REVISED : BIOLREPROD / 2007 / 065953 1 2 Gene - Environment Interactions , Not Neonatal Growth Hormone Deficiency , Time Puberty 3 in

24 The factors that influence the timing of puberty and the onset of adult fertility are poorly 25 understood. While focus on the juvenile period has provided insights into how growth-related 26 cues affect pubertal timing, growth velocity during infancy that is sustained into the juvenile 27 period may be important. On the other hand, social factors, specifically exposure to 28 psychosocial stressors, can delay sexual maturation, possibly by altering growth velocities 29 during development. Using female rhesus monkeys, the present study used a prospective 30 analysis to determine how neonatal growth hormone (GH) inhibition with a sandostatin analog 31 or suppression of the pituitary – gonadal axis with a GnRH analog affected growth and sexual 32 maturation. Secondly, a separate retrospective analysis was done assessing the effects of 33 social dominance status during development on pubertal timing. Because a specific 34 polymorphism in the gene encoding the serotonin (5HT) reuptake transporter increases 35 vulnerability to psychosocial stressors, females were also genotyped and were then classified 36 as socially dominant having both alleles for the long promoter variant or having at least one 37 allele for the short promoter variant or classified as socially subordinate having the long variant 38 or subordinate having the short variant. Neonatal treatments were not balanced for social 39 status or genotype so analyses were performed separately. Although the neonatal treatments 40 reduced GH secretion postnatally and through the juvenile period, neither growth nor sexual 41 maturation were affected. In contrast, the retrospective analysis showed sexual maturation was 42 delayed significantly in subordinate females carrying at least one allele of the short promoter 43 variant in the gene encoding the serotonin reuptake transporter and this delay was associated 44 with reduced GH and leptin secretion during the juvenile phase but not with differences in 45 growth velocities from birth. These data suggest that decreased neonatal GH secretion does not 46 adversely affect sexual maturation but that polymorphisms in the gene encoding the 5HT 47 transporter modulate the adverse consequences of social subordination on the timing of puberty 48 in female rhesus monkeys. 49 Social status and puberty timing page 3 Introduction 50 While the understanding of the common neural pathways [1-3] that drive the appropriate release 51 of GnRH from the medial basal hypothalamus to initiate puberty are well accepted [4-7], the 52 factors that influence the variation in pubertal timing are less clear. Prepubertal growth or 53 factors that regulate growth have long been considered important for timing maturation of the 54 reproductive axis. Indeed, changes in hormones that either stimulate skeletal or muscular 55 growth [8-12] or increase in response to accumulation of body fat [13-15] explain some of the 56 variation in puberty timing, suggesting these can act centrally to influence systems regulating 57 developmental increases in GnRH [16, 17]. 58 While focus on the juvenile period has provided insights into how growth-related cues affect 59 pubertal timing, birth weight and postnatal growth may also be important. Clinical data indicate 60 that low birth weight followed by rapid catch-up growth is associated with increases insulin-like 61 growth factor (IGF) I secretion and early puberty [18, 19]. These children are taller, heavier, and 62 have more fat than age match normal birth weight children [20]. Other epidemiological data 63 indicate that girls heavier at birth have early puberty compared with normal birth weight girls 64 showing similar patterns of growth during infancy [21]. In an analysis of birth weight and growth 65 patterns of female monkeys, low birth weight is associated with blunted postnatal and juvenile 66 growth and later age at fertility [22]. The key variable in these studies appears to be the growth 67 velocity during infancy that is sustained into the juvenile period. Additional support for the 68 notion that growth during infancy may be important is provided by studies showing the 69 experimental manipulation of the neonatal hypothalamic – pituitary – gonadal (HPG) axis in 70 male monkeys affects growth and puberty. Males treated with a GnRH analog during the 71 neonatal period have blunted growth and delayed puberty [23, 24]. The mechanism by which 72 GnRH analog treatment during the neonatal period reduces growth is not clear but could be due 73 to altered gonadal steroid stimulation of the growth hormone (GH) – IGF1 axis. 74 For children, social factors can also influence growth and, thus, puberty timing. Again, 75 epidemiological data on adolescents living in urbanized settings indicate girls in families with 76 Social status and puberty timing page 4 higher socio-economical status (SES) have earlier menarche preceded by accelerated growth 77 [19, 25-27]. While access to better nutrition may explain the positive relationship between 78 higher SES and early puberty [19], SES and caloric intake is complex. Indeed, other data 79 indicate that in industrial societies, children from families of low SES may consume high caloric, 80 inexpensive foods that accelerates weight gain and body fat accumulation [28-31], advancing 81 the age of puberty [32]. In these cases, indices of body fat, such as BMI, are better predictors 82 of puberty timing than SES [33]. However, adolescent girls who show an increased vulnerability 83 to psychosocial stress have an increased incidence of anovulation [34]. Similarly, studies of 84 socially-living rhesus monkeys, where diets are limited to standard low fat, high fiber meals, 85 indicate social status is a robust predictor of puberty timing [35] and reproductive success [36, 86 37]. Male rhesus monkeys from socially subordinate matrilines have delayed puberty [23, 38] 87 while females from subordinate matrilines show a significantly later age at first ovulation [35, 3988 41]. Although weight at first ovulation is similar between the later maturing subordinates 89 compared with the earlier maturing dominant females, it is not known whether these differences 90 are predicted from growth during infancy. 91 Although social status accounts for a significant amount of the variance in puberty timing in 92 rhesus monkey groups, there are nevertheless differences in the tempo of maturation between 93 females of similar social dominance ranks. For example, 42% of dominant and 88% of 94 subordinate females show a later age at first ovulation [35]. These data suggest that factors 95 that modify an individual’s response to social status may further explain variance in pubertal 96 timing. In this regard, the variable number tandem repeat polymorphisms in the length of the 97 promoter region of the SLC6A4 gene that encodes the serotonin reuptake transporter could be 98 important. The short (s) promoter length 5HTTLPR has diminished transcriptional activity 99 compared to the long (l) promoter length variant [42], and when present in humans, increases 100 the incidence of affective disorders in response to life stressors [43-46]. Homologous promoter 101 length variations in the SLC6A4 gene with reduced transcriptional activity are also present in 102 rhesus monkeys [42, 47]. The presence of the s 5HTTLPR is associated with greater emotional 103 Social status and puberty timing page 5 reactivity in juvenile monkeys [48-50] and potentiates the adverse consequence of social 104 subordination on metabolic and anthropometric measures in adult females [51]. Previous 105 studies of rhesus monkeys also indicate that females with the s variant 5HTTLPR show reduced 106 ovulatory frequency, lower body weights, and reduced serum levels of leptin compared with 107 those with homozygous for the long promoter variant [52] and the 5HTTLPR accounts for some 108 variation in the timing of reproductive output in males [53]. Because the 5HTTLPR is associated 109 with differences in reactivity to social environments, it is possible that these polymorphisms 110 interact with social status to influence the timing of reproductive maturation. 111 Using a rhesus monkey model, the present analysis combines both a prospective study to 112 determine how birth weight and growth during infancy predict the timing of pubertal events and 113 a separate retrospective study to determine how social status and SCL6A4 polymorphisms 114 influence pubertal timing. The neonatal period was manipulated in one of two ways compared 115 to control females. One group was treated continuously with a somatostatin analog to decrease 116 GH secretion while the other was treated continuously with a GnRH analog to block the 117 neonatal activation of the pituitary-gonadal axis. Although not as robust as occurs in males, the 118 HPG axis is active during the postnatal period in females [54, 55]. Because postnatal treatment 119 with a GnRH analog blunts growth in male monkeys [23, 24], we tested the hypothesis that such 120 treatment would slow growth in females in a manner similar to that as the inhibition of GH 121 secretion. In addition, given the importance of social status as a predictor of age at first 122 ovulation and the possibility that this may be influenced by 5HTTLPR polymorphisms, data were 123 analyzed separately to determine if growth and puberty is slowed significantly in subordinate 124 females with the s-variant 5HTTLPR genotype. 125 Social status and puberty timing page 6 Methods 126 Female rhesus monkeys (Macaca mulatta) were subjects. All females were members of 127 established social groups maintained at the Yerkes National Primate Research Center Field 128 Station. These groups varied in size from 25 to 85 animals and each contained one to two adult 129 males, multiple adult females and their juvenile or infant offspring. Animals were fed a standard 130 monkey diet (Purina Mills Lab Diet #5037, containing 13% of calories from fat, 18% from 131 protein, and 69% from carbohydrates) and received seasonal fruit and vegetables daily. The 132 Emory University Animal Care and Use Committee in accordance with the Animal Welfare Act 133 and the U.S. Department of Health and Human Services “Guide for Care and Use of Laboratory 134 Animals” approved the protocol. 135 On the day of birth, animals were randomly assigned to one of three treatment groups: control 136 (Con), depot Lupron treated (Lup), or Sandostatin LAR treated (GHx). Control animals (n = 6) 137 were weighed and injected SC were saline (0.25 ml) that continued every 25 days until the last 138 treatment at 225 days of age. Lup females (n = 6) were weighed and injected with 750 μg/kg 139 SC Depot Lupron (Tap Pharmaceuticals) every 25 days until the last treatment at 225 days of 140 age. This dose effectively blocks activation of the pituitary – gonadal axis in juvenile female 141 monkeys [56, 57] and arrests precocious puberty in girls [58]. GHx females (n = 6) were 142 weighed and injected with 625 μg/kg, IM of Sandostatin LAR (Sandoz Pharmaceuticals) every 143 25 days until the last treatment at 225 days of age. This dose produces GH deficiency in 144 juvenile female monkeys [56]. Animals received their respective treatments every 25 days with 145 the last treatment given at 225 days or 7.4 mo of age. Because both Depot Lupron and 146 Sandostatin LAR sustain serum levels of the drug for 28 days, treatments effectively ended at 147 8.3 mo of age (252 days of age). 148 The relative dominance status of each female in her social group was defined by the outcome of 149 dyadic interactions observed opportunistically throughout the study period [59]. Social status is 150 determined by membership in a matriline, as infants acquire the rank of their mothers [59]. 151 However, as the animals age and begin to engage in more agonistic interactions, their 152 Social status and puberty timing page 7 respective dominance positions become evident. Because the specific number of animals 153 within a group varied amongst the subjects, it was not possible to assign individual numerical 154 ranks to females. Consequently, females were classified as dominant or subordinate depending 155 whether their position was in the top half or bottom half of the group. It was not possible to 156 further categorize females as high, middle, or low ranking as the number of animals in some 157 cells in the social status by genotype matrix would have had one subject, precluding any 158 meaningful analysis. 159 5HTTLPR polymorphisms were determined as described previously for rhesus monkeys [42]. 160 Specifically, DNA was extracted from whole blood using the Pure Gene Blood Kit (Gentra, D161 4000). Polymorphisms in the promoter region of the SCL6A4 gene were identified following 162 amplification of the relevant gene segments by polymerase chain reaction using the 163 oligonucleotide primers fwd (cag ggg aga tcc tgg gag gga) and rev (ggc gtt gcc gct ctg aat gc) 164 based on the protocol described previously. The “s” amplicon (398bps) and the “l” amplicon 165 (419bps) were separated on an agarose gel containing ethidium bromide and identified by direct 166 visualization. Because the l/s genotype produces a similar phenotype as the s/s genotype on 167 most measures [47, 49], females carrying at least one s allele were categorized as having an “s168 variant” genotype. Because subjects were not selected a priori on the basis of social status and 169 5HTTLPR polymorphisms, social status classification and 5HTTLPR were not balanced across 170 neonatal treatments and the effects of each were analyzed separately (see below). A 171 description of the subjects is contained in Table 1. 172 Females were studied from birth through first ovulation. The primary outcome measures were 173 reproductive and anthropometric. Puberty was characterized in several ways. In addition to 174 menarche, assessed from daily observations, the age at the initial perineal swelling and 175 coloration was recorded. For rhesus monkeys, this typically proceeds menarche and is an 176 external indication estradiol secretion has commenced [60]. First ovulation was determined in 177 all females and the interval from menarche to first ovulation was considered as the tempo of 178 puberty [61]. Body weights were obtained at birth and monthly thereafter. Heights were 179 Social status and puberty timing page 8 obtained every 90 days while the females were anesthetized (10 mg/kg ketamine 180 hydrochloride). Females were placed in a supine position with the legs straightened on a long 181 piece of paper and a mark was made at the top of the head and at bottom of the heel. Two 182 individuals measured the distance using MHC vernier calipers and the mean of the two 183 measurements was calculated for an animal’s height. Body mass index was calculated as body 184 weight in kg divided height in m. 185 Serum samples were obtained by femoral venipuncture of infants following removal from her 186 mother. By 4 mo of age, females had become habituated to allow venipuncture of the 187 saphenous vein. Conscious venipuncture of captive acclimated rhesus monkeys does not 188 adversely affect limbic – HP – adrenal (LHPA) arousal, puberty, or reproductive performance 189 [62-64]. Morning, non-fasted samples were obtained once a month from birth through 18 190 months of age, once weekly until menarche, and twice weekly until first ovulation was 191 confirmed. Samples obtained at 6 and 7 mo were assayed for GH to determine whether either 192 of the neonatal treatments affected GH secretion. Nocturnal samples, collected at 18 and 24 mo 193 of age on each subjected were also assayed for GH. To better estimate GH secretion at these 194 ages, samples were obtained at 2200 ans 2230 hr and the average GH value between these 195 two time points was used for analysis. Other hormone analyses were focused on the interval 196 from 24 through ~32 mo of age, as this represents the age range from pre-menarche through 197 the earliest age of first ovulation in outdoor housed rhesus monkeys at our facility [64]. Daytime 198 samples obtained from 24 through ~32 mo of age were also assayed for leptin and cortisol. 199 Following menarche, all samples were assayed for progesterone. First ovulation was inferred 200 from a sustained rise in serum progesterone (>1 ng/ml for >5 days). Short luteal phase cycles 201 were identified by a blunted rise in progesterone (1 to 3 ng/ml) for 4 to 6 days followed by the 202 appearance of menstruation [8]. 203 All assays were performed in the YNPRC Biomarkers Core Lab. Progesterone was measured 204 using a previously described radioimmunoassay (RIA) that employs a commercially available kit 205 from Diagnostic Products Corporation (Los Angeles CA) [52]. The assay has a sensitivity of 206 Social status and puberty timing page 9 0.10 ng/ml with an interand intra-assay CV of 8.14% and 7.73%, respectively. Serum GH was 207 determined by a commercially available ELISA (Diagnostic Systems Laboratory (DSL), Webster 208 TX) having a sensitivity of 0.10 ng/ml using 20 μl of serum with an inter and intra assay CV of 209 9.13% and 6.33%, respectively. Serum leptin were measured by RIA using a commercially 210 available kit validated for nonhuman primates (Linco, St. Louis MO). Assaying 100 μl, the assay 211 has a range of 0.5 to 100 ng/ml. Intra-assay CVs were 5.5% and inter-assay were 8.8%. Serum 212 levels of cortisol were measured by RIA with a kit from DSL. The assay has a sensitivity of 0.10 213 ng/ml with an interand intra-assay coefficient of variation (CV) of 6.3% and 9.1%, respectively. 214 Data were summarized as the mean ± sem. As noted above, neither social status nor 215 5HTTLPR genotype was balanced across the neonatal treatment groups (Con, Lup, or GHx) so 216 data were analyzed separately. The first analysis tested the hypothesis that neonatal 217 treatments with Lurpon or Sandostatin were significantly slow growth and delay puberty. The 218 separate retrospective analysis tested the hypothesis that social subordination, exacerbated by 219 the presence of the s-variant allele in the SCL6A4 gene would significantly slow growth and 220 delay puberty. Data were analyzed with ANOVA models. Tests having a p < 0.05 were 221 considered significant. A Bonferroni adjustment (p = .05/n) was used for n pair wise 222 comparisons if interaction terms are significant. Data were transformed, as needed, to produce 223 homogeneity of variance. Because sample size for the social status by 5HTTLPR analyses was 224 small, we also calculated effects size that measures the magnitude of a treatment effect 225 independent of sample size. Effect sizes >0.8 are considered large, with percentage of overlap 226 in the distribution of measures between two populations becoming smaller as effect size 227 increases [65]. 228 Results 229 Consequences of neonatal treatments 230 As shown in Table 2, neonatal concentrations of GH were significantly affected by the 231 treatments (F2, 15 = 3.85, p = 0.04). Serum levels of morning GH were significantly higher in Con 232 compared to GHx females, with concentrations in Lup females being intermediate. While birth 233 Social status and puberty timing page 10 weight did not vary significantly between treatment groups (F 2, 15 = 3.15, p = 0.06), weight gain 234 during the 8 mo treatment interval was significantly greater in Con compared to Lup females, 235 with GHx females being intermediated (F 1, 15 = p = 0.03). In contrast, neither height at birth (F2, 236 15 = 0.62, p = 0.53) nor the increase in height during the 8-month treatment period (F2, 15 = 0.49, 237 p = 0.62) varied significantly between groups. 238 As can be seen in Table 2, the neonatal treatments had no effect on any of these same 239 parameters of puberty. Age at the appearance of secondary sexual characteristics, measured 240 by perineal swelling and coloration, did not vary among the three groups (F2, 15 = 0.18, p = 0.84). 241 In addition, neither age at menarche (F2, 15 = 0.68, p = 0.68) nor was age at first ovulation (F2, 15 242 = 0.21, p = 0.82) differed significantly as a function of the neonatal treatments. Consequently, 243 the tempo of maturation, defined as the interval from menarche to first ovulation, was also not 244 affected by the neonatal treatments (F2, 15 = 0.61, p = 56). 245 Body growth from the end of treatment at 8 mo through 30 mo of age was not affected by 246 neonatal treatments, as the increase in body weight (F2, 15 = 0.18, p = 0.84) and height (F2, 15 = 247 0.25, p = 0.78) was not significantly different among the groups. In contrast, a significant 248 neonatal treatment age interaction was observed for serum GH (F2, 15 = 8.36, p < 0.01). At 18 249 mo of age, nocturnal GH levels were significantly higher in Con compared with GHx females 250 that, in turn, were significantly higher than Lup-treated females. At 24 mo of age, nocturnal GH 251 concentrations were still significantly higher in Con compared with GHx females, with animals 252 treated neonatally with Lupron being intermediate. Serum leptin in the months prior to and 253 following menarche (25 through 30 mo of age) also did not vary significantly between the three 254 neonatally treated groups (F2, 15 = 0.43, p = 0.66, data not shown). Finally, serum levels of 255 cortisol did not vary significantly between the three groups during the neonatal treatment period 256 (Table 2; F 2, 15 = 0.52, p = 0.61). Serum levels did increase significantly in all females during 257 this period (F 5, 75 = 7.58, p < 0.05), from average concentrations at birth of 18.66 ± 2.22 μg/dl to 258 29.46 ± 2.63 μg/dl at the end of the neonatal treatment period. 259 Importance of social status – 5HTTLPR 260 Social status and puberty timing page 11 Reproductive maturation was significantly influenced by the interaction of social status and 261 5HTTLPR genotype (Figure 1). The age at the onset of perineal swelling was not influenced by 262 a female’s social status during development (F1, 14 = 0.32, p = 0.58), genotype (F1, 14 = 0.96, p = 263 0.35), nor the interaction of status and genotype (F1, 14 = 1.67, p = 0.22). Similarly, age at 264 menarche was not influenced by social status (F1, 14 = 1.02, p = 0.33), genotype (F1, 14 = 0.30, p 265 = 0.59), or a status by genotype interaction (F1, 14 = 0.46, p = 0.51). In contrast, age at first 266 ovulation occurred significantly earlier in dominant females (F1, 14 = 13.40, p < 0.01) and those 267 with an l/l genotype (F1, 14 = 13.82, p < 0.01). However, the variance in the age at first ovulation 268 was best explained by a significant status by genotype interaction (F1, 14 = 32.04, p < 0.01), as 269 subordinate females with the s-variant genotype had first ovulation at a significantly older age 270 than all other females. The computation of effect size for age at first ovulation for these 271 subordinate females compared to all other females was 1.81. Consequently, the tempo of 272 puberty was significantly longer (F1, 14 = 7.72, p = 0.02) in subordinate s-variant females (11.2 ± 273 1.3 mo) compared subordinate females with an l/l genotype (3.6 ± 1.8 mo), or dominant l/l (5.2 ± 274 1.8 mo) or s-variant females (3.1 ± 2.0 mo). The effect size for the tempo of maturation 275 between subordinate, s-variant females and all other females was also large (1.47). 276 Further analyses revealed that hormonal but not anthropometric measures were also affected 277 by status and 5HRRLPR. As illustrated in Table 3, birth weights and heights were not 278 significantly influenced by status (weight: F1, 14 = 0.46, p = 0.51; height: F1, 14 = 0.40, p = 0.54), 279 genotype (weight: F1, 14 = 1.79, p = 0.20; height: F1, 14 = 0.29, p = 0.60), or status by genotype 280 interaction (weight: F1, 14 = 0.10, p = 0.99; height: F1, 14 = 3.36, p = 0.09). Similarly, neither body 281 weight gain nor accumulation of height through 30 mo of age was influenced by status (weight: 282 F1, 14 = 0.04, p = 0.84; height: F1, 14 = 1.34, p = 0.27), genotype (weight: F1, 14 = 0.08, p = 0.78; 283 height: F1, 14 = 0.90, p = 0.36), or by a status genotype interaction (weight: F1, 14 = 1.49, p = 284 0.24; height: F1, 14 = 1.03, p = 0.33). Effect sizes comparing subordinate, s-variant females to 285 all other females was also small for the gain in both weight (0.21) and height (0.34). 286 Social status and puberty timing page 12 In contrast to the anthropometric data, nocturnal concentrations of GH (Figure 2) were 287 significantly influenced a status by 5HTTLPR interaction (F1, 14 = 9.32, p = 0.01) that did not vary 288 with advancing age (F1, 14 = 0.16, p = 0.70). As can be seen, the main effect of genotype is 289 significant (F1, 14 = 33.31, p < 0.01), with levels higher in females homozygous for the l-allele. 290 However, GH is significantly lower in subordinate s-variant females compared to all other 291 females and this was reflected in a large effect size of 1.25 for these females compared to all 292 others. Serum leptin also varied significantly between dominant and subordinate females 293 between 24 and 30 months of age (Figure 3; F6, 84 = 2.69, p = 0.02). Post hoc analyses 294 revealed that serum concentrations were significantly lower in subordinate, s-variant females 295 between 24 and 27 mo of age compared with all other females, again reflected in an effect size 296 of 0.98. 297 Analysis of developmental patterns of morning cortisol concentrations indicated that the 298 significantly age dependent increase during the adolescent period (Figure 4, F 13, 182 = 11.90, p < 299 0.05) was not affect by social status (F2, 15 = 1.00, p = 0.34) or 5HTTLPR (F2, 15 = 0.34, p = 0.57) 300 nor their interaction with age (F13, 182 = 0.98, p = 0.48). 301 Because the neonatal treatment groups were not formed on the basis of social status and 302 5HTTLPR, the interactive effects of these treatments with social status and genotype cannot be 303 statistically evaluated. However, it appeared that neonatal treatments would have had little 304 additive effect. Of the seven subordinate females with the s-variant genotype who ovulated 305 significantly later than all other females, two were control, three were Lupron treated, and two 306 were Sandostatin-treated. 307 Discussion 308 The prospective analysis revealed the neonatal suppression of the HPG or GH axis decreased 309 the developmental secretion of GH but had no adverse effects on growth or the timing of 310 puberty. However, reanalyzing the data to determine whether social status interacted with 311 polymorphisms in the gene encoding the 5HT transporter influenced development indicated the 312 socially subordinate females carrying the s-variant allele in the SCL6A4 gene have delayed 313 Social status and puberty timing page 13 puberty and this is associated with reduced concentrations of GH and leptin during the 314 prepubertal period. While these analyses do not support the hypothesis that perturbations in 315 neonatal GH secretion influence pubertal timing, they do show how social factors and genetic 316 polymorphisms can interact to influence the occurrence of first ovulation in female rhesus 317 monkeys and this effect is associated with significant differences in growth-related signals.. 318 The rationale for manipulating the HPG and GH axes neonatally was based on data from 319 children [20, 21] and monkeys [22] that postnatal growth velocities appeared to influence the 320 timing of puberty possibly, due to changes in GH and IGF1 [18, 19]. Furthermore, suppression 321 of the neonatal HPG axis in male monkeys slowed growth and delayed puberty [23, 24]. Thus, 322 our intent was to disrupt postnatal growth in females by suppressing the either the HPG or the 323 GH axis. However, we saw no effect of either the GnRH analog or somatostatin analog 324 treatments during the neonatal interval on the eventual parameters of growth or puberty in 325 female monkeys. While GH secretion was lowered significantly neonatally and during 326 prepuberty by both treatments compared to saline, no other effects were evident. Given the sex 327 differences in the neonatal activation of the HPG axis in rhesus monkeys [54, 55], perhaps it is 328 not surprising that the neonatal administration of Lupron had no effect on eventual puberty as 329 the axis shows much greater activity in males compared with females. What the data do 330 indicate that the lower serum GH concentrations throughout development produced by Lupron 331 and Sandostatin postnatal do not adversely affect growth or eventual puberty. Although 332 significantly lower than controls, these GH concentrations were not suppressed to the degree of 333 those produced in other monkey models of juvenile GH deficiency that does result in a slowing 334 of growth and a delay in first ovulation [10, 56]. Taken together, these data suggest there may 335 be a critical lower limit below of GH secretion below which normal growth and puberty is 336 compromised. Nevertheless, the data from the present study clearly indicate that perturbations 337 of the GH and HPG axes during the neonatal period have no adverse effects on maturation in 338 female monkeys. 339 Social status and puberty timing page 14 In contrast, the retrospective analyses not only confirms previous reports on male [23, 38] and 340 female monkeys [35, 39-41] that social subordination delays puberty but extends these findings 341 to indicate that this effect of subordination occurs predominantly in those subordinate females 342 having at least on s-allele in the encoding 5HT transporter. These data are consistent with a 343 recent report of an increased incidence of functional hypothalamic amenorrhea is girls 344 experienced an increased incidence of stress-induced psychopathology [34]. In macaque 345 groups, social subordination is largely maintained through continual harassment and the threat 346 of aggression from dominant towards subordinate animals [59]. As a result, socially subordinate 347 adult females are characteristically hypercortisolemic secondary to a dysregulation of the LHPA 348 axis [66-68]. Because the presence of the s allele is associated with the increased response to 349 psychosocial stressors in rhesus monkeys [47-50, 69], we expected that morning cortisol 350 concentrations would be highest in subordinate females with the s-variant genotype. Such a 351 finding would have supported that hypothesis that activation of the LHPA axis accounted for the 352 delayed puberty in this cohort [70, 71]. However, morning cortisol during the juvenile period did 353 not vary significantly between the groups. Recognizing that this measure is an inadequate 354 marker of LHPA activity, other assessments such as a dexamethasone suppression test to 355 determine glucocorticoid negative feedback, may have better differentiated the groups [66]. 356 Although anthropometric measures also did not differ as a function of social status and 357 5HTTLPR, morning GH and leptin concentrations were significantly lower in these slowly 358 maturing females. It is possible that increased psychosocial stress accounted for these 359 differences. CSF concentrations of somatostatin are higher [72] and the GH response to 360 clonidine blunted [73] in macaques raised in an unpredictable environment, possibly due to a 361 CRH increasing somatostatin [74-76]. CRH inhibits pulsatile GH release [77] and GHRH362 induced GH secretion in rats [78, 79] while a CRH antagonist increases GH secretion and 363 hypothalamic GHRH mRNA [80]. While the lower concentrations of serum leptin could be the 364 result of stress-induced attenuation of food intake, lowering body fat stores [81], no differences 365 in body weight or BMI scores were observed among the animals. Given similarities in body 366 Social status and puberty timing page 15 weight, it would seem differences in food intake and adequate nutrition would not explain the 367 delayed puberty in the subordinate, s-variant females. However, the present analysis also 368 cannot determine whether the lower levels of GH and leptin during in juvenile subordinates with 369 the s-variant genotype played any role in the delayed occurrence of first ovulation. As noted 370 above, experimentally induced deficits in juvenile concentrations of serum GH account for 371 variation in pubertal timing [10, 56] and leptin administration advances puberty in female [82] but 372 not male monkeys [83]. Furthermore, leptin administration restores LH secretion and ovulatory 373 function in some women with functional hypothalamic anovulation [84]. Obviously further 374 studies are needed to identify what signals mediate the slowing of puberty in these subordinate 375 monkeys. 376 This retrospective analysis must be interpreted cautiously as it was severely under-powered. 377 Furthermore, we must emphasize that our sample size is not adequate to determine the genetic 378 contributions to behavior and physiology and it is entirely likely that the reproductive phenotypes 379 examined in this study are influenced by many genes [85]. Indeed, recent analyses have begun 380 to show variations in puberty timing are explained by polymorphisms in genes whose protein 381 products affect GnRH secretion, including the insulin receptor subtype 1 (IRS1) [86], the gene 382 encoding leptin (LEP) [87], as well the gene encoding kisspeptin (KISS1) [88] and its receptor 383 (KISS1R) [89]. Furthermore, a polymorphism in the SHBG gene (SHBG), that may regulate 384 bioavailability of estradiol, is associated with the timing of menarche [90]. In contrast, attempts 385 to find associations between puberty timing and polymorphisms in the gene encoding the GnRH 386 receptor (GnRHR) have not been successful [91]. The data from the present study show that a 387 gene polymorphism that affects individual reactivity to socio-environmental stressors can also 388 affect puberty, presumably through activation of the LHPA axis. It is likely that this 389 polymorphism could act independently or synergistically with other gene polymorphisms to 390 influence the timing of puberty by acting at multiple levels of the HPG axis. These present data 391 can best serve as the foundation for broader linkage and association analyses to understand 392 how social context and genes interact to time the onset of puberty [92]. Nevertheless, the data 393 Social status and puberty timing page 16 confirm the critical importance of the psychosocial environment for development and illustrate394the potential of using socially housed macaques to understand gene by environment effects on395behavior and physiology [93].396Acknowledgements397We appreciate the expert technical assistance of Jeff Fisher, Kathy Chikazawa, and Juliet398Brown. This work was supported by HD35183, HD46501, and, in part, RR00165. The YNPRC399is fully accredited by the American Association for Laboratory Animal Care International.400 References1. Dungan HM, Clifton DK, Steiner RA. Minireview: kisspeptin neurons as central processors inthe regulation of gonadotropin-releasing hormone secretion. Endocrinology 2006; 147:1154-1158.2. El Majdoubi M, Sahu A, Ramaswamy S, Plant TM. Neuropeptide Y: A hypothalamic brakerestraining the onset of puberty in primates. Proc Natl Acad Sci U S A 2000; 97: 6179-6184.3. Kasuya E, Nyberg CL, Mogi K, Terasawa E. 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Mean ± SEM serum concentrations of nocturnal GH (ng/ml) at 18 and 24 months ofage for dominant (dom) or subordinate (sub) being homogenous for the long promoter lengthvariant or (l/l) or having at least one short promoter length allele (s-variant) polymorphism in theSCL6A4 gene. Different letters at each age indicate groups are significantly different (p < 0.05). Social status and puberty timingpage 27 Figure 3. Mean ± SEM serum concentrations of morning leptin (ng/ml) from 24 through 30months af age for dominant (dom) or subordinate (sub) being homogenous for the longpromoter length variant or (l/l) or having at least one short promoter length allele (s-variant)polymorphism in the SCL6A4 gene. Asterisks indicate subordinate, s-variant females hadsignificantly lower concentrations than other groups (p < 0.05).Figure 4. Mean ± SEM serum concentrations of morning cortisol (μg/dl) from 24 through 33.5months of age for dominant (dom) or subordinate (sub) being homogenous for the longpromoter length variant or (l/l) or having at least one short promoter length allele (s-variant)polymorphism in the SCL6A4 gene. Different letters at each age indicate groups aresignificantly different (p < 0.05). Social status and puberty timingpage 28 Table 1. Samples sizes for the prospective analysis of neonatal treatments and theretrospective analysis of social status – genotype effects on pubertal timing. Social Status X 5HTTLPRDominantSubordinateNeonatal Treatment Sample Size l/ls-variant l/ls-variant Control61032 Lupron-treated61203 Sandostatin Treated 62112 Sample sizes184347 Social status and puberty timingpage 29 Table 2. Developmental parameters (mean ± sem) for females treated neonatally with saline(Con), depot Lupron (Lup), or Sandostatin LAR (GHx). For a given measure, groupswith different letters are significantly different from one another (p < 0.05). ParameterConn = 6Lupn = 6GHxn = 6 Birth weight (kg)0.45 ± 0.020.43 ± 0.030.40 ± 0.01 Birth Height (cm)29.9 ± 0.530.1 ± 0.429.0 ± 0.4