The alpha-fetoprotein knock-out mouse model suggests that parental behavior is sexually differentiated under the influence of prenatal estradiol.

In rodent species, sexual differentiation of the brain for many reproductive processes depends largely on estradiol. This was recently confirmed again by using the alpha-fetoprotein knockout (AFP-KO) mouse model, which lacks the protective actions of alpha-fetoprotein against maternal estradiol and as a result represents a good model to determine the contribution of prenatal estradiol to the sexual differentiation of the brain and behavior. Female AFP-KO mice were defeminized and masculinized with regard to their neuroendocrine responses as well as sexual behavior. Since parental behavior is also strongly sexually differentiated in mice, we used the AFP-KO mouse model here to ask whether parental responses are differentiated prenatally under the influence of estradiol. It was found that AFP-KO females showed longer latencies to retrieve pups to the nest and also exhibited lower levels of crouching over the pups in the nest in comparison to WT females. In fact, they resembled males (WT and AFP-KO). Other measures of maternal behavior, for example the incidence of infanticide, tended to be higher in AFP-KO females than in WT females but this increase failed to reach statistical significance. The deficits observed in parental behavior of AFP-KO females could not be explained by any changes in olfactory function, novelty recognition or anxiety. Thus our results suggest that prenatal estradiol defeminizes the parental brain in mice.

Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens.

Two clearly opposing views exist on the function of alpha-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, in the sexual differentiation of the rodent brain. AFP has been proposed to either prevent the entry of estrogens or to actively transport estrogens into the developing female brain. The availability of Afp mutant mice (Afp(-/-)) now finally allows us to resolve this longstanding controversy concerning the role of AFP in brain sexual differentiation, and thus to determine whether prenatal estrogens contribute to the development of the female brain. Here we show that the brain and behavior of female Afp(-/-) mice were masculinized and defeminized. However, when estrogen production was blocked by embryonic treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione, the feminine phenotype of these mice was rescued. These results clearly demonstrate that prenatal estrogens masculinize and defeminize the brain and that AFP protects the female brain from these effects of estrogens.

Absence of gonadotropin-releasing hormone 1 and Kiss1 activation in alpha-fetoprotein knockout mice: prenatal estrogens defeminize the potential to show preovulatory luteinizing hormone surges.

Sex differences in gonadal function are driven by either cyclical (females) or tonic (males) hypothalamic GnRH1 release and, subsequently, gonadotrophin (LH and FSH) secretion from the pituitary. This sex difference seems to depend on the perinatal actions of gonadal hormones on the hypothalamus. We used alpha-fetoprotein (AFP) knockout mice (Afp(-/-)) to study the mechanisms by which estrogens affect the sexual differentiation of the GnRH1 system. Afp(-/-) mice lack the protective actions of AFP against estrogens circulating during embryonic development, leading to infertility probably due to a hypothalamic dysfunction. Therefore, we first determined whether Afp(-/-) females are capable of showing a steroid-induced preovulatory LH surge by FOS/GnRH1 immunohistochemistry and RIA of plasma LH levels. Because the KISS1/GPR54 system is a key upstream regulator of the GnRH1 system as well as being sexually dimorphic, we also analyzed whether Kisspeptin-10 neurons were activated in Afp(-/-) mice after treatment with estradiol and progesterone. We found that the GnRH1 and Kisspeptin-10 neuronal systems are defeminized in Afp(-/-) females because they did not show either steroid-induced LH surges or significant FOS/GnRH1 double labeling. Furthermore, Kisspeptin-10 immunoreactivity and neural activation, measured by the number of double-labeled FOS/Kisspeptin-10 cells, were lower in Afp(-/-) females, suggesting a down-regulation of GnRH1 function. Thus, the sex difference in the ability to show preovulatory LH surges depends on the prenatal actions of estrogens in the male hypothalamus and, thus, is lost in Afp(-/-) females because they lack AFP to protect them against the defeminizing effects of estrogens during prenatal development.

Evidence for a role of early oestrogens in the central processing of sexually relevant olfactory cues in female mice.

We previously found that female aromatase knockout (ArKO) mice showed less investigation of socially relevant odours as well as reduced sexual behaviour. We now ask whether these behavioural deficits might be due to an inadequate processing of odours in female ArKO mice. Therefore, we exposed female ArKO mice to same- and opposite-sex urinary odours and determined the expression of the immediate early gene c-Fos along the main and accessory olfactory projection pathways. We included ArKO males in the present study as we previously observed that they show female-typical detection thresholds of urinary odours, suggesting a role for perinatal oestrogens in these behavioural responses. No sex or genotype differences were observed in the olfactory bulb after urine exposure. By contrast, sex differences in c-Fos responses were observed in wild-type (WT) mice following exposure to male urine in the more central regions of the olfactory pathway; only WT females showed a significant Fos induction in the amygdala, central medial pre-optic area and ventromedial hypothalamus. However, ArKO females did not show a c-Fos response to male odours in the ventromedial hypothalamus, suggesting that the processing of male odours is affected in ArKO females and thus that oestrogens may be necessary for the development of neural responses to sexually relevant odours in female mice. By contrast, c-Fos responses to either male or oestrous female urine were very similar between ArKO and WT males, pointing to a central role of androgen vs. oestrogen signalling in the male circuits that control olfactory investigation and preferences.

Male aromatase knockout mice acquire a conditioned place preference for cocaine but not for contact with an estrous female.

We have previously shown that male mice carrying a targeted mutation in the Cyp19 gene which encodes the aromatase enzyme (aromatase knockout or ArKO), showed a reduced interest to investigate volatile odors from conspecifics in a Y-maze. We asked here whether the incentive value of reproductively relevant odors is reduced in ArKO males by comparing the ability of male wild-type (WT) and ArKO mice to learn a conditioned place preference using exposure to reproductively relevant odors as incentive stimuli. When the presence of an anesthetized estrous female or soiled bedding from estrous females was used as incentive stimuli, only WT and not male ArKO mice showed conditioned place preference suggesting that the reward value of these stimuli is reduced in ArKO males. However, ArKO males showed conditioned place preference when cocaine was used as incentive stimulus, indicating that ArKO males are able to learn the conditioned place preference procedure. These results thus further confirm the important role of estradiol in sexually related behavioral responses in male mice.

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.

Reduced prepubertal expression of progesterone receptor in the hypothalamus of female aromatase knockout mice.

Previous research using alpha-fetoprotein knockout and aromatase knockout (ArKO) female mice suggested that the developing hypothalamic mechanisms that later control feminine sexual behavior are protected prenatally from estradiol, whereas shortly after birth, they may be stimulated by this same sex hormone. In the present study, we found that the amount of progesterone receptor immunoreactivity (PR-ir) in the anteroventral periventricular nucleus and medial part of the medial preoptic nucleus was significantly lower in ArKO female mice than in wild-type (WT) females at several prepubertal ages including postnatal d 15 (P15), P15, P20, and P25 but not neonatally at P0, P5, or P10. Likewise, PR-ir in the lateral subdivision of the ventromedial hypothalamic nucleus was significantly lower at P25 in ArKO vs. WT female mice but not at earlier postnatal ages. PR-ir was consistently higher in male than in female WT mice in the anteroventral periventricular nucleus and medial preoptic nucleus over P0-P10 and in the ventromedial hypothalamic nucleus over P0-P20. In these brain regions across these latter ages, PR-ir in male ArKO mice was significantly lower than in WT males and resembled the values seen in WT females, confirming previous reports that estradiol formed in the developing male hypothalamus from testicular testosterone is responsible for male-typical levels of neural PR expression. Thus, estradiol induces both female- and male-typical expression of PR postnatally in the mouse hypothalamus. Future experiments will determine whether this estradiol-induced PR expression contributes to either female- or male-typical brain and behavioral differentiation.

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.

Destruction of the main olfactory epithelium reduces female sexual behavior and olfactory investigation in female mice.

We studied the contribution of the main olfactory system to mate recognition and sexual behavior in female mice. Female mice received an intranasal irrigation of either a zinc sulfate (ZnSO4) solution to destroy the main olfactory epithelium (MOE) or saline (SAL) to serve as control. ZnSO4-treated female mice were no longer able to reliably distinguish between volatile as well as nonvolatile odors from an intact versus a castrated male. Furthermore, sexual behavior in mating tests with a sexually experienced male was significantly reduced in ZnSO4-treated female mice. Vomeronasal function did not seem to be affected by ZnSO4 treatment: nasal application of male urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of ZnSO4 as well as SAL-treated female mice. Likewise, soybean agglutinin staining, which stains the axons of vomeronasal neurons projecting to the glomerular layer of the AOB was similar in ZnSO4-treated female mice compared to SAL-treated female mice. By contrast, a significant reduction of Fos in the main olfactory bulb was observed in ZnSO4-treated females in comparison to SAL-treated animals, confirming a substantial destruction of the MOE. These results show that the MOE is primarily involved in the detection and processing of odors that are used to localize and identify the sex and endocrine status of conspecifics. By contrast, both the main and accessory olfactory systems contribute to female sexual receptivity in female mice.

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.

The vomeronasal organ is required for the expression of lordosis behaviour, but not sex discrimination in female mice.

The role of the vomeronasal organ (VNO) in mediating neuroendocrine responses in female mice is well known; however, whether the VNO is equally important for sex discrimination is more controversial as evidence exists for a role of the main olfactory system in mate recognition. Therefore, we studied the effect of VNO removal (VNOx) on the ability of female mice to discriminate between volatile and non-volatile odours of conspecifics of the two sexes and in different endocrine states using Y-maze tests. VNOx female mice were able to reliably distinguish between male and female or male and gonadectomized (gdx) male volatile odours. However, when subjects had to discriminate between male and female or gdx male non-volatile odours, VNOx females were no longer able to discriminate between sex or different endocrine status. These results thus show that the VNO is primarily involved in the detection and processing of non-volatile odours, and that female mice can use volatile odours detected and processed by the main olfactory system for mate recognition. However, VNO inputs are needed to promote contact with the male, including facilitation of lordosis responses to his mounts. A single subcutaneous injection with gonadotropin-releasing hormone (GnRH) partially reversed the deficit in lordosis behaviour observed in VNOx females suggesting that VNO inputs may reach hypothalamic GnRH neurons to influence the display of sexual behaviour.

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.