Blockade of lordosis by androst-1,4,6-triene-3,17-dione (ATD) and tamoxifen in female hamsters primed with testosterone propionate

Normal female hamsters display lordosis after testosterone propionate (TP) plus progesterone (P) treatments. Such effect is probably mediated through aromatization of testosterone (T) into estradiol. If so, then an aromatase inhibitor (ATD) or an estrogen antagonist (tamoxifen, TAM) should be able to block the activational effect of T on lordosis. To test this hypothesis, 48 ovariectomized female hamsters were assigned into six groups which, according to treatments received, were ATD + TP, TAM + TP, OIL + TP, ATD + EB (estradiol benzoate), TAM + EB, and OIL + EB groups. The groups received assigned treatments for 2 days and were injected with P on the third day. Five minutes of behavior test was conducted 4 hr after P injection. The OIL + TP, OIL + EB, and ATD + EB groups all had averaged total lordosis duration (TLD) longer than 200 sec. The TLD of the TAM + EB group was only 117 sec. The ATD + TP and TAM + TP groups showed almost no lordosis. The results showed that the estrogen antagonist (TAM) impaired lordosis no matter whether the animals were primed with TP or EB, but the aromatase inhibitor (ATD) blocked lordosis only in TP primed females. It is concluded that the aromatization of T to estrogen is required for testosterone activation of lordosis in female hamsters.     

Testosterone implanted in the preoptic area of male Japanese quail must be aromatized to activate copulation

Intracranial implantation of minute pellets of gonadal steroids was combined with aromatase inhibitor treatment to determine if aromatization within the preoptic area (POA) is necessary for androgens to activate sexual behavior in the Japanese quail (Coturnix japonica). In this species, implantation of pellets of testosterone propionate (TP) or estradiol benzoate (EB) in the POA of castrated males restores male-typical copulatory behavior. In Experiment 1, adult male castrated quail were implanted intracranially with 200-micrograms pellets of equimolar mixtures of crystalline TP + cholesterol (CHOL), TP + 1,4,6-androstatriene-3,17-dione (ATD, an aromatase inhibitor), EB + ATD, or CHOL and behavior-tested with intact males and females. Copulation was stimulated by POA implants containing TP or EB (three of six CHOL + TP males and two of seven ATD + EB males copulated vs zero of four CHOL males), but copulation was not inhibited by combining ATD with TP (three of four ATD + TP males copulated). In Experiment 2, adult male castrated quail were injected systemically with ATD or oil for 6 days prior to and 14 days after intracranial implantation of 200-micrograms pellets containing the same amounts of TP or EB as in Experiment 1. The ATD injections completely blocked copulatory behavior in males with TP implants in the POA such that ATD/TP and Oil/TP mount frequencies differed significantly, but failed to block copulation in males with EB implants in the POA (proportions of males copulating were ATD/EB, 6/8; ATD/TP, 0/6; Oil/TP, 4/7). The cloacal foam gland, an androgen-sensitive secondary sex character, was unaffected by the dose of ATD used. We conclude that activation of copulatory behavior by TP implants in the POA is not due to nonspecific effects of high local testosterone concentrations but rather to aromatization. These results support the hypothesis that cells within the POA aromatize testosterone to estrogens, which directly stimulate the cellular processes leading to activation of male-typical copulatory behavior.     

Effects of ATD on male sexual behavior and androgen receptor binding: a reexamination of the aromatization hypothesis

The aromatization hypothesis asserts that testosterone (T) must be aromatized to estradiol (E2) to activate copulatory behavior in the male rat. In support of this hypothesis, the aromatization inhibitor, ATD, has been found to suppress male sexual behavior in T-treated rats. In our experiment, we first replicated this finding by peripherally injecting ATD (15 mg/day) or propylene glycol into T-treated (two 10-mm Silastic capsules) or control castrated male rats. In a second experiment, we bilaterally implanted either ATD-filled or blank cannulae into the medial preoptic area (MPOA) of either T-treated or control castrated male rats. With this more local distribution of ATD, a lesser decline in sexual behavior was found, suggesting that other brain areas are involved in the neurohormonal activation of copulatory behavior in the male rat. To determine whether in vivo ATD interacts with androgen or estrogen receptors, we conducted cell nuclear androgen and estrogen receptor binding assays of hypothalamus, preoptic area, amygdala, and septum following treatment with the combinations of systemic T alone. ATD plus T, ATD alone, and blank control. In all four brain areas binding of T to androgen receptors was significantly decreased in the presence of ATD, suggesting that ATD may act both as an androgen receptor blocker and as an aromatization inhibitor. Competitive binding studies indicated that ATD competes in vitro for cytosol androgen receptors, thus substantiating the in vivo antiandrogenic effects of ATD. Cell nuclear estrogen receptor binding was not significantly increased by exposure to T in the physiological range. No agonistic properties of ATD were observed either behaviorally or biochemically. Thus, an alternative explanation for the inhibitory effects of ATD on male sexual behavior is that ATD prevents T from binding to androgen receptors.     

Sexual differentiation in quail: Conversion of androgen to estrogen mediates testosterone-induced demasculinization of copulation but not other male characteristics

Previous research has shown that administration of either testosterone or estradiol to male quail embryos will demasculinize behavior and morphology. Six experiments in which embryos were treated were conducted to test the hypothesis that this testosterone-induced demasculinization is due to conversion of testosterone to estrogen (aromatization). In Experiment 1, dihydrotestosterone propionate, a nonaromatizable androgen, failed to demasculinize copulatory behavior, but did demasculinize crowing, strutting, and proctodeal glands. In Experiment 2, injection of the aromatizable androgens testosterone propionate (TP), testosterone, or androstenedione demasculinized copulatory behavior, the nonaromatizable androgen androsterone failed to have such an effect, and all androgens demasculinized proctodeal glands. In Experiment 3, Silastic implants of testosterone demasculinized all male characteristics, whereas implants of androsterone demasculinized only proctodeal glands. In Experiment 4, the antiestrogen tamoxifen prevented TP from demasculinizing copulatory behavior, but had no such effect with respect to crowing and strutting. In Experiments 5 and 6, the aromatization inhibitor 1,4,6-androstatrien-3,17-dione (ATD) prevented TP but not estradiol benzoate from demasculinizing copulatory behavior. Thus (1) in quail, testosterone-induced demasculinization of copulatory behavior is due to androgen aromatization, whereas testosterone-induced demasculinization of crowing, strutting, and proctodeal glands is not; (2) the distinct components of normal male reproductive behavior exhibit different patterns of steroid specificity during the organizational period, as was previously shown for the activational period; (3) the steroid specificity of crowing, strutting, and proctodeal glands changes between the organizational and activational periods. During organization, there is little specificity, whereas during activation, these characteristics respond only to androgens, never to estrogens. This difference suggests that developmental changes have occurred in the underlying biochemical substrates.     

Mechanisms involved in the ability of human chorionic gonadotropin to increase the responsiveness of the infant rat's testis to follicle-stimulating hormone

Pretreatment of 9-day-old rats for 3 days with human chorionic gonadotropin (hCG) increases the amount of estradiol secreted by the testis in response to in vivo or in vitro stimulation with follicle-stimulating hormone (FSH). Potential mechanisms for this sensitizing effect were studied by treating infant rats with a variety of agents and then using radioimmunoassay to determine testicular estradiol secretion. Substitution of 3 days priming with estradiol for hCG did not enhance subsequent in vitro responsiveness to FSH. Subcutaneous capsules of 1,4,6-androstatriene-3,17-dione (ATD) blocked stimulation of testicular aromatization in vivo by hCG or FSH. ATD capsules alone, or when combined with the antiestrogen tamoxifen, were not able to alter the ability of hCG pretreatment to increase responsiveness to in vitro FSH. It was concluded that estradiol was not involved in the sensitization caused by hCG in this model system. When gonadal tissue from 12-day-old rats was incubated in the presence or absence of 0.6 microM testosterone and various concentrations of FSH, more estradiol was secreted by testes in the containing testosterone. The amount secreted was not different from that noted after hCG priming. Priming of 9-day-old rats for 3 days with the nonaromatizable androgen 5 alpha-dihydrotestosterone did not influence the amount of estradiol secreted in response to FSH. It is further concluded that hCG augments the testicular aromatization response of infant rats to FSH by providing additional substrate for these reactions.     

Sex differences in the inhibition by ATD of testosterone-activated mounting behavior in guinea pigs

Adult male and female guinea pigs from a genetically heterogeneous stock were gonadectomized and tested for mounting behavior before and during various treatments with testosterone cypionate (TC) alone or in combination with an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). ATD was implanted subdermally in Silastic capsules (either 1 or 2 in females; 2 or 3 in males). In females 2 capsules of ATD completely blocked the behavioral effects of TC, and 1 capsule was an effective blocker in 58% of the females. The blocking effect was reversed by injection of diethylstilbestrol. In males, there was no measurable effect of ATD on mounting activity even when 3 capsules were implanted. Moreover, the TC induction of higher components of male sexual behavior (intromission and ejaculation) was also not impaired by ATD. Results are interpreted as indicating that either the process of male sexual differentiation or the male genotype eliminates the requirement for aromatization in androgenic activation of sexual behavior.     

Behavioral action of estrogen in male hamsters: effect of the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD)

This study examines three independent behavioral variables known to be activated by testosterone in the male hamster; namely, the tendency to approach the female, the tendency to leave the female, and the amount of sniffing directed to her. Both intact and testosterone-maintained castrated male hamsters were given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in subcutaneous, Silastic capsules. In intact males, there was an ATD dose-dependent increase in the tendency to leave the female and a decrease in the amount of olfactory investigation. The tendency of the male to approach the female was unaffected. The higher doses of ATD also abolished the ability of males to discriminate between females using vaginal odor cues. These results were confirmed in castrated males whose behavior was maintained at the intact level by testosterone implants. In addition, in both intact and castrated males, estradiol or diethylstilbestrol was able to reverse the behavioral changes induced by ATD. Our results indicate that estrogen produced by aromatization of testosterone activates behavior. We conclude that estrogen, by influencing some, but not all, components of masculine behavior, has a specific role in modulating male-female interactions.     

Aromatization: Important for sexual differentiation of the neonatal rat brain

This paper examines the hypothesis that testosterone (T) produces its differ-entiative effect on the neonatal rat brain after undergoing conversion in situ to estradiol-17β (E₂). We examined the abilities of an aromatizing enzyme inhibitor, (1,4,6-androstatriene-3,17-dione) (ATD), and an anti-estrogen, CI628, to inhibit sexual differentiation. Male and female rats were treated during the first few days of postnatal life with ATD or CI628, and females were treated on the following day with T in Silastic capsules or its propionate (TP) in oil. ATD and ATD+T females were normal with respect to time of vaginal opening, ovarian weight, ability to demonstrate an LH surge, and lordosis behavior. T and TP females were masculinized with respect to all these measures. CI628 and CI628+TP females had impaired ovarian function and intermediate lordosis quotients (LQs) compared to controls, though they were higher in both measures than T females. ATD males demonstrated high LQs in response to estradiol benzoate (EB) + progesterone, comparable to those of control females. CI628 males had intermediate LQs, which were significantly higher than those of control males. These results indicate that ATD can substantially protect the neonatal rat brain from the differentiative effects of exogenous or endogenous T, probably by blocking aromatization. CI628 affords only partial protection against T and produces a weak differentiative effect due to its own weak estrogenicity. Thus, aromatization of T in newborn rat brains appears to be essential if sexual differentiation is to occur.     

Role of aromatization in sexual differentiation: Effects of prenatal ATD treatment and neonatal castration

Pregnant female rats were administered either the aromatization inhibitor ATD (1,4,6-androstatriene-3,17-dione) or propylene glycol from Days 10 to 21 of gestation. On the day of birth one-half of the offspring from each group were gonadectomized. The remaining offspring were gonadectomized 35 days after birth. When adult the animals were given eight weekly mating tests following treatment with 2 or 8 μg of estradiol benzoate (EB) and 25 or 200 μg of progesterone (P). The probability of lordotic behavior as well as the frequency of ear-wiggle and hop and dart responses was measured. Prenatal ATD treatment resulted in a slight increase in lordotic behavior in the males. Lordotic potential was greatly facilitated by castration at birth. ATD treatment also increased the frequency of proceptive behaviors in males and combined ATD treatment and neonatal castration produced a dramatic increase in these behaviors. Prenatal ATD treatment and neonatal ovariectomy had only modest effects on the display of receptive and proceptive behaviors in females. Two weeks after the last test for female mating behaviors, the animals received daily injections of 200 μg of testosterone propionate. Four weekly tests for male-typical responses were given starting 1 week after the first injection. Prenatal ATD treatment did not markedly affect masculine behavior in the males. Castration at birth eliminated the ejaculatory response and reduced the frequency of mounting and intromission behavior. Prenatal ATD treatment and ovariectomy at birth had no appreciable effects on the display of male-typical behaviors in the females. Testosterone-stimulated masculine behavior of the female was similar to that of the male castrated at birth.     

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.     

Masculinization and defeminization induced in female hamsters by neonatal treatment with estradiol benzoate and RU-2858

To test the hypothesis that masculinization may be androgen dependent and defeminization, estrogen dependent, newborn female hamsters were administered 50–2000 ng of estradiol benzoate (EB), 0.05–50 ng of the synthetic estrogen RU-2858, or 1000 ng of testosterone propionate (TP). All three agents facilitated the display of male-type mounting responses in adulthood. A dose of 500 ng of EB was most potent in this regard, although significant masculinization was induced by 50 ng of EB and 0.05 ng of RU-2858 as well as by TP. TP did not inhibit adult lordotic behavior. Lordosis durations were reduced in a dose-dependent manner by EB, whereas the dose-response curve for RU-2858-induced defeminization was found to be nonlinear. The observation that picogram to nanogram amounts of estrogen can masculinize is consistent with the aromatization hypothesis of sexual differentiation.     

A prenatal source for defeminization of female rats is the maternal ovary

Sexual behavior in laboratory rats is influenced by a variety of factors in the perinatal environment. Male rats are masculinized and defeminized in response to circulating testosterone perinatally. Females undergo a process of feminization but in some cases are exposed to testosterone. Previous work has shown that during prenatal development female rats normally undergo a partial masculinization and defeminization of sexual behavior as reflected by altered responsiveness to gonadal hormones in adulthood. In the present study we investigated whether the maternal ovary influences adult females' responsiveness to gonadal hormones. Pregnant rats were ovariectomized on Day 10 of pregnancy and their offspring tested for sexual behavior in adulthood. Following ovariectomy pregnancies were maintained by administration of systemic progesterone. In addition the ovariectomized pregnant rats were given one of three daily treatments (Days 10-21): 0.2 microgram estradiol benzoate in sesame oil and 0.1 cc propylene glycol, 5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in 0.1 cc propylene glycol, or 0.1 cc propylene glycol. A control group was generated from SHAM operated mothers given daily control injections of propylene glycol and sesame oil. Offspring were ovariectomized in adulthood and tested for display of feminine sexual behavior in response to estradiol benzoate and progesterone or estradiol benzoate alone. Masculine sexual behavior was measured in response to testosterone propionate (TP). Feminine sexual behavior was enhanced in offspring from ovariectomized mothers given only progesterone replacement during pregnancy. Offspring from mothers treated with ATD displayed the greatest elevations in feminine sexual behavior. Estradiol treatments of ovariectomized mothers prevented the increase in feminine potential seen in offspring in the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of testosterone metabolites in the neonatal period on open-field behavior and lordosis in the rat

Two experiments were done to compare the effects of neonatal exposure to testosterone and its major metabolites, dihydrotestosterone (DHT) and estradiol (E₂), on the development of sex differences in open-field behavior in the rat. In Experiment 1 female rats administered either testosterone propionate (TP), DHT, or estradiol benzoate (EB) were found as adults to have low activity scores, more typical of adult males, when compared to the high scores of oil-treated females. In Experiment 2 the adult open-field behavior of female rats treated neonatally with testosterone or the metabolites was compared to that of male rats treated from Day 1 to 10 of life with the aromatizing enzyme inhibitor, androst-1,4,6-triene-3,17-dione (ATD). These same animals were later tested for lordotic behavior after gonadectomy and priming with EB and progesterone. All male animals and female animals exposed neonatally to testosterone or to either of the metabolites had suppressed open-field activity scores compared to oil-treated females. However, the lordotic behavior of females exposed to DHT and of males exposed to ATD was not defeminized and was comparable to that of oil-treated females. These observations were discussed in terms of a role for the androgenic actions of testosterone in establishing sex differences in nonreproductive behavior in the rat.     

Neonatal steroid exposure increases in vitro aromatization ability of peripubertal hamster ovaries

Neonatal female hamsters were exposed to doses of testosterone propionate or estradiol benzoate that would lead to behavioral masculinization and defeminization at adulthood. At Days 20, 30, or 40 of life, ovaries were removed and incubated in Kreb's Ringer bicarbonate for 4 h with or without the addition of 1 X 10(-9) M testosterone as a substrate. Incubation medium was assayed by radioimmunoassay (RIA) for the accumulation of estradiol. Ovaries from steroid-exposed animals aromatized testosterone to estradiol at a greatly increased rate compared to ovaries from oil-injected control animals. Serum from treated animals contained androgen, which could act as substrate for aromatization in vivo. Neonatally estrogenized females exhibited elevated circulating estradiol levels. Increased aromatizing ability could be due to early antral follicle formation as a result of elevated luteinizing hormone (LH) and/or follicle-stimulating hormone (FSH). Implications of increased aromatization ability of ovaries in the process of behavioral sexual differentiation are discussed.     

Conversion of testosterone to estradiol may not be necessary for the expression of mating behavior in male Syrian hamsters (Mesocricetus auratus)

Male sexual behavior is mediated in part by androgens, but in several species, mating is also influenced by estradiol formed locally in the brain by the aromatization of testosterone. The role of testosterone aromatization in the copulatory behavior of male Syrian hamsters is unclear because prior studies are equivocal. Therefore, the present study tested whether blocking the conversion of testosterone to estradiol would inhibit male hamster sexual behavior. Chronic systemic administration of the nonsteroidal aromatase inhibitor Fadrozole (2.0 mg/kg/day) for 5 or 8 weeks did not significantly increase mount latency or reduce mount frequency, intromission frequency, ejaculation frequency, or anogenital investigation relative to levels shown by surgical controls. However, Fadrozole effectively inhibited aromatase activity, as evidenced by the suppression of estrogen-dependent progesterone receptor immunoreactivity in the male hamster brain. The JZB39 anti-progesterone receptor antibody labeled significantly more neurons in brains of sham-treated hamsters than in brains of Fadrozole-treated hamsters. These data suggest that aromatization of testosterone to estradiol is not necessary for normal mating behavior in Syrian hamsters.     

Neonatal hormonal influences on the development of proceptive and receptive feminine sexual behavior in rats

The objective of this study was to examine the influence of androgen and of the inhibiting of aromatization of androgen to estrogen during the early neonatal period on the development of receptive (lordosis and acceptance of stimulus male mounting attempts) and proceptive (affiliation with and solicitation of stimulus males) feminine sexual behavior. Within 8 hr of birth, male rats were castrated or received subcutaneous implants of the aromatase inhibitor androst-1,4,6-triene-3, 17-dione (ATD) while females received injections of testosterone propionate (TP). At 90 days of age all treated animals and controls were tested for receptive and proceptive feminine sexual behavior. It was found that androgen present neonatally blocked proceptive as well as receptive behavior patterns in adult rats. The proceptive and receptive feminine sexual behavior patterns displayed by adult males deprived of the effects of androgen neonatally either by castration or by treatment with ATD were comparable to those of normal females.     

Cellular actions of testosterone in vascular cells: Mechanism independent of aromatization to estradiol

In this work we investigated the role of testosterone on cellular processes involved in vascular disease, and whether these effects depend on its local conversion to estradiol. Cultures of rat aortic endothelial and smooth muscle cells in vitro treated with physiological concentrations of testosterone were employed. Testosterone rapidly increased endothelial nitric oxide production. To evaluate whether this non genomic action was dependent on testosterone aromatization we used an aromatase inhibitor. Anastrozole compound did not modify the fast increase in nitric oxide production elicited by testosterone. The hormonal effect was completely blocked by an androgen receptor antagonist (flutamide); meanwhile it wasn′t modified by the presence of an estrogen receptor antagonist (ICI182780).The possibility of intracellular estradiol synthesis was ruled out when no differences were found in estradiol measurements performed in culture incubation medium from control and testosterone treated cells. The 5α-reductase inhibitor finasteride partially suppressed the enhancement in nitric oxide production, suggesting that the effect of testosterone was partially due to dihydrotestosterone conversion. Testosterone stimulated muscle cell proliferation independent of local conversion to estradiol. When cellular events that play key roles in vascular disease development were analyzed, testosterone prevented monocyte adhesion to endothelial cells induced by a proinflammatory stimulus (bacterial lipopolysaccharides), and prompted muscle cell migration in presence of a cell motility inducer. In summary, testosterone modulates vascular behavior through its direct action on vascular cells independent of aromatization to estradiol. The cellular actions exhibited by the steroid varied whether cells were under basal or inflammatory conditions.     

The effect of the antiestrogen CI-628 on androgen-induced aggressive behavior in castrated male mice

This study was conducted to determine whether or not the antiestrogen CI-628 would block testosterone-maintained fighting in castrated male mice. TO strain adult male mice were castrated and injected s.c. every day for 14 days with either (1) 75 μg testosterone or (2) 75 μg testosterone and 1 mg CI-628, and in addition 1 mg of CI-628 6 hr prior to each injection of antiestrogen and androgen. Vehicle-injected, castrated, and CI-628-injected animals were employed as controls. Testosterone-maintained intermale aggressive behavior was blocked by the antiestrogen CI-628. This study provides support for the hypothesis that testosterone exerts its effects on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites.     

Exposure of embryos to an aromatization inhibitor increases copulatory behaviour of male quail

This experiment examined the possibility that endogenous embryonic androgen contributes to sexual differentiation of behaviour in male or female quail ($\text{Coturnix}$$\text{coturnix}$$\text{japonica}$), and that it does so via aromatization (conversion to oestrogen). Eggs were injected on day 9 of incubation with oil or ATD (an aromatization inhibitor). As adults, males and females were exposed to short days, injected with testosterone propionate, tested for male-typical behaviour, then injected with oestradiol benzoate and tested for female-typical receptivity. ATD increased the level of male-typical copulatory behaviour in males. Male-typical behaviour in females was not affected, nor was female-typical behaviour in either sex. Thus normal male quail are actually slightly demasculinized by their own androgen during embryonic development, and this process is mediated by aromatization.     

Sex steroids modulate changes in social and sexual preference during juvenile development in zebra finches

Zebra finches, like many other animals, have close social relationships mainly with the family at young ages but begin to express interest in opposite-sex extra-family animals as they enter the late juvenile period and sexual maturity. This experiment tested a set of hypotheses that sex steroids are involved in this developmental transition. At 25–30 days, subjects were implanted subcutaneously with Silastic tubes that were empty (controls), filled with testosterone propionate, filled with estradiol benzoate, or filled with a combination of ATD (an aromatization inhibitor) and flutamide (an anti-androgen). Once a week between ages 30 and 90 days, they were given three-choice tests where the three stimulus types were the family members, unpaired males, or unpaired females. The preferred category was defined as the one adjacent to the proximity zone in which the subject spent the most time. Control males were more likely to prefer females and less likely to prefer the family as they got older, and control females were increasingly likely to prefer males. Males treated with testosterone or estradiol showed a premature increase in preferences for females. Females treated with ATD plus flutamide failed to show the normal increase in preferences for males shown by controls. These results indicate an involvement of sex steroids in the maturation of sexual preferences in a socially monogamous species that relies on visual and auditory, rather than olfactory, cues for sexual or other social behavior.