Molecular genetic analysis of the flagellar axoneme and fibrous sheath in sperm motility and male infertility

Sperm motility is a critical component of male fertility; however the mechanisms which underlie the normal assembly and function of the mammalian flagellum are not well-characterized. To better understand the genes and proteins involved in sperm motility, I utilized the AE24 mouse model in which a transgene insertion has mutated an unidentified endogenous gene. Sperm from these mice possess an unstable axoneme, resulting in flagellar paralysis. I attempted to clone the endogenous gene from wild-type and AE24 genomic libraries. Although regions of wild-type and mutant DNA surrounding the site of the transgene insertion were cloned, I have not yet identified a relevant open reading frame or a candidate gene. In addition, mapping the site of the transgene insertion using Fluorescence In Situ Hybridization (FISH) on metaphase chromosomes from AE24 cells was unsuccessful, probably due to the probe\u27s small size. Finally, I performed differential display analysis to compare mRNA expression between wild-type and AE24 testes. While several differentially expressed sequences were identified, northern analysis needs to confirm these results before studies are continued. As another approach to identifying proteins involved in flagellar function, I cloned the gene and cDNA for a major human sperm fibrous sheath (FS) protein, hAKAP82 and the gene for its mouse homologue. The human AKAP82 protein is tyrosine-phosphorylated in a capacitation-dependent manner. I showed that the human protein was an A-Kinase Anchor Protein (AKAP) and mapped its PK-A binding site. I demonstrated that both the mouse and human genes were alternatively spliced and that the human gene mapped to the X-chromosome by FISH. I showed that the protein was made as a precursor that is processed into the mature protein. Immunoblotting and immunofluorescence analyses showed that this processing was less complete in the human than the mouse. However, I found no evidence for an association between either processing or tyrosine phosphorylation of this protein and motility in sperm from normal men. In addition, I found no evidence to support a role for hAKAP82 in Dysplasia of the Fibrous Sheath, a naturally-occurring, heritable human disorder that results in abnormal development of the FS and infertility