The goal of this research is to elucidate the functional role and describe the factors that regulate the developmental appearance of a unique protein associated with mammalian spermatogenesis. during the prophase of the first meiotic division a number of gene products are expressed that are stage and/or cell specific. One of these is an isozyme of lactate dehydrogenase (LDH) that may play an important metabolic role during spermatogenesis. LDH-C4 has been well characterized in terms of its structure, biochemistry and genetics. An understanding of ldh-c gene regulation impinges on questions concerning sperm production from the standpoint of developmental processes and infertility as well as fertility control. Mouse and human ldh-c genes show tissue and temporal specificity in expression but species differences in transcription rates and mRNA stability. These properties will be exploited to identify DNA sequences important for gene regulation as well as DNA binding proteins and transcription factors involved in gene expression during spermatogenesis. there are three specific aims: (1) to identify DNA sequences that regulate ldh-c expression in spermatogenic cells. A germ cell culture system in which this gene can be expressed, has been established and transgenic mice will be used to confirm testis-specific expression. Methylation patterns will be compared in expressing and non-expressing tissues; deletion analysis will be used to establish the extent of sequence necessary for regulatory activity in transfection assays; precise regulatory sequences identified by protein-DNA interactions will be used as probes to screen testis expression libraries in order to clone DNA binding factors; (2) to examine the role of ldh-c mRNA stability and of the 3' untranslated region in regulating translation; (3) to disrupt the endogenous ldh-c gene locus by homologous recombination with DNA encoding a selectable neomycin resistance marker and thereby eliminate LDH-C4 protein production in these targeted transgenic mice. This will permit us to ask whether LDH-C4 is absolutely required during spermatogenesis and/or in the spermatozoan. These goals are important to obtain an understanding of specific gene expression during spermatogenesis. They also provide potential insights into similar developmental processes in other systems, e.g. haematopoiesis, oncogenesis and organogenesis.
