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.     

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.     

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.     

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue

Aromatase is the enzyme which catalyses the conversion of C₁₉ steroids into C₁₈ estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.     

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.     

Sexual differentiation of behavior in the zebra finch: effect of early gonadectomy or androgen treatment

Treatment of nestling zebra finches with estradiol benzoate (EB) has been shown to masculinize singing in females and demasculinize copulatory behavior in males, suggesting that sexual differentiation of these behaviors is under hormonal control such that testicular hormones induce the capacity for song and ovarian hormones suppress the capacity for mounting. Two experiments were carried out to obtain a more complete picture of sexual differentiation in this species. In Experiment 1, nestlings were injected daily for the first 2 weeks after hatching with testosterone propionate (TP), dihydrotestosterone propionate (DHTP), or a combination of DHTP and EB. As adults, birds were gonadectomized and implanted with TP prior to testing, then tested again after implantation with EB. Singing was not increased in females by any of the treatments. The only effect of either TP or DHTP given alone was defeminization of female proceptive behavior by DHTP. Thus androgens appear to have less influence than estrogens on sexual differentiation of behavior in this species. The combination of DHTP and EB demasculinized mounting in males. In Experiment 2, nestlings were gonadectomized at 7-9 days of age and implanted with TP prior to testing in adulthood. Early gonadectomy had little effect on later behavior; early castrated males sang, danced, and copulated normally and early ovariectomized females neither sang nor mounted.     

Impairment of spatial learning by estradiol treatment in female mice is attenuated by estradiol exposure during development

High doses of estradiol (E(2)) can impair spatial learning in the Morris water maze, in ovariectomized mice, but the same dose has no effect on adult castrated males. Here, we test the hypothesis that this sex difference is caused by neonatal actions of E(2). In Experiment 1, C57BL/6J pups were given daily estradiol benzoate (EB) or oil injections from the day of birth until postnatal Day 3. Adults were gonadectomized and received EB (s.c.) or oil 28 h before the first day of training, and 4 h before each of four daily training sessions on the Morris water maze. Females given oil as neonates, and EB prior to training displayed the poorest performance. Females that received EB as neonates and EB prior to training were insensitive to the deleterious effects of adult EB and performed better than males given the same hormone treatments. We conducted a second experiment using aromatase enzyme knockout (ArKO) mice. Adult male and female ArKO and wild-type (WT) littermates were gonadectomized and received either injections of oil or EB prior to and during water maze training (as described above). Hormone treatment failed to affect performance, yet, female but not male ArKO mice showed impaired learning compared to WT littermates. Thus, exposure to estradiol during neonatal development can counteract the deleterious effects of EB on adult spatial learning.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003     

Sex- and age-related response to aromatase deficiency in bone

Deficiency of sex steroids causes osteoporosis, but the relationship between estrogen and androgen is not clear because androgen is converted into estrogen by aromatase. In this study, we characterized bone metabolism in the aromatase-deficient (ArKO) mouse. At 9 weeks old, a marked loss of cancellous bone due to increased bone resorption was observed not only in female ArKO mice but also in males. The degree of bone loss in ArKO males was similar to that in females, and treatment with 17beta-estradiol completely restored the bone mass in both sexes. At 32 weeks old, female ArKO mice showed severe loss of cancellous and cortical bone. Male ArKO mice of this age also showed reduced bone mass, but the degree of bone loss in females was more marked than that in males. Here, we report sex- and age-related responses to aromatase deficiency in bone.     

Sexual partner preference requires a functional aromatase (cyp19) gene in male mice

Sexual motivation, sexual partner preference, and sexual performance represent three different aspects of sexual behavior that are critical in determining the reproductive success of a species. Although the display of sexual behavior is under strict hormonal control in both sexes, the relative roles of androgen and estrogen receptors in activating the various components of male sexual behavior are still largely unknown. A recently developed mouse model that is deficient in estradiol due to targeted disruption of exons 1 and 2 of the Cyp19 gene (aromatase knockout (ArKO) mice) was used here to analyze the role of estradiol in the control of all three aspects of male sexual behavior. When tested in a Y-maze providing volatile olfactory cues, male ArKO mice did not show a preference for the odors from an estrous female over those from an intact male, whereas wild-type (WT) and heterozygous (HET) males clearly preferred to sniff estrous odors. When provided with visual and olfactory cues, male ArKO mice also failed to show a preference for an estrous female when given a choice between an estrous female and an empty arm. However, sexual partner preferences of male ArKO mice were not sex-reversed: they did not prefer to investigate an intact male over an estrous female or empty arm. Thus, male ArKO mice seemed to have general deficits in discriminating between conspecifics by using olfactory and visual cues. Male coital behavior was also severely impaired in male ArKO mice: they displayed significantly fewer mounts, intromissions, and ejaculations than WT and HET males. Latencies to first mount or intromission were also significantly longer in ArKO males compared to WT and HET males, in addition to them showing less interest in investigating olfactory and visual cues in a Y-maze, suggesting that they were sexually less motivated. However, three out of seven male ArKO mice were capable of siring litters provided they were housed with a female for a prolonged period of time. In conclusion, aromatization of testosterone to estradiol appears to be essential for sexual motivation and sexual partner preference. By contrast, estradiol may play only a limited role in the expression of male coital behaviors.     

Female-typical sexual behavior of rhesus and defeminization by androgens given prenatally

Adult rhesus monkeys were observed in standardized tests for female-typical sexual and related social responses. In the first experiment reported, 7 castrated males and 5 spayed females were paired with each of 4 intact males on two occasions following intramuscular injection with estradiol benzoate (EB) (6 micrograms/kg X 14 days) and on two other occasions without such treatment. In tests without EB, males and females did not behave differently toward the intact male partners, and all responses were displayed at low frequencies. In tests with EB, females showed reliably higher frequencies than males of approaching, sitting close to, grooming, and soliciting, and they presented to a higher proportion of the male partner's sexual contacts. EB reliably increased the frequency of display of all of these same five responses in females but not in castrated males. The intact male partners displayed reliably fewer approaches, sexual contacts, mounts, intromissions, and ejaculations to castrated males than to spayed females regardless of estrogenization. In a second experiment 10 intact adult pseudohermaphroditic females and 6 intact control females were tested following EB injections with each of the same 4 intact males. Pseudohermaphrodites were experimentally produced by injecting pregnant females with either testosterone propionate (TP) or dihydrotestosterone propionate (DHTP). Pseudohermaphrodites, regardless of type of androgen used in their production, showed reliably fewer solicits than controls to male partners. Moreover, they displayed most of the other responses at lower average frequencies than controls. Frequencies of intromission and ejaculation by intact male partners were reliably lower with pseudohermaphrodites than with control females, but frequencies of approach, sexual contact, and mount were not reliably different. We conclude that in this testing and measurement situation male and female rhesus monkeys differ markedly in the degree of expression of female-typical sexual behaviors, and genotypic males are behaviorally less responsive to estrogens than females. Exposing genotypic females to androgens during fetal life decreases the expression of female-typical, estrogen-influenced responses, and the effect is most pronounced on those soliciting responses that subserve proceptivity.     

Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.     

Estrogen resistance and aromatase deficiency in humans

The primordial role of estrogens in female reproductive function is well known. The recent production of transgenic mice deficient in estrogen receptors (ERKO) or in aromatase (ArKO) and the discovery in man of inactivating mutations of the corresponding genes (ER) have contributed to the understanding of the role of estrogens in metabolic processes in female as well as male. To date 8 well documented cases (5 women and 3 men) of congenital deficiencies in estrogens have been reported. As mice deficient in ERa had been previously described, these cases definitely proved that estrogen absence was compatible with survival and disproved the "lethality concept" previously held because the role of estrogens in implantation and gestation maintenance. ERKO mice are phenotypically normal though sterile, but their bone density is lower (20-25%) than that of controls. Similarly, men with no aromatase or no ER display continuous growth, osteoporosis and also (but not necessarily) alterations in testicular functions. How much do primordial functions such as bone development, control of gonadotrophin secretion and lipid metabolism depend on estrogens? These interrogations, elegantly clarified following testosterone and estradiol treatment in an aromatase deficient man are considered in this present synthesis.     

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.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid β-oxidation. The impairment was reversed to the wild-type levels by treatment with 17β-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid β-oxidation and in maintaining lipid homeostasis.     

Behavioral demasculinization of female quail is induced by estrogens: studies with the new aromatase inhibitor, R76713.

The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males never show copulatory behavior even after administration of high levels of exogenous testosterone (T). It is usually assumed that such a demasculinization normally takes place in female embryos under the influence of endogenous estrogens but few experimental data are available to confirm the validity of this model. A series of four experiments was performed during which R76713, a triazole derivative that specifically inhibits aromatase (estrogen synthetase) activity, was injected into quail eggs at different stages of incubation to prevent the production of endogenous estrogens. The consequences of these embryonic treatments on the T-activated sexual behavior in adults were then quantified. When injected before Day 12 of incubation, R76713 completely blocked the behavioral demasculinization of females without affecting the behavior of the males. After a treatment with T, almost all R76713-treated females showed as adults a masculine copulatory behavior that was undistinguishable from the behavior of intact males. This effect was fully reversed by the injection in egg of EB demonstrating that the effects of R76713 were specifically due to the suppression of endogenous estrogens. Injection of R76713 during the late phase of the incubation (Day 12 or Day 15) only maintained weak copulatory behavior in females which confirmed that the behavioral demasculinization in quail takes place mainly though not exclusively during the early stages of ontogeny. In a last experiment, we combined an early R76713 treatment with an injection of EB either on Day 9 or on Day 14 of incubation. This showed that the sensitivity to differentiating effects of estrogens varies with age in a sexually differentiated manner. The EB injection on Day 9 demasculinized both male and female embryos. If this injection was delayed until Day 14, it was no longer effective in males but still caused a partial demasculinization of females. This demonstrates that even if females are not yet behaviorally demasculinized on Day 9 of incubation (suppression of aromatase activity at that age will maintain the behavior), their sensitivity to estrogens is already different from that of males.     

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.     

Sexual behavior and penile reflexes of neonatally castrated male rats treated in infancy with estrogen and dihydrotestosterone

Male rats castrated neonatally and treated with a combination of 0.5 μg estradiol benzoate (EB) plus 50μg dihydrotestosterone propionate (DHTP) for the next 14 days displayed normal sexual behavior when injected with testosterone propionate (TP) in adulthood. Neither EB nor DHTP alone had this developmental effect inasmuch as only 20–25% of the neonatal castrates treated with just 0.1, 0.5, or 10 μg EB, or 50 μg DHTP, displayed ejaculatory responses. The periodic application of mildly painful electric shock, which has been previously shown to markedly facilitate ejaculatory responding in normal male rats, failed to improve sexual performance in these latter subjects. This was true even of the castrates treated neonatally with DHTP which frequently intromitted. Castrates treated with EB or DHTP alone neonatally were subjected to spinal transection (after testing of sexual behavior) for examination of penile reflexes. Those treated with DHTP showed normal reflexes, characterized by numerous erections and flips, indicating the presumably nonaromatizable DHTP has developmental effects on penile reflexes similar to those of testosterone. Subjects treated with EB, including four animals that had ejaculated at least once, displayed very few, if any, erections on reflex tests and no flips. These results show that sometimes intromissive and ejaculatory patterns can occur even though the animal appears to have little or no capacity for penile reflexes.