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a Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310
b Yerkes Regional Primate Research Center, Atlanta, Georgia 30329
c Department of Obstetrics and Gynecology, Texas Tech University Health Science Center, Amarillo, Texas 79106
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leptin-deficient mice (ob/ob) are obese and infertile [6,7]. Both conditions were reversible with leptin administration [7]. Leptin treatment advanced sexual maturation in female mice [8,9]. If leptin serves as the trigger to initiate pubertal activation of the hypothalamic-pituitary-testicular axis, one would expect a rise in leptin secretion prior to this event. However, whether or not there is an increase in leptin secretion in humans before or in concert with pubertal development remains controversial. In girls, leptin concentrations in the circulation increased progressively from 5 to 15 yr of age, while in boys, levels rose from 5 to 10 yr of age and declined thereafter through 13 yr of age [10]. In a longitudinal study (2.5–5.1 yr) study of 8 prepubertal boys (Tanner's stage 1 or early 2), leptin concentrations were at or near a maximum value at the time of the initiation of the pubertal rise in testosterone (T) levels and testicular volume [11]. Mean levels of leptin in these boys rose significantly between the prepubertal period and the initiation of puberty, but then declined through midpuberty and postpuberty. In a larger, more comprehensive longitudinal study of 40 normal boys and girls [12], leptin did not differ between the sexes prior to the onset of puberty. During Tanner's stage 1 there was a gradual increase in leptin levels in both sexes, but then leptin levels declined in boys from stages 2 through 5 while levels in girls continued to increase. Similar changes in leptin levels during the peripubertal period in boys and girls were reported in an earlier cross-sectional study [13]. Conversely in another study, leptin levels did not change with pubertal stage in boys although they did increase in girls [14]. Moreover, there was no transient increase in leptin levels just prior to the onset of puberty as reported by Mantzoros and collaborators [11]. In addition, 24-h patterns of serum leptin did not differ between prepubertal and pubertal boys or girls [15], and leptin levels in patients with delayed puberty were significantly higher than those in normal prepubertal and midpubertal subjects [16]. Thus, the role that leptin plays in the initiation of puberty in humans remains unresolved.
Data in the rhesus monkey do not appear to support a role for leptin in initiating the onset of puberty in the male. In a cross-sectional study in monkeys (from birth until 20 yr of age), leptin levels overall were positively correlated with serum T concentrations [17]. Levels were elevated in the youngest animals, fell to lower values in juveniles, and then increased again with the onset of puberty. The rise in leptin secretion in these animals did not appear to precede activation of the testes at puberty. In a limited-length (from 18 to 30 mo of age), longitudinal study of intact and agonadal male monkeys, leptin levels did not change significantly during reactivation of the hypothalamic-pituitary-testicular axis at puberty [18].
Because the potential role of leptin in triggering the onset of puberty remains controversial, we undertook a detailed longitudinal study (from 10 mo until 7 yr of age) of developmental changes in leptin secretion in a group (N = 8) of male rhesus monkeys housed under natural environmental conditions. Because mature animals maintained under these conditions are seasonal, this study also provided the opportunity to assess changes in leptin secretion during seasonal increases and decreases in photoperiod and gonadal activity. We also assessed the relationship between body composition, leptin levels, and testicular activity in these animals once sexual maturity had been achieved.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments were performed according to the principles and procedures of the NIH Guidelines for the Care and Use of Laboratory Animals. The animals utilized in this study were born into a large social group of 75 animals being maintained in an outdoor compound (30 x 30 m) with an attached temperature-controlled, indoor area. The subjects were the controls from a larger study designed to examine the effects of neonatal treatment with a GnRH analogue on sexual and behavioral development [19]. The animals were maintained with their mother in the social group until they were 4 yr of age and then were relocated to a similar but smaller compound (15 x 15 m) as an all-male group.
Blood samples were drawn from all animals without anesthesia by saphenous venepuncture every other month from 10 mo until 30 mo of age and then monthly until 80 mo of age for assay of LH, T, and leptin. Peripubertal changes in serum LH and T in these animals were reported previously [19]. Body weights were recorded monthly throughout this study.
Body Composition Analysis
When animals were 4.5, 5.0, 5.5, and 6.0 yr of age, whole-body tissue analysis (total lean mass, total fat mass, and percent body fat) was performed on anesthetized (Telazol; Elkin-Sinn Inc., Cherry Hill, NJ; 3 mg/kg BW, i.m.) subjects using a Norland (Fort Atkinson, WI) XR26 dual-beam densitometer. The XR26 uses dual-energy x-ray absorptiometry to estimate body composition in vivo. The unit was equipped for high-speed scanning with a high-intensity x-ray source. The system utilizes the Norland revision 2.5.2 analytical software.
Assays
Serum LH concentrations were measured by the mouse interstitial-cell-testosterone-bioassay as modified by Steiner and Bremner [20]. LH data are expressed in terms of the monkey pituitary WP-XV-20 standard. Serum T was measured by RIA using a commercial kit (Diagnostic Products, Los Angeles, CA). Leptin was measured using a human leptin RIA kit (Diagnostics Systems Laboratories, Webster, TX) validated for the rhesus monkey. The intraassay and interassay coefficients of variation for the three assays, respectively, were 4.0 and 13.7%, 8.7 and 5.0%, and 5.0 and 12.9%.
Statistical Analyses
Data are presented as the mean ± SEM. Body weight, LH, T, and leptin levels were analyzed initially by one-way ANOVA with repeated measures over time followed, when appropriate, by Tukey's test for multiple comparisons (GB-Stat; Dynamic Microsystems, MD). During the juvenile period the relationships between leptin levels, body weight, and age were analyzed by Spearman's correlation test (SPSS; Advanced Statistics 6.1; SPSS, Chicago, IL). The same test was used to assess the relationship between leptin levels, total lean mass, total fat mass, and percent body fat at 4.5, 5.0, 5.5, and 6.0 yr of age.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in mean serum LH, T, and leptin during the peripubertal period (39–50 mo of age) are illustrated in Figure 4. Seven of eight animals achieved sexual maturation during the breeding season of the animals' fourth year (41–46 mo of age). Animals were considered sexually mature if they exhibited a pubertal increase in testicular volume, serum T, and LH and/or sperm was recovered upon electroejaculation [19]. Puberty was delayed in the other animal (Ym-3) until the subsequent breeding season. We did not detect any significant fluctuations in mean levels of leptin (Fig. 4) or in leptin concentrations from individual animals (Fig. 2, middle panel) prior to or in association with the pubertal rise in LH and T. Mean body weight increased by 2 kg over this period (data not shown). The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period, but in general this animal had the highest leptin concentrations throughout development.
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After the achievement of sexual maturity, mean leptin concentrations varied seasonally (P < 0.0001), with peak levels in the late winter (Jan–Mar) and a nadir in late summer (Aug–Sept; Fig. 5). During the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3–4 mo behind the seasonal increase in LH and T.
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Seasonal changes in leptin levels in individual monkeys are shown in Figure 2 (bottom). All animals appeared to exhibit seasonal excursions of leptin, although the magnitude of these changes varied substantially from animal to animal (% change between the peak and trough during Year 5 = 137 ± 46). Even though the animal (Ym-3) with delayed puberty became sexually mature during Year 5, leptin levels in this animal remained higher than in the other animals through 80 mo of age.
At 4.5 yr of age (corresponds to October of the animal's fifth year), serum leptin concentrations were positively correlated with percent body fat and the fat mass/lean body mass ratio (P = 0.04 for each; Fig. 6, top) and were inversely proportional to lean body mass (P = 0.03; Fig. 6, bottom). There was no significant association between leptin levels and serum T at this age. After this age (at 5.0, 5.5, and 6.0 yr of age), there was no consistent association between fat mass, percent body fat, lean body mass, serum T, and serum leptin (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of several studies in children [10–13] and lower mammals [5,8,9], it has been hypothesized that leptin serves as a metabolic signal to initiate events resulting in the achievement of sexual competence. The current study was initiated to further investigate a potential role for leptin in the initiation of pubertal development using the male rhesus monkey raised under natural environmental lighting conditions as an animal model.
The data from the current report do not support the hypothesis that leptin acts as a trigger to initiate peripubertal events in this primate species. Serum leptin levels were highest early in the juvenile period (10–38 mo of age) and declined gradually thereafter in negative association with increasing body weight (body mass more than doubled over this interval). Moreover, none of the seven animals that achieved sexual maturity during the breeding season of the animals' fourth year exhibited transient increases in serum leptin levels either preceding or in association with peripubertal changes in serum LH, T, or testicular volume. In addition, the one animal in which puberty was delayed for a year had the highest leptin concentrations and lowest body weight throughout the juvenile and peripubertal period (39–50 mo of age).
The reciprocal relationship between declining leptin levels and increasing body weight from birth until puberty in the rhesus monkey in the current study is essentially as described by others in a cross-sectional study in the same species [17]. However, these data run contrary to the positive correlation between increasing body weight and leptin levels reported in children (5–10 yr of age) [10]. Studies on the relationship between leptin and pubertal development in other nonhuman primate males do not appear to exist, nor have comparable studies been performed in the female rhesus monkey. Additional studies are necessary to elucidate apparent differences between the human and this nonhuman primate. We confirm in this report the earlier work of Plant and Durrant [18] that male rhesus monkeys do not exhibit significant excursions of serum leptin during the critical phase when the GnRH pulse generator is being activated in preparation for the onset of puberty. Of the seven animals that reached puberty during the breeding season (41–46 mo of age) of their fourth year, serum leptin in individual animals exhibited no significant fluctuations from 6 mo before until 6 mo after the peripubertal increase in serum LH and T. Thus, while our animals were maintained under environmental conditions different (natural vs. constant laboratory conditions) from those in the previous study [18], our results support the earlier work suggesting that leptin does not act as a metabolic trigger to initiate the onset of puberty in the male rhesus monkey. Perhaps even more revealing, the animal that showed a 1-yr delay in puberty exhibited the highest levels of leptin throughout the juvenile and peripubertal period when the other seven animals became mature. One note of caution, however, is that the current data are based on monthly AM blood samples over this period. Thus, it is possible that more frequent blood sampling or AM versus PM samples may have detected a transient rise in leptin during the period immediately before pubertal endocrine events. We believe this is unlikely, however, because no peripubertal rise in leptin was detected in weekly blood samples in the earlier study [18], and no differences were found in the nocturnal rhythm of leptin between prepubertal and pubertal boys and girls [15].
Not all studies in humans have shown an elevation of leptin prior to or in association with activation of the gonads, and there are other clinical studies that do not appear to support a role for leptin in the initiation of puberty in children. As mentioned previously, leptin levels did not change with pubertal stage in boys but did increase with pubertal development in girls [14]. The pattern of serum leptin over the 24-h day also did not differ between prepubertal and pubertal girls and boys [15]. Male patients with delayed puberty (constitutional or idiopathic hypogonadotropic hypogonadism) had higher levels of leptin than normal prepubertal or midpubertal boys [16]. During 120 days of GnRH treatment, gonadotropin and T concentrations and testicular size increased, but leptin levels did not change. At the end of treatment, levels were still higher than in midpubertal boys. During the GnRH treatment there was no correlation between changes in leptin levels and testicular volume, T, or body mass index. After long-term therapy of the patients with idiopathic hypogonadotropic hypogonadism, leptin levels did decline, leading the authors to suggest that time rather than increased testicular volume or T levels was responsible for the decrease at puberty.
It is interesting to speculate that the low levels of leptin present in nonhuman primates (vs. humans) may be a reflection of the small amount of adipose tissue present in laboratory-raised primates. These animals generally receive controlled amounts of monkey chow supplemented with fruits and vegetables, diets that are very low in fat; and indeed it becomes difficult to find appreciable amounts of adipose tissue in these animals. The higher levels of leptin and the increase in adipose tissue in children may be a reflection of a greater adiposity at all stages of development and may be a greater indicator of the effect of diet on the development of adipose tissue than of leptin as an initiator of pubertal development. It appears from our previous work [19,23,24] that a hierarchy of regulatory factors are involved in setting the rate of sexual maturation and the timing of puberty in the male monkey raised in a social group. One of the overriding factors is the social rank of the animal [19]. More high-ranking than low-ranking animals experienced puberty during their fourth year of life; and testicular size and serum LH and T were positively correlated with social rank during this period [19]. Higher-ranked animals had greater levels of T earlier during the breeding season of their fourth year, and levels remained higher for a longer period than in the lowest-ranking animal [25]. Testicular size, serum T, and body weight maintenance during the breeding season of Year 4 were positively associated with matriline rank [26]. However, regardless of social rank, the achievement of reproductive competence in these animals is confined to the fall breeding season when photoperiod length is declining [19,23,24]. Thus, when puberty was delayed, it was delayed for a full year. In addition to social rank and photoperiod, neonatal activity of the hypothalamic-pituitary-testicular axis may also influence the tempo of sexual maturation in the male primate. Fewer animals treated with GnRH analogues during the neonatal period to suppress this axis achieved sexual maturation during Year 4 [19,23].
The achievement of a critical body weight or fat mass may also play a role in the initiation of puberty [27], but what acts as a signal or how this signal is transmitted to the reproductive axis remains unknown. Although serum leptin levels do not increase prior to the onset of puberty ([18] and current study), and in fact decline throughout the juvenile period ([17] and current study) in the male monkey, leptin may still play a permissive role or act as a gate to signal that the body is capable of supporting sexual function as suggested recently in two reviews [28,29]. Thus, if nutrition is adequate (as signaled by leptin) and other more dominant factors (social rank and photoperiod) optimal, the onset of puberty would be triggered. Related to this issue and as yet unresolved is the potential involvement of insulin-like growth factor-1 (IGF-1) in this process. IGF-1 levels increase during development in a variety of primate species including the rhesus monkey [30,31]. Disruption of the gene coding the growth hormone (GH) receptor in female mice is associated with reduced IGF-1 levels and a delay in the onset of puberty [32]. Inhibition of GH and IGF-1 secretion with a somatostatin analogue induced delay in the onset of first ovulation, whereas IGF-1 infusion advanced first ovulation but had no effect on the age of menarche in juvenile female monkeys [33,34]. IGF-1 administration also accelerates the decrease in the sensitivity to the negative feedback effects of estradiol on LH secretion in adolescent ovariectomized female monkeys [31]. In the animals studied for the current report, both IGF-1 and IGF-binding protein 3 (IGFBP-3) and the ratio of IGF-1 to IGFBP-3 increased during development and peaked (approximately 38 mo of age) just prior to the beginning of the pubertal rise in testicular activity (39–40 mo of age; unpublished results). Moreover, the animal that exhibited a 1-yr delay in the onset of puberty in the current study in general had the lowest IGF-1 and IGFBP-3 levels throughout the juvenile period. IGF-1 stimulates IGF-1 receptor expression and GnRH secretion from the median eminence of immature rats [35,36], an effect that can be blocked via neutralization with GnRH antiserum [36]. Thus, the peaking of IGF-1 bioavailability late in the juvenile period may facilitate reactivation of the GnRH pulse generator during Year 4 in the male monkey provided that other more dominant regulator factors in this process (social rank and photoperiod) are optimal to allow its reemergence.
We are the first to demonstrate that there are seasonal fluctuations in leptin levels in male rhesus monkeys maintained under natural environmental conditions. Once sexual maturity was achieved, a small but distinct seasonal rhythm in serum leptin was evident during Years 5 and 6 in the animals, with a nadir in late summer and a peak in late winter. This leptin rhythm lagged 3–4 mo behind seasonal fluctuations in LH and T. It has been reported that T suppresses leptin production by adipose tissue [37–39]. This suppression of leptin by T may be partially responsible for the lower levels of leptin in men than in women [40,41] and the decline of leptin levels that begins in boys at the time of puberty [10–13,16]. The fact that seasonal peaks of T and leptin are separated by a 3- to 4-mo period suggests that seasonal changes in leptin may be driven by seasonal changes in testicular function. However, we also observed seasonal fluctuations (peak levels also occurred in late winter or early spring) of leptin in the animals during Years 2 and 3 before the pubertal activation of testicular function. The latter data suggest that other factors (e.g., photoperiod and/or seasonal changes in adiposity) may be responsible for seasonal changes in leptin secretion in the rhesus monkey. In this regard, however, there were no significant differences in fat mass at 4.5 (Oct), 5.0 (Apr), 5.5 (Oct), or 6.0 (May) yr of age in these animals. Thus, the factor or factors responsible for the seasonal changes in leptin remained undetermined.
At 4.5 yr of age, leptin concentrations were positively correlated with percent body fat and inversely related to lean body mass; but at 5.0, 5.5, or 6.0 yr of age there was no significant correlation between these measures of body composition and leptin levels. Moreover, we were unable to show any significant correlation between T and leptin levels at any of these ages. The loss of correlation between leptin and body fat and lean body mass after 4.5 yr of age in our animals is difficult to explain with currently available data but most likely reflects changes in body composition and endocrine interactions in the period immediately following the achievement of sexual maturation.
In summary, the data from the current study do not support the hypothesis that an increase in circulating leptin concentrations serves as a physiological trigger for initiating the onset of puberty in the male rhesus monkey. Leptin levels decreased with age throughout the juvenile period, and there was a high negative correlation with body weight. There was no detectable transient increase in leptin immediately before or in association with the peripubertal increase in gonadal activity as has been reported in children. Once sexual maturity was achieved there was a modest, but significant, seasonal fluctuation in leptin levels that was several months out of phase with the seasonal rhythm of sexual activity. Whether seasonal changes in photoperiod, testicular activity, and/or body composition or some other environmental factor drives this circannual rhythm of leptin remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Support was provided by NIH grants HD26423, RR03034, and RR00165. 
2 Correspondence: David R. Mann, Department of Physiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495. FAX: 404 752 1056; mann{at}msm.edu
Accepted: September 28, 1999.
Received: August 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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