MODIFICATION OF EGG PLASMA MEMBRANE
William H Kinsey, Professor
University Of Kansas Medical Center Msn 1039 Kansas City, Ks 66160
Grant 2R01HD014846-10A1 from National Institute Of Child Health And Human Development IRG: REB
Project start date: 1981-02-01
Project end date: 1991-07-31
Sponsored Links Excellgen http://Excellgen.com
Modification Of Egg Plasma Membrane
William H Kinsey, Professor
University Of Kansas Medical Center Msn 1039 Kansas City, Ks 66160
Grant 2R01HD014846-20A1 from National Institute Of Child Health And Human Development IRG: REB
Abstract: The long term objective of this proposal is to identify the biochemical steps which transduce the initial signal of sperm-egg fusion into the various responses of the egg to fertilization. It is now established that egg activation involves the action of protein tyrosine kinases (PTKS) which function to initiate the calcium transient, as well as regulate pronuclear migration and fusion, initiation of DNA synthesis, and mitosis. Recent studies have demonstrated that the Fyn protein tyrosine kinase plays a major role in triggering the calcium transient that follows sperm-egg fusion. The objective of this proposal is to identify the mechanism by which Fyn activity is stimulated at fertilization. Using the sea urchin egg as a model system, we propose that Fyn kinase is activated by a tightly coupled phosphotyrosyl phosphatase which relieves Fyn from inhibition via dephosphorylation of Tyr 527. Specific aim I will characterize the phosphatase that acts on Fyn in the sea urchin egg. The role of this phosphatase in egg activation will be determined through microinjection experiments using mRNA encoding recombinant fusion proteins and blocking antibodies as specific inhibitors of phosphatase function. Specific aim II will characterize the sites of interaction between Fyn and this phosphatase. We will also determine the size of the c-Fyn pool that is activated at fertilization and determine if an amplification event occurs. Specific Aim III will test the hypothesis that c-Fyn is activated by a P-Tyr displacement mechanism at fertilization. Further studies will attempt to design a c-Fyn agonist based on the P-Tyr displacement mechanism The results of this study will identify the specific regulatory mechanism by which Fyn is activated at fertilization. This information will further the understanding of the biochemical mechanism by which the metabolic pathways and developmental programs in the egg are activated at fertilization and thereby enhance our ability to promote successful development in humans.
Keywords: cell membrane, egg /ovum, enzyme activity, fertilization, protein tyrosine kinase, chimeric protein, enzyme inhibitor, enzyme substrate, intermolecular interaction, protein tyrosine phosphatase, microinjection, sea urchin, zebrafish
Project start date: 1997-01-01
Project end date: 2006-06-30
2R01HD014846-20A1 (2001): $218967
2R01HD014846-25 (2007): $241080
2R01HD014846-16A1 (1997): $155972
Grants awarded to William H Kinsey
Confocal Live Cell Imaging Instrument
William H Kinsey, Professor
University Of Kansas Medical Center Msn 1039 Kansas City, Ks 66160
Grant 1S10RR019279-01 from National Center For Research Resources IRG: ZRG1
Abstract: The purpose of this application is to acquire a confocal microscope system that will be specifically designed for imaging of live cells and embryos under controlled culture conditions. The growing importance of fluorescent protein variants which can act as in vivo reporters for protein expression and subcellular location has accelerated the demand for an instrument that will allow investigators at KUMC to make more use of this technology in live cells. The need to image intracellular calcium fluxes in eggs and embryos have also raised the demand for a system dedicated to live cell imaging. The equipment requested in this proposal has been selected to meet the needs of a group of twelve major users at KUMC and will be operated as a component of the Center for Reproductive Sciences Imaging Core. The request is for a Nikon multiplatform four line confocal microscope that includes a three PMT detector, calcium ratiometric imaging system, image acquisition and analysis software, an enclosed temperature/humidity control system, and dual micromanipulators for microinjection of eggs and single cells. The intended use of this system will be primarily for time lapse observation of live vertebrate cells or embryos for periods of up to 24 hrs, and it will be housed in a separate room dedicated to this purpose. The live cell imaging system will be available to all investigators within the University of Kansas System and to members of surrounding institutions. However, the majority of the research projects using this facility will come from twelve NIH-funded laboratories identified as major users. Dr. William Kinsey will use the instrument for imaging zebrafish and mouse eggs injected with fluorescent calcium reporter molecules. Dr. De Lisle will study secretory vesicle dynamics using GFP constructs. Dr. Godwin will require the ability to image GFP and RFP constructs in living zebrafish embryos. Drs. Hunt and Petroff will use the ratiometric calcium imaging capabilities as well as imaging of GFP constructs. Dr. Little and Dr. Rongish will be doing time-lapse analysis of vascular endothelial or cardiac cells in avian embryos and mammalian embryo explants. Dr. Sarras studies the role of extracellular matrix components in zebrafish development, and will use this facility for time lapse imaging of GFP constructs in live embryos. Dr. Soares will use the facility for microinjection of fluorescent enzyme inhibitors and for calcium imaging in trophoblast cells. Dr. Stephens will image GFP constructs as related to Golgi processing in the Vpu gene in HIV-1 pathogenesis models. Dr. Tash will use the equipment to inject and quantitate fluorescent inhibitors of spermatogenesis. Dr. Terranova will use the equipment to inject and image GFP constructs into mammalian oocytes in the study of the role of Src tyrosine kinase in early development. He will also study calcium transients in granulosa cells induced to divide in vitro with tumor necrosis factor alpha.
Keywords: biomedical equipment purchase, confocal scanning microscopy, cardiovascular imaging /visualization, green fluorescent protein, reproductive system, single cell analysis
Project start date: 2004-04-01
Project end date: 2005-03-31
1S10RR019279-01 (2004): $270214
REPRODUCTIVE BIOLOGY: EARLY PREGNANCY AND DEVELOPMENT
William H Kinsey, Professor
University Of Kansas Medical Center, Msn 1039, Kansas City, Ks 66160
Grant 5T32HD007455-15 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Abstract: The objective of this program is to prepare post-doctoral trainees to establish independent research careers in the areas of gonadal function, early pregnancy and embryonic development through implantation. The training program will consist of three elements; independent laboratory research, group seminars and other functions held jointly with the Center for Reproductive Sciences, and formal course work in research ethics and animal welfare, as well as subjects that will enhance the trainee´s research expertise. The independent research will occur in a laboratory selected by the trainee from a group of 12 scientists who are leaders in various aspects of reproductive and developmental biology. The members of this training group have well established NIH-funded programs and have trained more than 86 postdoctoral fellows. Areas of study include fertilization, pre-implantation development, trophoblast-uterine interactions, the immunobiology of implantation and early pregnancy, as well as regulation of gonadal function, gene expression during testis and embryonic development. The strength of this group is enhanced by collaboration among the members as evidenced by the record of joint publications, grants, and students. This program will take advantage of an existing Center for Reproductive Sciences comprising 21 principal investigators and 78 additional staff and trainees to provide an enhanced training environment. The Center for Reproductive Sciences sponsors an active seminar program and hosts a regional conference in reproductive biology, which provides additional opportunities for trainees to present their work. The recruitment, selection and guidance of trainees will be coordinated through an Internal Advisory Committee, with input from all participating faculty and in consultation with an External Advisor, who will meet with the group once each year. Applicants who have been awarded a Ph.D. degree in one of the biological sciences, or a D.V.M. or M.D. degree and who demonstrate a commitment to research in reproductive biology will be considered for entry into the program. An emphasis will be placed on recruiting under represented minorities into the program. The records of the six trainees selected during the first funding period are impressive, with two of the four trainees who have completed their training having assumed faculty positions, one tenure track and one research track. The overall success of the program will ultimately be measured by the contributions of these trainees after they have established independent research programs
Keywords: Development; Gestation; Pregnancy; Reproductive Biology
Project start date: 1996-05-01
Project end date: 2011-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
5T32HD007455-15 (2010): $114941
5T32HD007455-14 (2009): $79736
5T32HD007455-12 (2007): $114851
2T32HD007455-11 (2006): $72901
5T32HD007455-09 (2004): $34359
5T32HD007455-08 (2003): $38486
2T32HD007455-06 (2001): $76209
MODIFICATION OF EGG PLASMA MEMBRANE
William H Kinsey, Professor
Anatomy And Cell Biologyuniversity Of Kansas Medical Center
msn 1039
kansas City, Ks 66160
Grant 5R01HD014846-18 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development IRG: REB
Abstract: The long term objective of this proposal is to identify the biochemical steps which transduce the initial signal of sperm-egg fusion into the various responses of the egg to fertilization. It is now established that egg activation involves the action of protein tyrosine kineses (PTKs) which function to regulate pronuclear migration and fusion, initiation of DNA synthesis, and mitosis. In addition, recent studies have demonstrated that protein tyrosine kinase activity plays a unique and essential role in the fertilized egg to program developmental events at the gastrula stage. The objective of this proposal is to identify those processes in the egg that are regulated by the fertilization induced activation of Fyn kinase activity and to determine the mechanism by which they are controlled. Using the sea urchin egg as a model system, the PI proposes that Fyn kinase functions downstream of the initial signal for egg activation, acting to regulate one or more specific PTK-dependent functions which may include pronuclear migration, DNA synthesis, and mitosis. Specific aim I will characterize the egg isoform of Fyn kinase and study its expression in the egg. The role of Fyn kinase in egg activation will be determined through microinjection experiments using recombinant GST fusion proteins encoding the unique SH2 and SH3 domains of Fyn N-terminal as specific inhibitors of Fyn function. The hypothesis that Fyn stimulates DNA synthesis via RAS activation will be tested. Specific aim II will identify the protein substrates of Fyn kinase in the egg. This will allow the PI to establish the specific biochemical pathways that are activated in response to Fyn activity. Specific Aim III will study the mechanism by which Fyn kinase activity is stimulated at fertilization. Particular emphasis will be placed on the effect of other protein kinases and phosphatases in regulating Fyn catalytic activity
Keywords: cell cycle, cell membrane, egg /ovum, enzyme activity, fertilization, protein tyrosine kinase cell cycle protein, chimeric protein, early embryonic stage, egg protein, enzyme inhibitor, enzyme substrate, isozyme, nonmammalian vertebrate embryology, phosphorylation high performance liquid chromatography, laboratory rabbit, microinjection, molecular cloning, sea urchin
Project start date: 1997-01-01
Project end date: 2000-12-31
5R01HD014846-18 (1999): $155868
5R01HD014846-17 (1998): $151355
Sponsored Links Excellgen http://Excellgen.com
7R01HD014846-24 (2005): $202500
5R01HD014846-23 (2004): $202500
5R01HD014846-22 (2003): $202500
5R01HD014846-21 (2002): $202500
5R01HD014846-14 (1994): $124818
5R01HD014846-13 (1993): $120048
PYK2 FUNCTION DURING FERTILIZATION
William H Kinsey, Professor
University Of Kansas Medical Center, Msn 1039, Kansas City, Ks 66160
Grant 1R01HD062860-01A1 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Abstract: Fertilization involves a series of signal transduction events that serve to activate the block to polyspermy, induce resumption of the cell cycle, incorporate the paternal genome, and activate egg metabolism. Key steps in this process include the actin cytoskeleton modifications that physically incorporate the sperm nucleus into the egg and subsequently reorganize the egg cortex. Recent findings by this laboratory revealed that the cytoskeleton-associated PYK2 kinase is rapidly activated during fertilization and functional studies indicate a role for PYK2 in sperm incorporation and egg activation. The objective of the proposed study is to test the hypothesis that PYK2 kinase participates in the signaling events triggered by sperm-egg adhesion or fusion that are critical for sperm incorporation and modification of the egg cytoskeleton. The project will make use of the economical zebrafish fertilization system which provides unique advantages for identification of signaling events at the point of sperm-egg contact as well as for proteomic analysis of substrates of the PYK2 kinase. The first specific aim will identify the specific function(s) of PYK2 kinase in egg activation. The second aim will identify the signals which activate PYK2 at fertilization. The third aim will identify the protein targets phosphorylated by PYK2 in order to work out the downstream pathways that it controls. The results of this study will provide important details regarding control of the egg cytoskeleton at fertilization and possibly help resolve the question of signaling pathways activated by sperm-egg recognition and fusion. These findings will have significant application to the development of techniques for assisted reproductive technologies to enhance human fertility. Lay Summary This study will work out the mechanism by which the sperm activates the egg during fertilization. The specific role of PYK2 kinase in bringing the sperm nucleus into the egg will be analyzed with the ultimate aim of enhancing the success of human in vitro fertilization. Assisted reproductive technologies such as in vitro oocyte maturation, in vitro fertilization and intracytoplasmic sperm injection have remained fairly inefficient processes. The proposed experiments will provide basic knowledge regarding the signal transduction process initiated by sperm-egg contact which could be used to improve these clinical methods
Keywords: Actins; Adenosine 5`-(trihydrogendiphosphate), 2`-(dihydrogen phosphate), 5`-5`-ester with 3-carboxy-1-beta-D-ribofuranosylpyridinium hydroxide, inner salt; Adhesions; Antimorphic mutation; Assisted Reproduction Technology; Assisted Reproductive Technology; Binding; Binding (Molecular Function); Biochemical Pathway; Biosynthetic Proteins; Blood Coagulation Factor IV; Blotting, Western; Brachydanio rerio; CADTK; CAK beta; CAKB; Ca++ element; Calcium; Calcium Channel; Calcium Channel Antagonist Receptor; Calcium Ion Signaling; Calcium Signaling; Cell Communication and Signaling; Cell Cycle; Cell Division Cycle; Cell Nucleus; Cell Signaling; Cell membrane; Cells; Cellular Matrix; Chromatin; Clinical; Coagulation Factor IV; Competence; Cone; Cones (Eye); Cones (Retina); Cytoplasmic Membrane; Cytoskeletal Modeling; Cytoskeletal Organization; Cytoskeletal Organization Process; Cytoskeletal Reorganization; Cytoskeletal System; Cytoskeleton; Danio rerio; Development; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; EC 2.7; Event; Extracellular Matrix, Integrins; FADK2; FAK2; FAK2 protein, human; Factor IV; Fecundability; Fecundity; Fertility; Fertility/Fertilization; Fertilization; Fertilization failure; Fertilization in Vitro; Fertilized Egg; Fertilized Ovums; Gametes; Genome; Germ Cells; Germ-Line Cells; Goals; Human; Human, General; ICSI; IVF; Image; Immune Precipitation; Immunoprecipitation; In Vitro; Individual; Insemination; Integrins; Intermediary Metabolism; Intracellular Communication and Signaling; Intracytoplasmic Sperm Injections; Ion Channels, Calcium; Kinases; Knowledge; Lab Findings; Laboratories; Laboratory Finding; Left; METBL; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Membrane Proteins; Membrane-Associated Proteins; Metabolic Networks; Metabolic Processes; Metabolism; Methods; Mice; Modeling; Modification; Molecular Interaction; Murine; Mus; NAADP; Network Analysis; Nucleus; Oocytes; Ooplasm; Outcome Study; Ovocytes; Ovum, Fertilized; PKB; PTK; PTK2B; PTK2B Protein Tyrosine Kinase 2 Beta; PTK2B gene; PTK2B protein tyrosine kinase 2 beta protein, human; PTK2B protein, human; PYK2; PYK2 Kinase; Pathway Analysis; Pathway interactions; Performance; Phosphoproteins; Phosphorylation; Phosphotransferases; Photoreceptors, Cone; Plasma Membrane; Procedures; Process; Protein Phosphorylation; Proteins; Proteomics; RAFT kinase; RAFTK; Reagent; Receptors, Calcium Channel Blocker; Recombinant Proteins; Reproduction; Reproductive Cells; Retinal Cone; Role; Running; Series; Sex Cell; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Site; Sperm; Sperm Injections, Intracytoplasmic; Spermatozoa; Staging; Structure of zygote; Surface; Surface Proteins; System; System, LOINC Axis 4; Technology, Assisted Reproductive; Test-Tube Fertilization; Testing; Time; Translating; Translatings; Transphosphorylases; VDCC; Voltage-Dependent Calcium Channels; Western Blotting; Western Blottings; Western Immunoblotting; Work; Zebra Danio; Zebra Fish; Zebrafish; Zygote; base; biological signal transduction; cell adhesion kinase beta; cell type; cone cell; egg; experiment; experimental research; experimental study; focal adhesion kinase 2, human; functional restoration; gene product; human PTK2B protein; imaging; improved; inhibitor; inhibitor/antagonist; initial cell; intracellular skeleton; knowledge base; language translation; nicotinate adenine dinucleotide phosphate; nicotinic acid adenine dinucleotide phosphate; novel; nuclear transfer; nuclear transplantation; oocyte maturation; particle; pathway; plasmalemma; proline-rich tyrosine kinase 2; protein blotting; public health relevance; related adhesion focal tyrosine kinase; research study; response; restore function; restore functionality; restore lost function; sexual cell; social role; sperm cell; success; technique development; zoosperm; zygote
Relevance: Assisted reproductive technologies such as in vitro oocyte maturation, in vitro fertilization and intracytoplasmic sperm injection have remained fairly inefficient processes. The proposed experiments will provide basic knowledge regarding the signal transduction process initiated by sperm-egg contact which could be used to improve these clinical methods
Project start date: 2010-08-13
Project end date: 2015-05-31
Budget start date: 13-AUG-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
1R01HD062860-01A1 (2010): $270000
MODIFICATION OF EGG PLASMA MEMBRANE
William H Kinsey, Professor
University Of Kansas Medical Center, Msn 1039, Kansas City, Ks 66160
Grant 5R01HD014846-28 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Abstract: Fertilization in species that utilize external fertilization, including model systems such as sea urchin, frog, and fish, all exhibit rapid protein tyrosine kinase (PTK) activation as a critical step required for the sperm- induced calcium transient, exit from meiosis II, pronuclear congression, and mitosis. Recent studies have demonstrated that mammalian eggs differ from lower species as to the mechanism controlling the sperm- induced calcium transient. However, while studies in oocytes of mice and rats indicate that Src-family kinase activity is not required for calcium signaling, there is strong evidence that these kinases play a role in Mil resumption and pronuclear congression. The main goals of this proposal are to establish which Src-family kinases are required signaling pathways critical to MM resumption and pronuclear congression. The first specific aim will test the hypothesis that individual Src-family PTKs (Fyn, Yes, or Src) are required for resumption of meiosis and pronuclear congression. The experimental approach will use a combination of Src-family knockout mice and dominant-negative constructs to define the role of each kinase in these processes. The Second aim will identify the specific biochemical pathways in which these kinases participate to accomplish each function. Here biochemical analyses will establish which pathways have been interrupted by suppression of each individual kinase Fyn, Yes, and Src. The relationship of each pathway to MM resumption of pronuclear congression will be established. The third specific aim will test the hypothesis that Fyn, Yes, and Src are differentially regulated after fertilization and that their activity increases at specific points during egg activation that allow them to control MM resumption and pronuclear congression. This aim will also identify the regulatory mechanisms by which these kinases are activated in the fertilized egg. The results of this study will provide an understanding of the biochemical mechanisms regulating MM resumption and pronuclear congression and will lead to future studies enabling us to modulate these processes artificially. Lay Summary Fertilization requires that multiple enzymes become turned on in order for the egg to grow into an embryo. This study will use more sensitive, modern techniques to establish which protein kinases trigger the fertilized egg to eject extra chromosomes and move the male and female nuclei together
Keywords: ATP-protein phosphotransferase; ATP[{..}]protein-tyrosine O-phosphotransferase; Activation Analysis; Analyses, Activation; Anaphase; Antibodies; Antimorphic mutation; Biochemical; Biochemical Pathway; Biological Models; Blood Coagulation Factor IV; Breeding; Ca++ element; Calcium; Calcium Ion Signaling; Calcium Signaling; Cell Cycle; Cell Division Cycle; Cell Nucleus; Cell membrane; Chromosomes; Coagulation Factor IV; Common Rat Strains; Critical Paths; Critical Pathways; Cytoplasmic Membrane; Defect; Development; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; EC 2.7; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Echinoidea; Embryo; Embryonic; Enzymes; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Event; Exhibits; Factor IV; Family; Female; Fertility/Fertilization; Fertilization; Fertilized Egg; Fertilized Ovums; Fishes; Frog; Future; G2/Mitotic-Specific Cyclin B2; Goals; HEK3; ICSI; Immunofluorescence; Immunofluorescence Immunologic; Immunologic, Immunofluorescence; Individual; Intracytoplasmic Sperm Injections; Investigators; Kinases; Knock-out; Knockout; Knockout Mice; Lead; Length; Location; M Phase; M phase (cell cycle); Mammalia; Mammals; Mammals, General; Mammals, Mice; Mammals, Rats; Marine Invertebrates; Measures; Meiosis; Metabolic Networks; Method LOINC Axis 6; Methodology; Methods; Methods and Techniques; Methods, Other; Mice; Mice, Knock-out; Mice, Knockout; Mitosis; Mitosis Stage; Mitotic Anaphase; Model System; Models, Biologic; Modification; Murine; Mus; Nuclear Envelope; Nuclear Membrane; Nucleus; Null Mouse; Oocytes; Outcome Study; Ovocytes; Ovum, Fertilized; PTK; Pathway interactions; Pb element; Phase; Phosphorylation Site; Phosphotransferases; Plasma Membrane; Play; Procedures; Process; Programs (PT); Programs [Publication Type]; Protein Kinase; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Protein-Tyrosine Kinases, src; Proteins; Rana; Rana (genus); Rat; Rattus; Reagent; Regulation; Regulatory Pathway; Research Personnel; Researchers; Role; Sea Urchins; Signal Pathway; Sperm; Sperm Injections, Intracytoplasmic; Spermatozoa; Staging; Structure of zygote; System; System, LOINC Axis 4; Techniques; Testing; Time; Translating; Translatings; Transphosphorylases; Tyrosine Kinase; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; Vertebrate Animals; Vertebrates; Wild Type Mouse; Work; Zygote; anaphase-promoting complex; cycB2; cyclin B2; cyclosome; egg; functional restoration; gene product; glycogen synthase a kinase; heavy metal Pb; heavy metal lead; hydroxyalkyl protein kinase; hydroxyaryl protein kinase; insight; knowledge base; language translation; male; meiotic; novel; nuclear transfer; nuclear transplantation; pathway; phosphorylase b kinase kinase; plasmalemma; programs; restore function; restore functionality; restore lost function; social role; sperm cell; src Kinases; src Tyrosine Kinases; src-Family Kinases; src-Family Tyrosine Kinases; success; time interval; tyrosyl protein kinase; vertebrata; zoosperm; zygote
Project start date: 1997-01-01
Project end date: 2012-02-29
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5R01HD014846-28 (2010): $233896
5R01HD014846-19 (2000): $168939
Sponsored Links Excellgen http://Excellgen.com
William H Kinsey
University Of Kansas Medical Center, Msn 1039, Kansas City, Ks 66160
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5U54HD055763-04_9001 (2010): $83095
Reproductive Biology: Early Pregnancy And Development
William H Kinsey, Professor
University Of Kansas Medical Center Msn 1039 Kansas City, Ks 66160
Grant 7T32HD007455-10 from National Institute Of Child Health And Human Development IRG: CHHD
Project start date: 1996-05-01
Project end date: 2006-04-30
7T32HD007455-10 (2005): $56283