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.     

Regulation of primate testicular function by GnRH analogues

The potential of GnRH analogues for regulating testicular function is reviewed. Our experiments showed that constant infusion of GnRH agonists effectively suppressed testicular function in monkeys. In men, however, spermatogenesis could not be suppressed to achieve azoospermia uniformly. GnRH antagonists, although at much higher dosages than agonists, caused a more rapid and uniform inhibition of testis function. Spermatogenesis was reversibly disrupted at the spermatogonial level. Concomitant testosterone supplementation, used to maintain libido and potency, attenuated the antitesticular effects of GnRH analogues. In monkeys testosterone appears to stimulate spermatogenesis directly on the testicular level, while evidence has been obtained that in rats testosterone can also stimulate the release and synthesis of FSH under antagonist mediated blockage of pituitary GnRH receptors. When extrapolating to human studies special care has to be exerted in the selection of testosterone substitution regimens. Although the agonistic and antagonistic analogues of GnRH ultimately exert their antireproductive effects via inhibition of gonadotropin secretion the antagonists may have the greater potential for male fertility regulation due to quicker pituitary and testicular suppression.     

Accelerated maturation of primate testis by xenografting into mice

Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys (Macaca mulatta) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.     

Circadian and circannual changes in the testicular function of adult rhesus monkeys

The endocrine and gametogenic status of the testes were studied in 9 healthy adult rhesus monkeys of proven fertility throughout a one-year period. Testosterone levels were estimated by radioimmunoassay in blood samples collected at 4 h intervals over a 24 h period once a month. Semen samples and testicular biopsies were also examined once a month. A well-defined circandian rhythm was evident in serum levels of testosterone. The rhythmicity was less pronounced in February and September. The 24 h mean levels of serum testosterone were high between the months of August to March and low in the months of May to July. All animals did not uniformly respond to electro-ejaculation in April and May. Semen volume and total number of spermatozoa were maximal between September and March and least from April to August. Testicular biopsies indicated that all stages of spermatogenesis were evident between September and March and the spermatogenic activity was less evident between April and August. The contents of Sertoli cells showed a seasonal cyclicity; they were laden with lipid droplets during April to August when spermatogenesis was quiescent and vacuolated during September to March when spermatogenesis was active. These studies indicate that the testing of contraceptive drugs needs to be restricted to months of September to March in male rhesus monkeys otherwise, it is possible that the naturally occurring reproductive quiscence may be attributed to the effect of the drug being tested. The data accrued from the present studies also provide quantitative information on circulating levels of testosterone which could be used as a reference background while evaluating the contraceptive drug-effects in male rhesus monkeys.     

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.     

Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse

Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.     

Melphalan,alone or conjugated to an FSH-β peptide,kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of FSHβ. We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHβ consisting of amino acids 33 to 53 (FSHβ (33–53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33–53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33–53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33–53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33–53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33–53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.     

L'Inhibine B chez les blessés radiculo-médullaires. Résultats préliminaires

Spinal cord injuries in adult men sometimes results in semen alteration and in ejaculation missing. Testicular biopsies showed spinal cord injury is bound with testicular and germinal cells alterations. An endocrine profile for thirteen patients without semen at the time of the study is performed: total testosterone, bioavailable testosterone, FSH and inhibin B. Compared with seven normal controls, subjects with spinal cord injury had a significantly lower value of total testosterone, bioavailable testosterone and inhibin B. The FSH values higher in spinal cord injury are not significative. These results allowed to expect inhibin B like a good marker of testicular function and spermatogenesis. A longitudinal and on more subjects will answer to this question.     

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.     

Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.     

The effects of long-term administration of either a crude inhibin preparation or an antiserum to FSH on serum hormone levels, testicular function and fertility of adult male rats.

Adult male rats were given either daily injections of ram rete testis fluid for periods of up to 70 days or injections of an antiserum against FSH every 3 days for 90 days. Compared with the control groups, the rats injected with ram rete testis fluid had lowered serum FSH levels, but only at treatment periods of 30 days and less. The levels of LH and testosterone in serum, testicular fluid secretion, sperm counts, testis weights and fertility were not affected by rete testis fluid treatment. The rats injected with anti-FSH serum exhibited an impairment of fertility which was never complete and evident only after 49 days of treatment. After 90 days of anti-FSH treatment, testis weight and free serum FSH were reduced, but sperm counts, testicular fluid secretion and serum levels of LH and testosterone were not affected.     

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.     

Variations in seminal parameters over a 12-month period in captive bonnet monkeys

Semen samples were collected from adult fertile bonnet monkeys twice a month by penile electroejaculation for twelve consecutive months. Various parameters like semen volume, weight of ejaculate and coagulum, sperm count, sperm motility, sperm morphology, and functional parameters e.g. plasma membrane integrity,in vitro nuclear chromatin decondensation and acrosomal status were evaluated to assess within and between animal variations. Effects of seasonality, if any, on quantity and quality of semen were also studied. Considerable intra- and inter-individual variations in the geometric mean values were observed for semen volume, weights of ejaculate and coagulum, and sperm counts during the study period. On the other hand, sperm motility, morphology, and functional parameters showed less within and between animal variations. Results on motility, morphology, and functional parameters indicated that good semen quality was maintained throughout the year. Various routine and functional parameters did not show any annual variations. The diurnal rhythmicity in circulatory testosterone levels was observed throughout the year. The study shows lack of seasonality in exocrine and endocrine testicular functions and further suggests that motility, morphology, and functional parameters are better indicators of semen quality in captive bonnet monkeys.     

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Seasonally and experimentally induced changes in testicular function of the Australian bush rat (Rattus fuscipes)

Serum concentrations of LH, FSH and testosterone were measured monthly throughout the year in male bush rats. Testicular size and ultrastructure, LH/hCG, FSH and oestradiol receptors and the response of the pituitary to LHRH were also recorded. LH and FSH rose in parallel with an increase in testicular size after the winter solstice with peak gonadotrophin levels in the spring (September). The subsequent fall in LH and FSH levels was associated with a rise in serum testosterone which reached peak levels during summer (December and January). In February serum testosterone levels and testicular size declined in parallel, while the pituitary response to an LHRH injection was maximal during late summer. The number of LH/hCG, FSH and oestradiol receptors per testis were all greatly reduced in the regressed testes when compared to active testes. In a controlled environment of decreased lighting (shortened photoperiod), temperature and food quality, the testes of sexually active adult males regressed at any time of the year, the resultant testicular morphology and endocrine status being identical to that of wild rats in the non-breeding season. Full testicular regression was achieved only when the photoperiod, temperature and food quality were changed: experiments in which only one or two of these factors were altered failed to produce complete sexual regression.     

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse

Background  

The hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.     

Seasonal changes in plasma concentrations of immunoreactive inhibin and testicular activity in male Japanese monkeys

Seasonal changes in plasma immunoreactive (ir-) inhibin, testosterone, LH, and FSH concentrations were examined in five sexually mature male Japanese monkeys (Macaca fuscata fuscata) housed indoors individually, to explore the reproductive cyclicity in the male. Blood samples were collected monthly throughout one year, and testicular size, semen volume, and number of sperm in the semen were ascertained at the same time in the same animals. Semen samples were obtained by penile electrostimulation. The results showed a clear seasonal increase in all parameters: plasma ir-inhibin, testosterone, testicular size, semen volume, and total number of sperm in the liquid portion of the semen during the autumn and winter months in synchrony with the natural breeding season. In contrast, plasma LH and FSH remained unchanged throughout the year, although plasma FSH tended to increase during the breeding season concomitant with an increase in plasma ir-inhibin. A significant positive correlation between FSH and ir-inhibin was observed in two of five monkeys. The positive correlations between plasma ir-inhibin and testicular activities during both the developing and regressing phases of the testicular cycle indicate that plasma ir-inhibin is a useful indicator of testicular activity as well as an indicator of Sertoli cell function in the Japanese monkey.     

Gonadotropin secretion and testicular function in golden hamsters exposed to skeleton photoperiods with ultrashort light pulses

Both sexually mature and sexually regressed male golden hamsters were transferred to asymmetric skeleton photoperiods with night interruptions of varying duration, the short pulses occurring 14 h after "dawn." Testicular function and accompanying changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone and spermatogenesis were observed. Sexually regressed animals exposed to a night-break of 6 seconds (sec) or longer exhibited maximal testicular development with a rapid rise in FSH secretion followed by a slower, more variable rise in LH. Full testicular size was achieved after 8 weeks. Night-breaks of 250 milliseconds (msec) or 1 sec induced testicular development and spermatogenesis but at a slower rate: levels of FSH and LH were still rising at the end of the experiment. Complete testicular maintenance was achieved by night-breaks of 1 sec or longer. Partial testicular regression was observed with a night-break of 250 msec. Night-breaks (60 sec) given less frequently than daily also stimulated testicular function and a night-break every 7 days increased FSH and LH secretion in sexually regressed hamsters, causing testicular development at a submaximal rate. Night-breaks given more frequently induced rapid testicular growth. Almost complete testicular maintenance of sexually mature hamsters was achieved with a 60-sec night-break at weekly intervals. Symmetric skeleton photoperiods also triggered testicular development in sexually regressed hamsters, with two 1-sec light pulses (14 h apart) being almost as effective as a normal long day. No difference in reproductive function was observed between animals on long days (14L:10D) and those exposed to maximally stimulatory skeleton photoperiods.     

Andropause: Une histoire a suivre

In males, the endocrine and exocrine functions of testes alterate with aging. Though in males, there is not a sudden and definitive interruption of reproduction function, as at menopause in females, spermatogenesis decreases, as testosterone production. This testicular decrease is escorted by a relative decrease of gonadotropin release. So, testicular function, vulnerable to aging, is to be continued.     

Chronic ethanol perturbs testicular folate metabolism and dietary folate deficiency reduces sex hormone levels in the Yucatan micropig

Although alcoholism causes changes in hepatic folate metabolism that are aggravated by folate deficiency, male reproductive effects have never been studied. We evaluated changes in folate metabolism in the male reproductive system following chronic ethanol consumption and folate deficiency. Twenty-four juvenile micropigs received folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE or FDE) for 14 wk, and the differences between the groups were determined by ANOVA. Chronic ethanol consumption (FSE and FDE compared with FS and FD groups) reduced testis and epididymis weights, testis sperm concentrations, and total sperm counts and circulating FSH levels. Folate deficiency (FD and FDE compared with FS and FSE groups) reduced circulating testosterone, estradiol and LH levels, and also testicular 17,20-lyase and aromatase activities. There was histological evidence of testicular lesions and incomplete progression of spermatogenesis in all treated groups relative to the FS control, with the FDE group being the most affected. Chronic ethanol consumption increased testis folate concentrations and decreased testis methionine synthase activity, whereas folate deficiency reduced total testis folate levels and increased methionine synthase activity. In all pigs combined, testicular methionine synthase activity was negatively associated with circulating estradiol, LH and FSH, and 17,20-lyase activity after controlling for ethanol, folate deficiency, and their interaction. Thus, while chronic ethanol consumption primarily impairs spermatogenesis, folate deficiency reduces sex hormones, and the two treatments have opposite effects on testicular folate metabolism. Furthermore, methionine synthase may influence the hormonal regulation of spermatogenesis.