The function of the spermatozoon is not only to transfer the very tightly packaged genetic information to the egg, but to deliver the DNA in a structurally organized form so that the genetic information can be properly accessed by the developing embryo. We have previously described the organization of hamster sperm DNA, and the long term goal of this proposal is to elucidate sperm DNA organization at the molecular level.
The specific aims i n this proposal are designed to isolate and characterize the specific DNA sequences by which the entire sperm genome is bound to two structures we have previously described, the sperm nuclear matrix and the sperm nuclear annulus, and to characterize the proteins to which they are bound. Preliminary evidence suggests that sperm DNA is associated with both of these structures by specific sequences. For the sequences attached to the sperm nuclear matrix, two specific genomic fragments, one recognized by the cDNA of the alphaA-crystallin gene and another recognized by the 5S RNA gene, that are known to be associated with the matrix will be examined. These genomic fragments will be subcloned, and the parts of each of these fragments that are associated with the sperm nuclear matrix will be identified and sequenced. Based on similar studies in somatic cells, these sequences are expected to be approximately 200 bp. To identify DNA sequences that are specifically associated with the sperm nuclear annulus, DNA prepared from isolated nuclear annuli will be screened for sequences that are specifically associated with the nuclear annulus by subtraction hybridization. Preparations of hamster sperm DNA are now available that are devoid of nuclear annuli, and these will be used to screen a Lambda phage library of nuclear annulus enriched DNA. Clones that do not hybridize will be isolated and tested for specificity to the nuclear annulus enriched fraction. The proteins that bind to the DNA in both the nuclear matrix and the nuclear annulus will be isolated while they are still attached to DNA from CsCl density, gradients. Hamster sperm nuclei will be decondensed using reagents that disrupt protein-- protein interactions but not the DNA-protein interactions present in sperm nuclear matrix and nuclear annulus. The decondensed structures will be centrifuged in CsCl density gradients and the proteins that co-isolate with the DNA will be identified and characterized. This information should provide important insight as to how, DNA is organized within the sperm nucleus at a molecular level.
|Ward, W Steven (2010) Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod 16:30-6|
|Yamauchi, Yasuhiro; Ward, Monika A; Ward, W Steven (2009) Asynchronous DNA replication and origin licensing in the mouse one-cell embryo. J Cell Biochem 107:214-23|