PROJECT I: Thyroid Status and the Ontogeny of the of the Primate Hypothalamic GnRH Pulse Generator. David R. Mann, Professor of Physiology, MSM, Principal Investigator; Tony M. Plant, Professor of Cell Biology and Physiology, University of Pittsburgh Medical School, Co-Investigator.

The major goal of this project is to establish whether thyroid hormone is required for neurobiological processes that dictate the ontogeny of the GnRH pulse generator from birth to puberty in the male monkey. The GnRH pulse generator is the major driving force for postnatal sexual development in mammals. In the monkey, this system is fully functional during infancy, but during the transition to the juvenile phase of development, the GnRH pulse generator is arrested and remains quiescent until the onset of puberty. The project will test two hypotheses. The first is that normal thyroid status during infancy is necessary for the arrest of GnRH pulse generator activity during the transition to the quiescent juvenile phase of sexual development and/or for the reaugmentation of the GnRH pulse generator with the onset of puberty. An additional hypothesis is that the induction of a hypothyroidism during the juvenile period will alter the normal tempo of reactivation of the GnRH pulse generator at the onset of puberty. These hypotheses will be tested in the agonadal male monkey that is a well-established nonhuman primate paradigm for human sexual development. Thus, the project will lead toward a better understanding of the mystery of human puberty and disorders of sexual development in boys and girls.

		PROJECT III: Role of GnRH in Luteolysis of Monkey. Rajagopala Sridaran, Professor of Physiology, MSM, Principal Investigator; Anthony J. Zeleznik, University of Pittsburgh Medical School, Co-Investigator.

The administration of GnRH to pregnant rats is associated with a suppression of luteal steroidogenesis and the termination of pregnancy. GnRH has been shown to impair key steps in the steroidogenic pathway as well as induce apoptosis in lutein cells. This project intends to extend the previous work of the PI in the rodent to primates using the monkey model. Studies are proposed to initially attempt to determine whether chronic GnRH administration induces luteolysis in the female monkey as well as the rodent, and if so, whether this effect is mediated directly at the level of the ovary or indirectly via an alteration of pituitary gonadotropin secretion. Corpora lutea will be harvested and in vitro effects of GnRH on key steps in the steroidogenic pathway will be assessed. Studies will also be performed to determine whether GnRH induces apoptosis in primate tissue as it does in the rodent. The findings resulting from these studies will complement the previous work of the PI in the rodent and should add to our understanding of the potential role of ovarian GnRH in the regulation of luteal function in higher primates.

Project IV: Sp-Mediated Regulation of CREB and LDH Gene Expression during Spermatogenesis Kelwyn H. Thomas, Associate Professor of Anatomy and Neuro-Biology, MSM, Principal Investigator, William H. Walker, Assistant Professor of Cell Biology and Physiology, University of Pittsburgh Medical School, Co-Investigator.

The focus of this project is a detailed examination of the role of Sp transcripts in the regulation of stage specific gene expression in germ cells during murine spermatogenesis. LDH and CREB genes will serve as gene models for examining this phenomenon. The hypothesis to be tested is that temporal patterns of activation and deactivation of these genes are regulated via interactions between Sp variant transcription factors and GC-box cis-regulatory elements present in the individual promoters. Sp variants will initially be produced in Sp-deficient Drosophila S2 cells and the products will be used to synthesize specific antibodies for immunocytochemistry experiments. These antibodies will then be utilized to determine spatial and temporal expression patterns for these variants. The Sp transcription factors will also be utilized in order to determine their ability to activate CREB and LDH promotor/CAT reporter constructs in Drosophila S2 cells. A final aim will be to identify stage and cell type-specific coactivators that interact with Sp transcripts in the regulation of CREB and LDH promotor-directed CAT gene expression. These studies will provide important insights into the transcriptional basis for gene expression during early stages of spermatogenesis.