The development and evolution of inhibitory neurons in primate cerebrum.

Neuroanatomists have long speculated that expanded primate brains contain an increased morphological diversity of inhibitory neurons (INs)1, and recent studies have identified primate-specific neuronal populations at the molecular level2. However, we know little about the developmental mechanisms that specify evolutionarily novel cell types in the brain. Here, we reconstruct gene expression trajectories specifying INs generated throughout the neurogenic period in macaques and mice by analysing the transcriptomes of 250,181 cells. We find that the initial classes of INs generated prenatally are largely conserved among mammals. Nonetheless, we identify two contrasting developmental mechanisms for specifying evolutionarily novel cell types during prenatal development. First, we show that recently identified primate-specific TAC3 striatal INs are specified by a unique transcriptional programme in progenitors followed by induction of a distinct suite of neuropeptides and neurotransmitter receptors in new-born neurons. Second, we find that multiple classes of transcriptionally conserved olfactory bulb (OB)-bound precursors are redirected to expanded primate white matter and striatum. These classes include a novel peristriatal class of striatum laureatum neurons that resemble dopaminergic periglomerular cells of the OB. We propose an evolutionary model in which conserved initial classes of neurons supplying the smaller primate OB are reused in the enlarged striatum and cortex. Together, our results provide a unified developmental taxonomy of initial classes of mammalian INs and reveal multiple developmental mechanisms for neural cell type evolution.

Conserved expression of lincRNA during human and macaque prefrontal cortex development and maturation.

The current annotation of the human genome includes more than 12,000 long intergenic noncoding RNAs (lincRNA). While a handful of lincRNA have been shown to play important regulatory roles, the functionality of most remains unclear. Here, we examined the expression conservation and putative functionality of lincRNA in human and macaque prefrontal cortex (PFC) development and maturation. We analyzed transcriptome sequence (RNA-seq) data from 38 human and 40 macaque individuals covering the entire postnatal development interval. Using the human data set, we detected the expression of 5835 lincRNA annotated in GENCODE and further identified 1888 novel lincRNA. Most of these lincRNA show low DNA sequence conservation, as well as low expression levels. Remarkably, developmental expression patterns of these lincRNA were as conserved between humans and macaques as those of protein-coding genes. Transfection of development-associated lincRNA into human SH-SY5Y cells affected gene expression, indicating their regulatory potential. In brain, expression of these putative target genes correlated with the expression of the corresponding lincRNA during human and macaque PFC development. These results support the potential functionality of lincRNA in primate PFC development.

Development and three-dimensional morphology of the zygomaticotemporal suture in primate skulls.

Cranial sutures are an essential part of the growing skull, allowing bones to increase in size during growth, with their morphology widely believed to be dictated by the forces and displacements that they experience. The zygomaticotemporal suture in primates is located in the relatively weak zygomatic arch, and externally it appears a very simple connection. However, large forces are almost certainly transmitted across this suture, suggesting that it requires some level of stability while also allowing controlled movements under high loading. Here we examine the 2- and 3-dimensional (3D) morphology of the zygomaticotemporal suture in an ontogenetic series of Macaca fascicularis skulls. High resolution microcomputed tomography data sets were examined, and virtual and physical 3D replicas were created to assess both structure and general stability. The zygomaticotemporal suture is much more complex than its external appearance suggests, with interlocking facets between the adjacent zygomatic and temporal bones. It appears as if some movement is permitted across the suture in younger animals, but as they approach adulthood the complexity of the suture's interlocking bone facets reaches a level where these movements become minimal.

Talar maturity determined by epiphyseal closure of the calcaneus.

The talus is used in many kinds of studies on primates including fossil species, and most of the individuals studied are adults. One of the most important indicators of adult individuals is epiphyseal closure; however, because the talus has no epiphysis, it is difficult to determine the maturity of the talus. The calcaneus has one epiphysis, and it has been used along with the talus in some analyses. The objective of this study was to quantify the maturation trajectory of the talus using epiphyseal closure of the calcaneus as a benchmark. We used 71 skeletons of free-ranging Macaca fuscata fuscata males of known day-age. We did not identify any size increase with age in talar dimensions among specimens with complete calcaneal epiphyseal closure. Thus, in male M. fuscatafuscata, the maturation trajectory of the talus can be quantified using epiphyseal closure of the calcaneus as a benchmark.

Postnatal neurogenesis: of mice, men, and macaques.

Over the past 20 years, the conception of brain development has radically changed from a fixed and limited hierarchical process to a more plastic and continuous one. Most surprising, the field has learned that postnatal neurogenesis is not just a seasonal phenomenon in songbirds but a process that occurs across species and seasons. Astrocytes, whose primary role in the central nervous system was thought to be strictly supportive, have emerged as a heterogeneous population, a subset of which is the neural stem cell. Postnatal neurogenesis persists in specialized niches within the rostral subventricular zone and hippocampal dentate gyrus and, for a limited period, within the white matter tracts and external granular layer of the cerebellum. These specialized microenvironments are influenced by factors in the blood, cerebrospinal fluid, and local extracellular matrix. This article reviews the current understanding of adult neurogenesis, which is conserved across many vertebrate species, underscoring the value of animal models in past and present studies of human neurogenesis and neurogenic disease.

Expression of fatty acid-binding proteins in adult hippocampal neurogenic niche of postischemic monkeys.

Intracellular fatty acid (FA) chaperones known as FA-binding proteins (FABPs) are a group of molecules known to participate in cellular metabolic processes such as lipid storage, membrane synthesis, and ß-oxidation or to coordinate transcriptional programs. However, their role in adult neurogenesis still remains obscure. The FABPs expressed in the central nervous system (CNS) are heart-type (FABP3), epidermal-type (FABP5), and brain-type (FABP7). These three FABPs possess a differential affinity for polyunsaturated fatty acids (PUFAs). Recently, we reported that GPR40, a receptor for free FAs and particularly for PUFAs, is expressed in the CNS of adult monkeys and upregulated after transient global brain ischemia in the hippocampal subgranular zone (SGZ), a neurogenic niche in adulthood. The SGZ showed a peak proliferation of progenitor cells and maximal expression of GPR40 during the second week after ischemia. As both FABPs and GPR40 might be closely related to the adult neurogenesis, here, we studied the expression of FABP 3, 5, and 7 in the SGZ, comparing normal and postischemic adult monkeys. Immunoblotting revealed that FABP5 and FABP7, but not FABP3, were significantly increased on day 15 after ischemia when compared with the nonischemic control. Immunohistochemistry showed that FABP5 was almost undetectable in the control SGZ but was abundant on day 15 after ischemia. FABP 3, 5, and 7 were expressed in S-100ß-positive astrocytes and nestin-positive neural progenitors. However, only FABP 5 and 7 were found in bromodeoxyuridine (BrdU)-positive newly generated cells. FABPs were most frequently coexpressed with the S-100ß-positive astrocytes, whereas ßIII-tubulin-or polysialylated neural cell-adhesion molecule (PSA-NCAM)-positive newborn neurons in the vicinity of the astrocytes expressed none of the three FABPs. These results support a role of astrocyte- and/or neural progenitor-derived FABPs as components of the molecular machine regulating the progenitor cell niche in the adult primate brain.

Primate life histories and dietary adaptations: a comparison of Asian colobines and macaques.

Primate life histories are strongly influenced by both body and brain mass and are mediated by food availability and perhaps dietary adaptations. It has been suggested that folivorous primates mature and reproduce more slowly than frugivores due to lower basal metabolic rates as well as to greater degrees of arboreality, which can lower mortality and thus fecundity. However, the opposite has also been proposed: faster life histories in folivores due to a diet of abundant, protein-rich leaves. We compared two primate taxa often found in sympatry: Asian colobines (folivores, 11 species) and Asian macaques (frugivores, 12 species). We first described new data for a little-known colobine (Phayre's leaf monkeys, Trachypithecus phayrei crepusculus) from Phu Khieo Wildlife Sanctuary, Thailand. We then compared gestation periods, ages at first birth, and interbirth intervals in colobines and macaques. We predicted that heavier species would have slower life histories, provisioned populations would have faster life histories, and folivores would have slower life histories than frugivores. We calculated general regression models using log body mass, nutritional regime, and taxon as predictor variables. Body mass and nutritional regime had the predicted effects for all three traits. We found taxonomic differences only for gestation, which was significantly longer in colobines, supporting the idea of slower fetal growth (lower maternal energy) compared to macaques and/or advanced dental or gut development. Ages at first birth and interbirth intervals were similar between taxa, perhaps due to additional factors (e.g., allomothering, dispersal). Our results emphasize the need for additional data from wild populations and for establishing whether growth data for provisioned animals (folivores in particular) are representative of wild ones.

Establishment of vertebral heart scale in the growth period of the Japanese macaque (Macaca fuscata).

We performed a thoracic X-ray examination of 56 Japanese macaques to obtain normal reference values for vertebral heart scale (VHS). Mean VHS was 10.25 +/- 0.94 v. In males, mean VHS was 10.56 +/- 0.73 v, with no significant correlation to age or weight. In contrast, mean VHS in females was 9.97 +/- 1.03 v, and tended to decrease with increasing age and weight. These findings will facilitate the diagnosis of cardiac disease in Japanese macaques in the future.

Single-Cell RNA Sequencing of Human, Macaque, and Mouse Testes Uncovers Conserved and Divergent Features of Mammalian Spermatogenesis.

Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate spermatogenesis is limited to populations defined by state-specific markers from rodent data. As between-species differences have been reported in the duration and differentiation hierarchy of this process, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA sequencing to identify transcriptional signatures of major germ and somatic cell types of the testes in human, macaque, and mice. This approach reveals similarities and differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, markers of differentiation, potential regulators of meiosis, RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis.

Single-Cell RNA Sequencing of the Cynomolgus Macaque Testis Reveals Conserved Transcriptional Profiles during Mammalian Spermatogenesis.

Spermatogenesis is highly orchestrated and involves the differentiation of diploid spermatogonia into haploid sperm. The process is driven by spermatogonial stem cells (SSCs). SSCs undergo mitotic self-renewal, whereas sub-populations undergo differentiation and later gain competence to initiate meiosis. Here, we describe a high-resolution single-cell RNA-seq atlas of cells derived from Cynomolgus macaque testis. We identify gene signatures that define spermatogonial populations and explore self-renewal versus differentiation dynamics. We detail transcriptional changes occurring over the entire process of spermatogenesis and highlight the concerted activity of DNA damage response (DDR) pathway genes, which have dual roles in maintaining genomic integrity and effecting meiotic sex chromosome inactivation (MSCI). We show remarkable similarities and differences in gene expression during spermatogenesis with two other eutherian mammals, i.e., mouse and humans. Sex chromosome expression in the male germline in all three species demonstrates conserved features of MSCI but divergent multicopy and ampliconic gene content.

Development of contrast sensitivity in infant Macaca nemestrina monkeys.

During an infant monkey's first 28 postnatal weeks, the visual contrast sensitivity function develops its characteristic adult form. Contrast sensitivity is depressed relative to that of the adult for all spatial frequencies during the early postnatal weeks. Absolute sensitivity to frequencies below 5 cycles per degree approaches adult levels by 20 weeks after birth, whereas sensitivity to fine spatial detail continues to develop through 28 weeks. The results imply that the development of primate spatial vision is more complex than just an improvement in the ability to resolve acuity gratings.

Japanese macaques form a cross-modal representation of their own species in their first year of life.

We tested whether infant Japanese macaques (Macaca fuscata) have a cross-modal representation of their own species. We presented monkeys with a photograph of either a monkey or a human face on an LCD monitor after playing back a vocalization of one of those two species. The subjects looked at the monitor longer when a human face was presented after the monkey vocalization than when the same face was presented after human vocalization. This suggests that monkeys recall and expect a monkey's face upon hearing a monkey's voice.

EMMLi: A maximum likelihood approach to the analysis of modularity.

Identification of phenotypic modules, semiautonomous sets of highly correlated traits, can be accomplished through exploratory (e.g., cluster analysis) or confirmatory approaches (e.g., RV coefficient analysis). Although statistically more robust, confirmatory approaches are generally unable to compare across different model structures. For example, RV coefficient analysis finds support for both two- and six-module models for the therian mammalian skull. Here, we present a maximum likelihood approach that takes into account model parameterization. We compare model log-likelihoods of trait correlation matrices using the finite-sample corrected Akaike Information Criterion, allowing for comparison of hypotheses across different model structures. Simulations varying model complexity and within- and between-module contrast demonstrate that this method correctly identifies model structure and parameters across a wide range of conditions. We further analyzed a dataset of 3-D data, consisting of 61 landmarks from 181 macaque (Macaca fuscata) skulls, distributed among five age categories, testing 31 models, including no modularity among the landmarks and various partitions of two, three, six, and eight modules. Our results clearly support a complex six-module model, with separate within- and intermodule correlations. Furthermore, this model was selected for all five age categories, demonstrating that this complex pattern of integration in the macaque skull appears early and is highly conserved throughout postnatal ontogeny. Subsampling analyses demonstrate that this method is robust to relatively low sample sizes, as is commonly encountered in rare or extinct taxa. This new approach allows for the direct comparison of models with different parameterizations, providing an important tool for the analysis of modularity across diverse systems.

Assessing the Effects of Tourist Provisioning on the Health of Wild Barbary Macaques in Morocco.

Feeding wildlife is a very popular tourist activity, largely because it facilitates the close observation of animals in their natural habitat. Such provisioning may benefit animals by improving their survival and reproductive success, especially during periods of natural food shortage. However, provisioning by tourists may also have negative impacts on the health of the animals involved; to date such impacts are poorly understood. Here, we investigated the effects of tourist provisioning on the health of wild adult Barbary macaques, Macaca sylvanus, in Morocco. We compared health measures between a heavily provisioned group and a group that received negligible food from tourists and, in the former group, we also assessed health measures in relation to the intensity of provisioning. We used a broad range of non-invasive health measures relating to birth rate and survival, disease and injury risk, body size and condition, and physiological stress. Our findings indicate that feeding by tourists may overall have negative impacts on the health of Barbary macaques, being linked in particular to larger body size, elevated stress levels and more alopecia. Finally, we propose a framework to help consider the potential costs and benefits of provisioning, which may facilitate future research and management decisions on whether-and how much-provisioning is acceptable.

Changes in testicular and nipple volume related to age and seasonality in Japanese macaques (Macaca fuscata), especially in the pre- and post-pubertal periods.

We investigated, longitudinally and cross-sectionally, age and seasonal change in both the testis and nipple volume of Japanese macaques (Macaca fuscata) in relation to concentration profiles of gonadal steroids: testosterone (T) in males and progesterone (P) in females. Testicular volume (TV) and nipple volume (NV) showed rapid growth at puberty, 4.5 and 3.5 years of age in males and females, respectively, but in both sexes there were precocious individuals. The testis as a whole matures at about 10 years of age. TV change is closely related to T concentration profile. The pattern of TV change is composed of maturation and seasonal effects, with individual variation evident mainly in the latter. Some individuals show a simple pattern consisting of one peak in the breeding season (from summer to winter) and one trough in the non-breeding season. Other individuals exhibit a more complicated pattern composed of two or more peaks and troughs before and during the breeding season. The nipple matures at about 7 years but it is difficult to determine the exact maturational age as there are many confounding factors relating to NV. NV shows seasonal fluctuations similar to that of TV. Many animals have periods of substantial growth whereas others do not. The NV in adults from 10 to 25 years does not appear to change much with age, but animals older than 25 years of age have significantly smaller nipples. Seasonal fluctuation in NV mirrors that of the P level. Considered to be controlled by estrogen and P, the NV is a good indicator of the physiological status of reproduction, with its peak about 2 weeks earlier than that of P, that is, at the mid-follicular phase. NV and P level show a similar pattern in pregnancy; from conception, indicated by a P peak, NV and P concentration first decrease, then they increase until peri-parturition and slowly decrease again until the next breeding season.

Growth rates in a captive population of Tonkean macaques.

Measuring variations in body mass is necessary to gain a deeper understanding of the evolution of life-history patterns, and it provides information on the timing of sexual maturity and the development of sexual dimorphism. In this study, we collected longitudinal data on body mass from infancy to adulthood in a captive population of Tonkean macaques (Macaca tonkeana). Tests to evaluate whether social group, maternal age, and dominance rank influenced growth rates showed that they had no significant effect. We investigated the timing and magnitude of breaking points in the growth paths of males and females, and checked whether these breaking points could correspond to specific reproductive and morphological developmental events. We found that male and female Tonkean macaques have roughly equivalent body masses until around the age of four, when males go through an adolescent growth spurt and females continue to grow at a constant rate. Males not only grow faster than females, but they also continue to grow for nearly one and a half years after females have attained their full body mass. Growth rate differences account for approximately two-thirds of the body mass sexual dimorphism; only the remaining third results from continued male growth beyond the age where full body mass is reached in females. We also discovered remarkable correspondences between the timing of testicular enlargement and the adolescent growth spurt in males, and between dental development and slowdown breaking points in both sexes.

Developmental changes in the luteinizing hormone secretory pattern in peripubertal female rhesus monkeys: comparisons between gonadally intact and ovariectomized animals.

Developmental changes in LH release patterns were observed longitudinally in female rhesus monkeys at 10-65 months of age. The average ages of menarche and first ovulation in this experiment (n = 14) were 31.1 +/- 2.6 and 47.0 +/- 2.6 months (mean +/- SE), respectively. To assess the ovarian influence on developmental changes in LH, data were simultaneously obtained from neonatally ovariectomized animals at similar ages. The estimation of circulating LH was made with RIA as well as biological assay. During the prepubertal period (10-20 months of age), basal LH was very low, and there was no circadian fluctuation of LH in gonadally intact monkeys. During the early pubertal stage (20-30 months of age), before menarche, basal LH levels started to increase, and a circadian LH rhythm (nocturnal increases) appeared. At the midpubertal stage (30-50 months of age), a period between menarche and first ovulation, basal LH levels further increased, and the circadian LH rhythm was maximal. At the late pubertal stage (50-60 months of age), a period after the first ovulation during which the animals were not able to reproduce fully as adults, basal LH declined, and the circadian rhythm diminished. Similar but more exaggerated developmental changes in basal LH and the circadian fluctuation of LH were observed in females ovariectomized neonatally. Basal LH levels at 10-20 months were as low as those in intact animals with no circadian rhythm present. During the early pubertal period, a circadian fluctuation appeared at the time when a slight increase in the basal LH level occurred. Furthermore, the amplitude of circadian fluctuation (the difference between morning and evening LH values) increased linearly with the increase in basal LH during the midpubertal stage. These LH parameters in ovariectomized animals reached their peaks at 40-44 months, an age before the first ovulation in intact animals. As basal LH levels declined during the late pubertal stage to postpubertal stage, circadian fluctuation disappeared. The results suggest that the increase in LH output and concomitant circadian fluctuations occur in close association with the pubertal process, and this change in LH release is not dependent on the presence of the ovary. Therefore, we suggest that alteration of the LHRH release pattern during maturation, as reflected by LH release, rather than resetting of the gonadostat, is the key factor involved in the mechanism of the onset of puberty.

Gonadal steroids influence serum somatomedin-C concentrations in prepubertal female rhesus monkey.

The present study examined the influence of estradiol (E2) and androstenedione (delta 4) on the regulation of GH and somatomedin-C (Sm-C) secretion in prepubertal female rhesus monkeys. Ovariectomized animals (n = 9) received empty capsules (0) or capsules filled with E2 or delta 4 in successive 2-week treatment blocks as follows: 0, E2, E2 plus delta 4, 0, delta 4, delta 4 plus E2, and 0. Serum samples were collected twice weekly for determination of basal hormone levels. Implantation of delta 4 capsules elevated serum delta 4 and testosterone concentrations, but did not influence E2 levels. Conversely, treatment with E2 only elevated serum E2. Basal concentrations of serum GH were unaffected by steroid treatment as levels varied between 5.5 and 9.5 ng/ml during the study. In contrast, Sm-C concentrations were increased significantly by each steroid treatment. Levels of Sm-C were elevated by delta 4 treatment (450.7 +/- 33.2 ng/ml) compared to baseline (376.2 +/- 37.7 ng/ml), whereas concentrations were maximally increased by E2 administration (greater than 600 ng/ml). The addition of delta 4 to the E2 treatment regimen did not further increase Sm-C levels. Regression analyses revealed that neither delta 4 nor testosterone concentrations were related to Sm-C or GH levels. In contrast, levels of E2 (r = 0.81) and GH (r = 0.42), which were not related to one another, were significantly correlated with Sm-C concentrations. Indeed, the multiple regression of E2 and GH on Sm-C accounted for significantly more variance in Sm-C levels (R2 = 0.706) than either hormone alone. These data suggest that E2 is responsible for the steroid-induced increases in Sm-C secretion observed previously in prepubertal females. Since steroid treatment elevated Sm-C levels independent of consistent changes in basal GH secretion, the effect is probably the result of either a direct E2 action on Sm-C release in the presence of a normal GH secretory pattern or an exacerbation of the nocturnal secretion of GH.

The metabolic clearance rate and the production rate of estradiol in sexually immature and adult female rhesus monkeys.

The blood MCR of estradiol (MCRE2) was measured in 34 experiments with 10 adult (4.7-8.2 kg) and 13 prepubertal (1.8-3.0 kg; 13-23 months old) female rhesus monkeys using the constant infusion technique. Twenty-six of the studies were performed using an adult and an immature animal simultaneously. Twenty-four of the studies were performed in pentobarbital-anesthetized animals, while the remainder used conscious animals restrained in primate chairs. The blood MCRE2 in the adult female was 167.5 +/- 9.5 liters/day (mean +/- SE; n = 14) or 27.5 +/- 1.4 liters/day x kg BW, and was not altered by anesthesia, stage of the menstrual cycle, amenorrhea of more than 60 days duration, or the site of origin of the blood used to calculate the MCR (radial artery, femoral artery, femoral vein, or saphenous vein). While the absolute MCRE2 in the immature animal (either anesthetized or conscious) was less than that in the adult, when corrected for body weight, the relative MCRE2 (in liters per day/kg BW) of the conscious immature animal was double that seen in the adult [48.4 +/- 5.2 (n = 6) vs. 27.5 +/- 1.4 (n = 1.4)]. Anesthesia caused a profound depression of the MCRE2 in the immature animal, which could be prevented if the body temperature of the animal was maintained at 37 C during the prolonged period of anesthesia. The production rate of estradiol (PRE2) was calculated as the product of the serum estradiol concentration (in micrograms per liter; measured by RIA techniques) and the plasma MCRE2 (blood MCRE2 x 1 - hematocrit). In the adult animals, the PRE2 ranged from 1.9 - 35.5 micrograms/day, and was lowest in the amenorrheic animals and highest during the late follicular phase. The PRE2 in the immature animals ranged from unmeasurable to 1.7 micrograms/day, averaging 0.7 +/- 0.2 micrograms/day (n = 12) in those animals where it could be measured. These data support the hypothesis that the low circulating estradiol levels in the immature animal are the consequence of a low PRE2 coupled with a high MCRE2.

Changes in sex hormone-binding globulin binding capacity and percent free estradiol during development in the female rhesus monkey (Macaca mulatta): relation to the metabolic clearance rate of estradiol.

Circulating sex hormone-binding globulin (SHBG) binding capacity and percent free estradiol (% free E2) were measured in separate groups of female rhesus monkeys from 2 months of age through adulthood (greater than 4.5 yr old). The SHBG concentration alone was also measured in 11 adult and 6 sexually immature animals on the same day as the blood MCR of E2. Serum SHBG levels were the highest (range, 12-25 micrograms T/dl) and the % free E2 the lowest (0.88%) in animals 6 months old or less. After the age, serum SHBG binding capacity declined generally at an average rate of 0.11 SHBG U (microgram T/dl)/month toward a nadir in adulthood. There was no difference in the SHBG levels in the follicular or luteal phase of the menstrual cycle. The relative blood MCR E2 and circulating SHBG binding capacity were significantly greater (P less than 0.001) in the sexually immature animals [MCR E2, 48.4 +/- 5.2 (SEM) liters/day . kg body wt; SHBG, 9.8 +/- 1.0 microgram T/dl, n = 6] than in adult animals (MCR E2, 27.7 +/- 1.7 liters/day kg body wt; SHBG, 4.6 +/- 0.3 microgram T/dl, n = 11). There was no relation between the MCR E2 and circulating SHBG levels within each group of adult or immature animals. The mean % free E2 doubled (to 1.6%) between 1 and 54 months of age; there was no relation between total circulating E2 and % free E2. Although a high SHBG binding capacity and a low % free E2 in the circulation of the immature animal does not inhibit the metabolic clearance of E2; it remains possible that these factors (and others) may hinder the access of E2 to reproductive target tissues and thereby contribute to the slow acquisition of reproductive competence in this species.