Vitellogenesis, the process whereby yolk lipoproteins are synthesized by the liver and accumulated in the developing oocyte, is of fundamental importance for the successful reproduction of non-mammalian vertebrates. This process is considered an excellent model system for the hormonal regulation of sex-differentiated hepatic protein synthesis in vertebrates. Accordingly, the main objective of this proposal is to understand the hormonal interplay for the expression of female specific hepatic genes under the chronic stress of captivity in the lizard Anolis pulchellus. Of major relevance is that cardiovascular disease and reproductive malfunctioning in humans have been related to altered sex-linked hepatic protein metabolism. In addition, it has also been shown that exposure to stress triggers the onset of these malfunctions. Therefore, studies of the regulation of vitellogenin gene expression could provide important insights in the genesis and treatment of these hepatic diseases for which no experimental models are available in humans. In previous studies we have shown that anoline lizards undergo pronounced reproductive regression under captivity stress, including a drastic decrease of the synthesis of vitellogenin by the liver paralleled to a decrease in VTG mRNA levels, cessation of follicular growth and ovulation, increased follicular atresia, and degeneration of the oviductal glands. Estrogen therapy to captive female lizards greatly improves reproductive activity and prevents or reverts the negative effect of captivity on vitellogenin synthesis and secretion by the liver. Since stress responses have been related to the alteration of hormone levels of the pituitary-adrenal axis, we suggest that the detrimental effects of captivity evoked in female specific liver functions are mediated through altered levels of glucocorticoids which, in turn, result in the faulty expression of estrogen dependent genes. Three main mechanisms are considered in our research plan: 1) reduction of estrogen levels, 2) reduction of the hepatic estrogen receptor (HER) levels/activity, and/or 3) estrogen competitive interactions at the gene regulatory site. Therefore, we propose: 1) To analyze the mechanisms of VTG gene expression during captivity at the molecular level by determining VTG protein synthesis, mRNA levels and transcriptional rates and evaluate the effect of estrogen and the activity/levels of the hepatic estrogen receptor. 2) To characterize the multihormonal interplay involved in VTG gene expression under stress and different hormonal treatments in vivo and in vitro. 3) To initiate structural studies of the Anolis VTG genes including the isolation of the 5' regulatory regions using homologous cDNA probes recently cloned in our laboratory. This will lead to future analysis of the interaction of hormone-receptor complexes at the regulatory sites. This research proposal will provide a rich environment for student training in biomedical research.
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