W. Steven Ward
	Affiliation: School of Medicine, Institute for Biogenesis Research Position: Professor and Director Degree: PhD (Vanderbilt University) Phone: (808) 956 5189 Fax: (808) 956 7316 Email: wward@hawaii.edu Address: IBR, 1960 East-West Rd., UH-Manoa, Honolulu, HI 96822

The function and structure of mammalian sperm chromatin.

Our research is focused on the structure of mammalian sperm chromatin and how this is related to function. The main hypothesis that we have tested is that the sperm cell provided the newly developing embryo with more than just the genetic code in the DNA sequence; it also provides a three dimensional organization of the DNA that provides crucial information as to how to use the father's genetic code.

DNA is packaged very densely in the sperm nucleus in a manner that is different from any other cell type. Most of the histones are replaced by protamines, and the DNA is crystallized into dense toroids with roughly 50,000 bp of DNA, each. Protamine condensation protects the sperm DNA from damage from external insults, and prevents transcription or DNA replication from occurring. We have shown that one structural feature present in all other somatic cells is also retained in sperm chromatin – the organization of DNA into loop domains attached at their bases to the nuclear matrix. This organization is crucial to two aspects of sperm chromatin function. Shortly after fertilization, the protamines are removed from the sperm DNA and the chromatin is repackaged with histones. The DNA is then replicated, and we have demonstrated that this DNA synthesis requires the loop domain organization to be intact. On the other hand, spermatozoa have the ability to digest their own DNA through an apoptotic-like process in which the sperm DNA is degraded. This degradation occurs on the nuclear matrix.

Our current research efforts are focused on understanding how sperm chromatin structure is related to the events that occur shortly after fertilization and how the DNA packaging in the sperm cell contributes to embryonic development.

Figure 1: Sperm DNA Loop Domain Organization is Required for the First Round of DNA Synthesis in the One Cell Embryo.


When normal mouse sperm heads are injected into oocytes, male (m) and female (f) pronuclei form, and DNA is replicated in each pronucleus (B). When the protamines are removed, and the only chromatin structure that is left is DNA loop domain organization, DNA replication still occurs (E). If some of the loop organization is removed by restriction endonuclease digestion, DNA replication still occurs (H). DNA alone, injected into the oocyte, does not result in DNA replication (K). Note that in this case, the female pronucleus still replicates its DNA.




Figure 2: (A) We have published evidence that each protamine toroid in the sperm chromatin is equivalent to a single DNA loop domain.


(B) We have shown that when spermatozoa are induced to break their DNA at the nuclear matrix, they cannot support embryogenesis. When these sperm are injected into oocytes, they form normal pronuclei, but when DNA replication begins, the paternal DNA is degraded. One the other hand, when the loop DNA not directly associated with the nuclear matrix is digested with restriction endonucleses, spermatozoa can still support DNA replication. These data suggest that it is the association of DNA with the sperm nuclear matrix that is important for DNA replication of the paternal genome.

• Ward, W. S., Partin, A. W., and Coffey, D. S.: DNA loop domains in mammalian spermatozoa. Chromos. 98:153-159, 1989.
• Ward, W. S., and Coffey. D.S.: DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod. 44:569-574, 1991.
• Ward, W. S.: DNA loop domain tertiary structure in mammalian spermatozoa. Biol. Reprod. 48:1193-1201, 1993.
• Ward, W. S.: The structure of the sleeping genome: implications of sperm DNA organization for somatic cells. J. Cell. Biochem. 55:77-82 1994.
• Nadel, B., de Lara, J., Finkernagel, S. W., and Ward, W. S.: Cell specific organization of the 5S rRNA gene cluster DNA loop domains in spermatozoa and somatic cells. Biol. Reprod. 53;1222-1228, 1995.
• Ward, W. S., McNeil, J., de Lara, J,, Lawrence, J. "Localization of three genes in the hook shaped hamster sperm nucleus by fluorescent in situ hybridization." Biol. Reprod. 54(6):1271 1278, 1996.
• Ward, W. S., Kimura, Y., Yanagimachi, R., "An Intact Sperm Nuclear Matrix May be Necessary for the Paternal Genome to Participate in Embryonic Development". Biology of Reproduction, 60:702-706, 1999.
• Sotolongo, B., and Ward, W. S.. "DNA Loop Domain Organization: The Three Dimensional Genomic Code" Journal of Cellular Biochemistry, 35:23-26, 2001.
• Klaus, A. V., McCarrey, J. R., Farkas, A., and Ward, W. S.. "Changes in DNA Loop Domain Structure During Spermatogenesis and Embryogenesis in the Syrian Golden Hamster". Biology of Reproduction, 64:1297-1306, 2001.
• Mohar, I., Szczygiel, M. A., Yanagimachi, R., and Ward, W. S.. "Sperm Nuclear Halos Can Transform into Normal Chromosomes after Injection into Oocytes", Molecular Reproduction and Development, 63:416-420, 2002.
• Sotolongo, B., Lino, E., and Ward, W. S.. "Hamster Spermatozoa have the Ability to Digest Their Own DNA", Biology of Reproduction, 69, 2029–2035, 2003.
• Ward, M. A., and Ward, W. S.. "A Model for the Function of Sperm DNA Degradation", Fertility, Reproduction and Development, Vol. 16 (5): 547-554, 2004.
• Shaman, J. A., Prisztoka, R., and Ward, W. S.. Topoisomerase IIB and an Extracellular Nuclease Interact to Digest Sperm DNA in an Apoptotic-Like Manner. Biology of Reproduction, 75:741-748, 2006.
• Yamauchi, Y., Shaman, J. A., and Ward, W. S.. "Topoisomerase II Mediated Breaks in Spermatozoa Cause the Specific Degradation of Paternal DNA in Fertilized Oocytes". Biology of Reproduction, 76:666-672, 2007.
• Shaman, J. A., Yamauchi, Y., and Ward, W. S.. "The Sperm Nuclear Matrix is Required for Paternal DNA Replication". Journal of Cellular Biochemistry, 102:680-688, 2007.
• Yamauchi, Y., Shaman, J. A., Boaz, S. M., and Ward, W. S.. "Paternal Pronuclear DNA Degradation is Functionally Linked to DNA Replication in Mouse Oocytes". Biology of Reproduction, 77:407-415, 2007.
• Boaz, S. M., Dominguez, K., Shaman, J. A., and Ward, W. S.. "Mouse Spermatozoa Contain a Nuclease that Is Activated by Pretreatment with EGTA and Subsequent Calcium Incubation." Journal of Cellular Biochemistry, 103:1636-1645, 2008.
• Yasuhrio, Y., Ward, M. A., and Ward, W. S.. "Asynchronous DNA Replication and Origin Licensing in the Mouse One Cell Embryo" Journal of Cellular Biochemistry, 107:214-223, 2009.
• Dominguez, K., and Ward, W. S. "A Novel Nuclease Activity that is Activated by Ca2+ Chelated to EGTA", Systems Biology in Reproductive Medicine, in press, 2009.
• Ward, W. S., "Function of Sperm Chromatin Structural Elements in Fertilization and Development". Molecular Human Reproduction, in press, 2009.