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Testis; |
Department of Cell Biology and Physiology,3 Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Department of Physical Therapy,4 School of Health Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282
ABSTRACT
Testosterone shows circadian rhythms in monkeys with low serum levels in the morning hours. The decline relies on a diminished frequency of LH pulses. Inhibin B shows no diurnal patterns. In elderly men, the diurnal rhythm of testosterone is blunted and inhibin levels fall. Here we explore whether aging exerts similar effects in the rhesus monkey. We collected blood samples from groups of young (6–9 yr) and old (12–16 yr) male rhesus monkeys at 20-min intervals for a period of 24 h under remote sampling via a venous catheter. We determined moment-to-moment changes in plasma levels of testosterone, FSH, and LH by RIA, and of inhibin B by ELISA. We found significant diurnal patterns of testosterone in both groups. The circadian rhythm in testosterone was enhanced in older monkeys. Testosterone levels and pulse frequencies dropped significantly below those of young monkeys during midday hours. Diminished pulse frequency of LH appeared to be responsible for the midday testosterone decrease in old monkeys, while LH and testosterone pulse frequency did not change in young monkeys at corresponding time points. Old monkeys showed extended periods of LH-pulse quiescence in the morning and midday hours. Inhibin B and FSH levels were generally lower in old monkeys compared with the young group, but neither inhibin B nor FSH showed circadian rhythms. We conclude from these data that old rhesus monkeys have a more prominent circadian rhythm of LH and testosterone resulting from an extended midday period of quiescence in the hypothalamus-pituitary-gonadal axis.
aging, androgen, circadian rhythm, gonadotropins, inhibin, luteinizing hormone, monkey, testis, testosterone
Although the decline of androgen levels in elderly men and its clinical and therapeutic implications have been studied in detail (for review, see [1]), the existence of an andropause and the replacement of testosterone in middle-aged and elderly men remains a controversial topic [2]. Several studies have revealed an age-related decrease of serum testosterone when the analysis was performed in large cohorts of men [3, 4]. This has led to the conclusion that androgen levels decline with age in older men, and that this deficiency might need to be treated by replacement of androgens. Since profiles of testosterone were not determined in these studies. As such, this conclusion is only valid if the morning-time sampling of serum testosterone indeed reflects a general decline, and is not a consequence of changes to the well-described diurnal patterns of testosterone that may lead to pronounced changes at specific times of the day or night, but may not incur changes in the amount of testosterone released from the testis over a 24-h cycle. Strong circadian patterns of testosterone exist in healthy young men [5–7]. A blunting of this diurnal rhythm has been shown in aging men [5, 8–10], and was associated with age-related increase of serum LH and changes in LH pulse frequency [7]. Other studies have found no change in diurnal patterns, but a significantly decreased amplitude of free and bioavailable testosterone in older men [11]. It is important to note that all of these studies revealed, in older men, significantly lower levels of serum total, bioavailable, and free testosterone in the morning hours, which represented the sampling period used in the large-cohort studies. More recent clinical studies determining age-dependent changes of diurnal patterns used partial to complete suppression of the hypothalamic GnRH release and observation of an age-related difference in the LH and testosterone activity [12], or analyzed the LH-stimulated testosterone response under clamping [13]. These studies revealed that a multitude of age-dependent changes (among them, a decrease in GnRH outflow, LH drive of testosterone secretion, and feedback on GnRH-driven LH secretion) are responsible for lower androgen production in aging men. Mechanisms leading to diminished streoidogenic function of Leydig cells have been intensively explored in rats [14]. Many age-related defects were described and studied, which indicate that direct damage to Leydig cells by free radicals may be responsible for decreased steroid release in aging rats.
While age-related changes of diurnal patterns have been explored in men, a potentially similar effect of aging has never been explored in macaque monkeys. Striking diurnal variations in plasma LH and testosterone, with high levels in the night and lower levels during the day, have been described in adult rhesus monkeys [15–18]. Individuals showed 7–12 discrete peaks of serum testosterone over 24 h, which were preceded by peaks of serum LH. While pulse amplitudes were unchanged over a 24-h period, pulse frequency was reduced during the day and was lowest in the morning hours. A midmorning decrease of pulse frequency for both LH and testosterone was obvious in most monkeys. While extended periods of complete absence of LH pulses were recorded in some monkeys in the morning, a full inhibition of pulses for several hours was not observed in other monkeys.
In the present study, we analyze age-related moment-to-moment changes of FSH, LH, and testosterone in two groups of rhesus monkeys: young adults (6–9 yr) and aging adults (16–19 yr). We hypothesized that older monkeys show age-related changes to their pituitary-gonadal axis similar to those observed in aging men [1]. In addition, we also analyzed age-related patterns of inhibin B, which shows a diurnal pattern [19] and an age-related decline in men [20, 21]. In contrast, no diurnal rhythm of inhibin has been detected in monkeys [22].
Four young adult (6–9 yr) and four elderly (16–19 yr) male rhesus monkeys (Macaca mulatta) were subjects of the study. The animals were maintained in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the experimental procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The animals were housed singly in adjacent remote sampling cages under an artificial photoperiod (12L:12D; lights-on at 0700 h, lights-off at 1900 h) and controlled temperature and humidity. The animals had no exposure to natural light or other seasonally changing environmental conditions. Animal feeding and maintenance of cages were performed on a set schedule that did not vary from day to day. All experimental procedures reported in this article were performed between April and July. The individual adult monkey identification numbers, age, initial body weight, and initial testicular volume (determined by caliper) are presented in Table 1. In order to establish that all monkeys show complete spermatogenesis, electroejaculation was performed as described previously [23]. Initial ejaculate volumes and sperm counts are presented in Table 1. Body weight, testis volumes, and ejaculate parameters did not change during the course of the study.
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Implantation of Venous Catheters
Animals received 2 wk of training prior to the surgical implantation of catheters to allow them to adapt to the remote sampling cage, jacket, and tethering system. The monkeys were implanted with i.v. catheters, as has been described previously [24]. The Silastic catheters were tunneled s.c. from the site of venous insertion (internal jugular vein) to the midscapular region, where they were exteriorized via a small cutaneous fistula. Animals with catheters were fitted with a nylon jacket attached via a flexible stainless-steel tether to a swivel device mounted on top of the cage, permitting continuous access to the venous circulation without tranquilization and with minimal restraint. Postsurgically, all animals received a single i.m. injection of penicillin (300 000 U of Bicillin L-A; Wyeth Laboratories, Philadelphia, PA) and i.v. injections of a broad-spectrum antibiotic (100 mg of cefazolin sodium [Kefazol]; Apothecon, Princeton, NJ) and an analgesic (1 mg/kg body weight of meperidine hydrochloride [Demoral]; Elkins-Sinn, Cherry Hill, NJ) twice daily for 4 days. Maintenance of the monkeys with i.v. catheters has been described previously [24]. The catheter lines reached through small holes in the wall into the adjacent remote sampling room, which allowed constant access to the venous system without any disturbance to the animal.
Frequent Collection of Blood for Determination of 24-h Hormone Profiles
All animals were allowed at least 1 wk postsurgery for recovery before the sampling was initiated. Frequent blood samples (1.5 ml every 20 min) were drawn over a period of 24 h via the catheter into heparinized syringes. After collection of plasma, the pellet of blood cells was reconstituted in sterile saline and returned to the respective monkey via the venous catheter every 90 min. Hematocrit determinations before and after sampling revealed no changes in blood cell content. The sampling was performed in the adjacent room, avoiding any disturbance of the day-night rhythm and provoking minimal stress or disturbance to the monkeys. Plasma was separated and stored at –20°C until assayed. The starting point of the sampling was at either 0900 or 1200 h, and continued for 24 h. The sampling occurred in groups of three and five, and both sampling times included animals from both age groups.
Circulating concentrations of endogenous FSH and LH were determined with homologous (cynomolgous) RIA reagents supplied by the National Hormone and Peptide Program and standardized at the Assay Core of Center for Research in Reproductive Physiology (CRRP). The sensitivity of the FSH assay was 0.13 ng/ml, and the intra- and interassay coefficients of variation were <3% and <15%, respectively. The sensitivity of the LH assay was 0.11 ng/ml, and the intra- and interassay coefficients of variation were <4% and <9%, respectively. Plasma testosterone concentrations were measured with a commercially available solid-phase RIA kit (Total T [TKTT], Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA). The mean sensitivity of the assay was 0.014 ng/ml, and the intra- and interassay coefficients of variation were <9% and <10%, respectively. Circulating concentrations of inhibin B were determined by a specific two-site ELISA previously validated for the monkey [24]. The sensitivity of the inhibin B assay was less than 20 pg/ml, and the intra- and interassay coefficients of variation were 
5% and 7%, respectively.
Data Analysis and Statistical Tests
All hormone values are presented as single data points in 24-h profiles (Figs. 1 and 2). LH and testosterone pulses were detected by the pulse detection algorithm, Pulsar, with G values that produce a 1% false-positive error rate: G(1) = 4.4, G(2) = 2.6, G(3) = 1.92, G(4) = 1.46, and G(5) = 1.13 [25]. For each animal, this program was used to determine the number of pulses in the 24-hr sampling period (frequency), the average pulse amplitude, and the mean concentration of hormone in all samples of the series, and these data are presented as mean ± SEM. All identified pulses are indicated by arrows in Figure 1. For statistical analysis, all values for testosterone and gonadotropins were subdivided into sample periods of 2 h, and means and SD values were calculated and are presented in Figure 3. Two-way ANOVA and all pair-wise multiple comparison procedures (Holm-Sidak method) [20] were used to determine statistically significant diurnal and age-related differences. For analysis of changes in pulse frequencies, the number of pulses occurring over 6-h time windows were determined and expressed as means ± SD (Fig. 4). Student t-tests were applied to determine statistically significant differences in mean hormone levels and Pulsar parameters between both age groups.
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The individual 24-h profiles of testosterone and LH are shown in Figure 1. All monkeys showed a pulsatile release of LH and testosterone. The individual LH and testosterone pulses recognized by the Pulsar analysis are indicated by arrows in Figure 1. Open arrows denote a pulse of LH followed by a subsequent pulse of testosterone. Few LH pulses were detected without subsequent testosterone peaks (shaded arrows), and very few testosterone pulses were detected without a previous LH pulse (filled arrows). Over a 24-h period, young adult monkeys (left panel) showed 9–12 LH-induced testosterone pulses, and older monkeys (right panel) showed 4–10 LH-induced testosterone pulses. Pulsar analysis revealed no age-related differences in regard to mean LH and testosterone levels (testosterone: young, 4.02 ± 0.39 ng/ml; old, 3.49 ± 0.33 ng/ml; LH: young, 0.63 ± 0.16 ng/ml; old, 0.38 ± 0.04 ng/ml) and amplitudes (testosterone: young, 4.22 ± 0.3; old, 3.67 ± 0.7; LH: young, 1.06 ± 0.33; old, 0.60 ± 0.04) over 24 h. Testosterone levels reached maximal peak levels between 6 and 10 ng/ml in both age groups; however, the number of testosterone peaks was significantly higher in young monkeys (12.0 ± 0.58) compared to old monkeys (7.5 ± 1.3) over 24 h. The number of LH peaks over 24 h did not show statistically significant age-related changes (young, 12.5 ± 1.26; old, 11.0 ± 1.78).
Figure 3A illustrates the mean level of testosterone plotted as two-hour sampling intervals. While significant overall diurnal rhythms of testosterone were detected in both age groups, the diurnal change was much more pronounced in older monkeys, with significantly lower levels of plasma testosterone during daytime (1000–1600 h) than during nighttime (2000–0400 h). A significant age-related difference in plasma testosterone levels was determined between 1200 h and 1600 h. Comparisons of two-hour means for LH (Fig. 3B) revealed an overall significantly diurnal rhythm of LH in old monkeys, but not in young monkeys.
LH pulse frequency detected by Pulsar analysis was further analyzed in pulses analyzed over periods of 6 h (Fig. 4A). No significant diurnal change of LH frequency was detected in young adult monkeys. In older monkeys, LH pulses were significantly less frequent at midday (1000–1600 h) when compared with nighttime (2200–0400 h). A significant difference between both age groups was observed during midday, with lower LH pulse frequency in older monkeys. The frequency of testosterone pulses with a prior LH pulse (Fig. 1, open arrows) showed a significant diurnal change in the old monkeys, but not the young age group (Fig. 4B). In old monkeys, but not in young monkeys, testosterone pulse frequency was significantly reduced during the day (1000–1600 h) when compared with nighttime (2200–0400 h). Age-related significant differences in testosterone pulse frequencies were observed for the two intervals from 0400–1000 h and 1000–1600 h.
In all monkeys, FSH and inhibin B were released in irregular pulses throughout the entire 24-h sampling window (Fig. 2). While, in young monkeys, many of the peaks of FSH occur in parallel with LH pulses, in old monkeys, FSH appeared to be released independently from LH. Mean 24-h levels for FSH (young, 0.37 ± 0.15 ng/ml; old, 0.28 ± 0.07 ng/ml) and inhibin B (young, 555.5 ± 127.9 pg/ml; old, 457.8 ± 80.4 pg/ml) were significantly lower in old monkeys. No significant diurnal changes were detected for inhibin B or FSH in either age groups.
In nonhuman primates, testosterone levels are significantly lower in the morning and early afternoon when compared with nighttime levels due to a diminished frequency of LH pulses [15–17]. Our results confirm the presence of a diurnal rhythm of testosterone in young and old rhesus monkeys. Individual profiles in the previously published data, as well as those in the present study, reveal individually different patterns, with some monkeys showing continuous pulses over 24 h, and others showing extended periods of no activity, most commonly in the morning hours [16, 17].
Our data provide strong evidence that diurnal changes of testosterone become more pronounced during aging in rhesus monkeys. Although no significant age-related change of testosterone levels over 24 h was determined, the number of LH-induced testosterone pulses was significantly reduced during daytime in older monkeys. Since androgen levels over 24 h are only slightly, but not significantly, lower in older monkeys, our results indicate that the androgen status of young and old monkeys is similar, and that androgen-dependent organs should function normally in both age groups. However, old monkeys are exposed to significantly lower androgen levels during the day. The physiological significance of the diurnal rhythm of testosterone in monkeys is unknown. However, the more pronounced rhythm in older monkeys indicates that the diurnal rhythm of testosterone is an important parameter that is modulated throughout life. Our data indicate that interindividual differences reported in studies from the 1970s and 1980s [16, 17] might have resulted from different ages of the rhesus monkeys being studied. The exact age of monkeys in these early studies was not reported, because experimental animals were often caught in the wild as adults, and birth dates were not determined.
We detected a diurnal pattern of plasma LH levels, as well as a significant reduction of LH pulses at midday in old monkeys, but not in young monkeys. Our data provide strong evidence that the enhanced diurnal rhythm of testosterone in old monkeys is induced by a modified diurnal pattern of LH. A change in neuroendocrine regulation of Leydig cell function is also considered as a potential mechanisms leading to a decline of androgens in aging men (reviewed in [1]). Alternative mechanisms for a decline of androgens in aging men are primary testicular changes, an increase of serum hormone-binding globulin, or decreased adrenal androgen release. In our study, the decline of daytime testosterone levels appears to have been controlled centrally, and does not appear to have been mediated by the testis. Mechanisms responsible for disturbed steroidogenic functions of Leydig cells have been intensely explored in rats. A multitude of cellular defects have been observed in older rats, leading to the hypothesis that damage through reactive oxygen species is responsible for Leydig cell dysfunction (reviewed in [14]). In this study, we did not explore any potential age-related decline of Leydig cell function. However, we observed no increase of LH levels in older monkeys, which could be used as an indicator of a decline of Leydig cell function. We also observed no overall decline in androgen levels, and all monkeys had testicular volumes in the normal range, provided sperm in their ejaculates, and were healthy adult males, showing no symptoms of androgen deprivation. Since we did not perform LH-stimulation tests, we cannot assume that the aging Leydig cells in the monkey testis were functioning normally.
In two of the young monkeys, the LH-testosterone pulses occurred continuously every 2–3 h during the 24-h sample period. In all of the old monkeys, and in two of the young monkeys, a pattern was observed showing a constant decline of the pulse frequency after initiation of rapid LH-testosterone pulses in the early afternoon. This pattern is in accordance with the previously proposed model that testosterone exerts a decelerating effect on the LH pulse generator [26] (reviewed in [27]). Orchidectomy induced an acceleration of LH pulses to a frequency of 4 pulses/h, which reverted to an intact pulse frequency of about 1 pulse/h after administration of testosterone implants within 10 days after castration [28]. The author concluded that, in the rhesus monkey, the testis imposes a decisive retardation of the neural mechanisms that governs the timing of intermittent GnRH secretion. It appears from our individual profiles, as well as from the previously published 24-h profiles, that the deceleration effect of testosterone on LH pulse activity continues to build up throughout the night until the pulse generator may reach complete deceleration, resulting in quiescence of LH pulses for several hours in the late night/early morning hours. We propose here that the deceleration model is not only applicable to long-term changes, but might also be responsible for the occurrence of diurnal changes. One explanation for the more pronounced diurnal rhythm of older monkeys could be that the old testis exerts a stronger feedback action on the pulse generator, leading to complete cessation of the LH pulse generator activity in the morning. It is quite striking that, in the old monkeys, reinitiation of LH pulses after a sustained period of no activity always occurs in the late afternoon. The mechanism leading to this well-timed awakening remains unknown, but seems to be dependent on hypothalamic input.
Winters et al. [22] showed that, in contrast to testosterone and LH, inhibin levels do not show obvious circadian changes. We have confirmed this finding in the present study. Our data reveal an overall decline of inhibin in older monkeys, as was observed in men [19–21]. Since the regulation of inhibin in the monkey is not only controlled by FSH, but also by testosterone [24], it appears that the effects of androgens have no immediate action on inhibin B plasma levels. Similar to inhibin, FSH does not show any diurnal pattern. In respect to circadian patterns, it appears that FSH and inhibin are widely independent of the LH-testosterone axis. Interestingly, both axes are affected by age: the LH-testosterone axis primarily in regard to the intensity of the diurnal patterns, and the FSH-inhibin axis primarily in overall plasma levels.
Nonhuman primate studies have provided the foundation for the understanding of the hypothalamic-pituitary-testicular axis in primates [27, 29]. However, so far, nonhuman primate studies have not provided any input on the effect of aging on gonadotropin, inhibin, or testosterone patterns. The present study reveals a new and interesting concept on the effect of aging on androgen levels in primates. Controlled studies to explore diurnal rhythms and the effects of aging in humans are difficult to perform, and the results of these studies are difficult to interpret, since individuals are always exposed to stress and disturbed activity and sleeping rhythms in an unusual environment. Many factors, such as a very high intrasubject variability, ethnicity, heredity, body composition, diet, stress, and other lifestyle factors, affect blood androgen levels in healthy men (reviewed in [1]). The monkey model offers a unique opportunity to collect hormone data by remote sampling under controlled conditions. Studies with remote sampling strategies on nonhuman primates can create moment-to-moment profiles that reflect normal and undisturbed physiological rhythms. The clinical relevance of such data is dependant upon the similarity of the monkey model to the normal situation in humans. The monkey is considered an excellent model for the human in regard to various aspects of endocrine regulation of the gonads and testicular function [30, 31]. Here, we report similarities, but also striking differences, in age-related patterns between rhesus monkeys and men. The pattern of the diurnal rhythm of testosterone appears to be identical between men and rhesus monkeys, with lowest levels in the afternoon [32]. Monkeys show an obvious pulsatile pattern of testosterone. Testosterone pulses, however, are not clearly identifiable in peripheral blood in men [33]. Although circadian rhythms of testosterone exist in young men, they appear to be lost in aging men [5–11], which is in contrast to our findings in monkeys. Additional studies are needed to provide a better understanding of age-related changes in the diurnal patterns and regulatory mechanisms controlling gonadotropin and testosterone release in primates. Better knowledge of these mechanisms would allow a more profound understanding of the physiological reasons for and consequences of low androgen levels in older men, and would contribute to an evidence-based approach to androgen replacement therapy in those individuals.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of Rachel Roslund, Lisa Nieman-Vento, Mike Cicco of Core B of CRRP, and Jan-Bernd Stukenborg, Kirsi Jahnukainen, and Scott Hergenrother, as well as the helpful advice and discussions on the design and interpretation of the data from Dr. Tony Plant. We are thankful to the Assay Core (Carolyn Phalin) of the CRRP for hormone determinations.
FOOTNOTES
1Supported by grant 1R21 AG024914 and startup funds from the University of Pittsburgh School of Medicine and by NICHD/NIH through cooperative agreement U54 08160 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. 
Correspondence: 2Stefan Schlatt, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, B301 Magee Womans Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213. FAX: 412 641 7676; e-mail: schlatt{at}pitt.edu
Received: 16 October 2007.
First decision: 21 November 2007.
Accepted: 12 March 2008.
REFERENCES
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