Organizing action of prenatally administered testosterone propionate on the Tissues mediating mating behavior in the female guinea pig

Our “Organizing Action” paper published in 1959 put forward the concept that prenatal exposure to testosterone masculinized the behavior of genetic female guinea pigs. Specifically, we proposed that testosterone or some metabolite acted on the central nervous tissues in which patterns of sexual behavior are organized. We later went on to demonstrate similar effects in rhesus monkeys by showing that play behavior by female monkeys prenatally treated with testosterone was masculinized as well. These findings support the organizing actions of androgens as a general process of sexual differentiation.     

Sex and context: hormones and primate sexual motivation

Gonadal hormones regulate the ability to copulate in most mammalian species, but not in primates because copulatory ability has been emancipated from hormonal control. Instead, gonadal hormones primarily influence sexual motivation. This separation of mating ability from hormonally modulated mating interest allows social experience and context to powerfully influence the expression of sexual behavior in nonhuman primates, both developmentally and in adulthood. For example, male rhesus monkeys mount males and females equally as juveniles, but mount females almost exclusively as adults. Having ejaculated with a female better predicted this transition to female mounting partners than did increased pubertal testosterone (T). It is proposed that increased pubertal T stimulates male sexual motivation, increasing the male's probability of sexual experience with females, ultimately producing a sexual preference for females. Eliminating T in adulthood reduces male sexual motivation in both humans and rhesus monkeys, but does not eliminate the capacity to engage in sex. In male rhesus monkeys the effects of reduced androgens on sexual behavior vary with social status and sexual experience. Human sexual behavior also varies with hormonal state, social context, and cultural conventions. Ovarian hormones influence female sexual desire, but the specific sexual behaviors engaged in are affected by perceived pregnancy risk, suggesting that cognition plays an important role in human sexual behavior. How the physical capacity to mate became emancipated from hormonal regulation in primates is not understood. This emancipation, however, increases the importance of motivational systems and results in primate sexual behavior being strongly influenced by social context.     

Timing of prenatal androgen exposure: anatomical and endocrine effects on juvenile male and female rhesus monkeys

Prenatal androgen shapes genital differentiation. In humans, genital anatomy determines sex of rearing and subsequent behavioral development. Rhesus monkey genital anatomy and neuroendocrine function are sexually differentiated, and behavioral development occurs in a complex social environment. We investigated prenatal hormonal influences on sexual differentiation by suppressing or increasing androgens in male and female rhesus monkeys. Pregnant multiparous female rhesus monkeys received 35-40 days of testosterone enanthate (TE) treatment, androgen antagonist (flutamide, FL) treatment, or vehicle starting on gestation day (GD) 35 or 40 (early) or GD 110 or 115 (late). Exogenous androgen increased neonatal LH secretion in females when given early and altered female genital differentiation when administered either early or late. TE treatment, early or late in gestation, had no measurable effects on male genital differentiation or neuroendocrine function. Early FL treatment, however, radically altered male genital differentiation, producing in two cases males with a urethral opening separate from the glans. In females, early FL treatment produced detectable alterations in genitalia consistent with a reduced exposure to prenatal androgen, suggesting that female rhesus monkeys are naturally exposed prenatally to meaningful levels of T. Late FL treatment reduced male penis size and increased neonatal T secretion, but had no effect in females. This is the first study to block endogenous prenatal testosterone in rhesus monkeys, thereby altering sexual differentiation. These findings illustrate the complexity of prenatal influences on anatomical and neuroendocrine development. The relationship between the anatomical changes reported here and sex differences in behavior is currently under investigation.     

A ten-month study of endogenous testosterone levels and behaviour in outdoor-living female rhesus monkeys (Macaca mulatta)

Endogenous testosterone levels were measured in association with sexual, aggressive, and social/affiliative behaviors in 11 outdoor-housed female rhesus monkeys over a ten-month period. Several behaviors (sex directed toward the male, sex received from the male, aggression directed toward the male, submission directed toward the male, submission directed toward the female, and groom another female) were significantly (p<0.05) positively correlated with testosterone in from one to five females. No trends were strong enough across all females to suggest that any of these correlations have species-wide significance. Factor analysis revealed clearcut clusters of behaviors, but elevations in testosterone were not strongly associated with any of these clusters. It is concluded that endogenous testosterone levels have little measurable effect on overt behavior in female rhesus monkeys.     

Effect of prenatal androgens on click-evoked otoacoustic emissions in male and female sheep (Ovis aries)

Otoacoustic emissions (OAEs) were measured in male and female Suffolk sheep (Ovis aries). Some sheep had been administered androgens or estrogens during prenatal development, some were gonadectomized after birth, and some were allowed to develop normally. As previously reported for spotted hyenas, gonadectomy did not alter the OAEs for either sex; accordingly, the untreated/intact and the untreated/gonadectomized animals were pooled to form the control groups. The click-evoked otoacoustic emissions (CEOAEs) exhibited by the female control group (N = 12) were slightly stronger (effect size = 0.42) than those in the male control group (N = 15), which is the same direction of effect reported for humans and rhesus monkeys. Females administered testosterone prenatally (N = 16) had substantially weaker (masculinized) CEOAEs than control females (effect size = 1.15). Both of these outcomes are in accord with the idea that prenatal exposure to androgens weakens the cochlear mechanisms that underlie the production of OAEs. The CEOAEs of males administered testosterone prenatally (N = 5) were not different from those of control males, an outcome also seen in similarly treated rhesus monkeys. Males administered dihydrotestosterone (DHT) prenatally (N = 3) had slightly stronger (hypo-masculinized) CEOAEs than control males. No spontaneous otoacoustic emissions (SOAEs) were found in any ears, a common finding in non-human species. To our knowledge, this is the first ruminant species measured for OAEs.     

Peripherally administered non-peptide oxytocin antagonist, L368,899, accumulates in limbic brain areas: a new pharmacological tool for the study of social motivation in non-human primates

Central administration of oxytocin (OT) antagonists inhibits maternal and sexual behavior in non-primates, providing the strongest experimental evidence that endogenous OT facilitates these behaviors. While there have been a few reports that ICV administration of OT increases social behaviors in monkeys, no studies to date have assessed the effects of OT antagonists. Therefore, we studied in rhesus monkeys whether L368,899, a non-peptide antagonist produced by Merck that selectively blocks the human uterine OT receptor, penetrates the CNS after peripheral administration and alters female maternal and sexual behavior. In two studies in four male monkeys, L368,899 was injected iv (1 mg/kg) after which (1) CSF samples were collected at intervals over 4 h and (2) brains were collected at 60 min. Assay of samples confirmed that iv-administered L368,899 entered CSF and accumulated in the hypothalamus, septum, orbitofrontal cortex, amygdala and hippocampus, but not other areas. An adult female monkey was tested for interest in either an infant or sexual behavior, receiving a different iv treatment prior to each test (1 or 3 mg/kg of L368,899 or saline). OT antagonist treatment reduced or eliminated interest in the infant and sexual behavior. These results, although preliminary, are the first to directly implicate endogenous OT in activation of primate maternal interest and sexual behavior. While it remains to be empirically demonstrated that peripherally administered L368,899 blocks central OT receptors, our behavioral findings suggest that this non-peptide antagonist may facilitate testing OT involvement in a variety of social and other behaviors in primates.     

Cognitive performance in rhesus monkeys varies by sex and prenatal androgen exposure

Men and women differ on performance and strategy on several spatial tasks. Rodents display similar sex differences, and manipulations of early hormone exposure alter the direction of these differences. However, most cognitive testing of nonhuman primates has utilized sample sizes too small to investigate sexually differentiated behaviors. This study presents an investigation of sex differences and the effects of prenatal androgen on spatial memory and strategy use in rhesus monkeys. Monkeys prenatally exposed to vehicle, testosterone, or the androgen receptor blocker flutamide performed a search task in which 5 of 12 goal boxes contained food rewards. Spatial consistency and the presence of local landmarks were varied. Performance when both spatial and marker cues were available did not differ by sex or prenatal treatment. Contrary to predictions, females easily solved the task when local markers were removed, and their performance outscored males. Although eliminating spatial consistency and requiring subjects to use local markers impaired performance by all monkeys, females continued to locate correct goal boxes at higher than chance levels and scored better than males. Blocking prenatal androgen exposure in males improved use of local markers. These findings suggest that the tendency to attend to landmarks and to use them in solving spatial problems is typical of females across many species, including rodents, humans, and rhesus monkeys. In rhesus monkeys and rodents, developmental androgen eliminates this specialization. However, these results are the only known example of better performance of females than males when salient markers are removed.     

Sexual differentiation of pheromone processing: Links to male-typical mating behavior and partner preference

Phoenix et al. (Phoenix, C., Goy, R., Gerall, A., Young, W., 1959. Organizing actions of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369–382.) were the first to propose an essential role of fetal testosterone exposure in the sexual differentiation of the capacity of mammals to display male-typical mating behavior. In one experiment control male and female guinea pigs as well as females given fetal testosterone actually showed equivalent levels of mounting behavior when gonadectomized and given ovarian steroids prior to adult tests with a stimulus female. This finding is discussed in the context of a recent, high-profile paper by Kimchi et al. (Kimchi, T., Xu, J., Dulac, C., 2007. A functional circuit underlying male sexual behaviour in the female mouse brain. Nature 448, 1009–1014.) arguing that female rodents possess the circuits that control the expression of male-typical mating behavior and that their function is normally suppressed in this sex by pheromonal inputs that are processed via the vomeronasal organ (VNO)-accessory olfactory nervous system. In another Phoenix et al. experiment, significantly more mounting behavior was observed in male guinea pigs and in females given fetal testosterone than in control females following adult gonadectomy and treatment with testosterone. Literature is reviewed that attempts to link sex differences in the anatomy and function of the accessory versus the main olfactory projections to the amygdala and hypothalamus to parallel sex differences in courtship behaviors, including sex partner preference, as well as the capacity to display mounting behavior.     

Infant-directed behavior in young rhesus monkeys: Sex differences and effects of prenatal androgens

Young (3–4 years old) laboratory-reared rhesus monkeys were observed in five 15-minute tests with 1–15-day-old infants. Males and females were equally likely to investigate infants. Females communicated more with infants by grin-lipsmacking and gurgling–-gestures that were not shown by any males. More females presented the ventrum to infants than did males. Females contacted infants more than did males by grooming, crouching over, and having full body contact with them. To see whether prenatal androgens produced the male pattern of response, we conducted similar tests with pseudohermaphrodites (prenatally androgenized genetic females) and neonatally castrated males. On most sexually dimorphic behaviors, pseudohermaphrodites behaved more like females than like males. Castrated males, like females and pseudohermaphrodites, crouched over infants more than did intact males. Castrated males differed from females only on one infant-directed response, the grin-lipsmack. These comparisons showed that defeminization of the repertoire of infant-directed responses was measurable only in intact males. We conclude accordingly that prenatal androgens alone are not responsible for defeminization of this repertoire and that a contribution from postnatal androgens is likely to be necessary.     

Cholelithiasis in a male rhesus monkey (Macaca mulatta) fed a cholesterol-containing diet.

In this report we describe the development of cholelithiasis in a male rhesus monkey fed a cholesterol-containing diet for 24 months. This represents the only case of cholelithiasis in this species of nonhuman primate that we have observed out of a population of over 500 rhesus monkeys fed similar cholesterol-containing diets. Associated with the appearance of gallstones was the production of bile saturated with cholesterol, and the excretion of bile with an abnormal bile acid composition.     

Prenatal flutamide treatment eliminates the adult male rat's dependency upon vasopressin when forming social-olfactory memories.

The sexually dimorphic number of cells expressing arginine vasopressin (AVP) in the bed nucleus of the stria terminalis and the density of AVP fibers within the lateral septum appear to be organized by pre- and postnatal androgens. Social recognition behaviors are also sexually dimorphic and AVP-dependent. Whereas AVP antagonists prevent males from recognizing familiar intruders by olfactory investigation of the anal-genital area, they have no effect in females. To test the hypothesis that the male's dependency upon AVP to form social recognition memories begins prior to birth, we compared the effectiveness of an AVP antagonist to block social recognition in control males and females with that seen in male offspring whose mothers were treated prenatally with an androgen antagonist (flutamide). In an initial study we showed that while sexual experience may enhance social recognition in males, virgin males exhibit the ability to recognize conspecifics and are sensitive to the memory blocking actions of AVP antagonists. In a second experiment, pregnant rats were treated daily for the last 10 days of gestation with either flutamide (10 mg) or control vehicle. Within 12 h of birth, male offspring from flutamide litters were injected with either testosterone proprionate (50 microg TP) or vehicle control. AVP-antagonist treatment in adults eliminated the ability of control males to recognize familiar juvenile intruders, but had no effect on males exposed prenatally to flutamide, regardless of whether these males were treated with TP or vehicle on day 1 of life. These data support the hypothesis that the development of the male's dependency upon AVP to express social recognition memories begins with the organizational actions of prenatal androgens.     

Generation of transgenic monkeys with human inherited genetic disease

Modeling human diseases using nonhuman primates including chimpanzee, rhesus, cynomolgus, marmoset and squirrel monkeys has been reported in the past decades. Due to the high similarity between nonhuman primates and humans, including genome constitution, cognitive behavioral functions, anatomical structure, metabolic, reproductive, and brain functions; nonhuman primates have played an important role in understanding physiological functions of the human body, clarifying the underlying mechanism of human diseases, and the development of novel treatments for human diseases. However, nonhuman primate research has been restricted to cognitive, behavioral, biochemical and pharmacological approaches of human diseases due to the limitation of gene transfer technology in nonhuman primates. The recent advancement in transgenic technology that has led to the generation of the first transgenic monkey in 2001 and a transgenic monkey model of Huntington’s disease (HD) in 2008 has changed that focus. The creation of transgenic HD monkeys that replicate key pathological features of human HD patients further suggests the crucial role of nonhuman primates in the future development of biomedicine. These successes have opened the door to genetic manipulation in nonhuman primates and a new era in modeling human inherited genetic disorders. We focused on the procedures in creating transgenic Huntington’s disease monkeys, but our work can be applied to transgenesis in other nonhuman primate species.     

Assessing variation in the social behavior of stumptail macaques using thermal criteria

This paper reports a method for comparing the environments of nonhuman primates based on biophysical, thermal criteria. The method is applied to an analysis of behaviors exhibited by group-living stumptail macaques (Macaca arctoides), documented by a group-scan observation technique, to test the hypothesis that the expression of social behavior is dependent on thermal conditions. Thermal conditions are identified by considering sky cover and the relative cooling power of the environment. The results show that the rates of occurrence of affiliative, play, and solitary behaviors are altered significantly at a relative cooling power at or above 550 kcal/m2/hr under cloudy conditions and at or above 600 kcal/m2/hr under sunny conditions. In addition, the rates of occurrence of play, sexual, aggressive, and submissive behavioral states are also significantly different under cloudy, rather than sunny, conditions over particular ranges of cooling. It is possible to conclude that thermal criteria affect the expression of social behaviors by stumptail macaques. This is consistent with studies of huddling behavior exhibited by stumptail macaques and rhesus macaques (M. mulatta), and suggests that 1) certain changes in the expression of social behaviors may be thermoregulatory in at least some nonhuman primate species and 2) thermal criteria are likely to be useful tools when conducting comparative analyses of behavioral data collected on animals in outdoor environments.     

A review of sexual initiating behavior by male and female cynomolgus monkeys and some species comparisons

The sexual initiating behavior of male and female cynomolgus monkeys (Macaca fascicularis) observed during standard laboratory tests is reviewed and compared with that of rhesus monkeys (M. mulatta) observed under identical conditions. Species differences in sexual behavior are related here to differences in habitat, sexual dimorphism, and the dominance gradient between the sexes. Compared with rhesus monkeys, cynomolgus monkeys appear to be more arboreal, less sexually dimorphic, and have a smaller dominance gradient between the sexes. They exhibit a facultative single-mount copulatory pattern rather than the serial mount pattern of the rhesus monkey. Female cynomolgus monkeys are less dominated than rhesus females by their male partners. Direct aggression between mates is more frequent and redirected aggression occurs less often than in rhesus monkeys. These behavioral differences affect the interpretation of changes in initiation rates that occur (1) during the menstrual cycle, (2) when females are ovariectomized and given hormone replacement treatments, and (3) when males are castrated and treated with androgens. We conclude that estradiol in the female and testosterone in the male increase the sexual motivation of both the treated and the untreated partner. Valid interpretations of changes in initiation rates depend on accurate and exclusive definitions of behavior and on a consideration of the behavioral context in which they are made.     

Serum leptin in nonpregnant and pregnant women and in old and new world nonhuman primates

Leptin is a hormone that is produced during mammalian pregnancy in the placental trophoblast and other tissues, including! fetal and maternal adipocytes. Synthesis of the polypeptide and the presence of its specific receptors throughout the human maternal fetoplacental unit suggest direct effects on conceptus growth and development. However, both the physiologic roles of leptin and the mechanisms regulating leptin synthesis in human pregnancy differ from those in laboratory and domestic species, necessitating the development of non-human primate research models. Therefore, we compared serum leptin concentrations in nonpregnant and pregnant women with those in both old world nonhuman primates (i.e., baboon, rhesus monkey, cynomolgus monkey) and new world nonhuman primates (i.e., squirrel monkey, titi monkey). As expected, maternal leptin levels were elevated in human and baboon pregnancies (P < 0.05 and P < 0.001, respectively). Levels in both species of old world monkeys were also greatly enhanced (P < 0.001). Although maternal serum concentrations were slightly elevated compared to nonpregnant levels in both species of new world monkeys, overall concentrations were dramatically lower than for either old world primates or humans. Results provide comparisons of serum leptin concentrations in pregnant and nonpregnant humans and baboons with those in both old and new world monkeys and further characterize these nonhuman primates as models for the investigation of leptin dynamics in pregnancy.     

Behavioral defeminization by prenatal androgen treatment in rats can be overcome by sexual experience in adulthood

Exposure to testosterone during a critical period of prenatal development disrupts the normal display of sexual behaviors in adult ovariectomized (OVX) rats treated with estradiol benzoate (EB) followed by progesterone (P). The organizational hypothesis posits that prenatally androgenized females (PNAFs) are desensitized to EB. We tested this hypothesis by first treating PNAFs with varying doses of EB (2.5, 5, 10, 20 μg) followed by P (500 μg), and second by subjecting females to an established EB behavioral sensitization paradigm where females are first given sexual experience with EB (10 μg) and P prior to repeated sexual behavior testing with EB alone. Long-Evans females were androgenized in utero by a s.c. injection of 500 μg testosterone propionate or the oil control to pregnant dams on gestational day 18. Female offspring were OVX on postnatal day 80 and tested one week later in the unilevel 4-hole pacing chamber. Genital tissue was defeminized in PNAFs, and the lordosis quotient (LQ) and partial (i.e., hops/darts) and full solicitations were significantly lower, while defensive behaviors were higher, in PNAF females, relative to non-PNAF females regardless of the acute EB priming dose. However, repeated testing with EB alone (10 μg), or EB and P eliminated the differences between groups on LQ and hops/darts, indicating that the behavioral deficit can be overcome by sexual experience. These results suggest that PNAFs are not desensitized to EB, and despite disruptions in sexual differentiation of anatomical structures, the deficiency in sexual behavior in response to acute EB and P can be experientially overcome. PNAFs appear, however, to have a chronic deficit in the expression of full solicitations.     

Interleukin-1 induces sleep-like behavior and alters call structure in juvenile rhesus macaques

To date, there have been no investigations of the behavioral effects of interleukin-1 (IL-1) in nonhuman primates. In this study the locomotor behavior and vocalizations of juvenile rhesus monkeys were monitored for 45 minutes following intravenous injections of recombinant human IL-1 alpha. In addition, their reaction to a broadcasted recording of infant monkey distress calls was determined 20 minutes after the beginning of each test session. IL-1 induced sleep-like inactivity and significantly diminished the monkey's behavioral and vocal responses to the broadcasted calls. The coo calls uttered by the monkeys following IL-1 treatment also had a longer duration and lower fundamental frequency than calls during the control condition. As several studies have indicated that behavioral effects of IL-1 may be mediated by corticotropin-releasing hormone (CRH), a second group of rhesus monkeys was given injections of CRH. CRH did not alter behavior or call structure at the dose administered. These results extend previous research on the behavioral effects of IL-1 to include the nonhuman primate and provide the first evidence that cytokines can affect vocal communication in rhesus monkeys. © 1995 Wiley-Liss, Inc.     

Social and hormonal influences on behavior of adult male, female, and pseudohermaphroditic rhesus monkeys

We previously demonstrated that in a simple pair test situation the expression of adult male sexual behavior by rhesus monkeys depends on both prenatal (organizational) and adult (activational) androgen exposure. In the present study we used a more complex social situation (trio tests) to evaluate the behavior of males, females, and female pseudohermaphrodites. In these trio tests, the experimental subjects were tested with two estrogenized stimulus females simultaneously. Sex differences in behavior were made apparent by this complex testing situation that could not have emerged in the pair test. Gonadectomized males and female pseudohermaphrodites, but not ovariectomized females that were concurrently receiving TP, exhibited increased male sexual behavior in trio tests compared to pair tests. In trio tests, the males and pseudohermaphrodites showed evidence of partner preference by interacting almost exclusively with one of the two stimulus females. These "preferred females" in turn were responsible for the majority of the proceptive behavior exhibited in these tests. Ovariectomized females rarely displayed male sexual behavior in either test situation. These results further support the hypothesis that prenatal androgen exposure predisposes monkeys to exhibit masculine behavior traits when they reach adulthood and are exposed to the activational influences of androgens.     

Sex differences in interest in infants in juvenile rhesus monkeys: relationship to prenatal androgen

Yearling rhesus monkey females interact more with infants than do males. However, the continuity of this sex difference throughout the juvenile period is unknown. Human females display similar sexually differentiated interest in infants, and studies of girls with congenital adrenal hyperplasia suggest that this sex difference may be modulated by prenatal androgen exposure. We investigated infant interest in 1- to 3-year-old juvenile rhesus monkeys. Hormonal influences on this behavior were investigated by treating pregnant females with an androgen-receptor blocker (flutamide), testosterone enanthate, or vehicle, early or late in gestation. Subjects were reared in their well-established natal groups, composed of species-typical matrilineal social structures, including members of all ages. Yearling control females interacted with infants more than did yearling control males. At 2 and 3 years of age, the magnitude of the sex difference in interactions with infants increased markedly, producing effect sizes of more than 2.5 standard deviations. These effects are larger than those reported in humans. Androgen treatment did not affect male or female interactions with infants, but late gestation flutamide masculinized aspects of females' interest in infants. Although early flutamide prevented complete masculinization of male genitalia, this treatment was not accompanied by any alterations in the masculine pattern of infant interest. We found no evidence that the robust juvenile sex difference in frequency of infant interactions results from socialization. However, it was largely unaffected by our hormone manipulations. Whether this reflects characteristics of our specific treatments or is evidence of nonhormonal influences on infant interest remains unanswered.     

Tests of planning and the Bischof-Köhler hypothesis in rhesus monkeys (Macaca mulatta)

The Bischof-Köhler hypothesis posits that nonhuman animals cannot plan for future motivational states that differ from a current state. [Naqshbandi, M., Roberts, W.A., 2006. Anticipation of future events in squirrel monkeys Samiri scireus and rats Rattus norvegicus: tests of the Bischof-Köhler hypothesis. J. Comp. Psychol. 120, 345–357] found that two squirrel monkeys that were not thirsty at the time of choice reversed their preference for a larger amount of food when choice of a smaller amount alleviated future thirst. This apparent anticipation of future thirst contradicts the Bischof-Köhler hypothesis. We used the methods described by Naqshbandi and Roberts with rhesus monkey subjects and found that the monkeys did not alter their behavior in anticipation of future thirst. To assess which factors enhance and inhibit the ability to express planning, we then systematically modified the experimental design in four subsequent experiments and found that monkeys that were not thirsty acted to alleviate future thirst only when the delay between their behavior and the contingent outcome was brief. Taken together these results suggest that the inability of rhesus monkeys to display planning resulted from their inability to learn behavior-outcome associations across long-delay intervals as would be expected from traditional accounts of operant learning, rather than from failure to anticipate future motivational states as posited by the Bischof-Köhler hypothesis.