Roger A. Gorski - US grants

The influence of steroid hormones on basic mechanisms of neuronal development will be studied in a unique model system, the Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) of the rat hypothalamus. Although the SDN-POA of the male rat contains more neurons and is about five times larger in overall volume than the corresponding nucleus of the female, the key to the proposed studies is that this morphological sex difference is influenced by the perinatal hormone environment. In fact, prolonged treatment of the female with testosterone propionate perinatally actually sex-reverses the brain of the female in terms of SDN-POA volume and neuronal number. This ability to determine the development of the SDN-POA by manipulating the hormone environment provides a novel opportunity to study the regulation of neurogenesis, neuronal migration and neuronal survival during a perinatal period of neuronal death. This proposal will be focused on the latter parameter which currently is considered the most likely mechanism by which steroids influence neuronal development. We will identify the specific period of cell death during perinatal development and quantify differences in this process in males, females and the female in which the SDN-POA is sex-reversed by steroids. We will also determine the anatomical sites of cell death by comparing its incidence in the SDN-POA, a control region, and along the pathway of migration the neurons of the SDN-POA take from their ependymal origin to the nucleus. During this specific perinatal period we will evaluate the ultrastructure of neurons, glia and neuropil in those areas where cell death occurs. Parameters of intracellular (e.g., number of organelles, membrane surfaces) and intercellular (e.g., synapse number and type, fraction of neuronal surface occupied by synaptic contacts, neuropil volume and characteristics) maturation will be measured by quantitative morphometrics in males, females and the sex-reversed female to describe the ultrastructural changes which precede cell death and identify the morphological correlates of steroid action which promotes neuronal survival. Since hormones may serve as critical growth factors for neurons, the proposed studies should contribute new information which will lead to greater understanding of the interaction between genomic and environmental factors during normal brain development and serve as the foundation for an understanding of abnormal brain development and function.

The sexual differentiation of the mammalian brain is of basic importance to reproductive biology and developmental neuroscience. Since the discovery of structural sex differences in the brain and their modification by the gonadal hormonal environment perinatally, it has been assumed that structural sex differences underlie one or more of the functional sex differences. This laboratory has focused on two of the structural sex differences in the rat brain, the Sexually Dimorphic Nucleus of the Preoptic Area (SDN) and the Anteroventral Periventricular Nucleus (AVPV), the SDN because it is one of the most marked sex differences and the AVPV because it responds to gonadal hormones in a direction opposite to that exhibited by the SDN. Recent evidence, however, suggests that the AVPV differentiates much later, perhaps due to the activity of postpubertal gonads. One study (Specific Aim IV) is proposed to determine when the AVPV does become sexually dimorphic and if postpubertal hormonal activity is responsible for its differentiation. The other aims relate to the SDN, its function, developmental hormonal sensitivity and the question whether growth factors mediate steroid action on its differentiation. A unique animal preparation has been developed - an animal with a unilaterally masculinized SDN. Although thus far documented only in the genetic male, it is likely that we can produce a genetic female with one SDN feminine and the other masculinized. First, we will determine the reproductive physiological and behavioral potential of these unique animals through classical techniques of reproductive neuroendocrinology and then include these animals in electrical stimulation and in some cases, lesion studies to elucidate the possible function(s) of the SDN with particular emphasis on prolactin (Specific Aim I). Although the rat with the unilaterally masculinized SDN offers a unique model for functional studies, the nucleus still has to be further characterized (Specific Aim II). Since it is possible that there may be two critical periods for the masculinization of the SDN, we plan to study the SDN and the reproductive function of animals subjected to hormonal manipulations prenatally and late-postnatally (Specific Aim III). Finally, we plan using the unilaterally modified SDN as a model, to determine whether specific growth factors play a role in masculinization of the brain (Specific Aim V). Recently, the concept of sexual differentiation has been extended to the structure of the human brain and even to gender orientation. It is only by a clear understanding of this process in laboratory animals that sound conclusions can be made about our own species.

The sexual differentiation of the brain is of fundamental significance yet the mechanisms of gonadal hormone action are unknown. A recent advance has been the discovery that structural sex differences exist in the brain, which are dependent upon hormones perinatally, and may underlie the functional sexual differentiation of the brain. The research continues to be focused on the most marked structural sex difference, the Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA). Four Specific Aims are proposed. 1. Characterize the development of the SDN-POA and its modification by the hormonal and neurochemical environment. Temporal windows and dose requirements for steroid action in modifying SDN-POA volume will be determined in several preparations in which SND-POA volume is predictably altered. In addition, the influence of the hormonal environment on characteristics of individual neurons will be determined, including size, the number of neurons specifically and permanently labeled autoradiographically by exposure to tritiated thymidine on embryonic day 18, and the development of receptors for cholecystokinin and selective opiates as determined by the autoradiographic analysis of in vitro binding. 2. Elucidate the mechanisms of steroid action. Two approaches are proposed: to verify the pathway of migration taken by SDN-POA neurons and determine the influence of the hormonal environment, and an analysis of the development of the ability of neuroblasts, migrating or postmitotic neurons to take up and retain estrogen. Both approaches will utilize autoradiography: the first will focus on neurons permanently labeled by tritiated thymidine, and the second, the uptake of tritiated moxestrol. 3. Establish the functional role of the SDN-POA in reproduction by utilizing both the destruction and stimulation of the SDN-POA. 4. Compare and contrast the development of the anteroventral periventricular nucleus (AVPV). Available data suggest that the AVPV is structurally sexually dimorphic in a direction opposite to that of the SDN-POA. Moreover, steroids may be inhibitory to the development and differentiation of this nucleus. Since the AVPV is just rostral to the SDN-POA, both nuclei can be studied in the same animal under the same conditions to challenge the universality of the hypothesis that gonadal steroids are neuronotrophic during the process of sexual differentiation. Collectively, the proposed research represents a multifaceted study of the influence of gonadal hormones on the development, structure and reproductive function of the rat hypothalamus.

DESCRIPTION: (provided by applicant) Sexual differentiation of the brain in male mammals is dependent upon testicular production of testosterone (T). In the laboratory rat this process occurs perinatally. A surge of T on embryonic days 18/19 appears to sensitize the hypothalamus to exposure of T postnatally. The mechanism of action of this embryonic surge of T and its regulation are basically unknown. With respect to the latter, there is some evidence that at least the earliest secretion of T by the Leydig cells of the testes is independent of other factors and is possibly programmed genetically. However, even "programmed" genetic events are subject to modification. The objective of the present study is to determine whether or not the maternal hormonal environment plays any role in the prenatal events which lead eventually to the sexual differentiation of the brain. Pregnant female rats will be hypophysectomized on embryonic day 13, or sham hypophysectomized. In an attempt to identify purely fetal/maternal interactions, half the male and female pups will be gonadectomized on the day of birth or sham gonadectomized. At 50 days of age the experimental rats will be sacrificed and their brains removed and processed histologically. The volume of the sexual dimorphic nucleus of the preoptic area (SDN-POA), which is normally greater in the male rat, and that of the anteroventral periventricular nucleus (AVPV), which is normally greater in the female rat, will be determined. It is hypothesized that hypophysectomy of the pregnant female will inhibit the development of the SDN-POA in males but increase AVPV volume. Potential effects on the female are more difficult to predict because there is evidence both for the hormone- independent development of the female rat brain and its dependence on some exposure to gonadal hormones. The demonstration of a maternal contribution to the sexual differentiation of the fetal brain would be of considerable importance to our understanding of the fundamental concept of the sexual differentiation of the brain in mammal presumably including human beings.