The central dogma of sexual differentiation of the brain states that embryonic or fetal secretions of the gonads determine whether the brain will follow a masculine or feminine pattern of development In zebra finches, estrogen appears to be responsible for a masculine pattern of development of the neural song system. However, the major source of estrogen synthesis in males of this species is the brain. The brain takes up androgen from the blood, converts it to estrogen, and releases estrogen into blood to maintain the high plasma levels found in males. Thus, the brain may be the synthetic source of estrogen during sexual differentiation and at other times of life. The proposed studies seek to understand the expression of the gene for P45Oaromatase (estrogen synthetase). The long-term objective is to understand the process of sexual differentiation of the brain, to determine the functional significance of unusually high levels of estrogen synthesis in brain of this species, and to understand the regulation of estrogen action on brain. (1) Using the cDNA for zebra finch aromatase, the expression of aromatase will be measured at various ages throughout development in brain and peripheral tissues, using Northern blot analysis and in situ histochemistry. Antibodies against recombinant aromatase will be prepared and used for immunohistochemistry and Western blot analysis. These studies will establish differences in aromatase expression in brain according to cell type, brain region, sex, and developmental stage. (2) Preliminary evidence suggests that transcripts of the aromatase gene expressed in brain may differ from those expressed in ovary. The cDNAs of transcripts expressed in brain will be analyzed by DNA sequencing to determine their structural relationship to each other. (3) Because there is reason to believe that brain expresses different functional forms of aromatase, or that different promoters regulate expression of aromatase in brain and peripheral tissues, transcript-specific probes for aromatase will be used to determine where and when each transcript is expressed, according to cell type, brain region, sex, and developmental stage. Specific transcripts will be transfected into neuronal and non-neuronal cell lines to measure the enzyme kinetics and functional properties of the resultant isoforms of enzyme, and to examine the subcellular localization of the enzymes. (4) To understand further the pattern of alternative splicing or use of alternative promoters during expression of aromatase, the aromatase gene will be characterized by DNA sequencing, restriction enzyme analysis, primer extension and RNAse protection analysis. These analyses will map the intron-exon boundaries and the transcription start sites. The proposed research will contribute significantly to an understanding of the principles of sexual differentiation of the brain, mechanisms of steroid hormone action on brain, and the differential regulation of steroid hormone synthesis in various tissues. At issue are the molecular mechanisms that determine how the same class of hormones, estrogens, are synthesized independently in different tissues or at different developmental stages. The knowledge gained by these studies will enhance the understanding and treatment of various endocrine disorders, including abnormalities of steroid synthesis and secretion during early development, and abnormalities of steroid hormone synthesis in brain during adult life.
