REPRODUCTIVE BIOCHEMISTRY OF TESTIS-SPECIFIC LDH-X

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To understand the complexity of spermatogenesis it is necessary to examine the details of gene expression and ultimately correlate these with the multiplicity of morphological and biochemical changes which occur as spermatogonia differentiate into spermatozoa. The gene encoding testis-specific lactate dehydrogenase (ldh-c) uses a promoter not transcriptionally active in somatic cells. Ldh-c expression begins during prophase of the first meiotic division. A transgene consisting of an 100 bp ldh-c core promoter (murine) and lacZ, expressed beta- galactosidase only n pachytene spermatocytes from male offspring of founder mice. The murine ldh-c promoter contains a dual function palindrome sequence required for transcriptional initiation in germ cells and repression of gene expression in somatic tissues. There are several ubiquitous cis-regulatory elements as well as testis specific protein -DNA binding. We can determine the roles played by testis specific and non-specific transcription factors in regulating this gene. This in turn will allow us to select transcription factors as targets in reverse genetics experiments that should enhance our understanding of the molecular events involved on the path from stem cell to spermatozoan. To accomplish this we propose the following specific aims: (1) to study transcriptional regulation of ldh-c by deletion analysis of upstream sequences in transgene constructs. Selected protein binding sequences of the murine ldh-c promoter will be used as "bait" in the "yeast one-hybrid" system for transcription factor capture, cloning, identification and isolation; (2) to use genetically engineered mice as model systems for the study of transcription by expressing regulatory motifs as transgenes; (3) to disrupt the endogenous ldh-c gene locus by homologous recombination and thereby eliminate LDH-C4 synthesis in transgenic mice; and (4) to down-regulate transcription factor expression in primary spermatocytes with ribozyme transgenes expressed under control of the ldh-c promoter. These goals are important for understanding specific gene expression. They will take us to the next level to analyze signal transduction pathways involved in sperm cell: supporting cell interactions. Ultimately, these data will permit us to fully realize the potential of gene therapy in alleviating male infertility and for fertility control.