Angiotensin converting enzyme (ACE) is responsible for the conversion of angiotensin I into the potent vasoconstrictor angiotensin II. As early as 1971, researchers were surprised to find that testis contains large amounts of ACE, and that the protein found in this tissue was biochemically distinct from the ACE enzyme produced by endothelium and other somatic tissues. Subsequent analysis has demonstrated that developing male germ cells produce large amounts of ACE mRNA encoding a protein approximately half as large as that observed in somatic tissues. Despite the smaller size, testis ACE is fully catalytic. Within all mammals studied to date, the ACE gene is encoded by a single genetic locus. Developing germ cells are unique in that they begin mRNA transcription of ACE within the 12th intron of the somatic ACE gene. Transgenic mouse studies have demonstrated that a very small portion of DNA located within this 12th intron serves as a male germ cell specific promoter for testis ACE transcription. From this and other studies, it is clear that testis ACE transcription is truly tissue restricted and reflects the unique biochemistry within developing male germ cells. The first Specific Aim is to define the biochemistry of testis ACE promoter transcription and to characterize and clone the male germ cell transcription factors that lead to high level ACE transcription. One of the most important and least understood questions concerning testis ACE is the role of this protein in male germ cell development. There is evidence both for and against the view that testis ACE is functionally important. However, in my opinion the data are inconclusive in establishing whether ACE plays an important role in spermatogenesis or in some aspect of fertilization. We believe the only definitive way to investigate the role of testis ACE is to create mice, using the technique of targeted homologous recombination, that completely lack testis ACE. Thus the second Specific Aim of the proposal is to use targeted homologous recombination to generate mice that do not produce testis ACE. This will permit study of the role of testis ACE during male germ cell development.
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