These studies will examine regulation of heat-shock-70-like protein (hsp70) isoforms and mRNAs by reproductive hormones. Since the hsp70 family comprises several highly related but distinct beat- shock-induced and constitutive gene products, high-resolution biochemical techniques (2-D gels, Western, slot, and Northern blots) and high-resolution cytochemical techniques (in situ hybridization and immunocytochemistry) will be used in conjunction with 4 antibody and 2 cDNA probes assembled for this project, each with distinct specificities. Heat-shock gene products are induced during gametogenesis in ovaries, testes, and even sporulation, and during early embryonic development; inappropriate expression of heat-shock proteins can lead to developmental defects. In addition, oncogenes induce hsp70 gene products and these products are elevated in transformed cells. Some hsp70 isoforms facilitate transport of secreted proteins into the endoplasmic reticulum and remove clathrin from coated vesicles, suggesting a role in secretion, but the regulation and role of hsp70 gene products in reproduction are not understood. However, estrogen and prolactin induce hsp70 mRNA. Furthermore, the most prominent estrogen- induced protein in ventromedial hypothalamus (EI70) and LHRH- induced protein (LHRH70) in pituitary is apparently the same hsp70- like isoform. Thus hsp70 isoforms may be generally involved in reproductive neuroendocrine processes. (I) The relationship between EI70, LHRH70 specific hsp70 isoforms will be studied using ATP binding, co-migration, immunoblots, peptide maps, and protein sequencing. (II) The induction of hsp70 isoforms and mRNA by estrogen, LHRH, prolactin, and prostaglandin E2 in brain, pituitary, and uterus will be examined using 2-D gels, immunocytochemistry, Northern and slot blots, and in situ hybridization, and correlated with regulation of reproductive functions. (III) The subcellular localization of hsp70 proteins after hormonal stimulation will be assessed with immunocytochemistry and subcellular fractionation, since localization of hsp70 isoforms is regulated by beat-shock and during the cell cycle. (IV) If time permits, hsp24 and hsp108, whose mRNAs are regulated by reproductive steroids, will be studied similarly using available probes. These studies may provide better understanding of the reproductive regulation of heat-shock proteins and their functional relation to reproductive cancers, birth defects, and fertility.
