Gerald P. Schatten, PhD, University of California Berkeley 1975 - US grants

The High Voltage Electron Microscope Facility is a Biotechnology Resource of the Biotechnology Resources Branch of N.I.H. and is available to bio-medical researchers for ultrastructural studies where use of a high voltage electron microscope can provide new information. Investigations are directed especially towards analysis of relatively large and complex three-dimensional cell structures either in intact cells or isolated organelles, such as isolated flagella, chromosomes and Golgi complexes, or in sections up to 1 or 2 microns thick. The cold stage will undergo further development and modification. Freezing and cold sample handling procedures have been developed for viewing frozen sections, whole specimens, isolated cell components and aqueous suspensions. An improved type of image intensifier and digital image/memory processor has been installed and successfully used. This is part of a program to improve resolution and performance at high magnification in order to make possible high resolution studies of macromolecular crystals. The H.V.E.M. is particularly well suited to high resolution studies of this type because of its greater penetrating power, its greater depth of field and because of its greater specimen chamber size, one sufficiently large to permit introduction of ancillary equipment such a tilting/cooling stages which are necessary to preserve and orient these biological structures. Methods are being developed for immunocytochemical labeling of cell structures such as cytoskeletal elements, both in cell whole mounts and in thick sections.

The successful union of the parental genomes during mammalian fertilization requires intracellular architectural changes. In particular, microtubule- mediated motility is essential for the apposition of the sperm and egg nuclei, for the proper maturation of the ovulated oocyte and for the completion of the fertilization process at first division. This application proposes to investigate five question about microtubule organization and dynamics. 1. Are the dynamics of the microtubules participating in mammalian fertilization unusual? This might be expected due to metaphase arrest, and since the first mitotic spindle is organized without centrioles. 2. What are the in vivo functions of kinetochore proteins? Will microinjected kinetochore antibodies interfere with the anaphase separation of the chromosomes? Will they influence spindle organization at metaphase or spindle formation at exogenous centrioles? 3. Is the centrosome maternally inherited in this mammal? 4. When and where do centrioles arise during early embryogenesis? At which developmental stage will the embryonic cytoplasm tolerate the introduction of exogenous centrioles? 5. Is alpha-tubulin post-translationally modified by acetylation or detyrosination, and are these changes correlated with microtubule stability or age? The hypotheses to be tested are that: 1. while the metaphase-arrested oocyte appears dormant, its microtubules are highly dynamic; 2. kinetochore antibodies will prevent prometaphase congression and anaphase chromosome separation if introduced prior to metaphase; 3. the centrosomes in this mammal are maternally inherited; 4. centrioles appear late in embryogenesis and the oocyte cytoplasm has a centriole disassembly factor; and 5. the microtubules involved in pronuclear union are post- translationally detyrosinated and acetylated, and these modifications are correlated with the generation of differing classes of stable microtubules. This research will advance knowledge about the structural changes leading to genomic union, a central step in reproduction. By addressing fundamental questions regarding the union of the parental genomes at fertilization, this research may contribute to new approaches for treating infertility, designing contraceptive strategies and for avoiding or screening for birth defects.

Assisted Reproduction Technology; Assisted Reproductive Technology; Award; Baboons; CRISP; Cell Locomotion; Cell Migration; Cell Movement; Cellular Migration; Computer Retrieval of Information on Scientific Projects Database; Fertility/Fertilization; Fertilization; Funding; Grant; ICSI; Institution; Intracytoplasmic Sperm Injections; Investigators; Macaca; Macaque; Mediating; Methods; Modeling; Motility; Motility, Cellular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Papio; Papios; Research; Research Personnel; Research Resources; Researchers; Resources; Risk; Safety; Savanna Baboons; Source; Sperm Injections, Intracytoplasmic; Technology, Assisted Reproductive; United States National Institutes of Health; cell motility; egg; non-human primate; nonhuman primate

The High Voltage Electron Microscope Facility is a Biotechnology Resource of the Biotechnology Resources Branch of N.I.H. and is available to bio-medical researchers for ultrastructural studies where use of a high voltage electron microscope can provide new information. Investigations are directed especially towards analysis of relatively large and complex three-dimensional cell structures either in intact cells or isolated organelles, such as isolated flagella, chromosomes and Golgi complexes, or in sections up to 1 or 2 microns thick. The cold stage will undergo further development and modification. Freezing and cold sample handling procedures have been developed for viewing frozen sections, whole specimens, isolated cell components and aqueous suspensions. An improved type of image intensifier and digital image/memory processor has been installed and successfully used. This is part of a program to improve resolution and performance at high magnification in order to make possible high resolution studies of macromolecular crystals. The H.V.E.M. is particularly well suited to high resolution studies of this type because of its greater penetrating power, its greater depth of field and because of its greater specimen chamber size, one sufficiently large to permit introduction of ancillary equipment such a tilting/cooling stages which are necessary to preserve and orient these biological structures. Methods are being developed for immunocytochemical labeling of cell structures such as cytoskeletal elements, both in cell whole mounts and in thick sections.

This Americas Program award will support a laboratory-workshop course to familiarize young scientists with modern approaches for exploring structural events at the cell and molecular level. Using advanced imaging technologies on marine and mammalian gametes and embryos, dynamic events in the cell cycle and during development will be observed. The application of imaging equipment in the broad field of cell and developmental biology and the biological insights derived from such technology will continue to lead to important discoveries. This workshop, organized by Dr. Gerald Schatten of the University of Wisconsin, Madison and Dr. Claudio Barros, of the Catholic University, Chile, will take place in the Marine Laboratory of the Catholic University of the North, Coquimbo, Chile. The participants, young scientists on a postdoctoral level, will come from countries in South and Central America and from other countries. The program should result in a very successful exchange of scientific/technological information which could lead to long term interactions between the participants. The areas of development and reproduction are of general concern to scientists around the globe and sharing knowledge and technique in this area is essential. The global scientific community will most certainly benefit from this interaction.

This laboratory-based workshop and international symposium is jointly organized by Prof. Gerald Schatten of the Dept. of Zoology, University of Wisconsin at Madison, and Prof. Barry Fabian, of the Dept. of Zoology, University of Witwatersrand, in Johannesburg, South Africa. The event, to take place January 5-24, 1996, at the U. of Witwatersrand, has manifold objectives: to enhance the level of cell and developmental biology teaching and research; to encourage and give visibility to the work of scientists who are members of under-represented groups; to strengthen Africa regional networking through the formation of the African Society of Cell and Developmental Biology; and to stimulate collaborative research projects between US and African scientists. The organizers have marshalled the loan of demonstration advanced imaging equipment and personnel from an international array of manufacturers. By bringing advanced instrumentation and expertise to a central location, the workshop will enable some two dozen qualified young investigators from various parts of Africa to perform experiments themselves, become familiar with the techniques and capabilities of the technology, and, it is hoped, inspire them to pass on these tools to their own trainees. NSF is supporting the participation of five scientist-instructors (four from the U.S. and one from Kenya), plus five African trainee-participants, at the pre-doctoral, post-doctoral and young-investigator level, from throughout Africa. This activity will strengthen the capacity of young researchers in Africa to use advanced techniques; promote experimentation in novel animal and plant species; and stimulate beneficial networking between a center of excellence in South Africa and institutions in other African countries.

Flawless sperm functioning is essential for the successful completion of fertilization in humans. This functioning includes activities of the sperm prior to its meeting the oocyte, sperm-oocyte plasma membrane fusion, and the cytoplasmic events mediated by the sperm nucleus and centrosome that conclude the fertilization processCthe merging of the parental genomes in the activated zygote. Defects in sperm behavior at any stage result in the inability to complete fertilization, and quantitative analyses of these defects have been developed into assays for male infertility. These assays determine the extent of aberrations in sperm number, morphology, motility, the ability to undergo the acrosome reaction, the release of acrosome enzymes, and penetration assays into zona-free hamster oocytes or isolated zonae. This application investigating idiopathic male infertility studies the microtubule organizing capacity of the human sperm centrosome. To investigate the exte nt t o which defects in the human sperm centrosome are causes of male infertility, questions are posed in four specific aims using sperm from fertile and infertile men. Aim #1 Is centrin, a centrosomal protein, found in human sperm, and does its concentration vary predictably in sperm donated from men of differing fertility? Aim #2. Does the binding of maternal centrosomal proteins, especially ?-tubulin, vary predictably in sperm from men of differing fertility? Aim #3. Are there differences in the ability of sperm from men with varying fertility to nucleate and direct the assembly of microtubule-containing asters in extracts from Xenopus eggs? Aim #4. Will the sperm asters formed in pronucleate-stage mammalian oocytes, inseminated with sperm from men of varying fertility, predictably vary in their size and microtubule density? FUNDING NIH R01 HD32887, HD18185 PUBLICATIONS Schatten G, Hewitson L, Simerly C, Sutovsky P, Huszar G. Cell and molecular biological challenges of ICSI A.R.T. before science? J Law Med Ethics 26:29-37, 1998. Luetjens CM, Payne CJ, Schatten G. Are chromosomes in sperm nuclei non-randomly positioned? J Reprod Fertil 22:82, 1998. Hewitson L, Simerly C, Sutovsky P, Dominko T, Schatten G. Molecular reconstitution and inheritance of the centrosome during fertilization Implications for fertility. In Gametes Development and Function (A Lauria, F Gandolfi, G Enne, L Gianaroli, eds). Serono Symposia, pp 371-392, 1998.

Successful fertilization in mammals requires the union of the sperm and egg nuclei within the activated oocyte cytoplasm. Cytoskeletal assembly within the oocyte is required for sperm penetration, pronuclear decondensation and opposition as well as chromosome segregation during meiosis and mitosis.

women's health; structural biology; animal tissue; reproductive system; mother /infant health care; growth factor; genome; Primates;

women's health; structural biology; animal tissue; reproductive system; mother /infant health care; growth factor; genome; Primates;

Fertilin, originally named PH-30, is a heterodimeric cell surface protein identified on guinea pig, rabbit and human spermatozoa. The alpha subunit has a 22 amino acid hydrophobic peptide that is known to act as a Afusion peptide@ during sperm-egg fusion. The mature fertilin beta subunit has an amino-terminal 90-93 amino acid domain that has sequence homology with a class of known integrin ligands (the snake venom disintegrins), and it is suggested that this domain plays a pivotal role in sperm-egg binding as an integrin-disintegrin interaction. Based on these observations, fertilin and the egg receptor for fertilin could be a potential target for contraception. Some scientists have explored the possibility of using purified fertilin as an antigen for immuno-contraception. Three male guinea pigs received injections of fertilin showing that 100% of the males were infertile. Several control males received injections of adjuvent without protein and none of them were inf erti le. Other studies showed that fertilin beta is expressed only in testis in humans. According to the previous work, we want to design a NH2 terminal 93 amino acid fragment of human fertilin beta (fertilin beta-NTP93) that utilizes the integrin-disintegrin binding domain as an antigen of vaccine and observe the inhibiting ability of sperm-egg binding by anti-human fertilin ?-NTP93 antibody in vitro. FUNDING Mellon Foundation-CONRAD Program (#MFG-97-34) PUBLICATIONS Moreno RD, Ramalho-Santos J, Sutovsky P, Chang E, Schatten G. Molecular membrane marker dynamics during rhesus spermatogenesis Acrosome biogenesis, Golgi apparatus and mitochondrial differentiation. Mol Biol Cell 9:66a, 1998. Ramalho-Santos J, Moreno RD, Sutovsky P, Wessel G, Schatten G. Membrane fusion and fertilization Implications for the presence of SNARE proteins on mammalian spermatozoa. Mol Biol Cell 9:332a, 1998.

OBJECTIVE: To explore the pattern of centrosome inheritance at fertilization and understand the motility and architectural changes necessary for the successful completion of fertilization in nonhuman primates. RESULTS Microtubule imaging has demonstrated that centrosome inheritance in nonhuman primates is paternal in origin, making the rhesus monkey an excellent model for examining human reproduction. To understand the motility and architectural changes necessary for the successful completion of fertilization, we have asked 1. What characterizes the organization of microtubules during fertili-zation in rhesus monkeys, and does it suggest the maternal or paternal inheritance of the centrosome? This information is key to understanding centrosome heredity, fundamental aspects of the motility leading to genomic union, and perhaps the evaluation and alleviation of infertility. 2. Is there evi-dence for a paternal centrosome contribution during polyspermy? These experiments will show whether polyspermy is lethal solely because of a genomic imbalance, or if supernumerary centro-somes are also problematic in primates. 3. Will parthenogenetically-activated oocytes organize bi-polar mitotic apparatus at the time of first division? If so, this would provide an indication that the oocyte contains all the necessary constituents for spindle formation, as well as provide information on the mode of centrosome inheritance. 4. What is the role of microtubules during rhesus fertilization? Does taxol treatment support the hypothesis that the sperm contributes the dominant, and perhaps sole, centrosome? Will zygotes recovering from microtubule inhibition form bipolar spindles in association with the male, but not female, pronucleus? We will address these questions through the use of microtubule inhibitors. 5. What are the dynamic properties of microtubules in maturing and mature rhesus oocytes? This knowledge may help clinicians evaluate at which stage of meiotic matur-ation human oocytes should be frozen for possible later fertilization. This research will advance the understanding of genomic union during fertilization in primates, a key step in reproduction. FUTURE DIRECTIONS We plan to explore microtubule dynamics during novel methods of assisted reproduction, such as intracytoplasmic sperm injection, in rhesus monkeys. KEY WORDS imaging, fertilization, motility and development, mitosis, cytoskeleton

Successful fertilization, completed when parental genomes unite within an activated oocyte, is critically important for efficient reproduction. Investigations on the microtubule configurations in bovine and porcine zygotes have lead to the discovery that the sperm contributes the centrosome during fertilization in these domestic species. In addition, we have demonstrated that sperm from breeding bulls vary predictably in the ability to organize microtubules after sperm incorporation, and that variations in sperm centrosomes affect the outcome of fertilization. The goals of this project are to characterize, at the molecular level, the relative parental contributions to the bovine zygote's centrosome during fertilization. We have posed eight questions in two aims. Aim 1. Is the recruitment of maternal molecules to the sperm centrosome essential for the completion of fertilization? 1.1. Do sperm asters in inseminated oocytes, but not in unincorporated sperm, contain maternal centrosomal molecules? 1.2. Does nuclear mitotic apparatus (NuMA) behave atypically during the first cell cycle by being accumulated at the centrosome during interphase? 1.3. Are microtubules essential for their recruitment to, or retention at, the zygote's centrosome? 1.4. Is recruitment and/or retention of maternal molecules essential for the nucleation, organization and/or functioning of the sperm astral microtubules? 1.5. Are -tubulin, NuMA, pericentrin, and PCM-1 and PCM-2 found at the meiotic spindle poles in unfertilized oocytes? Aim 2. What is the molecular basis of the motility which unites the male and female pronuclei? 2.1. Will sperm astral microtubules, nucleated by Xenopus and/or bovine sperm centrosomes and assembling in cell-free extracts, capture female pronuclei and effect the pronuclear motions? 2.2. Can these cell-free sperm asters capture and translocate female pronuclei isolated from fertilized or activated bovine oocytes? 2.3. Are microtubule dynamics required for pronuclear migration in this cell-free model, and will antibodies to motors, centrosomal molecules, or nuclear envelope proteins interfere with this motility? The information obtained during these experiments will add to our fundamental knowledge about the molecular mechanisms leading to pronuclear apposition and genomic union during fertilization in cows, a key step in the reproductive process of all animals. Furthermore, it holds promise for novel assays to test male reproductive function in potential breeding studs. This research may also uncover a new site at which to assay for compounds affecting fertility, with applications for fertility assessment, reproductive management, and reproductive toxicology, as well as aid in developing new approaches for improving reproductive efficiency in domestic species.

The recent and unanticipated successes in both cloning sheep using nuclei from adult fibroblasts and in solving many forms of infertility by intracytoplasmic sperm injection (ICSI) now forces questions as to whether a combination of these approaches will result in reliable and efficient applications for propagating valuable research models, especially non-human primates. Spontaneous mutations resulting in unique animals to investigate diseases (e.g., retinitis pigmentosa) are periodically discovered in non-human primate colonies, and the ability to propagate these animals is vital for future investigations. While electrofusion of blastomere nuclei with enucleated rhesus oocytes resulted in two offspring (one male and one female) in 1996, last year=s attempts have not been successful. Intracytoplasmic nuclear injection (ICNI) holds bright promise as an alternative approach for propagating identical non-human primates. This project seeks to sequentially perfect IC NI c apabilities and develop new animal resources by addressing four specific aims. 1. Is intracytoplasmic sperm injection an effective and efficient method for propagating rhesus monkeys? 2. Is round spermatid injection (ROSI) an effective method for propagation? 3. Is intracytoplasmic nuclear injection, using blastomere nuclei obtained from rhesus monkey embryos injected into unfertilized enucleated oocytes, an effective and efficient method of propagation? 4. Is intracytoplasmic nuclear injection, using nuclei obtained from somatic cells cultured from adult and fetal rhesus tissues and injected into unfertilized enucleated oocytes, an effective and efficient method of propagation? ICNI may well find fantastic uses as a reliable, routine approach for propagating invaluable research animal resources of vital importance for the entire biomedical research community. FUNDING NIH R24 RR13632, HD18185 PUBLICATIONS Dominko T, Hewitson L, Simerly C, Luetjens M, Sutovsky P, Takahashi D, Schatten G. What is transferred during nuclear transfer? Imaging of mitochondrial, DNA, and centrosome dynamics in nuclear transfer rhesus embryos. Biol Reprod 58:99, 1998. Hewitson L, Takahashi D, Dominko T, Simerly CR, Schatten G. Assessing intracytoplasmic sperm injection Fertilization, embryo development, embryo transfer and pregnancy in the rhesus monkey. Fertil Steril 70:S242, 1998. Hewitson L, Takahashi D, Dominko T, Simerly C, Schatten G. Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey. Hum Reprod 13:3449-3455, 1998.

OBJECTIVE: To investigate the extent to which defects in the human sperm centrosome are causes of male infertility. RESULTS Imaging techniques have demonstrated that human sperm can be induced to nucleate microtubules in vitro in a cell-free Xenopus egg extract, and human sperm of varying fertility differs in its ability to nucleate microtubules. Successful human fertilization depends on flawless sperm functioning. This includes the sperm's activities before it meets the oocyte, sperm-oocyte plasma membrane fusion, and the cytoplasmic events mediated by the sperm nucleus and centrosome that conclude fertilization the merging of the parental genomes in the activated zygote. Defects in sperm behavior at any stage result in the inability to complete fertilization, and quantitative analyses of these defects have been developed into assays for male infertility. These assays determine the extent of aberrations in sperm number, morphology, motility, the ability to undergo the acrosome reaction, the release of acrosome enzymes, and penetration assays into zona-free hamster oocytes or isolated zonae. This application investigating idiopathic male infertility studies the microtubule organizing capacity of the human sperm centrosome. Our aims were as follows 1 Is centrin, a centrosomal protein, found in human sperm, and do its concentrations vary predictably in sperm donated from men of differing fertility? 2. Does the binding of maternal centrosomal proteins, especially -tubulin, vary predictably in sperm from men of differing fertility? 3. Are there differences in the ability of sperm from men with varying fertility to nucleate and direct the assembly of microtubule-containing asters in extracts from Xenopus eggs? 4. Will the sperm asters formed in pronucleate-stage mammalian oocytes, inseminated with sperm from men of varying fertility, predictably vary in their size and microtubule density? FUTURE DIRECTIONS We plan to explore the possibility of developing a simple assay to test for sperm microtubule nucleating ability in vitro. KEY WORDS human, centrosome, sperm, motility, cell-free extract, Xenopus

The deliberate creation of human zygotes for biomedical research is unlikely to ever be free of religious, ethical, moral, political and financial complexities. Nevertheless, a detailed understanding of the cellular and molecular events during human fertilization is essential for diagnosing and treating infertility, as well as for developing new approaches for managing reproduction. To address these issues, this project poses fifteen questions in three specific aims to understand egg-mediated motility during fertilization in clinically relevant systems. I. Is the recruitment of maternal molecules to the sperm centrosome essential for the completion of fertilization, and does paternal centrin sever the sperm tail from the head and midpiece in a Ca++-dependent manner? II. What is the molecular basis of the motility which unites the male and female pronuclei? III. How do centrosomal and microtubule molecules behave during intracytoplasmic sperm injection (ICSI) and round spermatid injection/round spermatid nuclear injection (ROSI/ROSNI), and are there clinically useful diagnostic probes for assessing and/or predicting the normalcy of fertilization? Tested hypotheses include that Maternal -tubulin is attracted to, but not retained at, the sperm centrosome by microtubules and is itself responsible for microtubule nucleation; nuclear mitotic apparatus (NuMA) is also attracted to the zygote's centrosome by microtubules and is responsible for organizing the -tubulin nucleated microtubules into the radially symmetric sperm aster; Pericentrin, PCM-1 and PCM-2 are components of the zygote centrosome's substructure; Paternal centrin severs the sperm tail microtubules; Pronuclear motility in a cell-free model will demonstrate motor protein and/or microtubule dynamics in effecting genomic union; Microtubule and centrosome dynamics differ during ICSI and ROSI/ROSNI; Some oocytes discarded by infertility clinics as either unfertilized or fertilization failures display centrosome defects. Together these experiments will advance clinically-relevant knowledge about genomic union during fertilization, providing information on the biological events occurring during the new and well accepted, but poorly understood, methods of assisted reproductive technologies (ART). By exploring defects in interactions of the gametes, as well as subtle aspects of sperm function not detected with existing methods, the results may translate into applications for the diagnosis and treatment of infertility. RELEVENT PUBLICATIONS

This application is an (R13) grant requests partial support for the newly inaugurated advanced training course entitled: FRONTIERS IN REPRODUCTION: MOLECULAR AND CELLULAR CONCEPTS AND APPLICATIONS (Fir) held annually from late May to July 4 at the MBL in Woods Hole. This course is an intensive six-week laboratory and lecture designed for young independent scientists and physicians and advanced post-doctoral fellows seeking comprehensive and sophistical training in research strategies and state-of-the-art methods on cellular, immunological and molecular biological approaches. Participants receive authoritative conceptual training in all aspects of reproduction ranging from gamete interactions, early development, implantation, parturition, immunology, and the molecular basis of reproductive hormonal regulations, in a unique physical and intellectual environment not duplicated int he Nation's Universities, Medical Schools or Research Institutes. The course consists of daily lectures from resident faculty and other invited speakers, discussions and informal seminars, laboratory exercises and demonstrations and one-on-one tutorials. The five modules are integrated to provide broad exposure to emerging problems: Module 1> Gametogenesis and Fertilization; 2. Embryogenesis and Stem Cells; 3. Reproductive Immunology; 4. Signal Transduction and Gene Expression; and 5: Genetic Manipulations in Reproductive Endocrinology. The course concludes with the 'Frontiers in Reproduction Symposium,' which draws international attention and honors "Pioneer in Reproduction." Drs. Anne MacLaren and Ralph Brinster were the first honorees. The extreme competition ensures that the selected participants represent an outstanding cadre of the most promising M.D./M.D..-Ph.D. and Ph.D. scientists, and also ensures that members of under-represented groups as well represented. In the first year of the proposal, it is limited to 16 participants, 16 in year -02, 20 in years -03 and -04 and then 24 participants in years -5. FIR is under the direction of Joan Hunt, Kelly Mayo, and Gerald Schatten, who serve under the guidance of the FIR Advisory Board composed of Charles Lockwood, Michael McClure, Michael Nelson, and Jerry Strauss. The inaugural offering was quite successful, and with the strong support marshalled from the biomedical community, there is every indication that it will continue to prosper and inspire the next generation of leaders in academic reproductive medicine.

Research has implicated two factors which act specifically on endothelial cells as key players in the development and maintenance of the vasculature vascular endothelial growth factors (VEGF), and angiopoietins (ANG). Studies were designed to determine (1) if VEGF or ANG expression increases in the primate follicle during the periovulatory interval in vivo, and whether their expression is regulated by the gonadotropin surge or local (i.e., steroids) factors, and (2) if VEGF or ANG is expressed in the primate corpus luteum throughout its lifespan in the menstrual cycle. First, female rhesus monkeys received gonadotropins to promote the development of multiple preovulatory follicles. Then animals received an ovulatory gonadotropin (hCG) bolus alone or with the steroid synthesis inhibitor Trilostane, with and without progestin replacement. Follicular fluid and granulosa cells were collected before (0 hr) and 12, 24, or 36 hr after administering the hCG bolus. VEG F, ANG-1 and ANG-2 mRNAs were detected in granulosa cells at all timepoints. Whereas no significant changes in VEGF or ANG-2 mRNAs were observed, ANG-1 mRNA decreased from 0 to 12 hrs and then increased 30-fold by 36 hrs. Steroid ablation prevented the 36 hr rise in ANG-1 mRNA, which was partially restored by progestin replacement. Although VEGF mRNA levels were unchanged, VEGF levels in follicular fluid increased 6-fold between 0-12 hrs post-hCG and remained elevated at 36 hrs. VEGF levels were not altered by steroid ablation. Second, the corpus luteum was collected in the early, mid, mid-late, and late luteal phase of the cycle. Whereas no significant changes in VEGF or ANG-2 mRNAs were observed, ANG-1 mRNA increased from early-to-mid luteal phase and then remained elevated through late in the cycle. Although VEGF mRNA levels were unchanged, immunocytochemical staining to VEGF protein in luteal cells was intense from early-to-mid luteal phase but slight by late luteal phase. Thus, there is transcriptional and translational control of ANG-1 and VEGF, respectively, in the ovulatory follicle and in the corpus luteum during the menstrual cycle. Endothelial-specific growth factors may be critical for ovulation of the follicle and for the development and maintenance of the microvasculature in the corpus luteum in primates. FUNDING NIH HD22408, HD18185 PUBLICATIONS Hazzard TM, Molskness TA, Chaffin CL, Stouffer RL. Changes in expression of VEGF and angiopoietin-1 in the periovulatory follicle of the rhesus macaque. In International Symposium on Ovulation Evolving and Clinical Concepts (held in Salt Lake City, UT, September 24-27, 1998), p 4 (abstract #4).

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This Americas Program award will support travel and related expenses for seven participants, including three US senior scientists and four US and LA post- and pre-doctoral students, to attend a workshop on the newest advances in developmental genetics, to be held in Santiago, Chile, January 11-23, 1999, in conjunction with an international symposium. Organizers are Dr. Gerald Schatten, Oregon Regional Primate Research Center, and Dr.Roberto Mayor, University of Chile. The objective of the workshop is to enhance the level of cell and developmental biology teaching and research through lectures, direct laboratory experimentation, and formal and informal discussions. Several innovative technologies, including advanced imaging, will be discussed to familiarize the participants with modern approaches for exploring structural events at the cell and molecular level.

The workshop will help foster the career development of promising students, both from the US and Latin America, and will help stimulate new collaborations among US and South American scientists.


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We recently determined that continual administration of low doses of the antiprogestin ZK 137 316 (Schering AG) to adult, female rhesus monkeys permits ovarian/menstrual cyclicity, but disrupts the growth and differentiation of the uterine endometrium. To determine if these (or other) changes in the reproductive tract are sufficient to prevent fertility (intrauterine pregnancy), female macaques (n=10/group) were treated daily with vehicle (controls), 0.01 or 0.03 mg ZK 137 316/kg body weight before and during 5 months of continual cohabitation with males of proven fertility. Mating behavior was confirmed by visual observation, and ejaculation occurred judged by the detection of sperm in the vagina. A cumulative pregnancy rate of 90% (9 of 10) was observed in controls. Of the animals receiving 0.01 mg/kg, 40% (4 of 10) conceived, with all conceptions occurring in the first 2 months of pairing. No pregnancies (0%, 0 of 10) occurred in the 0.03 mg/kg group. Although timely menstrual cyclicity occurred more frequently in the 0.01 mg/kg- than the 0.03 mg/kg-treated animals, ovarian cyclicity as judged from the observation of a corpus luteum on the ovaries was observed in both groups. Blood indices of circulating ions, liver function and blood cell constituents revealed no significant effects of chronic treatment. General toxicologic evaluation of major organs and tissues, either at a gross or microscopic level, did not identify any treatment-related changes. Thus, chronic administration of low dose antiprogestin prevents pregnancy in nonhuman primates, while permitting ovarian cyclicity and a high incidence of regular menstruation. Future studies are planned to determine the site(s) and mechanism(s) of contraceptive action of low-dose antiprogestin, e.g., in the uterus to prevent gamete transport or implantation. Also, experiments will examine whether the anti-fertility effects of ZK 137 316 are reversible. If so, this novel regimen of antiprogestin can be pursued as a potential contraceptive for women. RELEVANT PUBLICATIONS Zelinski-Wooten MB, Slayden OD, Chwalisz K, Hess DL, Brenner RM, Stouffer RL. Chronic treatment of female rhesus monkeys with low doses of the antiprogestin ZK 137 316 Establishment of a regimen that permits normal menstrual cyclicity. Hum Reprod, in press. Slayden OD, Zelinski-Wooten MB, Chwalisz K, Stouffer RL, Brenner RM. Chronic treatment of cycling rhesus monkeys with low doses of the antiprogestin ZK 137 316 Morphometric assessment of the uterus and oviduct. Hum Reprod, in press.

Since progesterone (P) receptors are present in luteal cells of rhesus monkeys, we are testing the hypothesis that actions attributed to luteinizing hormone (LH) and chorionic gonadotropin (CG) are mediated, at least in part, by P produced in response to these gonadotropins. Studies currently focus on whether P mediates LH actions leading to rupture and luteinization of the follicle. Experiments were designed (1) to elucidate the dynamics of cellular and molecular events during the Aperiovulatory interval,@ and (2) to determine if inhibition of steroid synthesis and/or progestin replacement alters these events in the presence of an ovulatory gonadotropin stimulus. Female rhesus monkeys received gonadotropins to promote the development of multiple preovulatory follicles. Then animals received an ovulatory gonadotropin (hCG) bolus alone or with the steroid synthesis inhibitor Trilostane, with and without progestin replacement. Granulosa cells were collected before (0 hr), or 12, 14 and 36 hr after administering the hCG bolus. First, the hCG bolus rapidly (? 12 hr) stimulated increases in mRNAs encoding the P receptor (PR) and androgen receptor (AR), whereas mRNAs for estrogen receptor (ER) ? and ? did not change or decreased, respectively, within 36 hr. Steroid ablation prevented the rise in PR mRNA and reduced ER? mRNA; these effects were reversed by progestin replacement. Second, levels of mRNAs encoding interstitial collagenase (matrix metalloproteinase-1, MMP-1), gelatinase-A (MMP-2), matrilysin (MMP-7), as well as their endogenous tissue inhibitors (TIMP-1 and -2), increased within 12 hr, whereas gelatinase B (MMP-9) increased by 36 hr post-hCG. Steroid ablation decreased mRNA levels for MMP-1, -7, and -9, as well as TIMP-1 and -2, and progestin replacement restored MMP-1 and TIMP-1 mRNA levels. Thus, (1) there may be diverse roles for progesterone, as well as androgens and estrogens, via receptor-mediated pathways, throughout the periovulatory interval, and (2) one of the primary actions of progesterone, and possibly other steroids, may be to promote the expression of specific collagenolytic proteases and their inhibitors. This research should provide novel insight into mechanisms leading to certain types of infertility and suggest unique methods for controlling fertility by preventing ovulation in women. FUNDING NIH HD20869, HD18185 PUBLICATIONS Chaffin CL, Hess DL, Stouffer RL. Dynamics of progesterone (P) and progesterone receptor (PR) expression in the primate follicle during the periovulatory interval. In Endocrine Society Program and Abstracts 80th Annual Meeting (held in New Orleans, LA, June 24-27, 1998), p 184 (abstract #P1-306). Chaffin CL, Stouffer RL. Progesterone (P) promotes the expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the primate periovulatory follicle. In Society for the Study of Reproduction Program and Abstracts 31st Annual Meeting (held in College Station, TX, August 8-11, 1998), p 110 (abstract #126). Duffy DM, Stouffer RL, Chaffin CL. Noncoordinated expression of estrogen receptor ? and ? in the ovulatory follicle and the corpus luteum of the rhesus monkey. In Society for the Study of Reproduction Program and Abstracts 31st Annual Meeting (held in College Station, TX, August 8-11, 1998), p 108 (abstract #118). Chaffin CL, Stouffer RL. Dynamics and steroid regulation of oocyte maturation in macaque follicles during the periovulatory interval. In International Symposium on Ovulation Evolving and Clinical Concepts (held in Salt Lake City, UT, September 24-27, 1998), p 33 (abstract #1).

Depression and anxiety adversely affect millions of individuals, causing significant mortality and morbidity. Clinical studies over several decades suggest that these behavioral disorders emanate from both genetic factors and exposure to environmental factors. The interrelationships between genetic predisposition to these disorders, and how environmental and social factors modify genetic predisposition is critical to the development of clinical strategies for preventing these devastating disorders. For this purpose we are using a monkey model, to examine how social stress during early development triggers anxious and depressive behaviors and how social support modulates the development of these behaviors. Infant Japanese macaques are being tested early in life for temperament and growth hormone responsiveness. Clinical studies show a correlation between inhibited temperament and blunted GH responsiveness, and the anxiety and depressive disorders. Infant monkey s ar e then separated from their mothers and reared in a normal social colony environment. Behavioral studies are being used to determine if infants who receive greater social support have a decreased probability of developing anxious and depressive behaviors, compared to infants who receive less social support. FUNDING Center-supported project PUBLICATIONS None

Ovulation is a major target for the control of fertility and infertility in humans and animals. Several neuropeptides and neurotransmitters, i.e., neuropeptide Y, b-endorphin, norepinephrine (NE), gamma aminobutyric acid and acetylcholine, influence the preovulatory secretion pattern of gonadotropin-releasing hormone (GnRH). The magnitude of gene transcription/translation in the neurons that express these chemicals may identify how and where they modulate GnRH release. To test this hypothesis, we utilized the expression of the early oncogene, cfos, as a marker to locate the onset of gene transcriptional events in brainstem and hypothalamus during the preovulatory GnRH surge. Coitus in female rabbits, like estrogen in monkeys, induces NE/GnRH release at a predicted time after the stimulus. Thus, rabbits were killed and their brains fixed by paraformaldehyde infusion at times before and 15-, 30-, 60-, 90- and 120-min after coitus for cfos mRNA quantitation using in s itu hybridization. Within 30 minutes after coitus, several noradrenergic-associated brainstem nuclei, lateral tegmentum (A1), nucleus of the solitary tract (A2) and locus coeruleus (A6) exhibited enhanced cfos mRNA expression in females. Also, two nuclei in the medulla oblongata that send projections to A6, the nucleus of praepositus hypoglossy and the paragigantocellular nucleus, showed increased c-fos expression only in coitally activated females. The septum also showed heavier 35S-cfos label by 30 min after coitus than septum from unmated control females. In the hypothalamus the anteroventral periventricular area (AVPV), arcuate, paraventricular and medial preoptic area showed enhanced cfos mRNA expression between 15 and 60 minutes postmating. The AVPV showed the most concentrated cfos expression. Whether these times after coitus and transcriptional changes in cfos signals can be utilized for predictions of where and how coital stimulation triggers NE/GnRH release is unclear, but these data are encouraging. Because a massive release of hypothalamic NE and a two-fold increase in brainstem NE (A6) gene expression occurred around the time of the estrogen-induced hypothalamic GnRH release, we suggest that the signaling mechanisms in rabbits may be useful in understanding those in primates. FUNDING NIH HD30316, HD18185 PUBLICATIONS None

DESCRIPTION (provided by applicant): Life begins in utero typically. Prenatal environmental exposures, coupled with each zygote's genetics and epigenetic imprints, trace a life history path of health outcomes. The central theme of the Pittsburgh Specialized Center of Research on Sex and Gender Factors Affecting Women's Health is the "Genetic And Environmental Causes Of Adverse Pregnancy Outcomes." This major, but still under investigated, priority for women's health urgently requires multidisciplinary research both for the health of adult women and also for the health of developing fetuses and infants. For women, recurrent spontaneous abortions (RSA) are devastating. We have identified transgenerational transmission of a 'miscarriage gene' that may be an extreme example of deviant genomic imprinting. The implications for fetal outcomes are also of great importance, since in utero development of the fetus, both female and male, establishes the very foundation of the infant, adolescent, and adult. Three research projects along with two research cores and an administrative core are proposed under the directorship of Gerald Schatten, Ph.D. and Sarah Berga, M.D., Clinical Director. Project I, "Pregnancy Loss: Genomic Imprinting and Skewed X-Inactivation" (J. Richard Chaillet, MD, Ph.D., P.1), investigates DNA methylation defects in mice responsible for genomic imprinting as well as skewed X-chromosome inactivation, responsible for RSA in women, Project 11, "Epigenetic, Genetic and Environmental Regulation of Pregnancy in Primates" (Gerald Schatten, Ph.D., P.I. and Steve Caritis, MD, Co-P.I.), imaging primate pregnancies and inflammatory responses, addresses sex-specific genomic imprints in genetically controlled and experimentally-manipulated pregnancies. Project III (Julie DeLoia, Ph.D., P.I.), "Maternal and Fetal Consequences of Tobacco Smoke Exposure", analyzes the consequences of smoke exposure in pregnant women and in murine models to understand the interaction of 'genetic variants that jeopardize fetal development and pregnancy. The Imaging Core A performs noninvasive micro-PET and MRI imaging with specific probes, including transgenic MR11PET reporters. The Pregnancy Core B establishes and maintains pregnancies through conventional and artificial reproductive technologies (ART) in non-human primates and mice. The Administrative Core fosters intra- and inter-SCOR cooperation to facilitate and accelerate basic and clinical research. The multi-disciplinary, interactive, and collegial environments the new Pittsburgh Development Center at Magee-Women's Research Institute right on the contiguous campuses of the University of Pittsburgh and Carnegie-Mellon University, and brings together accomplished teams of clinical and basic investigators inspiring innovations in non-invasive imaging of pregnancy outcomes. Taken together, this comprehensive investigation will answer major women's health problems regarding the dynamic interplay among fetal and maternal genetics, sex-specific genomic imprints and consequences of our first environmental exposures. As such, it is an appropriate and complementary contributor to the ORWH's new SCOR program.

DESCRIPTION (provided by applicant): The MWRI is a multidisciplinary research institute dedicated to women and infants' health. The majority of its 70 members are from three Departments of the Medical School of the University of Pittsburgh: Obstetrics, Gynecology, and Reproductive Sciences; Pediatrics; and Pathology. The remaining members of the Institute are from other departments in the Medical, Nursing, and Pharmacy Schools and from the Graduate School of Public Health of the University of Pittsburgh. Membership includes investigators with M.D., Ph.D. or both, who have expertise in clinical or basic research as well as epidemiology or health services research. The Institute emphasizes research and training in interactive, innovative, and translational research. On-going projects include studies in amenorrhea, pre-eclampsia, pregnancy loss, and sexually transmitted diseases. Currently, the investigators of the MWRI utilize mice, rats, and non-human primates as animal models of human disease. The PDC of the MWRI is actively researching contemporary topics of national importance including: the pluripotency of embryonic stem cells using non-human primates; the feasibility of a variety of transgenic approaches for disease modeling using primates; the outcome of Assisted Reproductive Technologies (ART) in macaque models; and the feasibility of cloning in non-human primates, either for production of identical research models or to evaluate 'therapeutic' cloning. All the non-human primate research performed at the MWRI is performed under the auspices of the PDC. This application requests funds to: 1) provide the PDC and MWRI vivarium with state-of-the-art non-human primate enclosures that exceed all new United States Department of Agriculture (USDA) directives for psychological enrichment; and 2) modernize the PDC and MWRI vivarium with a steam sterilizer.

[unreadable] DESCRIPTION (provided by applicant): This is the first renewal application of our T15 award to offer a Short-Term Course in hESC research. Frontiers in Human Embryonic Stem Cells (FrHESC) is a dynamic advanced training course that offers a fresh series of daily lecturers on emerging concepts, followed by extended discussion, laboratory research, technologically intense workshops and informal seminars over a three week period (June 11-28, 2006).The Course is directed toward young independent scientists, physicians and established investigators. It will address critical concerns in stem cell biomedicine and is organized around four modules: Module A. hESC Growth, Propagation, and Pluripotency; Module B. Transgenesis, Cloning and Epigenesis in hESCs; Module C. Realizing the Clinical Promise of hESCs and Module D. Continuing Education. The Frontiers in Human Embryonic Stem CellsTraining Course Program is directed by a Board of Scientific Counselors, whose members are renowned leaders in hESC research, proactively consider the national needs for this undertaking and solicit the intellectual and financial resources to realize the promise of Human Embryonic Stem Cell Research. The FrHESC training course itself is organized by two Course Directors, Gerald Schatten and Roger Pedersen, who are experts in molecular cell biology, gametogenesis, preimplantation embryogenesis and stem cell research. The laboratory and lecture faculty selected to participate in the course are internationally acknowledged leaders in their fields. The purpose of this training course is to provide comprehensive and sophisticated training in research strategies and state-of-the-art methods on cellular, molecular and genetic approaches for advancing the Frontiers in Human Embryonic Stem Cell Research. To do so, the course will provide hands-on experience using a panel of some of the following NIH- approved hESC cell lines: BG01, BG02, SA01, SA02, HES2, HESS, HES4, MI01, UC06, WA01, WA07, WA09. These lines, which are currently in use at the Pittsburgh Development Center, were chosen because FrhESC faculty has experience with these lines. Using lines that faculty has extensive experience with and that are already in place on the course site will ensure the success of the hands-on training for the students. [unreadable] [unreadable] [unreadable]

This research project builds on existing transgenic and cloning expertise using nonhuman primates (NHP) and NIH-approved human embryonic stem cells (hES cells) to develop novel Alzheimer disease models. We propose generate transgenic AD-models in hES cells, as well as non-human primate embryonic stem cells (nhpES cells), using lentiviral vectors (LV: Aim I) and mammalian artificial chromosomes (MAC: Aim II). The ES cells let us perform differentiation into AD neural tissues in vitro by determining amyloid overexpression in h/nhpES differentiated into neuroprogenitor cells (NP) in vitro, in three-dimensional embryoid body cultures (EB) and in xenografts into SCID mice. The cell lines, embryoid bodies and xenografts generated will be characterized for amyloid overexpression, plaque formation and neurofibrillary tangles. Aim III, investigated exclusively in nonhuman primates in accordance with US Federal guidelines, marries AD-transgenesis with cloning using both somatic cell nuclear transfer (SCNT) or embryonic stem cell nuclear transfer (ESNT) for the purpose of deriving nonhuman primate embryonic stem cells (NT-nhpES cells), to be tested in vitro, in xenografts and after cell transplantation to determine whether undifferentiated NT-nhpES or differentiated NT-nhpES neuroprogenitors are tolerated by the host monkey. By year five these experiments will provide proof of principle that transgenic AD-primate embryos can be generated through NT eventually leading to the live births of NHP-AD offspring. Overall, this interdisciplinary research project develops novel in vitro and the proof of principle for preclinical AD models.

This Program Project addresses among the most important questions in stem cell medicine and biology through responsible studies on the potentials of pluripotent stem cells from humans (hESC) and nonhuman primates (nhpESC) during development in vitro and in utero, and after transplantation in vivo. Conceptually, the overarching themes are the molecular foundations of ESC and Embryonic Germ [EG] cell properties of Pluripotency, Genomic Imprinting, and Differentiation, under the directions of Gerald Schatten, PhD (PD; Project I PI), Roger Pedersen, PhD (co-PD;Project II PI) and Peter Donovan, PhD (co-PD;Project III PI). Each of the three Research Projects, along with three Technological Cores and an administrative one, address interrelated fundamental and pre-clinical problems. Proj I: "Imaging nhpES cells in vitro, in chimerae, and after transplantation." (G Schatten, PI) investigates the developmental potentials of nhpESC and nhpES derived after nuclear transfer (NTnhpES) by examining chimerae and genomic imprinting. Proj II: "Differentiation and Epigenesis of Pluripotent Stem Cells" (R. Pedersen, PI) examines the earliest postimplantation embryos, pluripotent cells and their progeny for imprint normalcy, and to determine differentiation stability. Proj III: "Derivation and Safety Testing of Non-Human Primate Embryonic Germ Cell Lines" (P. Donovan, PI) investigates primordial germ cells in vivo, derives nhpEG and compares their developmental potentials with ESCs, while ANALYZING genomic imprinting in utero and in vitro during reproduction. Imaging Core A (E. Ahrens, Core Director) non-invasively images in utero development, ESC/EGs in vitro, and tracks transplanted nhp/hESC with specific probes in vivo. Primate Core B (L. Hewitson, CD) provides NHP embryos, maintains pregnancies, evaluates, and performs transplants. ADMIN Core C fosters communication, dissemination, and coordination within this P01 and throughout NIH's stem cell research communities by research planning, real-time data transfer, conferences, and scientific exchanges, as well as our advanced lab course 'Frontiers in Human Embryonic Stem Cell Research.'Stem Cell Core D maintains, preserves, and quality controls new and existing nhpES/EG cell lines, distributing them to the projects and freely to other academic investigators. Complementing NIH's new Stem Cell Centers, our multidisciplinary team will make major contributions towards swiftly and accurately addressing key questions whose answers are the essential foundation for stem cell clinical studies.

Abdomen; Abdominal; Animal Model; Animal Models and Related Studies; Autism; Autism, Early Infantile; Autism, Infantile; Autistic Disorder; Baboons; CRISP; Cardiac Diseases; Cardiac Disorders; Celioscopy; Cell division; Cloning, Embryo; Computer Retrieval of Information on Scientific Projects Database; Derivation; Derivation procedure; Diabetes Mellitus; Diagnosis, Ultrasound; Diagnostic Laparoscopy; Disease; Disorder; Drugs; ES cell; Echography; Echotomography; Embryo; Embryo Cloning; Embryonic; Female; Flushing; Flushings; Freezing; Funding; Genital System, Female, Uterus; Grant; Heart Diseases; Hormonal; Idiopathic Parkinson Disease; Immune; Institution; Invasive; Investigators; Kanner's Syndrome; Laboratories; Laparoscopy; Lewy Body Parkinson Disease; Medical Imaging, Ultrasound; Medication; Methods; Modeling; Mother Cells; NIH; National Institutes of Health; National Institutes of Health (U.S.); Normalcy; Normalities; Operation; Operative Procedures; Operative Surgical Procedures; Papio; Papios; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsons disease; Partner in relationship; Peritoneoscopy; Pharmaceutic Preparations; Pharmaceutical Preparations; Primary Parkinsonism; Production; Progenitor Cells; Protocol; Protocols documentation; Range; Research; Research Personnel; Research Resources; Researchers; Resources; Safety; Savanna Baboons; Source; Sperm; Spermatozoa; Staging; Stem cells; Surgical; Surgical Interventions; Surgical Procedure; Testing; Therapeutic; Time; Ultrasonic Imaging; Ultrasonogram; Ultrasonography; Ultrasound Test; Ultrasound, Medical; United States National Institutes of Health; Uterus; diabetes; diagnostic ultrasound; disease/disorder; drug/agent; egg; embryonic stem cell; hESC; heart disorder; human ES cell; human ESC; human embryonic stem cell; mate; model organism; non-human primate; nonhuman primate; sonogram; sonography; sound measurement; sperm cell; stem cell of embryonic origin; surgery; ultrasound; ultrasound imaging; ultrasound scanning; womb; zoosperm

Project I investigates embryonic stem cell pluripotency directly through the generation of chimeric primate blastocysts, fetuses and offspring with nonhuman primate ES cells (nhpES) - including nhpES derived from blastocysts generated from nuclear transfer (NTnhpES). Through sophisticated non-invasive imaging, this project responsibly answers crucial questions about: primate ES pluripotency during development (aim I);primate ES cell stability and utility after NT (aim II);genomic imprinting in NHPs to solve clinically urgent concerns regarding ART epigenetic consequences; and primate ES fate after transplantation (aim IV). Nine questions are posed in four Specific Aims: Aim I. To dynamically image nhpES cell contributions in developing primate chimerae: 1.1. Will nhpES cells contribute to chimeric blastocysts, fetuses and healthy offspring? 1.2.With tetraploid embryos (4N), will ES cells contribute primarily to the inner cell mass in chimeric blastocysts, fetus and offspring? Primate chimera are generated in four ways, and the fate of each chimera is followed in vitro during preimplantation development, determining the cellular contributions of the ES and tetraploids to the expanded blastocyst stage after differentially labeling ES or embryos with GFP-transgenes, as well as in utero during fetal development and in the offspring. Aim II. Are ES cells derived after nuclear transfer (NTnhpES cells) developmentally restricted? Two questions are posed: 2.1. Are NTnhpES cell lines stable? 2.2 Will NTnhpES cells contribute to chimeric fetuses and offspring? Aim III. Do Genomic Imprints in NHP Embryos, Fetuses, Placentae, Amniotic Cells and Offspring Differ between ART and NT-Chimerae versus Natural Matings? 3.1. What is the level of DNA methylation in embryos, fetuses, placentae, and offspring after ART, NT, or natural matings? 3.2. What are the parental expressions of selected imprinted genes during NHP preimplantation development? 3.3. What is the DNA methylation status of specific genes in rhesus preimplantation embryos? Aim IV. Dynamic imaging of ES cell fates after transplantation. 4.1. Are NT-derived nhp-ESCs tolerated when transplanted back into the female from which both the somatic cell and the oocyte were obtained? 4.2 Are allogenic ES cells rejected like allografts or do their pre-implantation origins confer immunological privilege? This aim will investigate the tolerance of nhpES in allografts and autografts. Taken together, this project will provide crucial information regarding ES cell pluripotency in nonhuman primates and evaluate the safety of stem cell transplantation in nonhuman primates.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. For this protocol, macaques will be hormonally stimulated with super-ovulatory drugs to produce oocytes that will be collected via laparotomy. These oocytes will be fertilized in the laboratory using fresh or frozen sperm collected by rectal probe electro-ejaculation. The collected gametes will be used for fertilization in the laboratory by intracytoplasmic sperm injection and the embryos cultured to the blastocyst stage. Once reaching this stage, several methods will be employed to attempt to derive embryonic stem cells. In addition, some oocytes may undergo somatic cell nuclear transfer to form cloned embryos. These embryos will also be cultured to the blastocyst stage in order to derive stem cells. Long term, we anticipate that this research will enable us to optimize methods for the derivation of macaque embryonic stem cells from both fertilized and SCNT embryos. However, for this pilot project, our goal is to simply obtain data on cyno follicular stimulation and determine whether or not cynos may be an ideal animal model for future work. Any established cell lines will undergo rigorous testing to determine their pluripotency and normalcy. While human embryonic stem cells may have incredible therapeutic value for treating a wide range of disorders such as Parkinson's diabetes, autism, heart disease, Alzheimer's etc., rigorous testing in animal models is necessary before any such trials can begin. Macaques provide a valuable non-human primate model in which to determine the safety and efficacy of immune-matched stem cells.

DESCRIPTION (provided by applicant): Pluripotent stem cells and regenerative medicine are rapidly emerging fields with enormous biomedical implications across the entire spectrum of NIH's clinical, translational, biomedical and behavioral portfolios. This new R13 application seeks five years of sponsorship for an advanced training course entitled: Frontiers in Stem Cells and Regeneration (FrSCR) to be held annually at the MBL in Woods Hole under the Co- Directorship of Gerald Schatten (Pittsburgh) and Ken Muneoka (Tulane). FrSCR developed from our currently funded T15 award to offer Short-Term Courses in Human Embryonic Stem Cell research protocols, which we offered successfully 7 times during the past 6 years. FrSCR builds on the significant strengths and successes of the existing course, while expanded to incorporate important biomedical concepts in regeneration and pluripotency. This course continues to enable participating trainees to start successful research programs in the fields of pluripotent stem cells (PSC) and regenerative medicine, including multipotent adult stem cells (ASC). We intend to give the trainees the necessary knowledge of laboratory techniques, career mentoring and ethical legal societal impact of PSC research to greatly enhance their successful entry into this field. In order to achieve this goal, we propose the following specific aims: 1. Provide training in the detailed methods for experimentation with regeneration and stem cells, including PSC and ASC, including culture methods, characterization, directed differentiation, and conceptual underpinnings of regeneration. This will include responsible conduct of research. 2. Provide trainees with specific opportunities to discuss career planning and collaboration with senior scientists to foster career development. 3. Educate trainees on the legal, ethical, and regulatory landscape in which regenerative medicine research occurs, so that trainees begin their research programs in the most ethical and responsible manner avoiding pitfalls and delays. FrSCR is a dynamic and evolving entity that each year offers a fresh series of daily lectures on emerging concepts, followed by extended discussions, laboratory research, technologically intense workshops and informal seminars over a ten-day period. The course is directed towards established investigators as well as advanced fellows and newly independent scientists who are committed to fundamental, translational or eventual clinical research studies. The course addresses major current problems, followed by critical discussions and laboratory experiments in which advanced new techniques are presented to explore these problems. Thus, this course will provide a significant benefit to biomedical research through the training of new investigators in the field of stem cells and regeneration. PUBLIC HEALTH RELEVANCE: Pluripotent stem cells and regenerative medicine hold enormous promises for understanding the root causes of diseases as well as designing innovative strategies for treating disabilities and other disorders. The field is hampered by a lack of highly trained scientists with expertise in stem cell technological methods and the conceptual foundations of regeneration. The Frontiers in Stem Cells and Regeneration Course at the MBL will contribute to advances in the field by bringing together experts in the field as faculty and course trainees in an advanced, hands-on training course and thus contribute to the next generation of highly trained stem cell scientists.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There major goal of this project is to understand the underlying biology of nuclear transfer, with the ultimate goal of furthering the understanding of embryonic stem cells and to perhaps produce a cloned non-human primate offspring. In order to achieve this, oocytes will be collected from baboons and then manipulated in various ways in the lab to produce cloned and/or control embryos. The majority of these embryos will be retained in the laboratory for use in in vitro studies. However, a portion of the embryos will be transferred into recipient females in order to establish pregnancies.

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. Our overall objectives are to understand the role of microRNAs during pluripotency, differentiation and pathology in cynomolgus macaques as an accessible, important and feasible model. This pilot project complements other on-going studies on miRNA regulation in pluripotent and differentiated stem cells in vitro as well as parallel studies on miRNA antagonist administration into mouse lungs. The complexity of the lung suggests that lung development involves the expression of many genes. Today, one can use microarrays to quickly study gene expression in the both the lung as well as its associated pathologies. However, most of our information relies on rodent models. This study will attempt to bridge the gap between rodent and human development by utilizing macaques as a research model. Micro RNAs (miRNAs) are being shown to be involved in many cellular processes such as cell cycle regulation, development timing, signal transduction, tissue differentiation and disease. miRNAs have also been proposed to regulate pluripotency and differentiation in vitro and in vivo. One such miRNA, let-7d, is highly expressed in lung cells in patients with Idiopathic Pulmonary Fibrosis (IPF). Recently, we have conducted a number of in vivo studies in mice, in which we have injected a short RNA sequence (antagomir;purchased from Dharmacon - part of Thermo Fisher Scientific Inc.) consists of a short RNA sequence that binds to the miRNA and causes it to undergo degradation. The antagomir was injected intra tracheal to mice lungs. We found an increase in the levels of mesenchymal markers in the lung, indicating that the antagomirs not only entered the lung epithelia, but could cause the alteration of gene expression in the lung. In this study, we propose to administer this let-7 antagomir into one lung of a monkey with the other as a control in which a scrambled miRNA is delivered to determine if the same process occurs in the NHP model as in the mouse. The oligo is very small, and should regionalize to the small airways very quickly. Although definitive studies have not been done to determine exactly how quickly the oligo is regionalized, this is not an anticipated problem. Additionally, we propose to explore the intravenous administration (IV) vs. intra-bronchial. The significance of such experiments is not only to study the ontology of disease in a model closely related to the human, but also as a step in developing treatment for an incurable disease and the molecular mechanisms of normal development.

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. Rectal probe electro-ejaculation of male baboons has proven reliable for producing fresh, high quality sperm samples for investigative use in assisted reproductive technologies (ART) studies. In order to obtain baboon sperm samples for developmental reproductive studies and stem cell research, assigned male baboons provided semen samples via rectal probe electro-ejaculation for sperm isolation. Five identified areas for the use of baboon sperm include: 1. In vitro fertilizations of oocytes collected via follicular aspirations from hormonally stimulated female baboons. (Sperm required day of aspiration procedures;intra-species fertilizations) 2. In vitro fertilizations of oocytes from unplanned necropsy ovaries. (Sperm required 48hr from oocyte isolations on necropsy date;intra &inter-species fertilizations) 3. Alternative sperm source for fertilization studies in either macaques (rhesus;cynomolgus) or chimpanzee oocytes when neither macaque nor chimpanzee sperm is unattainable on day of oocyte retrieval. (Sperm required day of oocyte collections;inter-species fertilizations) 4. Cryopreservation and banking of sperm in liquid nitrogen. 5. Fresh sperm collections for research investigations (i.e. testing new antibodies [immunocytochemistry];biochemistry studies;etc). The requested baboons will be allowed rest periods between ejaculation procedures as mandated by the current facility standard operating procedure and monitored by a veterinarian. For males that successfully produce stem cell lines, we may request 2-3 skin samples (~5mm biopsy punch), 3-5mls of whole blood in clot tube and/or 3-5mls of whole blood in EDTA tube for genotyping or other genetic comparisons between the male and the established cell lines.

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. Through responsible embryonic stem cell research in closely related species such chimpanzees, we hope to work towards cellular based therapies for many disorders including diabetes, Alzheimer's, Parkinson's, and cerebral palsy just to name a few. The overall goal of this pilot study is to derive and fully characterize stable cultures of chimpanzee embryonic stem cells (cESC). In order to do this, female chimps will be visually monitored in order to determine the stage of their menstrual cycle. Once at the proper stage, the female will be hormonally stimulated with a combination of follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG). This hormonal stimulation regimen increases the number of mature oocytes that will be produced during the animal's menstrual cycle. Once stimulated, oocytes will be harvested using a technique known as trans-vaginal, ultrasound guided oocyte aspiration or TVA. This non-invasive procedure utilizes a special ultrasound probe that is placed into the vaginal vault of the animal and allows for the visualization of ovarian follicles. Once follicles are visualized, a needle is extended from the ultrasound probe, through the vaginal wall and into the follicle. Suction is applied and the contents of the follicle, including the oocyte, are collected into a sterile tube. The procedure is repeated until all follicles are harvested. After all follicles are harvested, the collected aspirates will be taken to the laboratory, where they will be cleaned and searched for the oocytes. The oocytes will be isolated into a culture dish and separated based on their quality and maturity. On average, in rhesus and baboons, we obtain approximately 30 oocytes per stimulation of which 60% are typically mature/usable. Once the oocytes reach full maturity, they will be fertilized using chimp sperm collected the same day via rectal probe electroejaculation. After fertilization, the embryos will be cultured in the laboratory until they reach the blastocyst stage. At this point, immunosurgery will be performed on each embryo in order to isolate the inner cell mass. The isolated inner cell mass will then be cultured on a layer of feeder cells where it will, hopefully, continue its growth and form a stable colony of undifferentiated embryonic stem cells.

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. For this protocol, cynomolgus macaques will be hormonally stimulated with superovulatory drugs to produce oocytes that will be collected via laparotomy. These oocytes will be used for basic scientific investigations on fertilization and embryonic development after fertilization (ICSI) or somatic cell nuclear transfer to produce cloned embryos. Fertilization in the lab is accomplished using fresh sperm collected by rectal probe electro-ejaculation. Some early fertilized zygotes and/or cloned constructs will be fixed and imaged after staining with probes to detect cellular structures within the cell that are necessary to complete the fertilization or cloning process. Other embryos will be cultured and used for stem cell production. These cells are important and have the potential of treating a wide range of disorders such as Parkinson's diabetes, autism, heart disease, Alzheimer's etc.

DESCRIPTION (provided by applicant): This new application seeks NCI sponsorship for our innovative multidisciplinary advanced R25 Cancer Education Grant Program (CEGP) in FRONTIERS IN STEM CELLS IN CANCER (FriSC2) to train promising physician-scientists and other scientists from predominately underrepresented communities in sophisticated technologies using pluripotent stem cells for clinically relevant discoveries in cancer;it is prepared in response to PAR-08-120. FriSC2 offers dynamic advanced training courses that provide a fresh series of daily lecturers on emerging concepts, followed by extended discussion, laboratory research, technologically intense workshops and informal seminars over week-long periods (first offering March 14-19, 2011 at Howard), alongside ongoing research opportunities and mentoring. The Course is directed toward young independent scientists, physicians and established investigators. The primary aim is the development of two complementary short courses (entry-level lab demonstrations to up to 125 trainees, followed by an intense advanced laboratory-based training for up to 16 participants) to educate and update cancer researchers on the implications of stem cells for mechanistic discoveries and designing improved strategies for treating cancer, and then, through pilot research projects and on-going mentoring, to help launch and sustain their independent research careers on the clinical and translational aspects of cancer and stem cells. This CEGP also provides an innovative educational opportunity to motivate biomedical trainees to pursue scientific careers in cancer research. The target audience is young and established faculty already involved in cancer investigations, as well as advanced postdoctoral fellows eager to learn about the newest findings and controversies regarding the implications of stem cell discoveries for cancer. The courses are to be offered at predominantly minority institutions, including Howard University (years 1, 3 and 5) and Ponce School of Medicine and Health Science (years 2 and 4), under the overall directorship of Gerald Schatten, Ph.D. from the University of Pittsburgh School of Medicine along with Provost James Wyche from Howard University and Idhaliz Flores, Ph.D. from Ponce School of Medicine as Co-PIs. The three specific aims are to: I. Provide conceptual education and experimental training in the methods necessary for investigations of cancer and stem cells;II. Provide participants with specific opportunities to discuss career planning and collaboration with senior scientists in order to foster career development;and III. Educate participants on the ethical, social and legal implications (ELSI) and the regulatory landscape in which PSC research occurs, so that students begin their own research programs in the most ethical and responsible manner and avoid needless pitfalls and delays. The purpose of this training course is to provide comprehensive and sophisticated training in research strategies and state-of-the-art methods on cellular, molecular and genetic approaches for advancing the Frontiers in Stem Cells in Cancer Research. PUBLIC HEALTH RELEVANCE: Stem cell discoveries are increasingly seen as having major implications for understanding the underlying causes of many cancers, promising better treatments and even vaccines. This proposal seeks R25 sponsorship to offer annual Cancer Education short-courses as well as limited support for subsequent pilot research projects and on-going mentoring on the FRONTIERS IN STEM CELLS IN CANCER (FriSC2). These courses are to be offered at Howard University (years 1, 3 and 5) and the Ponce School of Medicine and Health Science (years 2 and 4), which are predominantly African-American and Hispanic-American universities, respectively. We are optimistic that this will greatly enhance the diversity of young scientists entering the field of cancer research.

DESCRIPTION (provided by applicant): This new application seeks NICHD Continuing Education Training Program (T15) sponsorship for an innovative advanced program entitled 'Rehabilitative and Regenerative Medicine for Minority Health and Health Disparities ([RMH]2D) to train and mentor promising physician-scientists and other researchers from predominately underrepresented communities in sophisticated technologies in rehabilitation research. [RMH]2D (pronounced 'remedy') offers dynamic training that provides a fresh series of daily lecturers on emerging concepts, extended discussion, laboratory research, workshops and informal seminars over week-long periods, followed by an annual symposium dinner at a scientific meeting/congress. The primary aim is the development of an intense laboratory-based training for 16 newly independent investigators per year to educate them on regeneration research strategies for CNS and skeletal-muscular rehabilitation, and to help launch and sustain their careers. The courses are offered at Howard (years 1), Morehouse (year 2) and Meharry (year 3), under the overall directorship of Gerald Schatten, Ph.D., from Pittsburgh along with Howard's Provost James Wyche, Ph.D., Winston Thompson, Ph.D. from Morehouse and Ayman Al-Hendy, M.D., Ph.D. from Meharry. The four aims are to: I. Provide conceptual education and experimental training in the sophisticated methods focused on diseases prevalent in African-American communities; II. Foster candid and meaningful conversations among all course participants regarding current barriers to the recruitment and retention of the most talented researchers to achieve full diversification of NIH's research workforce; III. Educate participants on minority health disparities in research, the ethical, social and legal implications (ELSI), and the regulatory landscape in which [RMH]2D investigations occur, so that trainees may begin their own research programs in the most ethical and responsible manner and avoid needless pitfalls and delays; IV. Encourage and empower the course participants through the most fragile stages of their careers as well as monitor and track their achievements comprehensively and longitudinally to ensure that these precious funds can be demonstrated to be wise, cost-effective and fruitful investments. Overall, the purpose of this NICHD Continuing Education Training Program is to provide comprehensive and sophisticated training and state-of-the-art methods on bioengineering, cellular, molecular and genetic approaches for advancing the Frontiers in Rehabilitative and Regenerative Medicine for rectifying Minority Health and Health Disparities. PUBLIC HEALTH RELEVANCE: Discoveries in regenerative medicine are having major implications for both understanding the underlying causes of many disorders which send patients, especially those from under-represented groups, into rehabilitation, and breakthroughs with stem cells, tissue engineering and epigenetics hold bright promises for superior treatments, and perhaps even cures, for conditions ranging from post-traumatic stress disorder, autism, stroke, mental illnesses, neurodegenerative diseases like Parkinson's, muscle wasting, and even the ravages of aging. This proposal seeks T15 sponsorship to offer annual NICHD Continuing Education Training Programs on Rehabilitative and Regenerative Medicine for Minority Health and Health Disparities ([RMH]2D): A Frontiers Advanced Training Course. These courses are to be offered at Howard University (year 1), the Morehouse School of Medicine (year 2) and Meharry Medical College (year 3), which are predominantly African-American Universities. The PIs are optimistic that this will greatly enhance the diversity of new scientists entering the fields of regenerative and rehabilitative research.

DESCRIPTION (provided by applicant): This R25 research education application entitled: 'Frontiers in Aging and Regenerative Medicine (FrARR),' responsive to PAR-12-016, trains and mentors promising junior and senior undergraduates from predominately underrepresented communities in sophisticated aging research, and encourages and supports them as they embark on graduate studies. FrARR offers: first, dynamic training courses, then nurtured Mentor-Trainee partnerships enhanced by guidance from Senior Mentors and peer-peer mentoring, enriched by annual symposia and scientific retreats, with quantitative tracking of career trajectories. The courses are offered at HBCU institutions, i.e. Xavier University (years 1 & 4), Morehouse (years 2 & 5) and Meharry (year 3), under the overall directorship of Gerald Schatten (Pittsburgh) and S. Michael Jazwinski (Tulane), with Shubha Kale Ireland (Xavier), Winston Thompson (Morehouse), and Ayman Al-Hendy (Meharry). Our Advisory Board of Scientific Counselors, composed of acclaimed experts in the fields of aging, regeneration and health disparities, oversee this initiative. The five specific aims are: I. Engendering the enthusiasm, passions and commitments to scientific careers embraced by the faculty provides intellectual and emotional underpinnings for supporting undergraduate trainees as they complete their MSTEM degrees. This is accomplished via conceptual education and laboratory training, focused on diseases and disorders relevant to aging and African- American communities. Didactic training includes: A. Biology of Aging and Stem Cell Models; B. Neurobiology of Aging and Alzheimer's disease; C. Muscle and Bone Aging; D. Reproductive Aging; and E. Epigenetics and Environmental Modulators of Aging, along with complementary labs. II. Fostering candid regarding current barriers to the recruitment and retention of our most talented researchers and training in responsible conduct of research, ethical, legal and societal implications, and especially problems of minority health disparities in research. III. Motivate, Enable and Sustain Trainees as they transition to professional careers, with mentored research projects and on-going career advice. Enhance their growing interests by their presentations at the Annual Reunion Symposia at Woods Hole contemporaneously with the MBL's Aging Course. Course alumni provide peer-peer mentorship. IV. Encouraging and empowering trainees and alumni by guiding them through the graduate application and matriculation processes so that they are rapidly and smoothly able to initiate doctoral studies in aging research and regenerative medicine. V. Evaluate, Improve and Monitor FrARR's strengths and avoid weaknesses with quantitative independent mechanisms, track trainees' achievements comprehensively and longitudinally, to ensure that these precious funds can be quantitatively demonstrated as wise, cost-effective and fruitful investments. In summary, our overall goals are to provide comprehensive sophisticated training in research strategies and career launching for underrepresented trainees to advance the scientific workforce pioneering the Frontiers in Aging and Regenerative Medicine.