Effects of a calcium supplement on bone mineral density in male cyclists.

AIM: The purpose of this study was to maintain or improve bone density in male road cyclists through provision of calcium and vitamin D3 supplementation ingested before cycling. METHODS: Participants were male cyclists (N=17), with a mean (±SD) age of 42.7 (9.4) years. Measurements of lumbar spine and hip areal bone mineral density (aBMD) were performed at the start and end of a cycling season. Cyclists were randomized into the calcium supplement (CAL) or the control group (CON) group based on lumbar spine T-scores. The CAL group was instructed to consume 1600 mg calcium and 1000 IU vitamin D3 prior to cycling for the 5-month period. RESULTS: Femoral trochanter aBMD significantly decreased during the 5 month cycling season. There was no difference in aBMD between CAL and CON groups. CONCLUSION: Negative effects of competitive cycling on aBMD in hip structures can be observed within 5 months. Calcium and vitamin D3 ingested prior to cycling does not ameliorate this effect. This proof of concept paper provides evidence that more work is needed to find mechanisms to protect cyclists from destructive bone loss in hip structures.

Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine function in golden hamsters.

Grafts of fetal tissue including the suprachiasmatic nucleus (SCN) of the hypothalamus restore locomotor rhythmicity to behaviorally arrhythmic, SCN-lesioned Syrian hamsters. We sought to determine whether such transplants also reinstate endocrine rhythms in SCN-lesioned hamsters. In Exp 1, SCN lesions interrupted estrous cycles in a 14 h light, 10 h dark photoperiod and locomotor rhythms in constant dim red light (DD). SCN grafts that reinstated behavioral circadian rhythms consistently failed to reestablish estrous cycles. After ovariectomy, estradiol implants triggered LH surges at approximately circadian time 8 in 10 of 12 brain-intact control females and 0 of 9 SCN-lesioned, grafted females. Daily rhythms of the principal urinary melatonin metabolite, 6alpha-sulfatoxymelatonin, were not reestablished by behaviorally functional grafts. In Exp 2, SCN lesions eliminated locomotor rhythmicity in adult male hamsters maintained in DD. Seven to 12 weeks after restoration of locomotor activity rhythms by fetal grafts, hosts and sham-lesioned controls were decapitated at circadian times 4, 8, 12, 16, 20, or 24. Clear circadian rhythms of both serum corticosterone and cortisol were seen in sham-lesioned males, with peaks in late subjective day. No circadian rhythms in either adrenal hormone were evident in serum from lesioned-grafted males. Testicular regression, observed in intact and sham-lesioned males maintained in DD, was absent not only in arrhythmic SCN-lesioned hamsters given grafts of cerebral cortex, but also in animals in which hypothalamic grafts had reinstated locomotor rhythmicity. The pineal melatonin concentration rose sharply during the late subjective night in control hamsters, but not in SCN-lesioned animals bearing behaviorally effective transplants. Even though circadian rhythms of locomotor activity are restored by SCN transplants, circadian endocrine rhythms are not reestablished. Endocrine rhythms may require qualitatively different or more extensive SCN outputs than those established by fetal grafts.

Neonatal hypothyroidism permanently alters follicle-stimulating hormone and luteinizing hormone production in the male rat.

Transient neonatal hypothyroidism, induced with the goitrogen 6-n-propyl-2-thiouracil (PTU), results in dramatic increases in both testis size and sperm production in the adult rat. The observed increases in testis size and function occur in the presence of normal circulating testosterone levels. However, circulating gonadotropin levels are chronically reduced by 30-50% at all times in treated males. To better understand the permanent reduction in serum gonadotropin levels following transient neonatal hypothyroidism, we conducted a series of experiments to evaluate pituitary and hypothalamic function in the adult male PTU-treated rat. PTU treatment led to a significant reduction in GnRH-stimulated LH production. Castration resulted in 3.9- to 8.5-fold increases in circulating gonadotropin levels in both treated and control males; however, the absolute increases were significantly reduced in treated males. In contrast to circulating levels, pituitary gonadotropin contents did not increase in treated males after castration. PTU treatment did not lead to a reduction in the density of either luteotropes or folliculotropes, and both cell types increased in size and density after castration. The relative concentrations of both gonadotropin beta-subunit messenger RNAs increased more slowly in treated males than in controls after castration. Thus, although treated rats have the intrinsic ability to produce normal circulating levels of LH and FSH, gonadal feedback and an overall reduction in gonadotrope synthetic ability combine to produce the chronically reduced circulating levels of these hormones.

Effects of photoperiod duration and melatonin signal characteristics on the reproductive system of male Syrian hamsters.

Three experiments tested effects of photoperiod and the pineal hormone melatonin (MEL) on reproductive function among male Syrian-hamsters. In Experiment 1, hamsters were exposed for 32 weeks to 1 of 4 short photoperiods which varied in duration (11.5 L; 10 L; 8 L; 6 L). A fifth group was shifted from 11.5 L to 6 L after 6 weeks. Shorter photoperiods were associated with more rapid regression of the testes, but all groups eventually regressed to the same extent. In contrast, the temporal profile of testicular recrudescence, expressed as males became photorefractory, was not significantly different between groups. A decrease in photoperiod from 11.5 L to 6 L after 6 weeks did not delay the onset of recrudescence. The 11.5 L group was subdivided at week 32 and transferred to either 13 L or 16 L for the next 8 weeks to break photorefractoriness. Upon subsequent exposure to 8 L, both subgroups regressed their testes in similar fashion over weeks 40-52, indicating that the two long photoperiods were equally effective in breaking photorefractoriness. Nevertheless, FSH and prolactin were more consistently suppressed in the 16 L group following the switch to 8 L. Experiment 2 tested whether differing durations of MEL, administered s.c. each night for 9 weeks, elicit graded rates of reproductive regression in pinealectomized males. Testicular regression was more rapid in the group receiving MEL for 12 h than it was in the group receiving MEL for 8.5 h, thus supporting the hypothesis that the faster rates of testicular regression in the shorter photoperiods of Experiment 1 were due to their concomitant longer durations of nightly MEL secretion. Experiment 3 tested the hypothesis that rates of testicular regression in males receiving exogenous MEL would be affected by their prior photoperiodic history. Males were exposed to 18 L or 14 L for 7 weeks, then pinealectomized and administered 9.5 h MEL infusions s.c. each night for 9 weeks. In contrast to predictions, photoperiodic history had only transitory effects on MEL-induced testicular regression. Although the differences in MEL duration that accompany different short photoperiods have reproductive consequences (Experiment 1), the extent to which MEL duration expands during the transition from stimulatory to inhibitory photoperiods appears to be a less significant variable (Experiment 3).

Food deprivation and the facilitatory effects of estrogen in female hamsters: the LH surge and locomotor activity.

Two experiments investigated short-term food deprivation effects on neuroendocrine processes influenced by estrogen. These studies were prompted by prior work indicating that food deprivation increased the number of immunocytochemically identified cells containing estradiol receptors in the medial preoptic area of ovariectomized female hamsters. Presumably, this is one way that changes in metabolic fuel availability might alter the responsiveness of one or more systems to estradiol. The purpose of this study was to investigate two effects of estradiol that might be affected by food deprivation; these were 1) the positive feedback effects of estradiol on the luteinizing hormone (LH) surge, and 2) the facilitating effects of estradiol on locomotor activity. In Experiment 1, ovariectomized hamsters were administered estradiol, before or after 48 h of food deprivation. Two days after hormone treatment, blood was obtained by cardiac puncture, once in the morning (1100 h) and twice during the afternoon (1600-1800 h). These times were chosen to best characterize the magnitude of the LH surge. Food deprivation enhanced the amplitude of the LH surge in response to estradiol when this treatment preceded, but not when it followed, the administration of estradiol. However, there was variability in the dose of estradiol at which this effect of food deprivation occurred. In Experiment 2, the locomotor (running wheel) activity of two groups of gonadally intact female hamsters was quantified; one group was tested during the early (days 1 + 2; low estradiol) part of the estrous cycle, and the other group was tested during the late (days 3 + 4; high estradiol part of the estrous cycle. In both groups, testing was performed first under ad lib feeding conditions and again during 48 h of food deprivation. On average, the days 3 + 4 group was more active than the days 1 + 2 group, reflecting their differing levels of endogenous estradiol. Food deprivation significantly increased locomotor activity, independently of the stage of the estrous cycle during which it was imposed. These results are discussed in terms of the influence that altered estradiol receptor expression in the medial preoptic area might play in generating the effects we observed following short-term food deprivation.

Effects of photoperiod and androgen on pituitary function and neuropeptide staining in Siberian hamsters.

Short photoperiods decrease gonadotropin secretion in Siberian hamsters, but it is unknown whether the negative feedback effects of androgens are amplified under such conditions, as is the case in other species. Photoperiod regulates the synthesis and secretion of gonadotropin-releasing hormone (GnRH), beta-endorphin, and arginine vasopressin (AVP), which influence gonadotropin release and sexual behavior but are themselves regulated by gonadal steroid hormones. To determine the role of androgen in these effects of daylength, immunostaining and gonadotropin concentrations were examined after 8 wk of exposure to long or short days (LD or SD). Animals were either left intact, castrated, or castrated with immediate or delayed replacement of testosterone (T). We also investigated effects of age on photoperiodic influences on brain peptides and serum hormone levels. Serum prolactin concentrations were regulated by photoperiod and by gonadal status in LD hamsters. Effects of T on follicle-stimulating hormone secretion were more pronounced in SD hamsters. Older hamsters were generally less responsive to effects of daylength on pituitary function. Photoperiod and gonadal status regulated the number of AVP-immunoreactive (ir) cells in the bed nucleus of the stria terminalis and the medial amygdala. Androgen treatment yielded more AVP-ir neurons in LD than in SD. Photoperiod influenced the number of GnRH-ir cells only in the medial septum of castrated hamsters. Daylength regulated beta-endorphin-ir neurons in intact hamsters, but not in castrates. Only among old hamsters did photoperiod affect the influence of T on beta-endorphin staining in neurons and fibers. Such fiber staining was unaffected by photoperiod in intact and T-treated castrate hamsters, but was reduced in SD castrates. We conclude that daylength modulates the effects of androgen on gonadotropin secretion and influences the effect of T on neuropeptide staining in regionally specific patterns that depend on the age of the animal and its history of prior steroid exposure.

Regulation of the phase and period of circadian rhythms restored by suprachiasmatic transplants.

The influence of exogenous signals on circadian rhythms restored by transplants of the suprachiasmatic nucleus (SCN) of the hypothalamus has received little study. The authors tested the responsiveness of hamsters bearing SCN transplants to photic and pharmacological treatments. Light intensities as high as 6,500 lux were insufficient to produce entrainment, although masking was observed frequently. Triazolam failed to produce statistically significant phase shifts when administered during the subjective day, but 2 animals bearing functional SCN grafts responded to this benzodiazapine during the subjective night. The authors next tested the hypothesis that the host can retain circadian aftereffects that influence the period of the circadian system reconstituted by the graft. Intact hamsters were entrained to light:dark cycles of short (23.25-h) and long (25-h) period (T) for at least 3 months. Control hamsters released into constant darkness exhibited profound and long-lasting aftereffects of entrainment to T cycles. Hamsters that received SCN lesions after exposure to these T cycles and SCN grafts 3 weeks later exhibited marginal but statistically significant aftereffects that disappeared within 3 months. On subsequent transfer to constant light, tau lengthened by 0.25 +/- 0.6 h in hamsters with intact SCN (p < .05). Animals bearing SCN grafts continued to free run in constant light but differed from intact animals in that circadian period did not lengthen. Functional SCN grafts contained vasoactive intestinal polypeptide (VIP), neurophysin (NP), and cholecystokinin (CCK) immunoreactive (ir) cells. Inputs of neuropeptide Y-and serotonin-ir fibers from the host brain to grafted SCN peptide cell clusters were variable. Limited observations using retrograde and anterograde tracers do not support the existence of extensive input to the graft. Retinal input overlapped only rarely with clusters of VIP-ir, CCK-ir, or NP-ir cells. The authors conclude that the circadian system reinstated by SCN transplants is relatively impervious to photic influences that exert parametric and nonparametric influences in intact hamsters. The transient expression of aftereffects induced in the host before transplantation indicates that extra-SCN systems of the host can influence the period of the reconstituted circadian system to at least a limited degree.

Effects of melatonin and time of day on in vitro pituitary gonadotropin basal secretion and GnRH responsiveness in the male golden hamster.

Melatonin, a pineal hormone, is known to play an important role in mediating changes in the reproductive system which occur in response to seasonal changes in the length of the day. An in vitro pituitary perifusion system has been used to examine both direct and indirect effects of melatonin on pituitary gonadotropin basal secretion and responsiveness to GnRH stimulation. Anterior pituitaries from adult male golden hamsters were perifused with an APS 10 perifusion system for 6 h. Both basal secretion and secretion in response to hourly GnRH stimulation (1 min, 35 ng/ml pulses) were evaluated for LH and FSH. In order to expose pituitaries to shorter and longer durations of melatonin, tissue was perifused with melatonin-containing medium following dissection in the daytime (when endogenous melatonin levels are low) and following dissection in the nighttime (when endogenous melatonin levels are high). Exposure of pituitary tissue from golden hamsters to melatonin during perifusion caused a decrease in basal secretion of LH but did not affect GnRH stimulated LH or FSH release regardless of the time of dissection. There was, however, an effect of some diurnal factor to lower FSH responsiveness to GnRH stimulation from tissue dissected in the nighttime. It is suggested that melatonin may be responsible for this diurnal difference. Therefore, melatonin can act directly at the anterior pituitary to lower basal LH secretion, and melatonin or some other diurnal factor can act in vivo to lower pituitary FSH secretion in response to GnRH stimulation during the night.

Interactive effects of food deprivation and exercise on reproductive function in female hamsters.

Two experiments evaluated the combined effects of food deprivation and runningwheel access on estrous cycles and estrous behavior of female hamsters. In experiment 1, food deprivation on days 1 and 2 of the estrous cycle disrupted the next expected ovulation, and this effect was more, rather than less, robust in females allowed to exercise in running wheels while they were deprived. In experiment 2, a similar protocol was used except the females were ovariectomized and received sequential injections of estradiol benzoate (EB; 5 micrograms) and progesterone (P; 200 micrograms) separated by 48 h to induce lordosis, which was tested 4-5 after P. Food deprivation concomitant with hormonal treatment diminished lordosis durations, but this effect was significant only among the females that were permitted to run in activity wheels. Previous findings demonstrated that access to running wheels attenuated the inhibitory effects of short photoperiod exposure on hamster estrous cycles. In contrast, the present results indicate that this same manipulation exaggerates rather than diminishes the inhibitory effects of food deprivation on estrous cycles and hormone-induced behavioral estrus.

Changes in serum immunoreactive inhibin-alpha during photoperiod-induced testicular regression and recrudescence in the golden hamster.

Serum immunoreactive inhibin-alpha (irI alpha), FSH, LH, and testosterone (T) were measured in male golden hamsters during short-day-induced testicular regression and during testicular recrudescence following the transfer from short to long days. Serum FSH levels were maximally suppressed within 2 wk of transfer to short days. In contrast to FSH, irI alpha levels were not fully reduced until after 6 wk of exposure to short days, closely paralleling the timing of testicular regression. LH and T levels were also reduced within two 2k, with maximal suppression observed between 6 and 8 wk. Conversely, when males with regressed testes were transferred to long days, serum FSH rose to peak (25 ng/ml) levels by 3 wk and then declined to usual long-day levels. In contrast, serum irI alpha levels rose gradually, reaching adult long-day levels following 10 wk of exposure. Serum LH and T levels rose to peak levels between 5 and 8 wk before declining to adult levels. FSH, LH, and irI alpha levels were also measured after castration in male hamsters maintained on long or short days. Twenty-four hours after castration, levels of irI alpha were reduced in long-day males to levels comparable to those observed in castrated short-day males. Serum irI alpha levels respond slowly to abrupt changes in FSH levels after transfer to either long or short days, suggesting that testicular irI alpha secretion may not be directly and immediately influenced by circulating FSH levels in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.

Photoperiodic differences in in vitro pituitary gonadotropin basal secretion and gonadotropin-releasing hormone responsiveness in the golden hamster.

In order to examine pituitary gonadotropin secretion and responsiveness to GnRH after photic-induced changes in reproductive condition, an in vitro pituitary perifusion system was established for male golden hamster tissue. Anterior pituitaries from adult males which had been maintained on 14 h light:10 h dark (long days) or 6 h light:18 h dark (short days) for 10 weeks were perifused using an Acusyst perifusion system. Perfusates from unstimulated tissue (basal secretion) and from tissue stimulated with hourly pulses of GnRH (25, 50, or 100 ng/ml) were assayed for LH and FSH by RIA. Tissue from short-day animals had lower basal LH secretion than tissue from long day animals, but there were no significant photoperiodic differences for GnRH-stimulated LH secretion. In contrast, there were no photoperiodic differences in basal FSH secretion, but tissue from short-day animals secreted more FSH than tissue from long-day animals when stimulated with GnRH. Bioactivity of a small number of perfusate samples was assessed using in vitro rat granulosa cell and mouse Leydig cell assays for FSH and LH, respectively, and did not show any photoperiodic differences in LH or FSH bioactivity for GnRH-stimulated tissue. These studies indicate that the pituitaries of gonadally regressed hamsters are capable in vitro of responding to GnRH with similar or greater levels of gonadotropin release compared to pituitaries from animals with functional gonads. Therefore, it appears that the lowered serum gonadotropin levels seen in vivo in gonadally regressed animals are not due to a reduction in intrinsic pituitary sensitivity to GnRH.

Influence of progesterone and oestradiol-17 beta on blastocysts of the tammar wallaby (Macropus eugenii) during seasonal diapause.

Forty tammar wallabies, presumed to be carrying quiescent blastocysts, were injected with progesterone and oestradiol alone, or in combination, during seasonal quiescence when the corpus luteum is inactive. Plasma progesterone concentrations were increased to values equivalent to those of late pregnancy for the duration of the treatment in progesterone-treated groups but otherwise remained at values equivalent to seasonal quiescence. Tammars treated with low doses of oestradiol showed no measurable increase in plasma oestradiol concentrations but in those treated with high doses plasma concentrations were increased to oestrous levels. At autopsy on Day 18 after the start of treatment the embryos and reproductive tracts were assessed. While progesterone alone caused reactivation of about 50% of the embryos, blastocysts in tammars treated with oestradiol alone remained in diapause (low dose) or disappeared from the uterus (high dose): 2 blastocysts collapsed after some slight expansion. No synergistic effect on pregnancy was noted in tammars receiving both oestradiol and progesterone. We conclude that oestrogen alone is not capable of stimulating normal growth of blastocysts, and its role during early pregnancy in tammars remains unclear.

Chemical dosimetry of ethyl nitrosourea in the mouse testis.

[3H-Et]Nitrosourea was administered to male (101 X C3H) mice by i.p. injection at exposure levels of 10 mg/kg or 100 mg/kg. At intervals from 1 h to 6 days following treatment, the ratio of O6-ethylguanine to N7-ethylguanine in testis DNA averaged 1.13 following the 100 mg/kg exposure and 0.72 following the 10 mg/kg exposure. The amount of O6-ethylguanine recovered after the 100 mg/kg exposure was 40% greater than predicted from a linear extrapolation of the amount of O6-ethylguanine recovered after the 10 mg/kg exposure. We suggest that the high (100 mg/kg) exposure to ethyl nitrosourea results in depletion of the O6-alkylguanine acceptor protein within the testis and permits O6-ethylguanine to persist at higher levels than would be predicted from lower exposure data. W.L. Russell et al. (1982), W.L. Russell (1984) have found that specific-locus mutation frequencies induced in mouse spermatogonial stem cells are 5.8-fold greater after a single 100 mg/kg exposure to ethyl nitrosourea than after 10 weekly exposures to 10 mg/kg. The finding that the corresponding ratio for O6-ethylguanine formed in the testis is only 1.4 may be interpreted in a number of possible ways. If O6-ethylguanine is an important lesion for producing specific-locus mutations, then its formation in the stem cells must be at least 4-fold greater than that for the whole testis as the ENU exposure goes from 10 to 100 mg/kg: alternatively, the rate of repair of this lesion by the stem cells must decrease at least 4-fold relative to the average testicular cell. Other explanations for the difference in mutation response of the stem cells to acute vs. chronic ethyl nitrosourea-exposures include the possibility that other DNA lesions may be responsible for many of the mutations or that two hits on the DNA may be required to produce an effect.

Prolactin as a factor in the uterine response to progesterone in rabbits.

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.