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.

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.

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.

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. © 2005 Elsevier Inc. All rights reserved.Keywords: Estrogens; Sex differences; Olfaction; Odor discrimination; Learning Sex differences have been reported in olfactory sensitivity, with females being better able than males to detect male-derived odors. For instance, sows are significantly better than boars at using decreasing concentrations of the volatile male pig pheromone, androstenone, as a discriminative stimulus in operant tests for a sucrose reward (Dorries et al., 1995). In addition, using measurements of regional cerebral blood flow with positron emission tomography (Savic et al., 2001), women but not men show a significant activation of the preoptic area and ventromedial nuclei of the hypothalamus when actively smelling androstadienone, an androgenic compound secreted by the axillary glands in much higher concentrations in men than in women (Gower and Ruparelia, 1993). These sex differences in olfactory sensitivity are not only restricted to the detection of opposite sex odors, but also involve same-sex odors. For instance, using habituation/dishabituation tests to determine odor attraction thresholds, female mice respond more reliably than male mice to low concentrations of volatile urinary odors from either sex (Baum and Keverne, 2002;Pierman et al., 20...

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.