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.

We used estrogen-deficient aromatase knock-out (ArKO) mice to determine whether estrogens contribute to the development of the brain and behavior in females. Female mice of three different genotypes [i.e., wild type (WT), heterozygous (HET), and homozygous (ArKO)] were ovariectomized in adulthood and subsequently tested for odor preferences (choice: intact male vs estrous female) in a Y-maze. When treated with testosterone, ArKO females spent significantly less time sniffing odors (both volatile and nonvolatile) from either male or female stimuli compared with WT and HET females. When given direct access to anesthetized stimulus animals or when given a choice between odor and visual cues from both stimulus animals, ArKO females continued to spend less time investigating the stimuli compared with WT and HET females. These defects in olfactory investigation of ArKO females were partially corrected with estradiol treatment in adulthood. Estradiol-treated ArKO females no longer differed from WT and HET females in the time spent investigating either nonvolatile odors or the anogenital region of anesthetized animals. However, ArKO females still investigated volatile odors and/or visual cues less than WT and HET females. Sexual receptivity was severely impaired in ArKO females after treatments with estradiol and progesterone that successfully induced receptivity in WT and HET females. Furthermore, ArKO females showed diminished levels of male sexual behaviors, whereas WT and HET females readily mounted an estrous female. Together, these findings demonstrate that estrogen is required for normal female development. The concept that the female brain develops in the absence of any hormonal stimulation should therefore be reconsidered.

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.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.