Increased proliferation and neuronal fate in prairie vole brain progenitor cells cultured in vitro: effects by social exposure and sexual dimorphism.

BACKGROUND: The prairie vole (Microtus ochrogaster) is a socially monogamous rodent that establishes an enduring pair bond after cohabitation, with (6 h) or without (24 h) mating. Previously, we reported that social interaction and mating increased cell proliferation and differentiation to neuronal fate in neurogenic niches in male voles. We hypothesized that neurogenesis may be a neural plasticity mechanism involved in mating-induced pair bond formation. Here, we evaluated the differentiation potential of neural progenitor cells (NPCs) isolated from the subventricular zone (SVZ) of both female and male adult voles as a function of sociosexual experience. Animals were assigned to one of the following groups: (1) control (Co), sexually naive female and male voles that had no contact with another vole of the opposite sex; (2) social exposure (SE), males and females exposed to olfactory, auditory, and visual stimuli from a vole of the opposite sex, but without physical contact; and (3) social cohabitation with mating (SCM), male and female voles copulating to induce pair bonding formation. Subsequently, the NPCs were isolated from the SVZ, maintained, and supplemented with growth factors to form neurospheres in vitro. RESULTS: Notably, we detected in SE and SCM voles, a higher proliferation of neurosphere-derived Nestin + cells, as well as an increase in mature neurons (MAP2 +) and a decrease in glial (GFAP +) differentiated cells with some sex differences. These data suggest that when voles are exposed to sociosexual experiences that induce pair bonding, undifferentiated cells of the SVZ acquire a commitment to a neuronal lineage, and the determined potential of the neurosphere is conserved despite adaptations under in vitro conditions. Finally, we repeated the culture to obtain neurospheres under treatments with different hormones and factors (brain-derived neurotrophic factor, estradiol, prolactin, oxytocin, and progesterone); the ability of SVZ-isolated cells to generate neurospheres and differentiate in vitro into neurons or glial lineages in response to hormones or factors is also dependent on sex and sociosexual context. CONCLUSION: Social interactions that promote pair bonding in voles change the properties of cells isolated from the SVZ. Thus, SE or SCM induces a bias in the differentiation potential in both sexes, while SE is sufficient to promote proliferation in SVZ-isolated cells from male brains. In females, proliferation increases when mating is performed. The next question is whether the rise in proliferation and neurogenesis of cells from the SVZ are plastic processes essential for establishing, enhancing, maintaining, or accelerating pair bond formation. Highlights 1. Sociosexual experiences that promote pair bonding (social exposure and social cohabitation with mating) induce changes in the properties of neural stem/progenitor cells isolated from the SVZ in adult prairie voles. 2. Social interactions lead to increased proliferation and induce a bias in the differentiation potential of SVZ-isolated cells in both male and female voles. 3. The differentiation potential of SVZ-isolated cells is conserved under in vitro conditions, suggesting a commitment to a neuronal lineage under a sociosexual context. 4. Hormonal and growth factors treatments (brain-derived neurotrophic factor, estradiol, prolactin, oxytocin, and progesterone) affect the generation and differentiation of neurospheres, with dependencies on sex and sociosexual context. 5. Proliferation and neurogenesis in the SVZ may play a crucial role in establishing, enhancing, maintaining, or accelerating pair bond formation.

In this study, researchers evaluated whether social interactions and copulation induce changes in the proliferation and differentiation of neural progenitor cells in adult male and female voles using an in vitro neurosphere formation assay. The following groups were assigned: control animals without any exposure to another vole outside their litter, another group with social exposure consisting of sensory exposure to a vole of the opposite sex and a third group with social cohabitation and copulation. Forty eight hours after social interactions, cells were isolated from the neurogenic niche subventricular zone (SVZ) and cultured to assess their self-renewal and proliferation abilities to form neurospheres. The results showed in the social interaction groups, a greater number and growth of neurospheres in both males and females. Differentiation capacity was assessed by immunodetection of MAP2 and GFAP to identify neurons or glia, respectively, arise from neurospheres, with an increase in neuronal fate in groups with social interaction. In the second part of the study, the researchers analyzed the effect of different hormone and growth factor treatments and found that the response in both proliferation and differentiation potential may vary depending on the sociosexual context or sex. This study suggests that social interactions leading to pair bond formation alter the properties of SVZ cells, whereby proliferation and neurogenesis may have an impact on the establishment and maintenance of pair bonding.

Participation of progesterone receptors in facilitation and sequential inhibition of lordosis response induced by ring A-reduced progesterone metabolites in female mice.

Sexual receptivity in female rodents induced by the sequential injection of estrogen and progesterone is followed by a period in which females do not respond behaviorally to a second administration of progesterone (P); this is known as sequential inhibition. It has been proposed that the induction of sequential inhibition by progesterone in rats depends on down regulation of the progesterone receptor (PR) in brain areas involved in the expression of female sexual receptivity. P is rapidly metabolized to a variety of 5α- or 5ß-ring A-reduced progestins (RPrg). These RPrg have little or no affinity for the PR. They stimulate sexual receptivity (lordosis) more potently than P itself in estrogen-primed rats and do not induce sequential inhibition. The purpose of the current study was to test the role of the PR in the facilitation of lordosis and sequential inhibition induced by P and the following RPrg: 5α-pregnandione (5α-DHP), 5α,3ß-pregnanolone (5α,3ß-Pgl), 5ß-pregnanedione (5ß-DHP), and 5ß,3α-pregnanolone (5ß,3α-Pgl) in ovariectomized (ovx) female mice primed with estradiol benzoate. The RPrg were tested in C57BL/6 mice and in a strain lacking the progesterone receptor expression (PRKO). Our results show that both facilitation and sequential inhibition of lordosis induced by progesterone require the presence of the progesterone receptor. Interestingly, some RPrg facilitate lordosis but do not induce sequential inhibition in female mice. Sexual receptivity induced by RPrg does not require the progesterone receptor. Thus, RPrg induce sexual receptivity, but they probably exert their effects through a different cellular mechanism that does not involve the progesterone receptor. (PsycINFO Database Record

A high level of male sexual activity is necessary for the activation of the medial preoptic area and the arcuate nucleus during the 'male effect' in anestrous goats.

In small ungulates such as sheep or goats, the introduction of a male among a group of anovulatory females during the anestrus season leads to the reactivation of the gonadotrope axis and ovulation, a phenomenon known as the 'male effect'. In goats, our previous studies have demonstrated the importance of male sexual activity for an efficient reactivation of the gonadotrope axis assessed through ovulation and blood LH pulsatility. In the present experiment, we assessed whether the level of male sexual activity would also induce differential activation of two brain regions of key importance for the reactivation of GnRH activity, namely the medial preoptic area and the hypothalamic arcuate nucleus. In both structures, we observed a differential activation of Fos in females, depending on the level of buck sexual activity. Indeed, goats unexposed to males showed low levels of expression of Fos while those exposed to sexually inactive bucks showed an intermediate level of Fos expression. Finally, the highest level of Fos expression was found in females exposed to sexually active males. However, and contrary to our initial hypothesis, we were not able to find any specific activation of kisspeptin cells in the arcuate nucleus following the introduction of highly sexually active males. As a whole, these results demonstrate that the level of male sexual activity is a key factor to stimulate brain regions involved in the control of the gonadotrope axis in the context of the male effect in goats.

Steroid Receptors and Aromatase Gene Expression in Different Brain Areas of Copulating and Sexually Sluggish Male Rats.

INTRODUCTION: Sexually sluggish (SS) males have been identified in several species of mammals including rats. These animals take more than 30 minutes to ejaculate; they do not ejaculate or do so inconsistently despite being tested repeatedly with sexually receptive females. Different brain areas and hormones play an important role in the control of male sexual behavior. AIMS: Determine gene expression for the androgen receptor (AR), the estrogen receptor alpha (ERα), the progesterone receptor (PR), and the aromatase enzyme (ARO), in brain regions important in the control of male sexual behavior including the medial preoptic area (MPOA), the amygdala (AMG), the olfactory bulb (OB), and, as a control, the cortex (CTX) of copulating (C) and SS male rats. METHODS: Males that ejaculated within 30 minutes in three tests with receptive females were included in the C group, while those males that ejaculated in one or none of the four tests were included in the SS group. RNA was isolated 1 week after the last test of sexual behavior, and cDNA was synthesized from the brain areas listed above. MAIN OUTCOMES MEASURES: Expression of the AR, ERα, PR, and ARO genes was determined by quantitative polymerase chain reaction (qPCR). Cyclophilin A (CycA) and tyrosine 3-monooxygenase-tryptophan activation protein zeta (Ywhaz) were housekeeping genes used to determine relative gene expression with the 2(-ΔΔCt) method. RESULTS: The expression of mRNA for AR and ARO increased in the MPOA of SS males. ARO mRNA was increased in the AMG of SS males. In the OB, ERα mRNA was increased and AR mRNA reduced in SS males. CONCLUSION: These results indicate SS and C males show differences in gene expression within brain regions controlling sexual behavior.

Infusion of endomorphin-1 (EM-1) in the MPOA and the Me modulate sexual and socio-sexual behavior in the male rat.

Several neurotransmitters, among them neuropeptides, have been implicated in the control of male sexual behavior. Opioids are involved in mediating the positive affective states generated by the execution of sexual behavior in both males and females. We have previously shown that intracerebroventricular administration of endomorphin-1 (EM-1), the specific ligand for the µ opioid receptor, increased the ejaculation latency and interintromission interval and reduced the number of ejaculations during the test. In the present study we evaluated the effect of EM-1 upon male sexual behavior and socio-sexual interactions when infused in the medial preoptic area (MPOA) or the medial amygdala (Me). The results indicate that the administration of EM-1 in the MPOA increased mount and intromission latencies while infusion in the Me increased the number of mounts before ejaculation as well as the ejaculation latency. With respect to socio-sexual interactions, the duration of pursuit was significantly increased after administration of EM-1 in the MPOA. The effects upon sexual behavior and socio-sexual interactions were blocked by administration of the opioid antagonist naloxone. These results indicate that EM-1 modulates the appetitive aspect of sexual behavior in the MPOA and the consummatory phase in the Me.

Effects of chronic estradiol or testosterone treatment upon sexual behavior in sexually sluggish male rats.

Sexually sluggish (SS) male rats represent a small proportion of animals that take a long time to ejaculate, or sometimes they do not ejaculate, when tested on repeated occasions with sexually receptive females. The current study was done in order to evaluate whether chronic estradiol (E(2)) or testosterone (T) treatment could induce consistent ejaculatory behavior in these male rats. Those males that displayed sexual behavior but did not ejaculate in at least four of five tests were classified as SS and included in the present experiment. They were implanted subcutaneously with Silastic capsules that were empty or filled with E(2) or T. Starting one week after the implant, subjects were tested weekly with sexually receptive females for seven consecutive weeks. At the end of the experiment all subjects were weighed and sacrificed to weigh the accessory sex glands. The results showed that SS males implanted with an empty or T-filled capsule remained as such; they displayed mounts and intromissions but not a consistent pattern of ejaculation despite the twelve weeks of testing (5 screening tests and 7 with the corresponding implant). In contrast, the percentage of SS males implanted with an E(2) capsule that displayed the ejaculatory pattern was significantly reduced. Subjects treated with E(2) also displayed a higher number of mounts than the empty capsule group during several weeks of testing. At the end of the experiment the weights of the body, testicles, epididymis, seminal vesicles, and prostate of the SS male rats chronically treated with E(2) were lower than those of the empty or T capsule groups. In addition, male rats with a T implant showed lower testicular weight than the empty capsule group. These results suggest that the lack of ejaculation patter in SS male rats is not due to estrogenic or androgenic alteration.

Different doses of estradiol benzoate induce conditioned place preference after paced mating.

The ovarian hormones estrogen and progesterone are required for the complete display of sexual behavior in female rats. Paced mating produces a reward state in intact cycling and ovariectomized (OVX), hormonally primed females as evaluated by the conditioned place preference (CPP) paradigm. Most of the studies that have evaluated CPP induced by paced mating in OVX females have used relatively high doses of estradiol benzoate (EB). In the present study we determined if different doses of EB, combined with progesterone (P), could induce CPP after paced mating. For this purpose OVX female rats were divided in five groups that received one of different doses of estradiol benzoate (5, 2.5, 1.25 or 0.625 µg estradiol+0.5mg of progesterone) before being allowed to pace the sexual interaction and conditioned in a CPP paradigm. We found that the lowest dose of EB used (0.625 µg) significantly reduced the lordosis quotient and the lordosis coefficient. Even though these females paced the sexual interaction, they didn't change its original preference, suggesting that sexual interaction did not induce a positive affective, reward state. Females allowed to pace the sexual interaction with higher doses of EB developed CPP after paced mating. These results indicate that a threshold of estradiol is required for paced mating to induce CPP.

Hormones and sexual reward.

There are different physiological processes that influence behavior. One of this processes that produces approach behavior to a stimuli that induces a positive affective (PA) state, commonly known as reward, plays an important role in modulating behavior. There is an extensive literature in which the rewarding effects of drugs have been investigated. Less research has been devoted to the study of naturally occurring behaviors that produce a PA or reward state. Hormones modulate different behaviors, including sex. However, little attention has been devoted to study the possible role of hormones in reward states. One of the methods most frequently used to study reward or PA states is the conditioned place preference (CPP) paradigm. Hopefully this review will encourage researchers to directly address the effects of hormones on reward, research that is much needed.

Olfactory, partner preference and Fos expression in the vomeronasal projection pathway of sexually sluggish male rats.

Sexually sluggish (S) male rats take a long time to ejaculate or sometimes they don't achieve ejaculation when tested on repeated occasions with receptive females. In order to further understand what factors might contribute to the inconsistent display of sexual behavior in these animals and determine if S and non-copulating males have a different neurobiological profile, the present study was design to characterize sex-related behaviors of S male rats. We tested their preference to physically interact between a sexually receptive female and a sexually active male. We also tested whether S males have a preference to investigate soiled bedding from females in estrous, anestrous or clean bedding. We also evaluated if the serum hormonal levels of testosterone (T) and estradiol (E) are different between copulating (C) and S male rats. Finally, we compared the neuronal activity of the vomeronasal projection pathway of C and S male rats exposed to estrous bedding. The results indicate that S male rats increased the percentage of animals displaying mounts and intromissions after repeated testing with receptive females but no increased was observed in the percentage of animals displaying ejaculations (a total of nine tests were performed). Both C and S males had a clear preference to interact with a sexually receptive female. Both groups showed a clear preference for bedding from estrous females as opposed to anestrous or clean bedding. However, the time investigating the estrous bedding was significantly lower for the S in comparison to C rats. No differences in the serum levels of T and E were found. The structures of the vomeronasal projection pathway were equally activated by estrous bedding in C and S male rats. The results indicate that S male rats do not have hormonal alterations or deficient processing of sexually relevant olfactory cues. Although S males showed a reduced preference for estrous bedding, no changes in preference for a receptive female were observed.

Sexual incentive motivation, olfactory preference, and activation of the vomeronasal projection pathway by sexually relevant cues in non-copulating and naive male rats.

There are some apparently healthy male rats that fail to mate after repeated testing with receptive females. We have previously shown that these "non-copulator (NC)" males show no partner preference for a receptive female when given the opportunity to physically interact with a sexually receptive female or a sexually active male. We also demonstrated that although NC males prefer odors from estrous females to odors from anestrous females, this preference is significantly reduced in comparison to the preference displayed by copulating (C) males. The aim of the present study was to evaluate in NC males sexual incentive motivation, that is, the approach behavior of male rats to either a sexually receptive female or a sexually active male in a test where the subjects can smell, hear, and see the stimulus animal but prevents their physical interaction. In addition, we determined whether NC rats have alterations in their ability to detect odors from conspecifics or odors related to food. In the detection of odors from conspecifics, we determined if these NC males are sexually attracted toward odors from receptive females or sexually active males. For food-related odors, we quantified the time it took the subjects to locate a hidden a piece of apple. Finally, using the induction of Fos-immunoreactivity (Fos-IR) as an index of neuronal activation, we compared the response of the vomeronasal projection pathway (VN pathway) of C and NC male rats exposed to estrous bedding. Males without sexual experience (WSE) were included in all experiments to determine the importance of previous heterosexual experience in the different behavioral tests and in the activity of the VN pathway. In the sexual incentive motivation test, we found that C and WSE male rats have a clear preference for estrous females over sexually active males, whereas NC male rats showed no preference. In odor tests, our results showed that C males had a clear preference for odors from estrous females as opposed to odors from sexually active males. Although NC and WSE male rats showed a preference for estrous female odors, this preference was significantly reduced compared to that shown by C males. No differences were found between WSE, C, and NC males in the detection of stimuli associated with food-related odors. A significant increase in Fos-IR was observed in the mitral cell layer of the accessory olfactory bulb in all groups when exposed to estrous bedding. However, only the C male rats exposed to estrous female bedding showed an increase Fos-IR in all structures of the VN pathway. An increase in Fos-IR was observed in the medial preoptic area (MPOA) of WSE males exposed to estrous bedding. No increases in Fos-IR were detected along the VN pathway in NC male rats. We proposed that NC male rats do not display sexual behavior due to a reduced sexual motivation that could be caused by alterations in the neuronal activity of the VN pathway during the processing of estrous odors.

Has dopamine a physiological role in the control of sexual behavior? A critical review of the evidence.

The role of dopaminergic systems in the control of sexual behavior has been a subject of study for at least 40 years. Not surprisingly, reviews of the area have been published at variable intervals. However, the earlier reviews have been summaries of published research rather than a critical analysis of it. They have focused upon the conclusions presented in the original research papers rather than on evaluating the reliability and functional significance of the data reported to support these conclusions. During the last few years, important new knowledge concerning dopaminergic systems and their behavioral functions as well as the possible role of these systems in sexual behavior has been obtained. For the first time, it is now possible to integrate the data obtained in studies of sexual behavior into the wider context of general dopaminergic functions. To make this possible, we first present an analysis of the nature and organization of sexual behavior followed by a summary of current knowledge about the brain structures of crucial importance for this behavior. We then proceed with a description of the dopaminergic systems within or projecting to these structures. Whenever possible, we also try to include data on the electrophysiological actions of dopamine. Thereafter, we proceed with analyses of pharmacological data and release studies, both in males and in females. Consistently throughout this discussion, we make an effort to distinguish pharmacological effects on sexual behavior from a possible physiological role of dopamine. By pharmacological effects, we mean here drug-induced alterations in behavior that are not the result of the normal actions of synaptically released dopamine in the untreated animal. The conclusion of this endeavor is that pharmacological effects of dopaminergic drugs are variable in both males and females, independently of whether the drugs are administered systemically or intracerebrally. We conclude that the pharmacological data basically reinforce the notion that dopamine is important for motor functions and general arousal. These actions could, in fact, explain most of the effects seen on sexual behavior. Studies of dopamine release, in both males and females, have focused on the nucleus accumbens, a structure with at most a marginal importance for sexual behavior. Since accumbens dopamine release is associated with all kinds of events, aversive as well as appetitive, it can have no specific effect on sexual behavior but promotes arousal and activation of non-specific motor patterns. Preoptic and paraventricular nucleus release of dopamine may have some relationship to mechanisms of ejaculation or to the neuroendocrine consequences of sexual activity or they can be related to other autonomic processes associated with copulation. There is no compelling indication in existing experimental data that dopamine is of any particular importance for sexual motivation. There is experimental evidence showing that it is of no importance for sexual reward.

Progestins and place preference conditioning after paced mating.

When female rats pace their coital interaction, a reward state evaluated by conditioned place preference is induced. Progesterone (P) is essential for the expression of proceptive behavior and for the induction of CPP. However, the functional significance of ring A reduction of P for the induction of this state during estrous is unsettled. In the present study, we evaluated whether ring A-reduced metabolites of P are involved in the reward state induced when the females are allowed to pace their sexual contacts. Ovariectomized (ovx) female rats treated with estradiol benzoate (EB, 5 microg) and P (13 microg), Megestrol acetate (MA; 13 microg ), 5 alpha-pregnan-20 dione (5 alphaDHP; 3 microg), or 5 beta-pregnan-3 alpha-ol-20-one (5 beta,3 alpha-Pgl; 3 microg) were used. Progestins were dissolved in propylene glycol and intravenously (iv) injected through an indwelling jugular catheter before females were tested for pacing behavior. After 15 intromissions or one ejaculation, females were gently placed in the nonpreferred compartment of a CPP box. Paced mating in all groups treated with progestins induced a clear change of preference. The administration of progestins alone did not induce CPP. These results suggest that P and ring A-reduced metabolites facilitate the reward state following pacing.

Sexual and olfactory preference in noncopulating male rats.

In some species including rats, mice, gerbils, and rams, apparently normal males fail to copulate when repeatedly tested with receptive females. These animals are called "noncopulators (NC)," and the cause of this behavioral deficit is unknown. It has been shown that NC rats do not have hormonal alterations or deficits in the mechanisms that control penile function. The present study was designed to examine (Experiment 1) whether NC male rats prefer receptive females to sexually active males. In addition, the olfactory preference for bedding soiled from estrous or for anestrous bedding was investigated. These tests were performed in NC and copulating (C) male rats when the subjects were intact, gonadectomized (GDX), or GDX and treated with high doses of testosterone propionate (TP). Our results demonstrate that NC rats do not display sexual behavior even after high TP treatment. While C male rats have a clear preference for receptive females as opposed to a sexually active male, NC rats do not. In all hormonal conditions, the preference shown by NC rats for estrous bedding was significantly reduced in comparison to that seen in C rats. TP treatment in NC rats did not modify either partner or odor preference. In Experiment 2, we evaluated if NC rats are feminized and if it could be easier to induce feminine-like behavior by hormone treatment with estradiol benzoate (EB) or with EB plus progesterone (P) (EB+P). Odor preference for estrous or male bedding under these hormonal conditions was also compared. No differences between NC and C rats were found in feminine sexual behavior. In the olfactory test, we found that NC rats prefer odors from receptive females as opposed to male odors, but this preference is reduced compared to that of C rats. Males treated with EB or EB+P show no preference for female odors. These results demonstrate that treatment with EB or EB+P does not increase feminine sexual behavior in NC rats.

Facilitation of male-like coital behavior in female rats by kindling.

Kindling is a model of epilepsy and brain plasticity. When applied to the medial preoptic area (MPOA) of non-copulating male rats kindling induces masculine sexual behavior. In order to test if kindling could facilitate male-like coital behavior in female rats, sexually naive females were ovariectomized and kindled in the amygdala (AMG) or the MPOA until an intermediate stage (between 1 and 3, MPOA1-3) or until stage 5 (MPOA5 group). Once kindling was established, females were treated with 2.5 mg/Kg of testosterone propionate (TP) for 15 days. Male-like coital behavior was evaluated on days 5, 10 and 15 of treatment. Subjects were then injected with a TP dose of 5 mg/kg for 15 days and tested in the same way as with the lower dose. The number of mounts was significantly increased and the mount latency was significantly reduced in the MPOA1-3 group when tested 5 days after treatment with the low dose of TP. The same facilitation was observed in MPOA1-3 and MPOA5 groups on day 10 of treatment with the low dose of TP. When the animals were under the high dose of TP treatment, the number of intromissions was increased in all experimental groups (including the AMG kindled group) in comparison to sham animals. In a second experiment we evaluated if the facilitation of male-like coital behavior induced by kindling was produced by a modification of the response of the vomeronasal system to sexually relevant cues. Ovariectomized females were stimulated until they reached kindling stage 2, then they were treated with 2.5 mg/kg of TP for 5 days. After animals were exposed for 90 min to clean sawdust or sawdust soiled by estrous females they were perfused. Fos was detected by immunocytochemistry along the vomeronasal pathway. No differences were found in Fos responses between sham and MPOA kindled females. The facilitation of masculine sexual behavior observed in AMG kindled females may be a consequence of the propagation of the AD to other brain regions involved in the expression of masculine sexual behavior. We propose that masculine sexual behavior is facilitated in MPOA kindled female rats by local neural changes produced by this kind of stimulation without modifying the response of the vomeronasal system to sexually relevant cues.

Effect of prenatal androgen receptor antagonist or aromatase inhibitor on sexual behavior, partner preference and neuronal Fos responses to estrous female odors in the rat accessory olfactory system.

Many socially relevant odors are detected in rodent species by the vomeronasal organ and subsequently processed by the accessory olfactory system (AOS). We previously found that gonadectomized male and female rats treated in adulthood with testosterone propionate (TP) showed equivalent Fos responses in the AOS to odors derived from estrous females. Likewise, in contrast with numerous other mammalian species, gonadectomized female rats show surprisingly high levels of male-typical mounting behavior in response to adult TP. We tested the hypothesis that prenatal testosterone (T) exposure, acting via androgen receptors (ARs) or via estrogen receptors, masculinizes the AOS in rats of both sexes. Pregnant dams were treated with either the AR blocker, Flutamide, the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), or nothing (control) to assess the role of prenatal androgen and estradiol receptor activation, respectively, in this masculinization. Beginning at birth, male and female offspring were injected subcutaneously (sc) every other day with either ATD (pre- and neonatal ATD group) or oil vehicle (Flutamide and control groups) until postnatal Day 12. Subjects were gonadectomized as adults, hormonally treated and tested for different behaviors before having their AOS Fos responses to estrous female odors assessed. Prenatal treatment with Flutamide (but not ATD) significantly decreased anogenital distance and severely impaired intromissive and ejaculatory behaviors in males tested after TP replacement without disrupting mounting capacity in either sex. Pre- and neonatal treatment with ATD (but not Flutamide) enhanced lordosis responsiveness in males tested after sc injections of estradiol and progesterone, whereas these perinatal treatments had no effect on any aspect of masculine coital performance in either sex. After TP treatment, male and female control subjects preferred to approach a tethered stimulus female as opposed to a male, and prenatal Flutamide or perinatal ATD treatments did not modify this pattern of partner preference. Neuronal Fos responses to estrous odors were (as in previous studies) identical in the AOS of gonadectomized TP-treated control males and females. Prenatal Flutamide or perinatal ATD treatments failed to disrupt consistently this profile of Fos responses to estrous odors in the AOS of rats of either sex. These behavioral and neuroanatomical findings raise the possibility that the similar level of male-typical responsiveness to social odors that occurs in male and female rats after adult TP treatment results from nonsteroid-hormone-dependent, species-specific factors that act perinatally in the brains of rats of both sexes.