Restoration of male sexual behavior by adult exogenous estrogens in male aromatase knockout mice

We previously found that male aromatase knockout (ArKO) mice that carry a targeted mutation in exons 1 and 2 of the CYP19 gene and as a result cannot aromatize androgen to estrogen show impaired sexual behavior in adulthood. To determine whether this impairment was due to a lack of activation of sexual behavior by estradiol, we studied here male coital behavior as well as olfactory investigation of sexually relevant odors in male ArKO mice following adult treatment with estradiol benzoate (EB) or dihydrotestosterone propionate (DHTP). Again, we found that gonadally intact ArKO males show pronounced behavioral deficits affecting their male coital behavior as well as their olfactory investigation of volatile body odors but not that of soiled bedding. Deficits in male coital behavior were largely corrected following adult treatment with EB and the androgen DHTP, suggesting that estradiol has prominent activational effects on this behavior. By contrast, adult treatment with EB to either castrated or gonadally intact ArKO males did not stimulate olfactory investigation of volatile body odors, suggesting that this impairment may result from a lack of proper organization of this behavior during ontogeny due to the chronic lack of estrogens. In conclusion, the present studies suggest that the behavioral deficits in sexual behavior in male ArKO mice result predominantly from a lack of activation of the behavior by estrogens. This is in contrast with earlier pharmacological studies performed on rats and ferrets that have suggested strong organizational effects of estradiol on male sexual behavior.     

Female mice deficient in alpha-fetoprotein show female-typical neural responses to conspecific-derived pheromones

The neural mechanisms controlling sexual behavior are sexually differentiated by the perinatal actions of sex steroid hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estradiol, that exposure to prenatal estradiol completely defeminized the potential to show lordosis behavior in adulthood. Furthermore, AFP-KO females failed to show any male-directed mate preferences following treatment with estradiol and progesterone, indicating a reduced sexual motivation to seek out the male. In the present study, we asked whether neural responses to male- and female-derived odors are also affected in AFP-KO female mice. Therefore, we compared patterns of Fos, the protein product of the immediate early gene, c-fos, commonly used as a marker of neuronal activation, between wild-type (WT) and AFP-KO female mice following exposure to male or estrous female urine. We also tested WT males to confirm the previously observed sex differences in neural responses to male urinary odors. Interestingly, AFP-KO females showed normal, female-like Fos responses, i.e. exposure to urinary odors from male but not estrous female mice induced equivalent levels of Fos protein in the accessory olfactory pathways (e.g. the medial part of the preoptic nucleus, the bed nucleus of the stria terminalis, the amygdala, and the lateral part of the ventromedial hypothalamic nucleus) as well as in the main olfactory pathways (e.g. the piriform cortex and the anterior cortical amygdaloid nucleus), as WT females. By contrast, WT males did not show any significant induction of Fos protein in these brain areas upon exposure to either male or estrous female urinary odors. These results thus suggest that prenatal estradiol is not involved in the sexual differentiation of neural Fos responses to male-derived odors.     

Enhanced urinary odor discrimination in female aromatase knockout (ArKO) mice

We asked whether odor discrimination abilities are sexually dimorphic in mice and, if so, whether the perinatal actions of estradiol contribute to these sex differences. The ability to discriminate different types of urinary odors was compared in male and female wild-type (WT) subjects and in mice with a homozygous-null mutation of the estrogen synthetic enzyme, aromatase (aromatase knockout; ArKO). Olfactory discrimination was assessed in WT and ArKO male and female mice after they were gonadectomized in adulthood and subsequently treated with estradiol benzoate. A liquid olfactometer was used to assess food-motivated olfactory discrimination capacity. All animals eventually learned to distinguish between urinary odors collected from gonadally intact males and estrous females; however, WT males as well as ArKO mice of both sexes learned this discrimination significantly more rapidly than WT females. Similar group differences were obtained when mice discriminated between urinary odors collected from gonadally intact vs. castrated males or between two non-social odorants, amyl and butyl acetate. When subjects had to discriminate volatile urinary odors from ovariectomized female mice treated with estradiol sequenced with progesterone versus estradiol alone, ArKO females quickly acquired the task whereas WT males and females as well as ArKO males failed to do so. These results demonstrated a strong sex dimorphism in olfactory discrimination ability, with WT males performing better than females. Furthermore, female ArKO mice showed an enhanced ability to discriminate very similar urinary odorants, perhaps due to an increased sensitivity of the main olfactory nervous system to adult estradiol treatment as a result of perinatal estrogen deprivation.     

Potential contribution of prenatal estrogens to the sexual differentiation of mate preferences in mice

The neural mechanisms controlling sexual behavior are sexually differentiated by perinatal actions of gonadal hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estrogens, that exposure to prenatal estrogens completely defeminized their potential to show lordosis behavior in adulthood. Therefore, we determined here whether mate preferences were also affected in female AFP-KO mice. We observed a robust preference for an estrous female over an intact male in female AFP-KO mice, which were ovariectomized in adulthood and subsequently treated with estradiol and progesterone, whereas similarly treated WT females preferred the intact male over the estrous female. Gonadally intact WT males preferred the estrous female over the male, but only when visual cues were blocked by placing stimulus animals behind opaque partitions. Furthermore, when given the choice between an intact male and a castrated male, WT females preferred the intact male, whereas AFP-KO females showed no preference. Finally when given the choice between an estrous female and an ovariectomized female, WT males preferred the estrous female whereas AFP-KO females preferred the ovariectomized female or showed no preference depending on whether they could see the stimulus animals or not. Taken together, when AFP-KO females are tested under estrous conditions, they do not show any male-directed preferences, indicating a reduced sexual motivation to seek out the male in these females. However, they do not completely resemble males in their mate preferences suggesting that the male-typical pattern of mate preferences is not solely organized by prenatal estrogens.     

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.     

Attraction thresholds and sex discrimination of urinary odorants in male and female aromatase knockout (ArKO) mice

We previously found that both male and female aromatase knockout (ArKO) mice, which cannot synthesize estrogens due to a targeted mutation of the aromatase gene, showed less investigation of volatile body odors from anesthetized conspecifics of both sexes in Y-maze tests. We now ask whether ArKO mice are in fact capable of discriminating between and/or responding to volatile odors. Using habituation/dishabituation tests, we found that gonadectomized ArKO and wild-type (WT) mice of both sexes, which were tested without any sex hormone replacement, reliably distinguished between undiluted volatile urinary odors of either adult males or estrous females versus deionized water as well as between these two urinary odors themselves. However, ArKO mice of both sexes were less motivated than WT controls to investigate same-sex odors when they were presented last in the sequence of stimuli. In a second experiment, we compared the ability of ArKO and WT mice to respond to decreasing concentrations of either male or female urinary odors. We found a clear-cut sex difference in urinary odor attraction thresholds among WT mice: WT males failed to respond to urine dilutions higher than 1:20 by volume, whereas WT females continued to respond to urine dilutions up to 1:80. Male ArKO mice resembled WT females in their ability to respond to lower concentrations of urinary odors, raising the possibility that the observed sex difference among WT mice in urine attraction thresholds results from the perinatal actions of estrogen in the male nervous system. Female ArKO mice failed to show significant dishabituation responses to two (1:20 and 1:80) dilutions of female urine, perhaps, again, because of a reduced motivation to investigate less salient, same-sex urinary odors. Previously observed deficits in the preference of ArKO male and female mice to approach volatile body odors from conspecifics of either sex cannot be attributed to an inability of ArKO subjects to discriminate these odors according to sex but instead may reflect a deficient motivation to approach same-sex odors, especially when their concentration is low.     

Adult partner preference and sexual behavior of male rats affected by perinatal endocrine manipulations

Intact adult male rats, in which aromatization of testosterone to estradiol was prevented pre- and/or neonatally by ATD (1,4,6-androstatriene-3,17-dione), were repeatedly tested for partner preference behavior (choice: estrous female vs active male). In consecutive tests increasing preference scores for the female were found. Neonatal ATD males showed significantly lower preference scores for an estrous female than controls or prenatal ATD males. Prenatal ATD caused preference scores only slightly lower than those of controls. Ejaculation frequencies were markedly reduced or even absent in neonatal ATD males. Prenatal ATD treatment only had no or a moderately lowering effect on ejaculation frequency. Lordosis behavior of adult intact males was more facilitated following neonatal ATD treatment than following prenatal ATD treatment. In a number of tests the serotonergic drug 8-OH-DPAT was injected prior to testing for sexual partner preference and copulatory behavior. DPAT significantly increased preference for an estrous female in all groups of males when interaction was possible, but had no effect when sexual interaction was prevented by wire mesh. DPAT was able to increase the number of ejaculators in nonejaculating groups (i.e., perinatally ATD-treated males). "Premature ejaculations," i.e., ejaculations with the first intromission, were frequently observed with DPAT treatment in all groups of males. In conclusion, the availability of neonatal estrogen (derived from testosterone) organizes, at least partially, the preference for an estrous female normally shown by adult male rats. The lack of neonatal estrogen causes males to be less masculinized, both in partner preference behavior and ejaculatory behavior, and less defeminized in lordosis behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavioral characterization of non-copulating male mice

Non-copulating (NC) males are those animals that do not mate in spite of repeated testing with sexually receptive females. They have been observed in several species including rats and mice. The present experiment was designed to perform a detailed behavioral characterization of NC male mice. Thus, we evaluated their sexual incentive motivation for a sexually receptive female or a sexually active male, olfactory preference for volatile and non-volatile odors from females or males, and olfactory discrimination between female and male volatile odors and food related odors (milk versus vinegar). We compared the activity of the accessory olfactory system (AOS) in copulating (C) and NC males in response to estrous bedding using the induction of Fos-immunoreactivity (Fos-IR) as a measure of neuronal activation. We also determined if estradiol or dopamine treatment could induce sexual behavior in NC males. Finally, we compared the testis weight and the number of penile spines in C, NC, and gonadectomized males. In the sexual incentive motivation test C males spend significantly more time in the female incentive zone than in the male incentive zone. On the other hand, NC males spend the same amount of time in both incentive zones. In tests of olfactory preference, NC males spent less time investigating estrous odors than C males. As well, NC males discriminate urine from conspecifics but they spend less time smelling these odors than C males. In addition, no increase in Fos expression is observed in NC males when they are exposed to odors from estrous females. Our data also suggest that the deficits observed in NC males are not due to lower circulating levels of gonadal hormones, because estradiol supplementation does not induce sexual behavior in these animals, and their testis weight and the number of penile spines are normal. The results suggest that NC males are not sexually motivated by the receptive females and their odors.     

Brain Serotonin Signaling Does Not Determine Sexual Preference in Male Mice

It was reported recently that male mice lacking brain serotonin (5-HT) lose their preference for females (Liu et al., 2011, Nature, 472, 95–100), suggesting a role for 5-HT signaling in sexual preference. Regulation of sex preference by 5-HT lies outside of the well established roles in this behavior established for the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Presently, mice with a null mutation in the gene for tryptophan hydroxylase 2 (TPH2), which are depleted of brain 5-HT, were tested for sexual preference. When presented with inanimate (urine scents from male or estrous female) or animate (male or female mouse in estrus) sexual stimuli, TPH2-/- males show a clear preference for female over male stimuli. When a TPH2-/- male is offered the simultaneous choice between an estrous female and a male mouse, no sexual preference is expressed. However, when confounding behaviors that are seen among 3 mice in the same cage are controlled, TPH2-/- mice, like their TPH2+/+ counterparts, express a clear preference for female mice. Female TPH2-/- mice are preferred by males over TPH2+/+ females but this does not lead to increased pregnancy success. In fact, if one or both partners in a mating pair are TPH2-/- in genotype, pregnancy success rates are significantly decreased. Finally, expression of the VNO-specific cation channel TRPC2 and of CNGA2 in the MOE of TPH2-/- mice is normal, consistent with behavioral findings that sexual preference of TPH2-/- males for females is intact. In conclusion, 5-HT signaling in brain does not determine sexual preference in male mice. The use of pharmacological agents that are non-selective for the 5-HT neuronal system and that have serious adverse effects may have contributed historically to the stance that 5-HT regulates sexual behavior, including sex partner preference.     

Brief Exposure to Female Odors “Emboldens” Male Mice by Reducing Predator-Induced Behavioral and Hormonal Responses

In rodents, where chemical signals play a particularly important role in determining intersexual interactions, various studies have shown that male behavior and physiology is sensitive to female odor cues. Here we examined the effects of brief (1 min) and more prolonged (60 min) preexposure to the odors of a novel estrous female on the behavioral and hormonal responses of sexually experienced and inexperienced male mice, Mus musculus, to subsequent predator (cat and weasel) odor exposure and potential predator risk. Brief, but not prolonged, preexposure to the odors of an estrous female decreased the aversion and avoidance responses of male mice to cat odor in a Y-maze preference test, with the extent of responses being affected by a males prior sexual experience. Similarly, brief, but not prolonged, preexposure to female odors markedly attenuated the analgesic responses elicited in male mice by weasel odor. Brief exposure to a novel estrous female by itself had no significant immediate effects on either corticosterone or testosterone levels in the males. However, brief, but not prolonged, preexposure to the odors of an estrous female attenuated the marked increase in corticosterone and decrease in testosterone that were induced in males by exposure to weasel odor. The decreases in aversive responses to, and effects of, predator odor exposure that are induced by brief exposure to a novel estrous female may reflect a greater risk taking and boldness in males that could directly facilitate access to an immediately, and possibly transiently, available novel sexually receptive female.     

Olfactory preference in the male rat depends on multiple chemosensory inputs converging on the preoptic area

Both volatile and nonvolatile molecules are involved in chemosensory communication in rodents. Volatile odors from physically inaccessible estrous females induced increased numbers of c-Fos-positive cells in the preoptic area (POA) and in the cortical nucleus of the amygdala (CoA) of male rats. The numbers of c-Fos-positive cells in the medial nucleus of the amygdala (MeA) increased in response to the nonvolatile odors of bedding soiled with the excreta of estrous females. In an alternate choice paradigm, male rats carrying ibotenic acid lesions in either the MeA or the CoA—or a combination of both—distinguished the odors of estrous females from those of males, although the time spent sniffing the stimuli was diminished. Males with POA lesions showed complete loss of this capability. Males carrying either of the lesions did not detect differences between estrous and anestrous females or between intact and orchidectomized males. Lesions in the POA or MeA severely impaired male sexual behavior, whereas a CoA lesion had no effects. Thus, c-Fos-positive cells in the CoA might be involved in chemosensory transmission relevant to certain social contexts, but not in the execution of male sexual behavior. The POA is indispensable for both olfactory preferences and sexual behavior. The residual olfactory preference in males with MeA or CoA lesions or the combination of both could reflect an additional route for chemosensory transmission from the main olfactory bulb to the POA.     

Sexual motivation in the male rat: the role of primary incentives and copulatory experience.

Two experiments explored the motivational impact of primary incentive female cues on the operant behavior of sexually naive and experienced male rats. In the first experiment, a straight-arm runway was used to assess the subjects' motivation to approach a goalbox containing either male or female "targets." Twelve sexually naive Long-Evans males ran for: (1) an empty goalbox; (2) a male conspecific; (3) an ovariectomized (OVX) female; (4) an OVX female given estradiol; (5) or an OVX female treated with estradiol and progesterone. A perforated Plexiglas partition in the goalbox prevented the subject males from physically interacting with the targets, although olfactory, visual, and auditory cues were accessible. We hypothesized that subjects would manifest shorter run times (reflecting greater motivation) when the goalbox contained a receptive/proceptive female as opposed to a nonreceptive female target. Subjects' run times were ordered depending on the nature of the target (from slowest to fastest): empty goalbox, male conspecific, OVX female, OVX + estradiol female, and OVX + estradiol + progesterone female. As predicted, subjects ran significantly faster for a receptive/proceptive female than for a nonreceptive female, indicating that sexually naive males are inherently motivated by female precopulatory cues. In the second experiment, 30 sexually naive male subjects ran for a goalbox containing either a nonestrous (OVX) or an estrous (OVX + estradiol + progesterone) female. Following six trials, 10 males were allowed one intromission with a receptive female, 10 males experienced one ejaculation, and 10 remained sexually naive. Only those males having experienced an ejaculation subsequently decreased their run times for both nonestrous and estrous females, indicating that sexual reinforcement produced by ejaculation, but not intromission, further enhances the motivational impact of female incentive cues.     

Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation-dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.     

Medial Preoptic/Anterior Hypothalamic Lesions Induce a Female-Typical Profile of Sexual Partner Preference in Male Ferrets

In T-maze tests given to gonadectomized ferrets treated daily with estradiol benzoate (EB), females consistently prefer to approach and interact sexually with a stud male whereas male subjects, on average, prefer an estrous female. In the present experiment this sexually allomorphic pattern of partner preference was changed in males given lesions of the medial preoptic area/anterior hypothalamus (mPOA/AH). Electrolytic lesions, which caused extensive bilateral damage to the mPOA/AH, including the sexually dimorphic male nucleus (MN) of the POA/AH, led males to shift their mean preference away from the estrous female to the stud male. Their postoperative profile of partner preference more closely resembled that of sham-operated females than that of sham-operated males or of males which sustained either partial or minimal bilateral damage to the mPOA/AH so as to spare the MN-POA/AH in one or both hemispheres. Males with extensive bilateral mPOA/AH lesions, like sham-operated females, showed an even stronger preference to approach the stud male during T-maze tests in which the subjects could smell, see, and hear the stimulus animals without physically interacting with them. After receiving testosterone propionate, male ferrets with either extensive or partial lesions of the mPOA/AH showed significant deficits in neck gripping and mounting performance in tests with either female or male stimulus animals which were sexually receptive after gonadectomy and EB treatment. The present results, coupled with those of a previous study using excitotoxic mPOA/AH lesions, suggest that the male-typical profile of preference for an estrous female depends on the functional integrity of sexually dimorphic mPOA/AH neurons and the reward engendered by coital interaction with such a female. When these neurons either are destroyed experimentally (as in male ferrets with extensive bilateral mPOA/AH lesions) or are absent (as in sham-operated females), subjects are attracted by distal (possibly chemosensory) incentive cues from a stud male.     

Conditioned odor aversion induces social anxiety towards females in wild‐type and TrpC2 knockout male mice

Female‐emitted pheromonal inputs possess an intrinsic rewarding value for conspecific males, promoting approach and investigation of the potential mating partner. In mice these inputs are detected mainly by the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). We investigated the role of VNO‐mediated inputs in experience‐dependent plasticity of reproductive responses. We applied a sex‐specific conditioned odor aversion (COA) paradigm on adult, wild‐type (WT) male mice and on male mice impaired in VNO‐mediated signal transduction (TrpC2^?/?). We found that WT males, which underwent COA to female‐soiled bedding, lost their innate preference to female odors and presented lower motivation to approach a sexually receptive female. COA also abolished the testosterone surge normally seen following exposure to female odors. Moreover, the conditioned males displayed impairments in copulatory behaviors, which lasted for several weeks. Surprisingly, these males also exhibited phobic behaviors towards receptive females, including freezing and fleeing responses. In contrast, WT males which underwent COA specifically to male pheromones showed no change in olfactory preference and only a marginally significant elevation in intermale aggression. Finally, we show that TrpC2^?/? males were able to acquire aversion to female‐soiled bedding and presented similar behavioral alterations following COA in their responses to female cues. Our results demonstrate that the intrinsic rewarding value of female pheromones can be overridden through associative olfactory learning, which occurs independently of VNO inputs, probably through MOE signaling.     

Neurological effects of aromatase deficiency in the mouse

In the brain, the conversion from androgen into estrogen is an important process for the differentiation of the brain function in male rodents. The aromatase is expressed in some nucleus of the brain. To assess the functional significance of the aromatase gene in development and activation of sex-specific behavior, we analyzed behavioral phenotypes of the aromatase knockout (ArKO) male mice. ArKO males obviously decreased their fertility and showed deficits in male sexual behavior including mount, intromission and ejaculation. Noncontact penile erection was not significantly affected by defect of the aromatase gene. A reduction of aggressive behavior against male intruders was also observed in ArKO males, while they tend to exhibit aggression toward estrous females during male copulatory tests. Moreover, the infanticide toward the pups was observed in the ArKO males, whereas characteristic parental behavior, but not infanticide was observed in wild-type males. These results indicate that aromatase gene expression is a critical step not only for motivational and consummatory aspects of male sexual behavior, but also for aggressive and parental behaviors in male mice.     

Estradiol deficiency during development modulates the expression of circadian and daily rhythms in male and female aromatase knockout mice

Gonadal steroids modify the phase, amplitude and period of circadian rhythms. To further resolve the role of estradiol, we examined daily patterns of activity, circadian free running period and behavioral responses to light pulses using aromatase deficient (ArKO) mice. These animals lack the enzyme necessary to produce estradiol. We hypothesized that circulating estrogens during development and adulthood modulate the amount of activity, the temporal relationship of activity patterns relative to a light:dark cycle, and the free running period. Intact and gonadectomized male and female ArKO and wildtype (WT) littermates were used. WT males, but not ArKO males, retained the ability to respond to steroid hormones; the time of activity onset, free running period in constant darkness, and total daily activity were significantly different in gonadectomized compared to intact males. In contrast, gonadectomy did not alter the expression of these variables in ArKO males. ArKO females had a longer free running period in constant darkness compared to WT females regardless of gonadal state. Ovariectomized ArKO females had a significantly delayed activity onset when compared to intact ArKO females and ovariectomized WT females, despite all 3 groups being estrogen deficient. Phase shifts in response to light pulses given at different times of the day revealed an interaction between genotype, sex, and circulating steroids. These results from ArKO animals strongly suggest an organizational effect of estradiol during a critical period of development on the expression of biological rhythms.     

Estrogen treatment during development alters adult partner preference and reproductive behavior in female laboratory rats

There is broad acceptance for the idea that during development estradiol ‘organizes’ many aspects of reproductive behavior including partner preferences in the laboratory rat. With respect to partner preference, this idea is drawn from studies where estrogen action was in someway blocked, either through aromatase or estrogen receptor inhibition, during development in male rats. The lack of estrogens neonatally results in a decrease in the male rat's preference for females. In this study, the effect of early postnatal estradiol treatment on the partner preferences of female rats was examined as a further test of the hypothesis that male-typical partner preference is dependent upon early exposure to estrogens. Our principal finding was that increased postnatal estradiol exposure during development affected partner preference in the expected direction, and this effect was seen under several adult hormonal and behavioral testing conditions. Female rats that received exogenous estradiol during development spent more time with an estrous female and less time with a sexually active male than did cholesterol treated females. The estradiol treatment also disrupted normal female sexual behavior, receptivity, and proceptivity.     

Sexual experience does not compensate for the disruptive effects of zinc sulfate--lesioning of the main olfactory epithelium on sexual behavior in male mice

Recent studies point to an important role for the main olfactory epithelium (MOE) in regulating sexual behavior in male mice. We asked whether sexual experience could compensate for the disruptive effects of lesioning the MOE on sexual behavior in male mice. Male mice, which were either sexually naive or experienced, received an intranasal irrigation of either a zinc sulfate solution to destroy the MOE or saline. Sexual behavior in mating tests with an estrous female was completely abolished in zinc sulfate-treated male mice regardless of whether subjects were sexually experienced or not before the treatment. Furthermore, zinc sulfate treatment clearly disrupted olfactory investigation of both volatile and nonvolatile odors. Destruction of the MOE by zinc sulfate treatment was confirmed by a significant reduction in the expression of Fos protein in the main olfactory bulb following exposure to estrous female urine. By contrast, vomeronasal function did not seem to be affected by zinc sulfate treatment: nasal application of estrous female urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of zinc sulfate- and saline-treated males. Likewise, the expression of soybean agglutinin, which stains the axons of vomeronasal organ neurons projecting to the glomerular layer of the AOB, was similar in zinc sulfate- and saline-treated male mice. These results show that the main olfactory system is essential for the expression of sexual behavior in male mice and that sexual experience does not overcome the disruptive effects of MOE lesioning on this behavior.     

Sexual experience interacts with steroid exposure to shape the partner preferences of rats

A three-phase experiment manipulated sexual experience and hormone exposure (perinatally and in adulthood) in female rats housed individually from weaning so as to limit peripubertal social and sexual experience. Noncontact partner preference for a male or estrous female rat was measured both before and after sexual experience, first while rats were under the influence of circulating testosterone propionate (TP) and later after priming them with ovarian hormones (estradiol benzoate and progesterone; EB & P). When implanted with TP capsules and tested while sexually naive, all groups of female rats preferred females to males without differing statistically. However, following three sexual experience sessions with estrous females, differences emerged between the masculinized and control groups in the magnitude of their female-directed preference, with masculinized females demonstrating a significantly greater preference for estrous females. Sexual experience with male rats under EB & P did not result in a significant shift in preference in any group. Histological assessment indicated that the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was increased by exposure to TP postnatally, and SDN-POA volume correlated positively with partner preference scores but only when rats were both sexually experienced and exposed to circulating TP in adulthood. These results suggest that sexual experience interacts with steroid exposure to shape partner preference.