Influence of melatonin on the testicular regression induced by subcutaneous testosterone pellets in male rats kept in long or short photoperiod

Daily afternoon injections of 25 micrograms melatonin for 12 weeks had no effect on testicular weights of male rats kept in long photoperiod (14L:10D); similarly, exposure of rats to short photoperiod (2L:22D) had no effect on gonadal weight. However, rats maintained in a long or short photoperiod and implanted every 2 weeks with a 15 mm Silastic pellet containing testosterone showed a significant reduction in testicular weight; this effect was more pronounced in rats exposed to a short photoperiod. Melatonin injections in testosterone-treated rats in a long photoperiod exacerbated the inhibitory effects of testosterone alone. Subcutaneous 2-weekly implants of a beeswax pellet containing 1 mg melatonin reversed the effects of the melatonin injections on relative testicular weights but not those due to short photoperiod exposure. Testosterone implants significantly reduced pituitary LH values in long and short photoperiod-exposed animals, more particularly in those exposed to short photoperiod. Melatonin injections alone or in combination with melatonin pellets did not further exaggerate the depression in pituitary LH due to testosterone alone in long photoperiod-exposed animals; similarly melatonin pellets did not reverse the depression in pituitary LH observed. No significant differences in plasma prolactin concentrations or in thyroxine concentrations or free thyroxine index were observed after any combination of treatments. We therefore suggest that the effects observed with short photoperiod may be due to melatonin.     

Splenic hypertrophy and extramedullary hematopoiesis induced in male Syrian hamsters by short photoperiod or melatonin injections and reversed by melatonin pellets or pinealectomy

Adult male Syrian hamsters either placed in a short photoperiod alone or kept in a long photoperiod and given daily afternoon injections of the pineal indole melatonin (25 micrograms) exhibited splenic hypertrophy and extramedullary hematopoiesis in addition to a marked regression in testicular weight. The testicular regression as well as the changes in spleen weight and histology could be prevented if the animals in short photoperiod were either pinealectomized or implanted subcutaneously with a pellet containing 1 mg melatonin. Female Syrian hamsters given afternoon injections of melatonin for 7 or 12 weeks had ovaries devoid of corpora lutea; additionally, these animals had reduced relative spleen weights compared to the control animals. In conclusion, it is apparent that spleen weight varies with the functional status of the gonads. Splenic hypertrophy accompanied by pineal-induced testicular regression in males may be related to splenic extramedullary hematopoiesis.     

Melatonin and 6-methoxy-2-benzoxazolinone (6-MBOA) alter the response of the male Syrian hamster to natural photoperiod

Adult male hamsters bearing either a blank beeswax, 6-methoxy-2-benzoxazolinone (6-MBOA), or melatonin pellet were exposed to 8 weeks (Oct. 6–Dec. 6) of natural autumn decreasing photoperiod (<11 h light) and temperature conditions (mean 10°C for last 4 weeks) or to a 14 h light/10 h dark (14L10D) photoperiod and controlled temperature (20°C). Melatonin but not 6-MBOA pellets partially prevented the combined effects of short photoperiod and cold temperatures on the testes and accessory organs. However, both 6-MBOA-and melatonin-treated hamsters maintained outdoors had significantly higher pituitary follicle stimulating hormone (FSH) values compared to their respective indoor-treated controls or to the animals kept outdoors and treated with a blank beeswax pellet. When one compares the various effects of 6-MBOA and melatonin (2 mg/month) on the reproductive system of the male hamster, 6-MBOA is not as effective as melatonin in altering reproductive responses to short photoperiod and cool temperatures at the dose administered.     

Melatonin-pineal relationships in female golden hamsters.

Light deprivation by blinding in female hamsters was followed by a regression of the reproductive organs, an elevation of pituitary LH concentration and a depression of pituitary prolactin levels. Pinealectomy negated almost completely the effects of light deprivation on the neuroendocrine-reproductive axis. Weekly subcutaneous implants of a melatonin:beeswax pellet completely prevented the pineal gland from inhibiting reproductive physiology in blinded hamsters. The findings suggest that melatonin is not pineal antigonadotrophic factor in female golden hamsters. Melatonin implanted hamsters also had higher than normal levels of plasma prolactin.     

Effects of long-term treatment with melatonin or melatonin plus ectopic pituitary transplants on testicular LH/hCG and prolactin receptors in juvenile Syrian hamsters (Mesocricetus auratus)

Juvenile hamsters were injected daily with melatonin and some were also given transplants of 2 pituitaries under the kidney capsule. Weights of the testes and the accessory reproductive glands were reduced after 8 and after 12 weeks of melatonin treatment, but remained unaltered in animals treated with ectopic pituitary transplants. Levels of testicular LH/hCG receptors were significantly reduced by daily melatonin injections for 8 and 12 weeks. The presence of pituitary transplants in melatonin-injected hamsters prevented these reductions, and increased LH/hCG receptors above control levels. These changes in testicular LH/hCG receptors were closely related to alterations in serum prolactin concentration induced by melatonin and pituitary transplants. After 8, but not after 12 weeks of treatment, testicular prolactin receptor levels were reduced by melatonin and maintained by the presence of pituitary transplants. We conclude that: juvenile male hamsters become sensitive to the effects of daily melatonin injections when they reach maturity; daily melatonin injections can reduce the levels of testicular LH/hCG and prolactin receptors; and the effects of melatonin on LH/hCG and prolactin receptors are probably due to suppression of endogenous prolactin release.     

A study of indoles which inhibit pineal antigonadotrophic activity in male hamsters.

Blinding adult male golden hamsters led to involution of the testes and accessory sex organs (seminal vesicles and coagulating glands) and to a regression in pituitary prolactin levels within 8 weeks. The subcutaneous implantation of either melatonin or 6-hydroxymelatonin (1 mg/wk in beeswax) prevented the atrophy of the reproductive organs and the decrease in the stores of pituitary prolactin. Two other indoles, N-acetylserotonin and 5-hydroxytryptophol, failed to counteract the reproductive effects of blinding. Both melatonin and 6-hydroxymelatonin significantly elevated plasma LH titers.     

Studies on the minimal dosage of melatonin required to inhibit pineal antigonadotrophic activity in male golden hamsters.

Blinding adult male golden hamsters was followed by atrophy, within 12 weeks, of the testes and accessory sex organs (seminal vesicles and coagulating glands) and by a significant reduction in pituitary prolactin levels. In experiment 1 blind hamsters received subcutaneously implanted melatonin-beeswax (1:24 mg) pellets at the following intervals: once per week, per 2, 3, 4, 6 weeks, or only one pellet during the 12-week experimental period. The melatonin-beeswax pellets, regardless of the frequency of implantation, overcame completely the inhibitory effects of blinding on reproduction and nearly completely the depressant action of light deprivation on pituitary prolactin levels. In the second study the melatonin-beeswax pellets were implanted subcutaneously into blind hamsters every 2 weeks. The pellets contained either 1 mg, 500, 100, 50, or 1 mug melatonin. With the exception of the 1-mug dosage, melatonin again negated almost totally the inhibitory action of darkness on the gonads and accessory organs and also, for the most part, prevented the drop in pituitary prolactin levels. Based on these studies, when melatonin is chronically administered subcutaneously in a beeswax pellet the minimal dosage of melatonin required to counteract the inhibitory effect of darkness on reproduction seems to be less than 3.6 mug/day. The effects of chronic melatonin treatment are similar to those of pinealectomy.     

Diurnal Sensitivity of the Neuroendocrine-Reproductive Axis to the Antigonadotrophic Influence of Melatonin in Male Syrian Hamsters with Experimentally Altered Cortisol Rhythms

Two different experimental models were used to test if a temporal relationship exists between the rhythm of adrenal steroid secretion and the vulnerability of the hamster reproductive system to short photoperiod exposure or to the daily afternoon injection of melatonin. In the first experiment adrenalectomized hamsters were implanted with a Cortisol pellet to provide a sustained, rather than rhythmic, level of the hormone. The animals were either placed in short photoperiod or given a daily afternoon melatonin injection. In both cases the gonads underwent atrophy. In the second experiment adrenalectomized hamsters were given a Cortisol injection either in the morning (approx. 8 hr before the subsequent afternoon injection of melatonin) or in the afternoon (approx. 1 hr before the subsequent melatonin injection). Measurements of testicular and accessory organ weights 7 weeks later indicated regression of the reproductive system in both the groups when compared with their appropriate controls. Depressed levels of plasma LH. PRL, testosterone and thyroxine (T4) in these animals confirmed the melatonin induced gonadal collapse. The results suggest that apparently there is no temporal correlation between the rhythm of secretion of the adrenal steroids and the responsiveness of the reproductive system to late afternoon injection of melatonin. Interestingly, all the adrenalectomized Cortisol injected control animals (not receiving melatonin) had depressed plasma LH and PRL while the testicular weights and plasma testosterone titers remain unaffected.     

Effect of testosterone replacement on the alteration of steroid metabolism in the hypothalamic-preoptic area of male hamsters treated with melatonin.

Adult male hamsters were maintained under 14 hours of light per day and randomly assigned to groups that received daily afternoon melatonin (25 micrograms) or vehicle injections. Animals from both groups were killed following 4, 8, and 12 weeks of treatment. By 12 weeks, the melatonin-treated hamsters had significant reductions in the weights of the testes and seminal vesicles, serum testosterone levels, and activities did not differ between groups. In a second experiment, hamsters were hypothalamic-preoptic area (HPOA) aromatase activities. Hypothalamic-preoptic area 5 alpha-reductase activities did not differ between groups. In a second experiment, hamsters were again treated with melatonin or vehicle for 12 weeks prior to being killed. After 10 weeks of treatment, groups of melatonin-treated animals received subcutaneous silastic capsules (5, 10, or 20 mm) filled with testosterone. Animals in two other groups were given blank implants or no implants at all. Two weeks later, at autopsy, reproductive organ weights, serum testosterone levels, and HPOA aromatase activities were significantly suppressed by melatonin administration. 5 alpha-Reductase activity in the HPOA was not affected. Hamsters that had been given the 10- and 20-mm testosterone implants exhibited normal seminal vesicle weights and HPOA aromatase activities. These results suggest that melatonin-induced reduction of HPOA aromatase activity is mediated by decreased circulating levels of testosterone.     

Restoration of fertility in light-deprived female hamsters by chronic melatonin treatment

Summary Blinding young adult female hamsters was followed by functional involution of the ovaries and uteri and by the cessation of cyclic vaginal phenomena. Light deprivation was also accompanied by elevated plasma and pituitary levels of luteinizing hormone and depressed levels of prolactin in both the blood and the pituitary gland. Only one of 15 blinded hamsters became pregnant when they were exposed to fertile males for 30 days. Both pinealectomy or chronic melatonin treatment (1 mg melatonin implanted subcutaneously per week in beeswax) prevented the changes in the reproductive organs and in pituitary hormone levels attendant on light-deprivation. Both treatment also returned vaginal cycles to normal and restored plasma prolactin titers. Unlike hamsters that were blinded only, light deprived hamsters that were either pinealectomized or melatonin treated were capable of reproducing when they were caged with fertile males. The reproductive capability (i.e., percent of animals that become pregnant and the sizes of their litters) of these animals was equivalent to that of the untreated control hamsters. This is the first report that chronic melatonin treatment restores fertility in blinded female hamsters.Supported by Grant GB-43233X from the National Science Foundation     

A novel melatonin antagonist, N-(2,4-dinitrophenyl)-5-methoxytryptamine neutralizes some effects of melatonin in the female Syrian hamster

In this present study we evaluated the ability of a recently synthesized melatonin antagonist, N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23), to antagonize the effects of afternoon injections of melatonin on the reproductive and thyroid axes in the female Syrian hamster. Thirty-six animals were divided into four groups and treated daily for 13 weeks with an afternoon injection of melatonin (25 micrograms/injection) or saline diluent. ML-23 was given via the drinking water to both melatonin- and saline-treated groups. The experiment was continued until 78% of melatonin-treated animals exhibited acyclicity. The results show that ML-23 partially reversed the effects of melatonin on pituitary follicle-stimulating hormone concentrations but was without effect on the decreased pituitary and plasma prolactin concentrations induced by melatonin treatment. Furthermore, ML-23 antagonized the effects of melatonin on plasma thyroxine levels and significantly increased plasma triiodothyronine concentrations and the free triiodothyronine index when used in combination with melatonin. The decrease in ovarian weight and plasma estradiol, but not progesterone, obtained with melatonin treatment also was reversed by ML-23. Our data suggest that ML-23 prevents the effects of melatonin treatment on ovarian weight, pituitary follicle-stimulating hormone levels, plasma estradiol, and thyroxine concentrations in the female Syrian hamster. Since ML-23 did not prevent the effects of melatonin on pituitary weight, plasma luteinizing hormone and prolactin, and pituitary prolactin concentrations, the actions of ML-23 may involve only peripheral sites of action of melatonin. Alternatively, the dose of ML-23 may not have been optimal to prevent all of the central effects of the indoleamine.     

The effects of melatonin and hypothyroidism on estradiol and gonadotropin levels in female Syrian hamsters

Since melatonin injections administered near the end of the daily photoperiod influence both gonadal and thyroid hormones in the female hamster, the present study was designed to compare the effects of melatonin and hypothyroidism on the reproductive system and to determine whether thyroid status influenced the action of melatonin on the regulation of the hormones of reproduction. The effects of daily melatonin injections were determined in control hamsters, in hamsters rendered hypothyroid with thiourea, and in hypothyroid hamsters receiving thyroxin (T4) hormone replacement. As previously reported, melatonin injections disrupted estrous cyclicity, disrupted the normal pattern of gonadotropin secretion, and resulted in atrophy of the uterus and vagina. These changes coincided with depressed serum and pituitary prolactin (PRL), and depressed levels of estradiol. The effects of melatonin on uterus, vagina, ovary, and on gonadotropin levels were not prevented by T4 replacement, with the exception of a melatonin-induced increase in serum follicle-stimulating hormone (FSH). This suggested that the cessation of estrous cyclicity was not primarily a result of thyroid deficiency. Hypothyroidism, however, like melatonin, resulted in a reduced number of developing and mature follicles and corpora lutea in the ovaries, and in reduced uterine weight. It also produced follicular atresia, reduced the circulating levels of estradiol, and resulted in reduced incidence of estrus smears. T4 replacement, for 2 weeks, prevented the decline in mature follicles and corpora lutea, reduced the extent of follicular atresia, increased circulating levels of estradiol, and increased uterine weight. PRL and luteinizing hormone (LH) data also provided evidence for antagonistic effects of melatonin and T4 in female hamsters. These data raise the question whether melatonin-induced changes in circulating levels of T4 play a role in the seasonal cycles of reproductive competence in the female hamster.     

Partial maintenance of testes and accessory organs in blinded hamsters by homoplastic anterior pituitary grafts or exogenous prolactin

When blinded, golden Syrian hamsters undergo marked gonadal atrophy. This phenomenon is prevented by pinealectomy. The mechanisms involved in this pineal-mediated response were investigated through either the transplantation of pituitary homografts or treatment of blinded, male hamsters with exogenous prolactin. It was found that anterior pituitary homografts placed beneath the kidney capsule on the day of bilateral optic enucleation partially maintained testicular and accessory organ weights. Serum prolactin levels were reduced in blinded animals below that of intact controls. On the other hand, blinded hamsters bearing anterior pituitary homografts showed serum prolactin levels comparable to those of intact controls. In other experiments, the injection of either 3.2 or 6.4 I.U. of ovine prolactin/hamster/ day for a period up to seven weeks partially inhibited the atrophy of testes and accessory organ weights in blinded hamsters. These data suggest a possible role for prolactin in the pineal-mediated atrophic response to light deprivation.     

Melatonin: Failure of pharmacological doses to induce testicular atrophy in the male golden hamster

Effects of graded doses of melatonin on reproductive function of the male golden hamster were studied. Daily injections of melatonin at 17:00 h brought about complete testicular regression when the hamster received 10, 25, and 100 ug melatonin; larger doses of 1,000 ug or 2,500 ug had a partial or no effect on testicular or accessory sex organ weights. The dose response relationship of testicular and accessory sex organ weights to melatonin treatment somewhat resembled an inverted bell-shaped curve. Plasma, LH and prolactin concentrations followed the pattern of the testicular and accessory sex organ weights; small doses of melatonin suppressed plasma, LH and prolactin while larger doses had no suppressive effects. These results indicate that melatonin can exert a reproductive inhibitory effect if injected with small, but not with larger doses.     

Refractoriness to the antigonadotrophic effects of melatonin in male hamsters and its interruption by exposure of the animals to long daily photoperiods

Exposure of adult male Syrian hamsters to artificially shortened photoperiods in the laboratory was followed by gonadal involution. Return of these animals to long days (light:ddark cycles of 14:10) resulted in regrowth of the reproductive organs, following which the animals were refractory to the normally antigonadotrophic effects of daily melatonin injections given late in the afternoon. When hamsters were kept under naturally shortening photoperiods beginning on October 19, their reproductive organs involuted and remained infantile until the following spring when they again recrudesced to adult size. These animals too were refractory to the inhibitory effects of daily afternoon melatonin injections. The refractory period to melatonin was interrupted by exposure of the animals to long days for 22 weeks.     

Beta-adrenergic blockers prevent short photoperiod-induced gonadal regression, but not melatonin-induced regression in male Syrian hamsters

Pineal melatonin synthesis is regulated by norepinephrine at beta-adrenergic receptors on pinealocytes. Melatonin released from the pineal is believed to be responsible for short photoperiod-induced gonadal regression. By blocking melatonin production at the beta-adrenergic receptor with beta-adrenergic antagonists, short photoperiod-induced gonadal regression was prevented. Propranolol or nadolol injected daily in the late afternoon prohibited short photoperiod-produced testicular regression in male Syrian hamsters. Likewise, propranolol and nadolol pellets were able to block short photoperiod-induced gonadal regression. Interestingly, in the presence of beta-adrenergic antagonists, gonadal regression was still produced by daily afternoon injections of melatonin. These results indicate that propranolol or nadolol can be used to "pharmacologically pinealectomize" hamsters and block the physiological action of the pineal.     

Hormonal consequences of subcutaneous 6-methoxy-2-benzoxazolinone pellets or injections in prepubertal male and female rats

Prepubertal 27-day-old female rats maintained in a 14L:10D cycle (lights on 06:00 h) were injected s.c. at 13:00 h with saline or 2, 20 or 200 micrograms 6-methoxy-2-benzoxazolinone (6-MBOA) and killed 25-27 h later. No significant differences in body, pituitary or ovarian weight were noted. Differences in uterine weight (mg/100 g body weight) and in circulating free thyroxine index fit the pattern of a reduction after the lower doses with reversal of this effect after the highest dose. A dose-related rise in plasma prolactin concentration was accompanied by a significant increase in pituitary prolactin at the lowest (2 micrograms) dose. When 27-day-old prepubertal male and female rats maintained in a 14L:10D cycle were implanted with a beeswax pellet or a wax pellet that contained 100 micrograms or 1 mg 6-MBOA and killed 3 days later between 14:00 and 16:00 h, body and absolute ventral prostate weights (but not weights of other accessory organs, testes or relative ventral prostate weights) in males were lower. Pituitary (but not plasma) prolactin concentrations were higher after the lower dose compared to the controls; pituitary and plasma values of LH and FSH were unchanged. In females, reproductive variables were unchanged except for a reduction of pituitary prolactin after the 1 mg dose. Triiodothyronine and its free index were elevated after the higher dose in males and the lower dose in females. The free thyroxine index appeared raised after the larger dose only in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin decreases estrogen receptor expression in the medial preoptic area of inbred (LSH/SsLak) golden hamsters.

Daily late afternoon injections of melatonin (25 micrograms/day s.c.) were found to reduce the number of cells expressing estrogen receptor immunoreactivity in the medial preoptic area of ovariectomized inbred (LSH/SsLak) golden hamsters. Employing immunocytochemical analysis with the H222 monoclonal antibody to the human estrogen receptor, we examined the effects of melatonin on estrogen receptor expression in the hypothalamus, particularly the medial preoptic area, of ovariectomized virgin female hamsters. Analysis of the results showed that melatonin administration induced a 50-70% decrease in numbers of estrogen receptor-immunoreactive neurons in the medial preoptic area of ovariectomized female hamsters. Furthermore, an overall qualitative decrease in the intensity of estrogen receptor immunoreactivity was observed. In intact regularly cycling female hamsters used to monitor the efficacy of melatonin treatment, there were significant reductions in the serum levels of FSH, LH, and prolactin as measured by radioimmunoassay and in uterine and pituitary weights after 8 wk of melatonin treatment. These results suggest that melatonin may exert its anti-reproductive effects in hamsters by modulating estrogen receptor levels in medial preoptic area neurons, thus influencing steroid feedback mechanisms.     

Melatonin-induced suppression of gonadotropin titers in male golden hamsters: Effect on gonadal feedback mechanisms

Daily subcutaneous injections of melatonin cause testicular regression and a decline in circulating gonadotropin levels in male hamsters maintained on long photoperiods. We examine here if a reduction in gonadotropin levels also occurs in castrates administered melatonin and if melatonin-regressed hamsters respond to castration with an increased release of pituitary gonadotropins — a typical “castration response.” Groups of intact and castrated male hamsters maintained on a photoperiod of LD 14:10 received subcutaneous injections of 15 ug melatonin/day. Controls received vehicle only. After 7 weeks of injections the intact males were castrated. All animals were sacrificed a few days later and serum was assayed for gonadotropin titers. Melatonin injections caused a marked decline in serum gonadotropins in castrates and in intact males also caused testicular regression. In the latter, no “castration response” was observed upon removal of the testes. Thus, daily injections of melatonin depress serum gonadotropins in castrate and intact males and block the castration-associated rise in circulating gonadotropins in the latter.     

Effect of melatonin implants on gonadal weights and pineal gland fine structure of the golden hamster

Weekly subcutaneous implants of melatonin in a beeswax pellet prevented the testicular regression which normally occurs in hamsters exposed to short photoperiod for 8 weeks. Normal (14L:10D) hamster testes were indistinguishable from the testes of melatonin-treated (1L:23D) hamsters. The exogenous melatonin had varied effects on the fine structure of the golden hamster pineal gland. Pinealocyte nuclear characteristics of melatonin-treated hamsters (smaller average diameter, less polymorphism, and more heterochromatin) as well as apparent reductions in the amounts of hypertrophic SER and lipid moieties seemed to indicate that melatonin caused inhibition of pineal gland activity, and in this respect counteracted the effects of short photoperiod. However, an apparent increase in the number of large mitochondria, membrane whorls and dense-cored secretory vesicles in pinealocytes of melatonin-treated hamsters suggests enhanced pineal gland activity.