ORGANIZATION OF DNA IN THE MAMMALIAN SPERM NUCLEUS
William S Ward
Anatomy, Biochemistry & Physiologyuniversity Of Hawaii At Manoa
2530 Dole Street, Sak D-200
honolulu, Hi 96822
Grant 7R01HD028501-09 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development IRG: REB
Abstract: In this twice amended renewal proposal, the P.I. requests $507,142 in direct costs for a period of three years to investigate the organization of DNA in the mammalian sperm nucleus. Prior work by the P.I. has defined two kinds of sperm chromosomal organization chromosomal anchoring at the sperm nuclear annulus, and DNA loop domains. The P.I. has isolated four distinct short clones of DNA specifically associated with the sperm nuclear annulus. In Specific Aim 1, he proposes to study them further and will a) isolate and characterize longer clones from a genomic library, b) determine if the sequences are associated with more than one chromosome by FISH mapping to mitotic chromosomes, and c) use FISH techniques to determine if the sequences begin to co-localize at the same time at the nuclear annulus to provide support for the idea that the annulus is involved in the chromatin condensation that occurs as spermatids differentiate. The P.I. has previously shown that the DNA loop domain is different, and smaller, in sperm cells than in somatic cells. In Specific Aim 2, the P.I. will test the hypothesis that the sperm DNA loop domain is unique to the germ cell lineage and is passed to the embryo. He will do this by using five probes for different gene sequences to map loop domain structure in spermatogonia, spermatocytes and spermatids; in oocytes; and, finally, to determine if it is inherited, in fertilized eggs and in early and late-stage embryonic cell lines. In Specific Aim 3, the hypothesis that one of the sperm DNA loop domains can function as a replicon will be examined by using an "in vitro" DNA replication system
Keywords: DNA, chromatin, embryogenesis, nucleic acid sequence, sperm, spermatogenesis DNA replication, cell nucleus, cytogenetics, digital imaging, egg /ovum, imaging /visualization, nucleic acid structure, replicon Xenopus oocyte, confocal scanning microscopy, electron microscopy, fluorescent in situ hybridization, hamster
Project start date: 1991-08-01
Project end date: 2002-02-10
7R01HD028501-09 (2000): $96697
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
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
William S Ward, Professor
University Of Hawaii At Manoa, 2530 Dole Street, Sak D-200, Honolulu, Hi 96822
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. The goal of COBRE is to transform the existing Institute for Biogenesis Research (IBR) at the John A. Burns School of Medicine (JABSOM) in the University of Hawaii at Mnoa (UH-Mnoa) into an interdisciplinary, translational research institute for reproductive biology. The IBR was established in 1999 by the State of Hawaii. Its founding rationale was to continue the legacy of reproductive biology research pioneered by National Academy member Dr. Ryuzo Yanagimachi. 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. The IBR´s research focus lies in early molecular events in mammalian embryogenesis and their effects on later stages of development. This COBRE supports five junior investigators whose research expertise is directly related to the IBR´s research focus, and the creation of a Transgenic Mouse Core. Through partnership with the Pacific IVF Institute and the clinical Department of Obstetrics and Gynecology, we will support translational projects designed to advance the science of reproduction and reduce both infertility and birth defects for children. We seek to accomplish these objectives through the following specific aims 1) the IBR COBRE will contribute to infrastructure at UH-Mnoa by enhancing Core resources available for investigators across campus, and 2) the IBR COBRE will support talented junior faculty toward independence as they conduct innovative basic, clinical and translational studies designed to produce data relevant to reproductive health
Keywords: 0-11 years old; Academy; Biogenesis; Birth Defects; Burn injury; Burns; COBRE; CRISP; Cannot achieve a pregnancy; Center of Biomedical Research Excellence; Centers of Research Excellence; Child; Child Youth; Children (0-21); Clinical; Cloning; Computer Retrieval of Information on Scientific Projects Database; Congenital Abnormality; Congenital Anatomic Abnormality; Congenital Anatomical Abnormality; Congenital Defects; Congenital Deformity; Congenital Malformation; Data; Development; Difficulty conceiving; Discipline of obstetrics; Embryo Development; Embryogenesis; Embryonic Development; Event; Faculty; Fertilization in Vitro; Funding; Goals; Grant; Gynecology; Hawaii; Health, Reproductive; Human, Child; ICSI; IVF; Infertility; Infrastructure; Institutes; Institution; Intracytoplasmic Sperm Injections; Investigators; Mammalia; Mammals; Mammals, General; Mediating; Mice, Transgenic; Molecular; Molecular Genetic Abnormality; NIH; National Institutes of Health; National Institutes of Health (U.S.); Obstetrics; Origin of Life; Reproduction; Reproductive Biology; Reproductive Health; Research; Research Design; Research Infrastructure; Research Institute; Research Personnel; Research Resources; Researchers; Resources; Schools, Medical; Science; Source; Sperm Injections, Intracytoplasmic; Staging; Study Type; Test-Tube Fertilization; Transgenic Mice; Translational Research; Translational Research Enterprise; Translational Science; United States National Institutes of Health; Universities; children; design; designing; infertile; innovate; innovation; innovative; medical schools; member; study design; translation research enterprise; translational study; unable to bear children; youngster
Project start date: 2009-07-01
Project end date: 2010-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PAR-07-229
5P20RR024206-02_6620 (2009): $519459
William S Ward, Professor
University Of Hawaii At Manoa, 2530 Dole Street, Sak D-200, Honolulu, Hi 96822
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. The Transgenic Mouse Core will draw upon the expertise of Institute for Biogenesis Research (IBR) faculty members to develop a core facility that augments institutional capacity at the University of Hawai
Keywords: i, who have the expertise to develop such a core
Relevance: i at Manoa (UH Manoa). We will create a state-of-the-art Transgenic Mouse Core (TGMC) through using technology developed at the IBR. Four of the five junior investigators, Drs. Alarcon, Moisyadi, M. Ward, and Yamazaki, have mastered the techniques required for mouse transgenesis that were developed at the IBR by Dr. Ryuzo Yanagimachi. More recently, junior investigator Dr. Stefan Moisyadi has developed several modifications of ICSI-Tr that employ transposases to increase the efficiency of transgenic techniques. This will be the focus of Dr. Moisyadi´s Project 5. Finally, junior investigator, Dr. Yamazaki, is very proficient in growing and manipulating mouse embryonic stem cells, which are critical to the production of so-called "knock-out mice" in which a gene is removed from the mouse rather than added. The rationale for developing the Transgenic Mouse Core is based on the current needs of both the junior investigators and other researchers at UH-Mnoa and on the unique expertise that already exists in the IBR. A Transgenic Mouse Core would be a unique mechanism to enhance the institutional capacity of the entire University of Hawaii while providing an important resource to the junior investigators of the IBR COBRE. Moreover, the IBR contains the only group of scientists within UHMnoa and indeed in the State of Hawai
Project start date: 2009-07-01
Project end date: 2010-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
5P20RR024206-02_6621 (2009): $541874
CONTRIBUTION OF SPERM NUCLEUS TO PATERNAL DNA REPLICATION
William S Ward, Professor
University Of Hawaii At Manoa, 2530 Dole Street, Sak D-200, Honolulu, Hi 96822
Grant 1R01HD060722-01A1 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Abstract: The mammalian sperm cell offers a unique opportunity to understand the principles of molecular inheritance beyond the genetic code, and this knowledge has a direct bearing on human assisted reproduction practices. The compact sperm chromatin is devoid of virtually all enzymatic machinery associated with normal cellular processes, and only maintains the epigenetic and genetic components that are necessary to mediate inheritance. The hypothesis which we propose to test in this application is that in addition to the father´s DNA the sperm nucleus provides molecular components that are essential for the proper initiation and regulation of paternal DNA replication. Without these elements, our published data suggest that the paternal DNA would never be replicated, and embryonic development would not be possible. Significance In this era of increased ART, more and more human embryos that will become children undergo this first, defining round of DNA synthesis in vitro after clinical manipulation of the gametes. We understand how to keep the DNA intact during gamete storage, but we do not know what parameters are necessary to maintain proper origin recognition sites. There are also important biological implications as the mammalian one-cell embryo is a particularly suitable model for understanding how cells demarcate and license DNA replication origins. DNA replication mechanisms are also important targets for many human diseases. The molecular machinery for the initiation, control, and completion of the complicated process by which the 6 billion base pairs of mammalian DNA is replicated is now well understood. Each origin of replication is "licensed" by the binding of series of proteins, beginning with the origin recognition complex (ORC) made up of six proteins, ORC1L - ORC6L. When licensed origins enter S-phase, another host of proteins associate in a specific manner to eventually recruit DNA polymerases. Licensing ensures that each component of the entire genome is replicated only once per cycle. In mammalian cells ORC2L-5L remain bound to replication origins throughout the cell cycle, while ORC1L is recruited to the origin in G1, and degraded during S-phase (the fate of ORC6L through the cell cycle is unknown). In the mammalian one cell the paternal and maternal genomes are replicated independently, and asynchronously, and our preliminary data suggest that initiation of DNA synthesis of two pronuclei in the same oocyte cytoplasm can vary by as much as three hours. In Specific Aim 1 we will determine the timing of licensing in the mouse one cell embryo using pronuclear transfer experiments. In Specific Aim 2 we will test the prediction of our hypothesis that ORC2L-5L are already present on the maternal chromatin before fertilization, but load onto the male chromatin after fertilization. In Specific Aim 3 we will test whether ORC2L-5L or ORC1L binds directly to the sperm nuclear matrix, using ORC-GFP fusion proteins. Future studies will identify the epigenetic component in sperm chromatin to which ORC1L-5L proteins bind. The 30,000 or so genes that make up the human genome physically reside in DNA, a long tape-like molecule. Each cell in the body has the entire DNA sequence, and it takes roughly 6 feet of DNA to encode all 30,000 genes. At fertilization, there are only two copies of this DNA, one from the mother and one from the father, and both need to be replicated flawlessly. This application proposes to test the idea that the sperm cell brings to the egg more than only the DNA, itself, but instructions on how to replicate it properly in the first embryonic cell division
Keywords: 0-11 years old; Base Pairing; Binding; Binding (Molecular Function); Biological; Blastocytes; Blastomeres; CDC18L; CDC6; CDC6 Homolog; CDC6 gene; CDC6/Cdc18; CDT1; CDT1 Gene; Cell Cycle; Cell Cycle Controller CDC6; Cell Division Cycle; Cell Function; Cell Nucleus; Cell Process; Cell division; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Child; Child Youth; Children (0-21); Chimera Protein; Chimeric Proteins; Chromatin; Clinical; Complex; Cytoplasm; DNA; DNA Polymerases; DNA Replication; DNA Sequence; DNA Synthesis; DNA biosynthesis; DNA replication origin; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Data; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxyribonucleic Acid; EC 2.7.7.7; Elements; Embryo; Embryo Development; Embryogenesis; Embryonic; Embryonic Cell; Embryonic Development; Ensure; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Event; Fathers; Fertility/Fertilization; Fertilization; Foot; Fusion Protein; Future; Gametes; Genes; Genetic; Genetic Code; Genome; Genome, Human; Germ Cells; Germ-Line Cells; HCDT1; Hour; HsCDC18; HsCDC6; Human; Human Genome; Human, Child; Human, General; Immunofluorescence; Immunofluorescence Immunologic; Immunologic, Immunofluorescence; In Vitro; Injection of therapeutic agent; Injections; Instruction; Knowledge; Licensing; Life; Mammalia; Mammalian Cell; Mammals; Mammals, General; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Messenger RNA; Mice; Mice, Transgenic; Modeling; Molecular; Molecular Interaction; Mothers; Murine; Mus; Nuclear Matrix; Nuclear Scaffold; Nucleus; ORC protein; ORC1; ORC1L; ORC1L gene; Oocytes; Ovocytes; Pathway interactions; Pes; Phase; Pressure; Pressure- physical agent; Process; Protein Binding; Proteins; Publishing; RNA, Messenger; Recruitment Activity; Regulation; Replication Licensing; Replication Origin; Reproductive Cells; Safety; Series; Sex Cell; Site; Source; Sperm; Spermatozoa; Structure; Structure of blastomere; Subcellular Process; Testing; Time; Transgenic Mice; assisted reproduction; blastomere structure; children; egg; embryo cell; experiment; experimental research; experimental study; foot; gene product; human disease; inhibitor; inhibitor/antagonist; initial cell; mRNA; male; ori Region; origin recognition complex; pathway; pressure; public health relevance; recruit; research study; sexual cell; sperm cell; youngster; zoosperm
Relevance: The 30,000 or so genes that make up the human genome physically reside in DNA, a long tape-like molecule. Each cell in the body has the entire DNA sequence, and it takes roughly 6 feet of DNA to encode all 30,000 genes. At fertilization, there are only two copies of this DNA, one from the mother and one from the father, and both need to be replicated flawlessly. This application proposes to test the idea that the sperm cell brings to the egg more than only the DNA, itself, but instructions on how to replicate it properly in the first embryonic cell division
Project start date: 2010-04-19
Project end date: 2015-02-28
Budget start date: 19-APR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
1R01HD060722-01A1 (2010): $246060
INSTITUTE FOR BIOGENESIS RESEARCH: COBRE
William S Ward, Professor
University Of Hawaii At Manoa, 2530 Dole Street, Sak D-200, Honolulu, Hi 96822
Grant 5P20RR024206-03 from National Center For Research Resources
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
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PAR-07-229
5P20RR024206-03 (2010): $2097037
5P20RR024206-02 (2009): $2098681
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
Sponsored Links Excellgen http://Excellgen.com
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
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
William S Ward
University Of Hawaii At Manoa
Project start date: 2010-04-19
Project end date: 2015-02-28
Sponsored Links Excellgen http://Excellgen.com