counts continued declining and reached a nadir of 1.4 million by week 20. Sperm motility and viability in this subject were completely suppressed after week 14. Sperm counts returned to baseline levels 12-14 weeks after the end of Nal-Glu administration. The mean serum LH level of the first six subjects decreased from 3 +/- 03. U/L at baseline to less than 0.1 U/L until week 20, and then levels returned to baseline. FSH levels similarly decreased from a combined mean of 3.6 +/- 0.9 U/L at baseline to below 0.3 U/L after 4 weeks of Nal-Glu administration. Serum mean testosterone levels between weekly injections of testosterone enanthate ranged from 27.4 +/- 5.9 to 4.8 +/- 1.4 nmol/L, but remained in the hypogonadal range (less than 10 nmol/L) for 4 of the 7 days. None of the subjects, however, complained of decreased libido or potency, as assessed by a questionnaire. No systemic or significant local side-effects were observed, other than a minimal reaction at the injection site. These data suggest that complete sustained azoospermia can be achieved in man, without loss of libido, by chronic administration of a GnRH antagonist plus testosterone.     

Testosterone prevents complete suppression of spermatogenesis in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis)

We studied the effects of administration of a GnRH antagonist combined with testosterone (T) as an approach to male contraception as well as the role of intratesticular androgens in spermatogenesis using a nonhuman primate model. Three groups of five adult cynomolgus monkeys (Macaca fascicularis) received daily sc injections of 420-460 micrograms/kg GnRH antagonist ([Ac-D2Nal1,D4ClPhe2,DPal3,Arg5,DGlu6(AA), DALa10]GnRH) for a period of 15 weeks. T supplementation, commencing on the first day of GnRH antagonist administration, was provided by single im injection of 40 mg (group 2) or 200 mg (group 3) of the long-acting testosterone ester testosterone-trans-4-n-butylcyclohexancarboxylate (20-Aet-1). Serum LH bioactivity was undetectable within 1 week of GnRH antagonist administration in all monkeys. GnRH antagonist administration alone (group 1) reduced serum T levels into the castrate range. Forty milligrams of 20-Aet-1 maintained serum T levels in the upper range of normal monkeys, while 200 mg 20-Aet-1 maintained serum T levels about 1.5-fold above normal. The response to electroejaculation was fully maintained in all T-treated monkeys. Sperm counts in the ejaculates dropped to zero among group 1 animals within 7-10 weeks of GnRH antagonist administration. In groups 2 and 3 consistent azoospermia could not be induced, and the sperm counts were significantly (P less than 0.05) higher in group 3 than in group 2. Histologically, spermatogenesis in group 1 was arrested at the spermatogonial level in 75% of seminiferous tubules. In group 2, spermatogenesis proceeded to spermatocytes in 50% of tubules and to elongated spermatids in 10% of tubules, while in group 3 elongation of spermatids occurred in 75% of tubules. The mean T and dihydrotestosterone concentrations in baseline testicular biopsies (n = 15) were 43.8 +/- 6.8 (+/- SE) and 5.7 +/- 1.5 ng/g, respectively. After GnRH antagonist with or without T administration, the mean (n = 15) intratesticular T and dihydrotestosterone levels were reduced to 20.3 +/- 4.9 and 3.2 +/- 0.5 ng/g, respectively, and differed little among the three groups. No correlation, however, could be established between testicular androgen levels and spermatogenic status (P greater than 0.30) or sperm counts (P greater than 0.60). These results demonstrate that administration of a GnRH antagonist in the presence of constant serum T levels does not induce consistent azoospermia, and that the supporting effects of T on spermatogenesis cannot be explained exclusively on the basis of the testicular androgen concentrations.     

Acute and chronic effects of a gonadotrophin-releasing hormone antagonist on pituitary and testicular function in monkeys

The effects of a potent gonadotrophin-releasing hormone (GnRH) antagonist, (N-Ac-D-rho-Cl-Phe1,2,D-Trp3-D-Arg6-D-Ala10)-GnRH (Org 30276), on pituitary and testicular function of adult macaque monkeys were investigated. After a study to find the correct dose in castrated monkeys, five intact adult male animals were treated with daily s.c. injections of 5 mg antagonist for 9 weeks. The treatment resulted in an immediate decline in serum LH and testosterone in three out of five animals. The two hormones remained suppressed during the 9-week treatment period. Testosterone and LH responses to a bolus injection of GnRH (50 micrograms i.v.) were blunted or abolished during the antagonist treatment. Testicular volumes decreased markedly and ejaculates obtained at the end of treatment were azoospermic or contained only few dead sperm. Histological examination of the testes showed complete disruption of seminiferous epithelium in these animals. A decrease of body weight was observed in the treated animals. When the treatment was ceased, all inhibitory effects of GnRH antagonists were reversible. In the other two animals no consistent suppression of pituitary or testicular function could be observed during this period, nor was a doubling of the treatment dose for a further 8 weeks capable of fully suppressing endocrine and seminal parameters in these monkeys. It is concluded that GnRH antagonist treatment is capable of rapidly decreasing serum LH and testosterone and disrupting spermatogenesis in this primate species. Suppression effected by antagonist treatment is more rapid than that caused by GnRH agonists. The individual responses to the tested doses, however, vary markedly.     

Hormonal effects of gonadotropin-releasing hormone (GnRH) agonist in men: effects of long term treatment with GnRH agonist infusion and androgen

Constant infusion of GnRH agonist (GnRH-A) leads to far greater suppression of spermatogenesis and gonadotropins in the monkey than its intermittent administration. We assessed if greater suppression of gonadotropins and spermatogenesis could also be achieved in man by continuous GnRH-A administration. Seven normal men were given 400 micrograms GnRH-A daily by constant sc infusion using a mechanical pump device and bimonthly injections of 200 mg testosterone (T) enanthate for 16 weeks. Basal serum LH, FSH, T, and estradiol concentrations were measured every week during a 5-week control period, daily on treatment days 0, 1-11, 14, 18, 22, 26, 28, and every week thereafter until day 56, and every 2 weeks thereafter during the remainder of the treatment phase and during the 14-week recovery phase. Detailed analysis of LH and FSH secretion during the 24-h period was performed by multiple blood sampling on days 0, 1, 10, 28, 56, 84, and 112. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. The mean sperm count declined by 93% to a nadir of 6 +/- 3 (+/- SE) million/mL between weeks 14-16. Four men had sperm counts less than 1 million/mL, and three subjects were azoospermic during treatment. Basal serum immunoreactive LH concentrations, after an early increase, declined to near baseline by day 14. The basal and 24-h integrated serum LH concentrations and 24-h urinary LH excretion were not significantly lowered by treatment. Bioassayable serum LH concentrations, however, after an early rise, declined significantly below baseline by day 28 and remained low thereafter. The frequency and amplitude of LH pulses were reduced by GnRH agonist infusion. Basal and 24-h integrated serum FSH concentrations, after a brief initial increase, declined to baseline by day 10, but were not significantly below baseline by day 112. Serum T concentrations did not fall into the hypogonadal range at any time during the treatment period. After discontinuation of treatment, serum LH and FSH and sperm counts returned to normal in all men. Thus, this regimen, employing constant infusion of 400 micrograms GnRH agonist daily plus T led to a greater suppression of spermatogenesis than the previous regimen employing single daily injections of 200 micrograms of the same agonist plus T. Whether the higher dose or the constant infusion was responsible for the greater inhibition of spermatogenesis is not clear. It is conceivable that a still higher dose of the agonist, given by constant infusion, might induce azoospermia in all men.     

Influence of simultaneous gonadotropin-releasing hormone agonist and testosterone treatment on spermatogenesis and potential fertilizing capacity in male monkeys

We examined the effect of continuous sc infusion of a GnRH agonist (Ag) and testosterone (T) supplementation on spermatogenesis and the potential fertilizing capacity of sperm in 15 rhesus monkeys. The monkeys were divided into 3 groups of 5 animals each. Groups 1 and 2 received 25 micrograms/day Ag for 44 weeks. Group 2 also received T replacement therapy [sufficient to maintain serum T values within the normal range; 4-5 ng/mL (13.9-17.3 nmol/L)]. Group 3 received a low dose of the Ag (5 micrograms/day for 24 weeks and then 10 micrograms/day for 20 weeks) to prolong the oligospermic phase before the onset of azoospermia. In groups 1 and 3, there was an initial transient (1 week) rise in serum LH and T levels which then fell below pretreatment value where they remained throughout the treatment period. The serum LH and T levels were lower in the group treated with 25 micrograms/day Ag. Similar changes in serum LH levels occurred in group 2, but T supplementation maintained serum T in the physiological range. The decline in serum LH levels was associated with reduced sensitivity to GnRH, since the responses of serum LH and T to GnRH were either abolished or greatly reduced in the 2 groups treated with 25 micrograms/day Ag and were less than 50% of the pre-Ag responses in monkeys treated with 5-10 micrograms/day Ag. Four of five monkeys treated with 25 micrograms/day Ag alone became azoospermic within 21 weeks. All five animals receiving Ag and T supplementation became azoospermic (mean time to onset, 12.6 weeks). Four of five monkeys treated with 5-10 micrograms/day Ag also had azoospermic ejaculates during the late treatment and early recovery period. Sperm counts recovered to the pretreatment levels in most monkeys by 10 weeks of the recovery period. The quality of semen samples taken from oligospermic monkeys was greatly reduced. The percentage of motile and percentage of live sperm per ejaculate, the net negative surface charge on sperm, and the scores of sperm in the hamster oocyte penetration test were subnormal. T supplementation did not improve these measures of semen quality. Testicular biopsies taken at the end of the Ag administration period from monkeys given 25 micrograms/day Ag showed diffuse atrophy of the seminiferous tubules, which contained primarily Sertoli cells and a few spermatogonia and spermatocytes, but no spermatids. The tubular atrophy and the suppression of spermatogenesis did not appear to be influenced by T replacement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone antagonist plus testosterone: a potential male contraceptive

No effective hormonal contraceptive has yet been devised for men. Through their suppressive effect on gonadotropin secretion, GnRH antagonists inhibit both testosterone (T) production and spermatogenesis in animals. Long term administration of an antagonist alone would result in androgen deficiency; this would cause unacceptable physiological and behavioral sequellae in men. Therefore, androgen replacement must be included in any GnRH antagonist regimen used in human male contraception. We tested the hypothesis that the combination of a GnRH antagonist plus T would suppress spermatogenesis in the male primate to azoospermic levels while maintaining normal serum T levels. We examined the effects of the GnRH antagonist Deterelix [N-Ac-DNal(2)1-DpCl-Phe2-DTrp3-DhArg(Et2)6 -DAla10-GnRH], alone and with simultaneous T replacement, on sperm production and serum T levels in adult male monkeys (n = 22). After 12 weeks of daily sc antagonist injection, all animals that received antagonist alone (n = 5) and those that 750 micrograms/kg.day antagonist plus T (n = 5) were azoospermic. After 16 weeks, four of five animals that received 250 micrograms/kg.day antagonist plus T became azoospermic. Control animals (n = 7) received daily injections of vehicle; sperm counts increased somewhat during the study period in that group. Castrate range T levels were achieved in animals receiving antagonist alone. T levels in the groups that received T supplementation and in the control group were in the normal male range throughout the treatment period. Sperm counts returned to the pretreatment range in all animals during the recovery period. We conclude that the combination of a GnRH antagonist plus T can induce azoospermia reversibly in this nonhuman primates species, and that a similar combination may be an effective contraceptive regimen in men. The GnRH antagonist alone may be an effective treatment for androgen-dependent neoplasia.     

Testosterone and dihydrotestosterone, but not estradiol, selectively maintain pituitary and serum follicle-stimulating hormone in gonadotropin-releasing hormone antagonist treated male rats

Recently it has been found that testosterone can maintain and restimulate serum and pituitary follicle-stimulating hormone (FSH) in the gonadotropin-releasing hormone (GnRH) antagonist treated adult male rat. The present investigation was undertaken to determine (1) which metabolite of testosterone, dihydrotestosterone (DHT), or estradiol accounts for the effects of testosterone in GnRH antagonist suppressed rats and (2) whether these effects of testosterone are influenced by other testicular factors. Eight groups of 6-8 adult male Sprague-Dawley rats were subjected to the following treatments: vehicle, GnRH antagonist (75 micrograms/day s.c.), testosterone-filled Silastic implants (3 x 5 cm, s.c.), DHT-filled Silastic implants (3 x 5 cm, s.c.), estradiol benzoate (15 micrograms/day s.c.), and combined administration of GnRH antagonist with either steroid. In addition, the GnRH antagonist/testosterone treatment regimen was applied to rats orchidectomized 72 h prior to initiation of treatments. After 3 weeks of treatment, serum was analyzed for concentrations of luteinizing-hormone (LH), FSH, testosterone, DHT, and estradiol. Pituitary extracts were analyzed for LH and FSH content. Except for the vehicle-treated groups, serum and pituitary LH concentrations were markedly suppressed by all treatments. In intact rats treated with GnRH antagonist alone and/or estradiol, the pituitary FSH level was reduced by more than 70% relative to controls, while both testosterone and DHT maintained pituitary FSH. Similarly, testosterone and DHT, but not estradiol, delayed the decline of serum FSH induced with GnRH antagonist alone. In orchidectomized animals, testosterone was also capable of preventing a reduction of pituitary FSH despite concomitant GnRH antagonist administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal effects of gonadotropin-releasing hormone (GnRH) agonist in the human male. III. Effects of long term combined treatment with GnRH agonist and androgen

Chronic treatment with agonist analogs of GnRH results in reversible oligospermia in man, but leads to impotence and decreased libido due to a concomitant fall in serum testosterone (T) concentrations. We, therefore, assessed the effects of combined treatment with a potent GnRH agonist and T on gonadotropins and spermatogenesis in normal men, anticipating that addition of androgen would prevent agonist-induced changes in libido. Seven normal men were treated with 200 micrograms of the GnRH agonist D-(Nal2)6GnRH (GnRH-A), sc, daily for 16 weeks. In addition, 200 mg T enanthate were administered every 2 weeks for the entire 16-week treatment period. Basal LH, FSH, and T concentrations were measured every week during a 5-week control period, daily on treatment days 0, 1-10, 14, 18, 22, 26, and 28, every week thereafter until day 56, and every 2 weeks thereafter for the remainder of the treatment and recovery phases. Detailed analysis of LH and FSH over the 24-h period was performed by multiple blood sampling on days 0, 1, 10, 28, 56, 84, and 112. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. The mean sperm count declined by 83%, to a nadir of 16.6 +/- 6.2 (+/- SEM) million/ml. One subject had no significant decrease in sperm count. Azoospermia was not achieved in any subject. Basal serum LH concentrations, after an early phase of stimulation, declined to near baseline by day 14. However, basal, 24-h integrated serum LH concentrations, and 24-h urinary LH excretion were not significantly lowered by combined treatment. Bioassayable serum LH concentrations, however, declined significantly from 20.4 +/- 6.3 to 4.5 +/- 0.5 mIU/ml, and the bioassayable to immunoassayable LH ratio decreased from 2.1 +/- 1.0 to 0.7 +/- 0.1 after 16 weeks of GnRH-A treatment. Basal and 24-h integrated FSH concentrations, after an initial period of stimulation, declined progressively to baseline by days 5-6 and were significantly below baseline by day 112. Serum T concentrations did not fall into the hypogonadal (less than 250 ng/dl) range in any subject at any time during the treatment period. After discontinuation of treatment, LH, FSH, and sperm counts returned to normal in all subjects. Thus, single daily injection of GnRH-A and T failed to predictably induce azoospermia in normal men over the 16-week treatment period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of continuous gonadotropin-releasing hormone (GnRH) agonist treatment on luteinizing hormone and testosterone secretion, the response to GnRH, and the testicular response to human chorionic gonadotropin in male rhesus monkeys

Five adult male rhesus monkeys were continuously infused for 56 days with 25 micrograms/day of GnRH agonist (Wy-40972; Ag) using an implanted osmotic pump. Bioassayable serum levels of LH were elevated 8-fold on the second day of Ag treatment and then declined precipitously to below pretreatment levels by day 15. Serum levels of testosterone (T) changed similarly during Ag treatment, except that the fall in serum LH levels preceded the decline in serum levels of T by at least 2 days. Ag administration also eliminated the diurnal variation in serum LH and T. GnRH administration (50 micrograms) induced a 13- to 20-fold rise in serum LH and a 3- to 7-fold increase in serum T in control monkeys. After 4 weeks of Ag administration , none of the animals responded to GnRH. Both control and experimental monkeys had a rise in serum T in response to hCG after 7 weeks of Ag treatment. Basal levels of LH and T returned to normal by 12 days posttreatment, and the serum LH and T responses to GnRH were normal 19 days posttreatment. These results indicate that 1) continuous administration of Ag is an effective method of inducing antiferility effects in male rhesus monkeys; 2) pituitary desensitization is a major factor involved in Ag-induced gonadal dysfunction in this species; and 3) the method of administration may be the critical factor in determining the effectiveness of GnRH agonists.     

Intramuscular testosterone undecanoate and norethisterone enanthate in a clinical trial for male contraception

Recent trials for hormonal male contraception are based on gestagens or GnRH antagonists combined with oral or injectable testosterone substitution. However, the efficacy of most trials remained disappointing. Norethisterone enanthate (NETE) has been used as a long-acting injectable female contraceptive and has shown sustained suppression of spermatogenesis in male monkeys and prolonged suppression of gonadotropins in men. This study was designed to prove the efficacy of the long-acting testosterone undecanoate ester (TU) alone or in combination with NETE in a phase II clinical trial. Fourteen healthy men received injections of 1000 mg TU in combination with injections of 200 mg NETE every 6 weeks over a period of 24 weeks, followed by a control period of 28 weeks. Another 14 volunteers received TU alone. During the study semen variables, reproductive hormones, clinical chemistry and lipid parameters, well-being, and sexual function were monitored. Scrotal content and prostates were checked sonographically. During the entire treatment period mean testosterone serum concentrations remained within the normal limits. Marked suppression of gonadotropins in both treatment groups resulted in azoospermia in 7 of 14 and 13 of 14 volunteers and in oligozoospermia in 7 of 14 and 1 of 14 in the groups given TU only or TU/NETE, respectively. However, the highest azoospermia rate in the TU/NETE group was achieved 8 weeks after the end of the treatment period, and 1 volunteer with very high initial sperm counts (mean, 190 million/mL at baseline) remained oligozoospermic (10.2 million/mL). From week 20 to week 24 there was a significant, fully reversible maximum weight gain of 3.7 kg, on the average, in the NETE group. In the NETE and TU alone groups there were significant 26.6% and 11.5% maximum decreases in high density lipoprotein cholesterol compared with baseline values during the treatment period. A significant elevation of low density lipoprotein and a decrease in lipoprotein(a) were detected in the TU/NETE group. In conclusion, combination treatment with NETE showed suppression of spermatogenesis comparable with results using testosterone esters in combination with GnRH antagonists or cyproterone acetate, but had more favorable injection intervals and better efficacy. Because of its long-lasting, profound suppression of spermatogenesis and the absence of serious side-effects, the combination of TU and NETE can be considered a first choice for further studies of hormonal male contraception.     

Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis)

The role of FSH in spermatogenesis was investigated in nonhuman primates depleted of testosterone by GnRH antagonist treatment. The GnRH antagonist antide (Nal-Lys; [N-acetyl-D-2-naphthyl-Ala1,D-4-chloro-Phe2,D-pyridyl-Ala3, nicotinyl-Lys5,D-nicotinyl-Lys6,isopropyl-Lys8,D-Ala10 ]-GnRH) was used at a daily dose of 450 micrograms/kg to suppress endogeneous gonadotropin and androgen production. Four groups of five cynomolgus monkeys (Macaca fascicularis) were subjected to the following treatment throughout a 16-week period: vehicle (group 1), GnRH antagonist (group 2), and GnRH antagonist plus human FSH (Fertinorm; 2 x 15 IU/day.animal; hFSH) during weeks 0-8 (group 3) or 8-16 (group 4). Testicular biopsies were performed before and after 4, 8, and 16 weeks of treatment. The tissue was analyzed by light microscopy and flow cytometry. Serum testosterone levels were suppressed into the range of orchidectomized animals in all GnRH antagonist-treated groups. In the absence of hFSH, serum inhibin levels were also markedly lowered. Concomitant administration of hFSH attenuated the GnRH antagonist-induced reduction of testicular size, while delayed treatment with hFSH failed to restimulate testicular volume. Numbers of A-dark spermatogonia, the reserve stem cells, were not altered by any of the treatments. hFSH either fully maintained or increased the counts for A-pale spermatogonia (renewing stem cells). The development of pachytene spermatocytes and round and elongated spermatids was markedly reduced or inhibited by the GnRH antagonist within 6-18 weeks. In contrasts, hFSH maintained these cell types at about 50% of baseline for 8 weeks. After 8 weeks of GnRH antagonist administration, hFSH stimulated A-pale spermatogonia and spermatocytes 2- to 3-fold with only minor effects on spermatid numbers. By means of flow cytometry, testicular cells were quantified according to DNA content. Within 8-16 weeks of GnRH antagonist treatment the percentage of 4C (mainly primary spermatocytes), 1C (round spermatids), and 1CC cells (elongated spermatids) had fallen from 65-75% to 5-25%. hFSH completely maintained the relative number of these cells, but failed to significantly restimulate the formation of 1CC cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacokinetics and pharmacodynamics of testosterone enanthate and dihydrotestosterone enanthate in non-human primates

The pharmacokinetics and pharmacodynamics of testosterone enanthate and dihydrotestosterone-enanthate were compared in orchidectomized cynomolgus monkeys (Macaca fascicularis) and in intact GnRH agonist-suppressed rhesus monkeys (Macaca mulatta). Following a single im injection of 32.8 mg testosterone enanthate or 32.7 mg dihydrotestosterone-enanthate, i.e. 23.6 mg of pure steroid, in the orchidectomized cynomolgus monkeys, serum testosterone and dihydrotestosterone levels rose to 400 and 800% of baseline, respectively, within 24 h. Androgen levels remained in that range for 3-5 days followed by a continuous decline until baseline values were attained after 4-5 weeks. The areas under the testosterone- and dihydrotestosterone-curves did not differ significantly 2290 +/- 340 (dihydrotestosterone-enanthate) vs 2920 +/- 485 (testosterone-enanthate) suggesting that similar amounts of steroid had been released from the respective ester preparation. Mean half-life estimates of the terminal elimination phase were 4 and 7 days for testosterone-enanthate and dihydrotestosterone-enanthate, respectively. In a second experiment rhesus monkeys received, at 4-weekly intervals, sc implantation of a biodegradable polylactic:polyglycolide rod loaded with the GnRH agonist buserelin. The last injection was given during week 20. GnRH agonist treatment suppressed serum bioactive LH, testosterone and dihydrotestosterone levels, testicular size, sperm production, and seminal carnitine content. The ejaculatory response to electrostimulation and the masturbatory behaviour were abolished. Testosterone or dihydrotestosterone injections at the same doses as above were given in week 10, 14, 17 and 20 of GnRH agonist treatment. Serum testosterone and dihydrotestosterone levels were stimulated 9- and 4-fold, respectively. Mean half-life estimates for testosterone-enanthate and dihydrotestosterone were 5 and 7 days, respectively. Both ester preparations completely restored the ejaculatory response, ejaculate size, masturbatory behaviour, and seminal carnitine levels. In conclusion, androgen substitution with dihydrotestosterone-enanthate, in equivalent doses, is as effective as testosterone-enanthate in restoring reproductive functions in hypogonadal monkeys.     

Effect of long-term testosterone oenanthate administration on male reproductive function: clinical evaluation, serum FSH, LH, testosterone, and seminal fluid analyses in normal men.

The effect of long-term testosterone administration on male reproductive function has been investigated in seven healthy young men age 20 to 27 years. Testosterone oenanthate (TOe) was administered in doses of 250 mg per week for 21 weeks. No toxic side-effects were observed. Libido, sexual potency, frequency of sexual intercourse and body hair development generally remained unaffected, but there was a reversible mean weight gain of 3.6 kg during TOe administration. Seminal fluid parameters and radioimmunoassayable serum FSH, LH, testosterone, and androstenedione levels were monitored before, during, and after TOe administration. The serum testosterone rose approximately by a factor of two, while the serum FSH and LH were rapidly suppressed after the initiation of the TOe therapy. The mean sperm concentration fell to values below three million spermatozoa per ml, and changes in sperm motility, the percentage of normal sperm morphology, and seminal fructose concentrations generally paralleled those of the mean sperm concentrations. In contrast, the mean seminal fluid volume and serum androstenedione levels did not change significantly during TOe administration. The mean sperm concentration showed a marked recovery 13 to 16 weeks after TOe withdrawal, but sperm counts remained below pre-treatment levels in three out of seven subjects 25 to 28 weeks after discontinuation of TOe.     

Exogenous gonadotrophin-releasing hormone (GnRH) stimulates LH secretion in male monkeys (Macaca fascicularis) treated chronically with high doses of a GnRH antagonist.

We reported previously that after a single injection of a gonadotrophin-releasing hormone (GnRH) antagonist to male monkeys, exogenous GnRH stimulated LH secretion in a time- and dose-dependent manner, indicating that GnRH antagonist-induced blockade of LH secretion resulted from pituitary GnRH receptor occupancy. The present study was performed to investigate whether GnRH can also restore a blockade of LH and testosterone secretion during chronic GnRH antagonist administration. Four adult male cynomolgus monkeys (Macaca fascicularis) received daily s.c. injections of the GnRH antagonist [N-Ac-D-pCl-Phe1,2,D-TRP3,D-Arg6-D-Ala10]-GnRH (ORG 30276) at a dose of 1400-1600 micrograms/kg for 8 weeks. Before the GnRH antagonist was given and during weeks 3 and 8 of treatment, pituitary stimulation tests were performed with 0.5, 5, 50 and 500 micrograms synthetic GnRH, administered in increasing order at intervals of 24 h. At 8 weeks, a dose of 1000 micrograms GnRH was also given. All doses of GnRH significantly (P less than 0.05) stimulated serum concentrations of bioactive LH (3- to 8-fold) and testosterone (2.6- to 3.8-fold) before the initiation of GnRH antagonist treatment. After 3 weeks of GnRH antagonist treatment, only 50 and 500 micrograms GnRH doses were able to increase LH and testosterone secretion. Release of LH was significantly (P less than 0.05) more elevated with 500 micrograms compared with 50 micrograms GnRH. After 8 weeks, only the highest dose of 1000 micrograms elicited a significant (P less than 0.05) rise in LH secretion. Basal hormone levels just before the bolus injection of GnRH were similar (P greater than 0.10-0.80).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of pituitary-testicular function with [D-Trp6] luteinizing hormone-releasing hormone in rhesus monkeys

The effect of [D-Trp6]LHRH, a potent agonist of LHRH, on pituitary-testicular responses was investigated in male rhesus monkeys. Acute administration of 5, 25, or 500 micrograms [D-Trp6]LHRH produced dose-related increases in serum testosterone and bioassayable LH levels. The administration of 500 micrograms [D-Trp6]LHRH twice weekly for 12 weeks led to a 75% decrease in the LH responses to successive doses of this peptide; testosterone responses in these animals were unchanged, however. The lack of any change in the motility or sperm number in semen obtained by electroejaculation suggested that there was no effect on spermatogenesis during the twice weekly administration. These animals were then treated with [D-Trp6]LHRH (500 micrograms daily) for 16 weeks. This caused dramatic decreases in the LH responses to the agonist in all four animals. The testosterone response was reduced in two and abolished in two animals. These latter two animals also lost their electroejaculatory response. During the recovery period following the cessation of treatment, these two animals produced ejaculates with no sperm, indicating that a transient period of azoospermia was achieved. The results of these studies suggest that 1) the responsiveness of the pituitary is more susceptible to desensitization by [D-Trp6]LHRH than that of the testes; and 2) even though chronic administration of [D-Trp6]LHRH suppressed the pituitary-Leydig cell axis in all monkeys, seminiferous tubular function was reduced only in those animals with very low androgen levels.     

Effect of administration of testosterone and gonadotrophin-releasing hormone (GnRH) antagonist on basal and GnRH-stimulated gonadotrophin secretion in orchidectomized monkeys

The aim of the present investigation was to investigate the effects of testosterone on basal and gonadotrophin-releasing hormone (GnRH)-stimulated gonadotrophin secretion in the presence and absence of a GnRH antagonist in a non-human primate model (Macaca fascicularis). Orchidectomized animals were used in order to avoid interference by testicular products other than testosterone involved in gonadotrophin feedback. Concomitant and delayed administration of testosterone at doses that provided serum levels either within the intact range (study 1) or markedly above that range (study 2) did not influence the suppression of basal gonadotrophin release induced by the GnRH antagonist during a 15-day period. To assess the possible effects of testosterone treatment at the pituitary level (study 3) GnRH stimulation tests (500 micrograms) were performed before and on days 8 and 15 of treatment with high-dose testosterone and GnRH antagonist alone or in combination. Testosterone alone abolished the gonadotrophin responses to exogenous GnRH observed under pretreatment conditions. With GnRH antagonist alone, an increased responsiveness (P less than 0.05) to GnRH was seen on day 8 and a similar response compared with pretreatment on day 15. Following combined treatment with GnRH antagonist and testosterone, GnRH-induced gonadotrophin secretion was consistently lower compared with that after GnRH antagonist alone (P less than 0.05), but was increased compared with that after testosterone alone (P less than 0.05). Thus, in the presence of a GnRH antagonist the feedback action of testosterone on LH and FSH was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression and recovery of pituitary gonadotrophin secretion in intact and orchidectomized rats treated neonatally with a gonadotrophin-releasing hormone antagonist

Suppression of neonatal rat pituitary-testis function by gonadotrophin-releasing hormone (GnRH) antagonists results in delayed sexual maturation and infertility. Since the mechanism is not understood, the acute effects of a GnRH antagonist on gonadotrophin secretion in neonatal male rats has been studied in more detail. Treatment with a GnRH antagonist analogue, N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 -GnRH (2 mg/kg per day) on days 1-10 of life had prolonged effects on gonadotrophin secretion; serum LH and FSH recovered in 1 week, but the pituitary content took 2 weeks to recover. Likewise, LH and FSH responses to acute in-vivo stimulation with a GnRH agonist were still suppressed 1 week after the treatment. Interestingly, a rebound (86% increase) in basal serum FSH was found 16 days after treatment with the antagonist. Whether testis factors influence gonadotrophin secretion during treatment with the GnRH antagonist and/or in the subsequent recovery period was also assessed. Neonatal rats were castrated on days 1, 5 or 10 of the 10-day period of antagonist treatment. Orchidectomy on days 1 and 5 only marginally affected gonadotrophin secretion. When orchidectomy was performed at the beginning of the recovery period, no effects on pituitary recovery were seen within 1 week of castration. After 16 days, serum LH and FSH in the antagonist-treated and control castrated rats were equally increased but the pituitary contents of the antagonist-treated rats were still suppressed. Finally, the effect of testosterone treatment on the recovery of gonadotrophin secretion after antagonist suppression was studied in intact and orchidectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)