Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print December 29, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027755
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
72/5/1087    most recent
biolreprod.104.027755v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.
Agricola
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.
BIOLOGY OF REPRODUCTION 72, 1087–1094 (2005)
DOI: 10.1095/biolreprod.104.027755
© 2005 by the Society for the Study of Reproduction, Inc.

Factors Regulating the Timing of Puberty Onset in Female Rhesus Monkeys (Macaca mulatta): Role of Prenatal Androgens, Social Rank, and Adolescent Body Weight1

Julia L. Zehr 2 , Page E. Van Meter , and Kim Wallen 

Psychology Department, Emory University, Yerkes National Primate Research Center, Atlanta, Georgia 30322


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated whether prenatal androgen exposure, social rank, and body weight are factors regulating pubertal development in outdoor-housed female rhesus monkeys. Subjects' mothers received injections of testosterone enanthate (20 mg/ wk), flutamide (an androgen receptor blocker, 30 mg/kg twice daily), or vehicle during Gestational Days 35/40–70 (early) or Days 105/110–140 (late). Monitoring of pubertal development began around 28 mo of age during the fall breeding season, with frequent assessment of menstruation, circulating steroids, and weight. Menarche occurred 1.5 mo later in females treated late in gestation than in females treated early in gestation. This short menarche delay occurred in females treated with androgen, flutamide, or vehicle. No effect of prenatal manipulations on first ovulation were found. Social rank was related to first ovulation but not menarche, with low-ranked females less likely than high- or middle-ranked females to ovulate at 2.5 yr than at 3.5 yr of age. Females ovulating early, around 2.5 yr, had higher pubertal body weights and body mass indexes (BMI) than did females ovulating later, suggesting that better nutritional reserves or positive energy balance affect pubertal development. Thus, social rank and likely nutritional status influenced pubertal development in this study. Hormonal manipulations had no detectable effect; instead, handling late in gestation, which may have increased maternal adrenal activity, delayed menarche. This finding contrasts with earlier studies that showed that prenatal androgens delay menarche by 4–6 mo on average. This study supports late gestation as a period of increased sensitivity to environmental insult and demonstrates that multiple factors, including prenatal programming, modulate the specific timing of pubertal events.

early development, menstrual cycle, neuroendocrinology, puberty, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Puberty is a process typically defined by the attainment of adult reproductive function. In primate species, adult reproductive function includes menstruation, ovulation, and successful reproduction. The timing of this developmental process varies widely, ranging from months to years between individual female rhesus monkeys. Female rhesus monkeys first menstruate around 2.5 yr of age [1–3] and subsequently experience a variable period of adolescent sterility [4], in which menstrual cycles are irregular and anovulatory [1]. While first and subsequent menstruations occur throughout the year, first ovulation in outdoor-housed rhesus females only occurs during the fall and winter months [3, 5]. Even if a young female attains pubertal age or weight during the nonbreeding season, she will not ovulate until the next breeding season [3]. This produces a bimodal distribution in age at first ovulation, with approximately 20–40% of outdoor housed females ovulating in the fall breeding season around 2.5 yr of age and with the remaining females ovulating for the first time a year later [3, 5, 6]. What regulates the timing of pubertal events remains poorly understood. We examined whether the timing of menarche and first ovulation varied with prenatal exposure to androgens, social rank, or adolescent body weight in female rhesus monkeys living outdoors in large social groups.

Previous studies found that exposing fetal female rhesus monkeys to high levels of prenatal androgen that markedly masculinized the females' genitalia also delayed the occurrence of first menstruation [7, 8]. However, the effects of this high level of androgen varied with the gestational timing of fetal androgen exposure [9]. Female rhesus monkeys treated with supraphysiological levels of androgens early in gestation, when genital differentiation is occurring, had later menarche than either control females or females treated with androgens late in gestation, when genital differentiation is complete and unaffected by exogenous androgens [9]. Although previous studies confirmed that prenatally androgenized females ovulate during late adolescence [7] and during adulthood [10] regardless of the timing or dose of androgen, no study has investigated whether prenatal exposure to exogenous androgen alters when first ovulation occurs.

Previous research has suggested that a critical weight must be reached for the occurrence of menarche [11]. Excess prenatal androgen may affect growth and thus affect the timing of puberty. Androgenized female rhesus monkeys had slower developmental increases in body weight and later menarche than did control females [12]. Alternatively, attainment of a critical weight is likely less important for reproduction than a positive energy balance because reproduction occurs only when individuals have available more energy than they expend [13]. Female monkeys fed high-fat diets were both younger and lighter at first ovulation than were females eating normal diets [14], supporting the hypothesis that positive energy balance is more important than absolute body weight for the initiation of puberty. However, androgenized females, while delayed in both developmental increases in body weight and menarche, weighed more at menarche than did controls [12], suggesting that prenatal androgens may delay puberty onset independent of their effects on body weight and/or positive energy balance.

Social rank also influences the timing of pubertal development in primate females living in species-typical, age-graded, stable social groups. High-ranked rhesus monkey females were more likely to conceive early, around 2.5 yr of age, than were low-ranked females [15–17]. Similarly, first menses, first sexual swelling, and first parturition, all indicators of puberty onset and pubertal maturation, occurred earlier in high-ranked than in low-ranked Savannah baboon females [18, 19]. However, the relationship of social rank to the timing of first ovulation, an important marker of puberty onset, remains uninvestigated.

The present study examined the relationship between prenatal steroids and puberty onset in rhesus monkey females living in large, age-graded social groups housed outdoors, where developing females occupied distinctly different social ranks. This article reports on female subjects who were part of a larger study examining the role of prenatal androgens in both male and female behavioral and endocrine development [20–22]. Female subjects' mothers received testosterone enanthate, flutamide (a nonsteroidal androgen receptor blocker), or vehicle during early or late gestation [20]. However, unlike previous studies that administered very large doses of androgen [e.g., 9], the androgen dose used in the present study did not masculinize female genitalia but did alter neonatal neuroendocrine function [20]. Thus, our treatments used androgen doses with the potential to affect neuroendocrine function but which provide no external indication that the developing fetus was exposed to excess androgen. This allowed us to investigate the effect of the timing of androgen exposure on pubertal development without the confounding effect of masculinized genitalia. While the primary purpose of prenatal flutamide treatment was to study its effects on males, prenatal flutamide treatment of females permitted examination of possible endogenous androgen influences on variation in female puberty onset. Although prenatal androgen levels are much lower in fetal females than males, they are exposed to detectable levels of testosterone prenatally [23]. In rats, females are also exposed to detectable testosterone levels [24]. Perinatal flutamide treatment of females significantly shortened anogenital distance [25], increased female sensitivity to estradiol for inducing female sexual behavior, and reduced mounting by females [26], suggesting that the low levels of endogenous androgens to which female rats are exposed have the capacity to partially defeminize and masculinize developing females. While we anticipated that flutamide would have a more profound effect in males, flutamide treatment of females would address whether the endogenous androgens to which females are exposed influence pubertal events. We predicted that prenatal androgen treatment early, but not late, in gestation would delay first menses and possibly first ovulation. It was expected that prenatal flutamide treatment at either gestational time would have little if any effect on pubertal onset.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and Prenatal Androgen Manipulations

Subjects (N = 27) were the offspring of rhesus macaque females who received one of several hormonal or control manipulations during gestation. Subjects were born in two cohorts of animals separated in age by 1 yr. All subjects and their mothers lived in large, age-graded social groups that had been formed more than 30 yr before the study's start and that reflected a species-typical social organization. Physical, endocrine, and behavioral development has been investigated previously in these same subjects [20–22]. All research followed guidelines in the NIH Guide for the Care and Use of Laboratory Animals, and the Emory University Institutional Animal Care and Use Committee approved all research. The Yerkes National Primate Research Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

A complete description of the prenatal androgen manipulations is provided elsewhere [20]. Briefly, mothers of subjects were injected with testosterone enanthate, flutamide, or vehicle during one of two prenatal time periods, Gestation Days 35 or 40 to Day 70 (early) or Gestation Days 110 or 115 to Day 145 (late). Gestational age was estimated using a combination of the mothers' sexual behavior, which accurately predicts conception day in group-living rhesus monkeys [27] and ultrasonically determined prenatal growth [27, 28]. Testosterone-treated mothers received a single injection of testosterone enanthate (#T3006; 20 mg/wk dissolved in 0.2 ml sesame oil; Sigma Chemical Corp.) once weekly and twice daily dimethyl sulfoxide (DMSO) vehicle injections (#D5879, Sigma Chemical Corp.). Flutamide-treated mothers received twice-daily injections of flutamide (#F9397; 30 mg/kg dissolved in DMSO; Sigma Chemical Corp.) throughout the treatment period. Vehicle-treated mothers received injections of DMSO (0.25 ml) twice daily. All mothers of subjects were trained to leave their social group, enter an indoor capture area, and present a leg for injection. Injections were given intramuscularly 7 days/wk, 2 times/ day, between 0730 and 1130 h and between 1330 and 1830 h. Mothers quickly habituated to the handling procedure, resulting in typical handling times per female of less than 2 min. Mothers were assigned randomly to androgen, flutamide, or control groups within mothers from high, medium, and low social ranks. Thus, treatments were balanced by maternal social rank. Maternal levels of circulating testosterone in early- and late-treated androgen subjects were markedly elevated by the injections [29] and did not differ by timing of treatment (early androgen: 6.80 ± 0.41 ng/ml; late androgen: 6.19 ± 1.02 ng/ml; t = 0.51, not significant). Circulating testosterone levels in flutamide and control-treated mothers never exceeded 0.45 ng/ml and were below assay sensitivity (<0.20 ng/ml) in almost 80% of maternal blood samples.

Subjects that contributed to this study were distributed across early vehicle (n = 2), late vehicle (n = 2), early androgen (n = 5), early flutamide (n = 6), late androgen (n = 6), and late flutamide (n = 6) treatment groups. The study design called for equal numbers of early- and late-handled vehicle controls; however, vehicle-control subjects were lost from illnesses unrelated to the experimental manipulations, reducing the sample size to two early- and two late-handled controls.

Constraints on scheduling treatments of pregnancies with confirmed conception dates resulted in an unexpected difference in birthdates between early- and late-treatment groups (early: 19 May ± 4 days; late: 30 April ± 4 days; F(1,19) = 10.74, P ≤ 0.005), which did not differ by type of treatment (androgen: 14 May ± 4 days; flutamide: 4 May ± 4 days; F(1,19) = 3.39, P ≤ 0.10). Birthdates for vehicle females were intermediate to those of the hormonally manipulated females (6 May ± 3 days). Because season modulates neuroendocrine activity [3, 30] and treatment groups differed in age relative to seasonal cues, birthdate was used as a covariate in analyses involving the timing of pubertal events.

One female (Tc6, early flutamide) was injured and housed indoors between the ages of 3.40 and 3.62 yr (40.8 and 43.4 mo); first ovulation occurred in this female during this period. This female was classified as a later maturer (definition below) because she was much older than 2.5 yr of age at first ovulation. Tc6 was included in categorical analyses of first ovulation but not in analyses requiring an exact age at first ovulation because indoor housing may have altered the exact timing of first ovulation.

Housing

All subjects lived in large, outdoor-housed, social groups at the Yerkes National Primate Research Center in Lawrenceville, Georgia. Animals received Purina monkey chow twice daily, oranges once daily, and had free access to water. Social groups consisted of 60–120 individuals, including adult males, adult females, and their offspring, and had been formed more than 30 yr before the study's start. Outdoor enclosures varied in size from 650 m2 to 1465 m2 with an attached, temperature-controlled, indoor enclosure.

Data Collection Procedure

Access of subjects for blood sampling and data collection occurred a minimum of 3 times/wk with no more than 2 days between accesses. Additional accesses occurred if a female was observed engaging in sexual behavior. Accesses typically began between 1200 and 1330 h EST. During accesses, subjects ran into a small capture unit attached to their outdoor compound and were individually transferred to a laboratory housing cage modified with front openings that allowed subjects to extend a leg. After training, subjects extended a leg through the front opening and remained still throughout the procedures, which averaged 3 min/subject. While one researcher gently held the leg of the female, a second researcher collected a blood sample from the saphenous vein of the unanesthetized subjects and checked for the presence of menstrual blood by inserting a moistened cotton swab into the female's vagina. Weights and hematocrits were obtained weekly to monitor growth and health.

Rhesus monkeys accessed using these handling techniques quickly habituate to the procedures with no evident distress or discomfort. Using similar handling procedures in group-housed rhesus monkeys, capture-naive females showed elevated prolactin responses to capture when compared with capture-acclimated females, suggesting that, after acclimation, these handling procedures have little impact on endocrine measures [31]. Furthermore, long-term and frequent accessing of females does not affect conception rate or gestation length [32] and thus was unlikely to have affected the measures of reproductive physiology used in the present study.

Hormone Assays

Blood samples were collected in serum separating tubes (Vacutainer SST Gel & Clot Activator, #367974; Becton Dickinson, Franklin Lakes, NJ) and centrifuged. Serum was removed from the tubes and frozen at –20°C until assayed. Estradiol and progesterone assays used commercially available radioimmunoassay kits produced by Diagnostic Systems Laboratories (estradiol: #DSL-4400; Webster, TX) and Diagnostic Products Corp. (progesterone: #TKPG5; Los Angeles, CA). Estradiol and progesterone assays were conducted in two laboratories (D. R. Mann, Morehouse School of Medicine; Yerkes Assay Services, Yerkes National Primate Research Center). A common set of samples was assayed in both laboratories, and the values were highly correlated (estradiol: r = 0.83; progesterone: r = 0.996). The inter- and intraassay coefficients of variation (CV) between the two labs were also comparable (estradiol: Yerkes, 17.3%, 7.7%, Mann, 9.2%, 5.3%; progesterone: Yerkes, 13%, 8.4%, Mann, 7.0%, 7.3%).

Determination of Menarche and First Ovulation

Menarche was defined as the first day on which menstruation was observed, and age at menarche was calculated from birthdate. First ovulation was determined based on the combined hormonal profiles of estradiol (sampled 3 times/wk) and progesterone (sampled 1–2 times/wk). In adults, estradiol peaks just before ovulation [33, 34]. Because blood samples were not collected on every day, the periovulatory estradiol peak (>250 pg/ml) could be missed. Thus, progesterone levels greater than 3 ng/ml were taken as evidence of luteal progesterone, even when observed without a corresponding periovulatory estradiol peak. Age at first ovulation was calculated from birthdate. Subjects were categorized as early maturers if they ovulated for the first time in the breeding season around 2.5 yr of age or were categorized as later maturers if they ovulated for the first time during the breeding season around 3.5 yr of age [5].

Determination of Social Rank

Female rhesus monkeys attain adult social rank around puberty and usually inherit a rank just below that of their mother [35, 36]. At the start of puberty, subjects were assigned their mother's rank for analysis purposes. For each subject, a proportional rank was calculated as the rank of their mother in the adult social hierarchy divided by the total number of adult females in the group. Proportional ranks ranged from 0 to 1, with the highest ranked females having the lowest values. Proportional ranks were used because subjects came from social groups differing in the total number of adult females. For categorical analyses, females were also categorized as high-, middle-, or low-ranked based on whether their proportional rank fell within the upper, middle, or lower third of the hierarchy.

Maternal rank in the adult social hierarchy was defined through a combination of historical records and opportunistic recording of agonistic interactions during breeding-season behavioral observations. Specifically, all adult females were rank ordered in a linear hierarchy based on the direction of aggressive behaviors (threats, chases, and contact aggression) and the direction of submissive behaviors (grimace, withdrawal, and screams). Adult daughters who did not display aggression toward their mothers or sisters were ranked below their mothers in a reverse age order [35, 36].

Determination of Body Weight

Prior to puberty, at 24 mo of age, and during puberty, at 30 and 36 mo of age, body weight, crown-rump length, and crown-heel length were measured from female subjects anesthetized with ketamine (10 mg/kg). Body mass index (BMI) was calculated for females at these three ages using two methods. First, BMI was calculated using a method commonly used in studies of puberty onset in rhesus macaques (body weight [kg] divided by the squared crown-rump length [m2] [37, 38]). Second, BMI was calculated using the method for human BMI (body weight [kg] divided by the squared crown-heel length [m2]). At 36 mo of age, one early androgen female did not have these measurements taken. Measures of BMI were not taken from females at 42 and 48 mo of age because pregnancy in some females interfered with accurate BMI assessment. In addition to measures at 24, 30, and 36 mo of age, weights were taken on all females weekly. As a result, weights at first menarche and first ovulation were recorded within 4 days for all females by using the closest of weekly weight measures.

Statistical Analysis

All analyses were conducted using SPSS for Windows (Version 10.0.5, SPSS Inc.). The effects of prenatal manipulations on average age at first menses and first ovulation were analyzed using a two-factor analysis of variance (ANOVA; timing by type of prenatal manipulation) with birthdate as a covariate. Estimated marginal means are presented in Figure 1, showing the age at menarche for each treatment group adjusted for differences in birthdate. Because first ovulation occurred in a bimodal pattern by season, G-tests were also used to test for the effects of prenatal treatment on age at first ovulation in early and later maturers (defined above) [39]. The G-statistic approximates a chi-square distribution but is appropriate for small sample sizes. Like chi-square, it tests the independence of two characteristics (e.g., treatment group and early vs. later first ovulation) [39]. High-, middle-, and low-ranked females were compared for differences in age at first menses and in pubertal weight measures using a one-factor ANOVA and for differences in age at first ovulation using the G-test [39]. Rank was also correlated with age at first menses using Pearson product-moment correlations. Differences between early and later maturers in weight measures were assessed using student t-tests, and differences between high-, middle-, and low-ranked females in weight measures were assessed using a one-factor ANOVA. By design, subject rank was balanced across the different prenatal treatment groups, and the small number of subjects available precluded an analysis of potential interactions between prenatal treatment, social rank, and pubertal maturation. A P ≤ 0.05 was considered significant, and a P = 0.10 was considered a trend.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1. Mean and individual ages at menarche among outdoor-housed female rhesus monkeys exposed to different prenatal treatments. Birthdate was entered in the analysis as a covariate, and estimated marginal means show the mean age at menarche for each group adjusted for differences in birthdate. Late-treated females had significantly later onset of menarche than did early-treated females (P ≤ 0.05)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Prenatal Androgen Manipulations

In a two-factor ANOVA comparing the effects of timing (early vs. late) and type (androgen, flutamide, or vehicle) of treatment on first menses using birthdate as a covariate, first menses varied by the timing but not the type of prenatal manipulation (Fig. 1). First menstruation was later in females treated late in gestation, regardless of treatment, than in females treated early in gestation (main effect of timing: F(1,20) = 7.69, P ≤ 0.01). The type of treatment administered (androgen, flutamide, or vehicle) did not affect age at menarche (F(1,20) < 1, not significant [NS]) and there was no interaction (F(1,20) < 1, NS).

Adolescent subjects showed a bimodal pattern of first ovulation, but prenatal hormone manipulations were unrelated to the timing of first ovulation. Across all subjects, 33% (9 of 27) of females were early maturers and 67% (18 of 27) of females were later maturers. Individual ages at first ovulation for females in each treatment group are depicted in Figure 2. Females did not differ in the likelihood of early first ovulation based on prenatal treatment group (G(5) = 2.5, N = 27, NS) or based on the timing (early vs. late; G(1) = 0.1, N = 27, NS) or type (androgen, flutamide, or vehicle; G(2) = 2.1, N = 27, NS) of prenatal manipulations. Timing and type of prenatal manipulation also did not affect the average age of first ovulation when birthdate was entered as a covariate (F values <1). Prenatal hormone manipulations did not affect measures of body weight or BMI at menarche or first ovulation (data not presented).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 2. Individual ages at first ovulation among outdoor-housed female rhesus monkeys exposed to different prenatal treatments. First ovulation occurred in a bimodal pattern, but females did not vary in the timing of first ovulation based on prenatal manipulations. Because age at first ovulation is bimodally distributed, the mean age at first ovulation for the treatment groups is not plotted

Body Weight and BMI

Neither prepubertal nor pubertal measures of body weight or BMI correlated with age at first menses (all r < 0.34, NS). However, early and later maturing females differed in weight and BMI before puberty and differed in weight at menarche and at first ovulation (Table 1). For prepubertal measures and for weight at menarche, later maturing females were smaller than early-maturing females. Early-maturing females, who ovulated 1 yr earlier than later maturing females, weighed significantly less than later maturing females at first ovulation.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Weight and body-composition differences between early and later maturing female rhesus monkeys before and during puberty.*

Social Rank

Social rank was related to age at first ovulation but not age at first menses. Only high- and middle-ranked females ovulated in early adolescence (Fig. 3, G(2) = 10.70, N = 27, P ≤ 0.005). Age at menarche did not correlate with rank (r = –0.12, N = 27, NS) and did not differ in high-, middle-, and low-ranked females (high rank: 2.48 ± 0.03 yr; middle rank: 2.43 ± 0.04 yr; low rank: 2.45 ± 0.04 yr; F(2,24) ≤ 1, NS).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Percent of rhesus monkey females ovulating around 2.5 yr of age (early maturers) and around 3.5 yr of age (later maturers) from high, middle, and low social ranks. Females from low social ranks were less likely to ovulate early (P ≤ 0.05)

Social rank also affected pubertal body weights (Table 2). Prior to puberty, at 24 mo of age, middle-ranked females had significantly higher body weights and crown-rump BMI than females from high or low social ranks (Table 2). At menarche, 30 mo of age, and 36 mo of age, body weights tended to be lower overall in females from low social rank (Table 2). When low-ranked females were compared directly with high- and middle-ranked females, low-ranked females were smaller at menarche (t(25) = 2.49, N = 27, P < 0.05). Finally, high-, middle-, and low-ranked females did not differ in weight at first ovulation or in any measures of BMI based on crown-heel length (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Weight and body-composition differences in high-, middle-, and low-ranked female rhesus monkeys before and during puberty.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Females handled twice daily, 7 days/wk, late in gestation, whether treated with androgen, flutamide, or vehicle, had their menarche delayed in the masculine direction by approximately 1.5 mo compared with females handled similarly early in gestation. This suggests that mild maternal stress during late gestation, resulting from daily handling, can delay menarche in primates. Prenatal exposure to exogenous glucocorticoids or maternal stress delays puberty onset in rodents [40, 41], but this is the first study to report such an effect in primates. In previous studies of these same subjects, prenatal androgen treatments altered physiological and behavioral development [20–22]. For example, early-androgen-treated females had altered responses to neonatal GnRH challenges [20], higher juvenile LH secretion [20], and masculinized vocal behavior [21]. Surprisingly, flutamide-treated females showed some masculinization of vocal behavior and infant-directed behavior [21, 22]. Although this experiment directly manipulated prenatal androgens, either testosterone or its metabolites, particularly estrogens, could affect sexual differentiation. Because prenatal flutamide treatment almost doubled maternal androgen levels [20], flutamide-treatment may also have partially masculinized these females in this and other studies. Although flutamide was effective peripherally [20], flutamide may not have been concentrated enough in the fetal brain to block elevated maternal androgens. Alternatively, increased androgen levels in flutamide- and androgen-treated subjects may have been aromatized to estradiol, resulting in masculinization through estrogen receptors in both treatments. Thus, this experiment cannot distinguish between two alternative hypotheses: namely, that maternal stress during gestation alters pubertal development or that both androgen- and flutamide-treated females were partially masculinized. Regardless of mechanism, these results demonstrate that late gestation is a developmental period when the fetus is exquisitely sensitive to environmental perturbation and when neural modifications occur without leaving external anatomical indicators of prenatal programming. While these results do not resolve whether androgens regulate the timing of puberty onset, they stand in contrast with previous reports.

Although menarche differed significantly in early- and late-treated females, this difference was small in magnitude compared with previous studies. Goy and colleagues reported that exogenous androgens early in gestation markedly delayed menarche in females reared indoors. Specifically, menarche in Goy's early androgen-treated females occurred 4–6 mo later than in controls [7–9] or our early-androgen-treated females. In contrast, menarche in Goy's late-androgen-treated females did not differ from menarche in controls [8, 9] or our late-androgen-treated females. While this difference between studies is undoubtedly related to the higher doses of androgen used previously, the delayed menarche observed by Goy and colleagues [9] may also have resulted from androgen acting on genital tissues rather than on the developing nervous system. Our early-androgen-treated females had no genital masculinization [20], whereas Goy's subjects had a penis with no external vaginal opening [9]. Thus, Goy's subjects menstruated through their penis instead of vaginally [7]. This extreme genital masculinization could have interfered with the detection of menses, if light menstrual flow was undetected and heavier flow, reflecting higher ovarian hormone secretion, was recorded as menarche. The high androgen levels used previously may also have reduced endometrial sensitivity to estradiol and progesterone, reducing menstrual flow and making menarche a less accurate indicator of central neuroendocrine activity. Interestingly, Goy's androgenized females menstruated infrequently as adults [10], a finding consistent with decreased endometrial responsiveness to ovarian hormones.

Although high levels of prenatal androgens do not prevent ovulation in adulthood [10], the timing of first ovulation could be altered. We found no evidence that our androgen treatments affected the timing of early adolescent ovulation in our intact subjects. Similar to previous reports [3, 5, 6, 15], one third of subjects ovulated in the fall breeding season at 2.5 yr of age. Because ovulation is seasonally restricted, the remainder ovulated 1 yr later [5, 6]. These data on menarche and first ovulation argue against a profound effect of prenatal androgens on pubertal timing in female primates. Although males experience puberty later than females, sex differences in puberty onset may be unrelated to sex differences in androgen exposure. Alternatively, timing mechanisms may not be particularly sensitive to androgen levels below those experienced by males.

The failure of prenatal hormone manipulations to dramatically masculinize pubertal timing parallels recent studies in sheep. In early sheep studies, puberty was defined by decreased sensitivity to estradiol negative feedback and increased circulating LH levels in gonadectomized subjects receiving low-dose estradiol replacement [42]. Using this model, prenatal androgens clearly masculinized the timing of neuroendocrine puberty [42]. However, the same hormonal treatments did not masculinize puberty onset in intact females [43], suggesting that the feedback systems of the hypothalamic-pituitary-gonadal axis compensate for variability in sensitivity and/or neuroendocrine output to produce normal endocrine phenotypes. In sheep, this compensation cannot be maintained throughout the lifespan [44]. Similarly, aged rhesus monkeys exposed to prenatal androgens produced nonfunctional oocytes more often than controls, suggesting long-term negative consequences of early androgen exposure [45]. Accumulating evidence from both monkey and sheep models suggests a role for prenatal androgen excess in the etiology of polycystic ovarian syndrome (PCOS) [46]. However, women with PCOS do not have masculinized genitalia and typically are diagnosed with PCOS in adulthood. Thus, the present and previous findings are relevant to reproductive variation in humans who are exposed to androgenic or antiandrogenic compounds that do not produce obvious evidence of exposure.

Low social rank delayed puberty onset. In the present study, none of the low-ranked females ovulated at 2.5 yr of age, while approximately half of high- and middle-ranked females ovulated at this age. Wilson and colleagues [15] did not measure ovulation, but found that the majority of females who first conceive at 2.5 yr of age were high ranking. Social rank may also affect sexual maturation in males because pubertal testicular growth is correlated with social rank [47]. These associations between social rank and pubertal development demonstrate a strong influence of social context on reproductive physiology. Better nutrition throughout development is one mechanism through which rank may affect reproductive development [18]. Our data are consistent with this mechanism because low-ranked females had lower body weights during early puberty and at menarche. Interestingly, rank effects are observed in captive groups, where individuals are not energetically challenged. Although captive populations receive adequate food for the entire group, low-ranking individuals may wait for access to food. Because delaying a single meal alters gonadotropin secretion [48], repeated delays in food access may ultimately affect maturation.

Previous research has proposed that a critical weight or amount of body fat must be achieved before puberty can occur [11]. In stumptail macaques, a nonseasonally breeding species, age at first ovulation correlated with body weight at 3 yr of age [49]. In rhesus macaques, seasonal cues can suppress reproductive function independent of body weight [5, 50], making it difficult to show direct associations between weight and age at first ovulation. Females ovulating early weighed more and had higher BMIs during early puberty and at menarche than females ovulating later, a finding consistent with regulation of puberty onset by body weight. By 36 mo of age, early and later maturing females did not differ in BMI, suggesting that early-maturing females grow faster during early puberty and that later maturing females catch up over time. However, a recent review argues that the relationship between puberty and measures of body weight, growth, and energy reserves are more relevant to laboratory populations than to wild populations [51]. In wild populations, instability of food sources and fluctuating energy reserves argue against the evolution of mechanisms delaying puberty until a critical weight is achieved [51]. Furthermore, hypothalamic-pituitary-gonadal function is suppressed when energy balance is disrupted rather than when fat stores are depleted [13]. Thus, weight differences in females ovulating early likely reflect long-term differences in energy balance that resulted in both faster growth and earlier maturation.

In summary, the timing of pubertal events is modulated by at least four factors: season, prenatal environment, social environment, and nutritional status. With the power to override individual variability in pubertal maturation, seasonal cues are either permissive of pubertal development or shut down neuroendocrine activity [3]. Manipulation of factors controlling puberty onset in nonseasonal species may produce more variability than in seasonal species, and in this study, photoperiod may have limited the window within which prenatal manipulations and other factors could alter pubertal timing. However, even within this limited seasonal window, both high social rank and higher pubertal body weight related to earlier first ovulation. Social context may influence maturation directly by altering neuroendocrine function or indirectly by altering nutrition or access to food. Prenatal perturbation of the hypothalamic-pituitary-gonadal axis may produce variation in puberty onset and has the potential to affect reproductive function [9, 10, 45]. Although sufficient to alter neonatal neuroendocrine function [20], the doses of androgens we used did not produce the dramatic effects on menarche previously reported [7–9]. Instead of producing large alterations in neuroendocrine function, moderate prenatal androgen exposure may modulate development by producing small changes in a variety of endpoints. Furthermore, the hypothalamic-pituitary-gonadal axis may compensate for small alterations in sensitivity or output, which only become evident across the lifespan [44]. Surprisingly, we found evidence that maternal handling during late gestation produced small delays in menarche not evident in response to the same maternal handling in early gestation, suggesting that some aspect of maternal stress alters pubertal timing. That prenatal hormone manipulations less profoundly affected pubertal maturation than did social rank reflects the importance of investigating development in relation to social context. Taken together, the results of this study support the model that multiple internal and external factors interact with a developmental clock to regulate the timing of puberty onset [52].

Interestingly, prenatal handling and social status affected menarche and first ovulation differently. In this regard, menarche, which has a lower energetic cost than does ovulation, may reflect a prenatally organized timing mechanism that is relatively insensitive to female energetics. In contrast, the timing of first ovulation may be primarily determined by female energetics acting in concert with permissive timing signals such as seasonal photoperiod. This suggests that these two markers of puberty onset are dissociable and that they are regulated differently during development by the environment, experience, and current physiological state. While the exact mechanisms of this differential sensitivity are unknown, the differential effects of prenatal handling and social status on menarche and first ovulation observed here raise the possibility that the factors determining endometrial build up and sloughing in adolescent monkeys share little in common with those determining the first functional occurrence of positive feedback and first ovulation.


   ACKNOWLEDGMENTS
 
The authors thank Jessica Ganas, Anne Graff, Stephanie Allard, and the members of the Wallen laboratory for their assistance in data collection. We thank Yerkes Assay Services and the D.R. Mann laboratory at Morehouse School of Medicine for conducting hormone assays. The authors also thank Dr. Mark E. Wilson for valuable discussions of experimental results.


   FOOTNOTES
 
1 Supported in part by NIH grants R01-MH50268 and K02-MH01062 (K.W.), by an NCRR grant RR-00165 to the Yerkes National Primate Research Center, and by an NSF Graduate Fellowship (J.L.Z.). Back

2 Correspondence: Julia L. Zehr, Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, MI 48824. FAX: 517 432 2744; zehrj{at}msu.edu Back

Received: 24 January 2004.

First decision: 18 February 2004.

Accepted: 16 December 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Foster DL. Luteinizing hormone and progesterone secretion during sexual maturation of rhesus monkey—short luteal phases during initial menstrual cycles. Biol Reprod 1977 17:584-590[Abstract]
  2. Resko JA, Goy RW, Robinson JA, Norman RL. The pubescent rhesus monkey—some characteristics of the menstrual cycle. Biol Reprod 1982 27:354-361[Abstract]
  3. Wilson ME, Gordon TP. Season determines timing of first ovulation in rhesus monkeys (Macaca mulatta) housed outdoors. J Reprod Fertil 1989 85:583-591
  4. Hartman CG. On the relative sterility of the adolescent organism. Science 1931 74:226-227[Free Full Text]
  5. Wilson ME, Gordon TP, Blank MS, Collins DC. Timing of sexual maturity in female rhesus monkeys (Macaca mulatta) housed outdoors. J Reprod Fertil 1984 70:625-633
  6. Wilson ME, Gordon TP, Collins DC. Ontogeny of luteinizing hormone secretion and first ovulation in seasonal breeding rhesus monkeys. Endocrinology 1986 118:293-301[Abstract]
  7. Goy RW, Resko JA. Gonadal hormones and behavior of normal and pseudohermaphroditic nonhuman female primates. Recent Prog Horm Res 1972 28:707-733
  8. Goy RW, Uno H, Sholl SA. Psychological and anatomical consequences of prenatal exposure to androgens in female rhesus. In: Mori T, Nagasawa H (eds.), Toxicity of Hormones in Perinatal Life. Boca Raton, FL: CRC Press, Inc.; 1989:127–142
  9. Goy RW, Bercovitch FB, McBrair MC. Behavioral masculinization is independent of genital masculinization in prenatally androgenized female rhesus macaques. Horm Behav 1988 22:552-571[CrossRef][Medline]
  10. Dumesic DA, Abbott DH, Eisner JR, Goy RW. Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinizing hormone levels in adulthood. Fertil Steril 1997 67:155-163[CrossRef][Medline]
  11. Frisch RE, Revelle R. Height and weight at menarche and a hypothesis of critical body weights and adolescent events. Science 1970 169:397-399[Abstract/Free Full Text]
  12. Wilen R, Goy RW, Resko JA, Naftolin F. Pubertal body weight and growth in female rhesus pseudohermaphrodite. Biol Reprod 1977 16:470-473
  13. Schneider JS, Wade GN. Inhibition of reproduction in service of energy balance. In: Wallen K, Schneider JS (eds.), Reproduction in Context: Social and Environmental Influences on Reproduction. Cambridge, MA: The MIT Press; 2000:35–82
  14. Schwartz SM, Wilson ME, Walker ML, Collins DC. Dietary influences on growth and sexual maturation in premenarchial rhesus monkeys. Horm Behav 1988 22:231-251[CrossRef][Medline]
  15. Wilson ME, Walker ML, Gordon TP. Consequences of first pregnancy in rhesus monkeys. Am J Phys Anthropol 1983 61:103-110[CrossRef][Medline]
  16. Paul A, Thommen D. Timing of birth, female reproductive success and infant sex-ratio in semifree-ranging Barbary macaques (Macaca sylvanus). Folia Primatol (Basel) 1984 42:2-16
  17. Bercovitch FB, Berard JD. Life-history costs and consequences of rapid reproductive maturation in female rhesus macaques. Behav Ecol Sociobiol 1993 32:103-109[CrossRef]
  18. Altmann J, Hausfater G, Altmann SA. Determinants of reproductive success in Savannah baboons, Papio cynocephalus. In: Clutton-Brock TH (ed.), Reproductive Success: Studies of Individual Variation in Contrasting Breeding Systems. Chicago: The University of Chicago Press; 1988:403–418
  19. Bercovitch FB, Strum SC. Dominance rank, resource availability, and reproductive maturation in female Savanna baboons. Behav Ecol Sociobiol 1993 33:313-318
  20. Herman RA, Jones B, Mann DR, Wallen K. Timing of prenatal androgen exposure: anatomical and endocrine effects on juvenile male and female rhesus monkeys. Horm Behav 2000 38:52-66[CrossRef][Medline]
  21. Tomaszycki ML, Davis JE, Gouzoules H, Wallen K. Sex differences in infant rhesus macaque separation-rejection vocalizations and effects of prenatal androgens. Horm Behav 2001 39:267-276[CrossRef][Medline]
  22. Herman RA, Measday MA, Wallen K. Sex differences in interest in infants in juvenile rhesus monkeys: relationship to prenatal androgen. Horm Behav 2003 43:573-583[CrossRef][Medline]
  23. Resko JA, Ellinwood WE. Testicular hormone production in fetal rhesus macaques. In: Novy MJ, Resko JA (eds.), Fetal Endocrinology. New York: Academic Press; 1981:253–267
  24. Baum MJ, Woutersen PJ, Slob AK. Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females. Biol Reprod 1991 44:747-751[Abstract]
  25. Clemens LG, Gladue BA, Coniglio LP. Prenatal endogenous androgenic influences on masculine sexual behavior and genital morphology in male and female rats. Horm Behav 1978 10:40-53[CrossRef][Medline]
  26. Gladue BA, Clemens LG. Androgenic influences on feminine sexual behavior in male and female rats—defeminization blocked by prenatal anti-androgen treatment. Endocrinology 1978 103:1702-1709[Abstract]
  27. Zehr JL, Tannenbaum PL, Jones B, Wallen K. Peak occurrence of female sexual initiation predicts day of conception in rhesus monkeys (Macaca mulatta). Reprod Fertil Dev 2000 12:397-404[CrossRef][Medline]
  28. Tarantal AF, Hendrickx AG. Prenatal growth in the Cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta)—a comparison by ultrasonography. Am J Primatol 1988 15:309-323[CrossRef]
  29. Stanczyk FZ, Hess DL, Namkung PC, Senner JW, Petra PH, Novy MJ. Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques. Biol Reprod 1986 35:126-132[Abstract]
  30. Gordon TP. Reproductive behavior in the rhesus monkey—social and endocrine variables. Am Zool 1981 21:185-195
  31. Blank MS, Gordon TP, Wilson ME. Effects of capture and venipuncture on serum levels of prolactin, growth-hormone and cortisol in outdoor compound-housed female rhesus monkeys (Macaca mulatta). Acta Endocrinol 1983 102:190-195
  32. Walker ML, Gordon TP, Wilson ME. Reproductive performance in capture-acclimated female rhesus monkeys (Macaca mulatta). J Med Primatol 1982 11:291-302[Medline]
  33. Weick RF, Dierschke DJ, Karsch FJ, Butler WR, Hotchkiss J, Knobil E. Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology 1973 93:1140-1147[Medline]
  34. Lasley B, Hendrickx AG, Stabenfeldt GH. Estradiol levels near the time of ovulation in the Bonnet monkey (Macaca radiata). Biol Reprod 1974 11:237-244[Abstract]
  35. Datta S. The acquisition of dominance among free-ranging rhesus monkey siblings. Anim Behav 1988 36:754-772[CrossRef]
  36. Datta SB, Beauchamp G. Effects of group demography on dominance relationships among female primates. 1. Mother-daughter and sister-sister relations. Am Natur 1991 138:201-226[CrossRef]
  37. Eisner JR, Dumesic DA, Kemnitz JW, Abbott DH. Timing of prenatal androgen excess determines differential impairment in insulin secretion and action in adult female rhesus monkeys. J Clin Endocrinol Metab 2000 85:1206-1210[Abstract/Free Full Text]
  38. Bercovitch FB, Widdig A, Trefilov A, Kessler MJ, Berard JD, Schmidtke J, Nurnberg P, Krawczak M. A longitudinal study of age-specific reproductive output and body condition among male rhesus macaques, Macaca mulatta. Naturwissenschaften 2003 90:309-312[CrossRef][Medline]
  39. Sokal RR, Rohlf FJ. Biometry: The Principles and Practice of Statistics in Biological Research. San Francisco: W.H. Freeman and Company; 1969
  40. Smith JT, Waddell BJ. Increased fetal glucocorticoid exposure delays puberty onset in postnatal life. Endocrinology 2000 141:2422-2428[Abstract/Free Full Text]
  41. Marchlewska-Koj A, Kruczek M, Kapusta J, Pochron E. Prenatal stress affects the rate of sexual maturation and attractiveness in bank voles. Physiol Behav 2003 79:305-310[CrossRef][Medline]
  42. Wood RI, Foster DL. Sexual differentiation of reproductive neuroendocrine function in sheep. Rev Reprod 1998 3:130-140[Abstract]
  43. Sharma TP, Herkimer C, West C, Ye W, Birch R, Robinson JE, Foster DL, Padmanabhan V. Fetal programming: prenatal androgen disrupts positive feedback actions of estradiol but does not affect timing of puberty in female sheep. Biol Reprod 2002 66:924-933[Abstract/Free Full Text]
  44. Birch RA, Padmanabhan V, Foster DL, Unsworth WP, Robinson JE. Prenatal programming of reproductive neuroendocrine function: fetal androgen exposure produces progressive disruption of reproductive cycles in sheep. Endocrinology 2003 144:1426-1434[Abstract/Free Full Text]
  45. Dumesic DA, Schramm RD, Peterson E, Paprocki AM, Zhou R, Abbott DH. Impaired developmental competence of oocytes in adult prenatally androgenized female rhesus monkeys undergoing gonadotropin stimulation for in vitro fertilization. J Clin Endocrinol Metab 2002 87:1111-1119[Abstract/Free Full Text]
  46. Abbott DH, Dumesic DA, Franks S. Developmental origin of polycystic ovary syndrome—a hypothesis. J Endocrinol 2002 174:1-5[Abstract]
  47. Mann DR, Akinbami MA, Gould KG, Paul K, Wallen K. Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank. J Endocrinol 1998 156:493-501[Abstract]
  48. Cameron JL. Regulation of reproductive hormone secretion in primates by short-term changes in nutrition. Rev Reprod 1996 1:117-126[Abstract]
  49. Nieuwenhuijsen K, Lammers A, de Neef KJ, Slob AK. Reproduction and social rank in female stumptail macaques (Macaca arctoides). Int J Primatol 1985 6:77-99
  50. Wilson ME. Relationship between growth and puberty in the rhesus monkey. In: Delemarre-van de Waal HA (ed.), Control of the Onset of Puberty III. New York: Elsevier Science Publishers; 1989:137–149
  51. Bronson FH. Puberty and energy reserves: a walk on the wild side. In: Wallen K, Schneider JS (eds.), Reproduction in Context: Social and Environmental Influences on Reproduction. Cambridge, MA: The MIT Press; 2000:15–34
  52. Sisk CL, Foster DL. The neural basis of puberty and adolescence. Nat Neurosci 2004 7:1040-1047[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
72/5/1087    most recent
biolreprod.104.027755v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.
Agricola
Right arrow Articles by Zehr, J. L.
Right arrow Articles by Wallen, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS