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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.

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.

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.

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.