Institute For Biogenesis Research: COBRE
William S Ward
Anatomy, Biochemistry & Physiologyuniversity Of Hawaii At Manoa
Grant 5P20RR024206-02 from National Center For Research Resources IRG: ZRR1
Abstract: The John A. Burns School of Medicine (JABSOM) at the University of Hawaii at Manoa (UH Manoa) is submitting this application in response to National Institutes of Health (NIH) PAR-07-229. With a Center of Biomedical Research Excellence (COBRE) award, we seek to transform JABSOM´s Institute for Biogenesis Research (IBR) into an interdisciplinary, translational research institute for reproductive biology. The IBR was established in 1999 by the State of Hawaii and UH Manoa in JABSOM´s Department of Anatomy & Reproductive Biology. Its rationale was to continue the legacy of reproductive biology research pioneered by National Academy member Dr. Ryuzo Yanagimachi. Dr. Yanagimachi and his team produced major breakthroughs in reproductive biology, including the development of intracytoplasmic sperm injection (ICSI), the principles underlying in vitro fertilization (IVF) in mammals, the first demonstration of repetitive mammalian cloning, and ICSI-mediated transgenic mice. We propose expansion of the IBR through collaboration, interdisciplinary mentoring, and creation of a transgenic Core. Such expansion will build upon the IBR´s foundation of outstanding basic science while directing its research emphasis toward interdisciplinary, translational research in reproductive biology. The IBR´s research focus lies in early molecular events in mammalian embryogenesis and their effects on later stages of development. Through partnership with the clinical Department of Obstetrics and Gynecology and Pacific IVF Institute, we will support translational projects designed to advance the science of reproduction and reduce infertility and birth defects for children through the latest molecular, cellular, and micro-techniques, many of which were developed at UH. We seek to accomplish this objective through the following specific aims. In Specific Aim 1, we will transform the Institute for Biogenesis Research (IBR) into a nationally competitive, interdisciplinary, translational reproductive biology research center. The focus of Specific Aim 2 will be to advance the science of reproduction and infertility and improve institutional capacity by engaging senior scientists to mentor junior investigators through interdisciplinary research studies and career development activities. In Specific Aim 3 we will improve institutional capacity by drawing upon the center´s expertise to develop a transgenic animal Core that will benefit investigators throughout the university. The IBR COBRE will contribute to infrastructure at UH Manoa by enhancing Core resources available for investigators across campus, and will support talented junior faculty toward independence as they conduct innovative basic, clinical and translational studies designed to produce data relevant to reproductive health
Project start date: 2008-09-29
Project end date: 2013-06-30
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to William S Ward
INSTITUTE FOR BIOGENESIS RESEARCH: COBRE
William S Ward, Professor
University Of Hawaii At Manoa, 2530 Dole Street, Sak D-200, Honolulu, Hi 96822
Grant 3P20RR024206-02S1_6796 from National Center For Research Resources
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To advance the science of reproduction and infertility by engaging senior scientists to mentor junior investigators through interdisciplinary research and career development studies
Keywords: Biogenesis; COBRE; CRISP; Cannot achieve a pregnancy; Center of Biomedical Research Excellence; Centers of Research Excellence; Computer Retrieval of Information on Scientific Projects Database; Difficulty conceiving; Funding; Grant; Infertility; Institutes; Institution; Interdisciplinary Research; Interdisciplinary Study; Investigators; Mentors; Multidisciplinary Collaboration; Multidisciplinary Research; NIH; National Institutes of Health; National Institutes of Health (U.S.); Origin of Life; Reproduction; Research; Research Personnel; Research Resources; Researchers; Resources; Science; Senior Scientist; Source; Study, Interdisciplinary; United States National Institutes of Health; career development; infertile; unable to bear children
Project start date: 2009-08-24
Project end date: 2011-08-23
Budget start date: 24-AUG-2009
Budget end date: 23-AUG-2011
PFA/PA: PAR-07-229
3P20RR024206-02S1_6796 (2009): $154231
3P20RR024206-02S1 (2009): $154231
1P20RR024206-01A1 (2008): $1831026
FUNCTIONAL ASPECTS OF SPERM CHROMATIN STRUCTURE
William S Ward
Anatomy, Biochemistry & Physiologyuniversity Of Hawaii At Manoa
2530 Dole Street, Sak D-200
honolulu, Hi 96822
Grant 7R03HD038080-02 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development IRG: REB
Abstract: The goal of this proposal is to test the hypothesis that the fully mature mammalian spermatozoon has a limited capacity to manipulate its chromatin in response to its environment, providing a mechanism to prevent the propagation of DNA that has been damaged during the life of the cell. Specifically, we propose that the spermatozoon can undergo a type of apoptotic DNA degradation, and a consequence of this ability is that the sperm cell also interacts with exogenous DNA. This has significant implications for research in many areas such as the manipulation of spermatozoa for in vitro fertilization (IVF), the development of male contraceptives, sexual transmission of diseases, the production of transgenic animals, and gene therapy. We have proposed a model for sperm chromatin structure that provides a possible mechanism for the spermatozoa-specific apoptosis and interaction with exogenous DNA, in which the protamine bound DNA toroids are linked by histone bound "spacer" DNA segments. We propose that these histone bound spacers, which we term active chromatin foci, are the sites where apoptotic DNA degradation takes place, and the sites of sperm chromatin interaction with exogenous DNA. We will test the specific hypothesis that mammalian spermatozoa have the ability to undergo a specific type of apoptotic DNA degradation and can interact with DNA, and that these activities occur at the histone bound active chromatin foci. We will first determine the distribution of histone bound DNA by pulse-field gel electrophoresis and Southern blot analysis, then compare this distribution to the apoptotic DNA degradation pattern. We will then determine how mouse spermatozoa interact with exogenous DNA. Our preliminary data suggests that live spermatozoa bind to and incorporate exogenous DNA onto a specific region of the nuclear matrix. We will determine whether this DNA is integrated into the sperm genome. Finally, we will isolate and characterize a topoisomerase II- like protein that we have identified in hamster sperm nuclear matrices. This is an enzyme that might be expected to be involved in both apoptosis, DNA binding, and integration. This application represents a new direction for our laboratory that will lead us to focus on therapeutic and contraceptive studies
Keywords: DNA, chromatin, genetic regulation, nucleic acid structure, programmed cell death, sperm DNA gyrase, chemical binding, nuclear matrix southern blotting, western blotting
Project start date: 1999-09-01
Project end date: 2002-08-31
7R03HD038080-02 (2000): $112573
3R03HD038080-01S1 (1999): $35885
1R03HD038080-01 (1999): $79500
ORGANIZATION OF DNA IN THE MAMMALIAN SPERM NUCLEUS
William S Ward
Surgeryuniv Of Med/dent Nj-r W Johnson Med Sch
robert Wood Johnson Medical Sch
piscataway, Nj 088548021
Grant 5R29HD028501-04 from National Institute Of Child Health And Human Development IRG: REB
Abstract: 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 in 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
Keywords: DNA, DNA binding protein, cell nucleus, nucleic acid structure, sperm molecular cloning, nuclear matrix, protein purification, protein structure density gradient ultracentrifugation, gel mobility shift assay, hamster, nucleic acid sequence, southern blotting, subtraction hybridization, western blotting
Project start date: 1991-08-01
Project end date: 1996-07-31
5R29HD028501-04 (1994): $100250
5R29HD028501-03 (1993): $94834
7R01HD028501-09 (2000): $96697
5R01HD028501-08 (2000): $90576
5R01HD028501-07 (1999): $181818
2R01HD028501-06A2 (1998): $176520
Importance Of The Sperm Nuclear Matrix For Embryogenesis
William S Ward
University Of Hawaii At Manoa 2530 Dole Street, Sak D-200 Honolulu, Hi 96822
Grant 5R01HD028501-13 from National Institute Of Child Health And Human Development IRG: REB
Abstract: Adapted from s ) The major hypothesis of this application is that the mammalian sperm nuclear matrix, and the epigenetic organization of the paternal DNA into loop domains by this structure, are essential for the paternal genome to participate in normal embryogenesis. Understanding the functional roles of this sperm DNA organization has been the long-term goal of this grant. We have demonstrated that mammalian sperm DNA is organized into loop domains that are attached at their bases to the protein skeleton of the sperm nucleus, the nuclear matrix, in a cell specific manner that changes during spermiogenesis and embryogenesis. Recently, we reported evidence that mouse oocytes injected with sperm nuclei with intact nuclear matrices develop into live births, but those injected with sperm nuclei with unstable nuclear matrices do not. These data led us to our current hypothesis that the sperm nuclear matrix and its organization of DNA into loop domains is essential for normal embryogenesis. DNA organization into loop domains in somatic cells functions in the control of transcription and DNA replication, and somatic cell nuclear matrices interact directly with several transcription factors. Therefore, one implication of our hypothesis is that the sperm nuclear matrix helps define which parts of the genome are replicated and transcribed first in the pronucleus. Maintaining this functional organization of the paternal genome has implications for clinical reproductive centers as well as for understanding the role of the paternal genome in development. We will test the hypothesis that the sperm nuclear matrix is essential for normal embryogenesis by three different approaches. First, we will test whether mouse sperm nuclei with disrupted DNA loop domain organization but intact DNA can participate in normal embryogenesis when injected into oocytes by ICSI. We have preliminary data to support that they do not. Second, we will test whether the embryo inherits the DNA loop domain organization, directly, using FISH of on pronuclei, and early embryonic cells. Finally, we will test whether the sperm nuclear matrix attracts transcription factors to the male pronucleus after fertilization.
Keywords: DNA, chromatin, embryogenesis, nucleic acid sequence, sperm, spermatogenesis, DNA replication, cell nucleus, cytogenetics, developmental genetics, fertilization, genome, nuclear matrix, nucleic acid structure, replicon, transcription factor, egg /ovum, embryonic stem cell, laboratory mouse
Project start date: 1991-08-01
Project end date: 2006-12-31
5R01HD028501-13 (2005): $214673
5R01HD028501-12 (2004): $248862
5R01HD028501-11 (2003): $247985