Michael Baum - US grants

Previous work by the applicant suggested that in ferrets, as in monkeys and man, a perinatal period exists during which exposure to androgen causes behavioral masculinization. However, in none of these species has a perinatal period yet been identified during which androgenization causes behavioral defeminization. Experiments are proposed which will search further for defeminizing effects of perimatally administered sex steroids on ferrets' coital behavior, and which explore the possible contribution of two metabolites of testosterone (T), estradiol (E2) and 5a-dihydrotestos-terone (DHT), to the differentiation of masculine coital behavior and socio-sexual orientation in this species. These studies will: (1) measure plasma levels of T in ferrets of both sexes during perinatal life, (2) attempt to induce behavioral defeminization in female ferrets by giving T during perinatal periods other than those previously studies, (3) compare the ability of T, E2, and DHT to cause the development of masculine coital behavior and of a preference to approach the estrous female, and test the ability of various drugs which inhibit the formation or neural binding of E2 and DHT to block behavioral masculinization in normal males, and (4) determine whether estrogen or androgen binding molecules are present perinatally in cytosol fractions prepared from ferret hypothalamus + preoptic region of each sex. Previous research also indicated that metabolites of T, (E2 and DHT) formed in the forebrain may normally contribute to the activation of sexual behavior in the male rat, perhaps in part by facilitating functional activity in ascending dopaminergic neurons.

Support is requested to continue experiments on the hormonal regulation of behavioral sexual differentiation in the ferret. Studies are proposed to determine (a) whether administering testosterone (T) to female ferrets on postnatal days 5-15, in concentrations which mimic the known plasma concentrations of T normally present in males during this neonatal period, will cause behavioral masculinization equivalent to that present in control males, (b) if particular sites in the female ferret forebrain can be identified into which T implanted neonatally enhances their ability to display masculine coital behaviors in adulthood, (c) whether neonatal administration of T, either s.c. or intracerebrally, influences the preference of female ferrets to approach male versus female sexual partners, (d) what the plasma concentrations of estradiol and estrone are in male and female ferrets during perinatal development and whether sex differences in the activity of aromatase in hypothalamus + preoptic area exist at the same perinatal ages, (e) whether the ability of male and female ferrets to display feminine sexual behavior in adulthood can be reduced by perinatal treatments which restrict the amount of estrogenic stimulation available to the developing brain.

DESCRIPTION (adapted from applicant's abstract): The proposed research seeks to understand how sexually dimorphic aspects of forebrain structure control sexually dimorphic aspects of psychosexual and reproductive function in a carnivore, the ferret. The central hypothesis to be tested is that the processing of volatile odors, arising from sexually active conspecifics, by neurons located in different segments of the main olfactory projection to the hypothalamus, differs in males and in females. Studies are proposed to compare the effects of olfactory bulb deafferentation on sexual partner preference in male and female ferrets and to determine whether exposure to male odors augments the capacity of vaginal-cervical stimulation to activate mediobasal hypothalamic LHRH neurons in females. Additional studies will determine whether prenatal inhibition of estradiol biosynthesis in the male ferret brain results in a female-like pattern of sexual partner preference in adulthood while attenuating the differentiation of a sexually dimorphic cluster of galanin neurons in the dorsal preoptic area/anterior hypothalamus (POA/AH). Other studies will assess the effects of infusing galanin or a galanin antagonist or of excitotoxic lesioning of the dorsomedial POA/AH on ferrets' odor and sexual partner preferences. The effects of these treatments on odor-induced expression of the immediate-early gene, c-fos, in neurons located at different stages of the main olfactory projection to the hypothalamus will also be compared in males and females. Finally, the density of projections from the medial amygdala to several hypothalamic regions will be compared in the two sexes using combined immunocytochemistry for the retrograde tracer, cholera toxin B, and Fos protein. Understanding how the male and female nervous systems process reproductive odors differently in a higher mammal like the ferret could provide new insights into sexually dimorphic brain-behavior relationships in man.

This is a Research Experiences for Undergraduates project. It will provide research experiences for eight students at Boston University. Students will participate in projects involving neurobiology, regulatory biology, cell physiology and plant and animal ecology.

DESCRIPTION (applicant's abstract): The vomeronasal organ (VNO) projection pathway mediates the effects of pheromones on several essential psychosexual and neuroendocrine functions in a variety of vertebrate species. In preliminary studies with rats and mice we have used the immediate-early gene product, Fos, as a nuclear marker of neuronal activation in different segments of the VNO pathway. In both species the ability of odors from sexually active males to augment neuronal Fos in the VNO pathway was found to be sexually dimorphic. New studies are proposed to exploit the mouse as a model system in which to study pheromonal communication and to manipulate genes governing the development of the nervous system. These experiments will establish whether pheromones derived from male and female urine activate different populations of sensory neurons in the VNO epithelium which, in turn, project to different rostral-caudal subdivisions of the accessory olfactory bulb and whether these actions of urinary pheromones on neuronal Fos in the terminal regions (bed nucleus of the stria terminalis; medial preoptic area) of VNO pathway in male and female subjects and correlate these results with animals' preference to investigate these respective stimuli. The possible contribution of perinatal aromatization of testosterone to the male- typical organization of VNO-projection pathway function will be assessed by comparing urine-induced neuronal Fos responses and olfactory preferences in wild-type and transgenic mice with a null mutation of the CYP19 gene, which are aromatase deficient. A final study will determine whether the robust neuronal Fos responses to male urinary odors, normally present at all levels of the female's VNO pathway, are attenuated in the most central portion of this pathway in cycling females by the receipt of mating stimulation from the male. Such an effect of mating could explain the 'Bruce effect' in which urine from a strange male, but not from a familiar, recent mating partner, blocks the establishment of pregnancy. The results of these experiments should identify sites in a well-characterized anatomical pathway at which sex hormones and social experience affect the detection and/or processing of olfactory signals.

DESCRIPTION (provided by applicant): Current dogma states that testosterone secreted perinatally by the testes promotes male brain sexual differentiation in mammals whereas the female brain develops in the absence of any sex hormone action. We challenge this dogma by hypothesizing that estradiol makes an essential contribution to the differentiation of neuroendocrine mechanisms controlling female-typical olfactory mate recognition and sexual behavior. First we will compare the number of progesterone receptor immunoreactive cells in hypothalamic nuclei of perinatal female mice with a null mutation either of the estrogen biosynthetic enzyme, aromatase (ArKO) or of the plasma estradiol binding protein, alpha feto protein (AFP-KO) and of wild type controls to determine whether there is a critical early postnatal period when estradiol normally acts in the female hypothalamus. Next, we will determine whether treatment of ArKO female mice with estradiol at early postnatal times specified by the outcome of Aim 1 will reverse adult deficits (previously seen in ArKO females) in the investigation of urinary odors and in sexual behavior. We will also determine whether treatment of wild type females with an aromatase inhibitor, ATD, over this same early period disrupts later female-typical behaviors. We recently showed that the discrimination of closely related urinary odors was significantly better in ArKO than in wild type female mice. Others had previously shown that forebrain estradiol receptor expression is upregulated in ArKO mice. We will test the hypothesis that adult treatment with estradiol more effectively enhances the discrimination of urinary odors needed for sex discrimination and/or individual mate recognition in ArKO than in wild type females. Finally, we will compare the ability of estradiol vs no steroid treatments of adult ovariectomized ArKO and wild type female mice to stimulate the birth and/or survival of neurons in the anterior portion of the subventricular zone which are destined to migrate along the rostral migratory stream into the main olfactory bulb where they may enhance olfactory discrimination. These studies should provide new information about the contribution of estradiol to the development of essential aspects of female-typical neural and behavioral functions. Affirmation of a perinatal role for estradiol in brain sexual differentiation in female mice would raise the question of whether estradiol plays a similar role in female-typical psychosexual differentiation in women.