The overall objective of the proposed research is to gain insight into the biochemistry, function, and physiological roles of steroid 5alpha- reductase, an enzyme that plays crucial roles in the biosynthesis and breakdown of steroid hormones. In the first granting period, we isolated and characterized cDNAs and genes that encode two distinct isozymes of 5alpha-reductase, designated type 1 and type 2, which have different biochemical properties and tissue distributions. These findings raise a number of questions relating to the physiological roles of the two isozymes and how their different biochemical properties influence these roles. Our initial goal in the proposed studies will be a detailed analysis of the biochemical properties of the two isozymes. Two experimental approaches will be taken to this end, including the purification of the isozymes to homogeneity and the identification and characterization of naturally occurring mutations in the type 2 isozyme in subjects with the genetic disease 5alpha-reductase deficiency. Our second objective is to create and analyze mice that are deficient in the 5alpha-reductase type 1 isozyme. As detailed in the Preliminary Results section, methods of targeted gene disruption have been used to generate mice that are heterozygous for a null allele at the type 1 locus. These animals will be bred to produce homozygous males and females that will be characterized at several different levels to determine the physiological role of this isozyme. Our third goal is to use similar methods to produce and characterize animals that are deficient in the 5alpha-reductase type 2 isozymes. Males homozygous for a type 2 null allele should represent a phenocopy of the human genetic disease of 5alpha-reductase deficiency, which is characterized by abnormalities in male phenotypic sexual development. Characterization of the type 2 deficient mice will provide insight into the physiological role of this isozyme in the development of the male phenotype and in the metabolism of steroid hormones. The final objective is to produce mice that are deficient in both 5alpha-reductase genes by crossing animals generated in Specific Aims 2 and 3. The resulting 5alpha-reductase deficient animals will be used to define the role of testosterone versus dihydrotestosterone in androgen action, to determine how fluctuations in the ratio of testosterone to estrogen impact on endocrine physiology, and to provide insight into the relative role of 5alpha-reduction in steroid hormone breakdown.
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