MOUSE SPERM CRYOPRESERVATION Release Date: October 15, 1998 RFA: HD-98-012 P.T. National Institute of Child Health and Human Development National Center for Research Resources Letter of Intent Receipt Date: December 4, 1998 Application Receipt Date: January 12, 1999 PURPOSE The National Institute of Child Health and Human Development (NICHD) and the National Center for Research Resources (NCRR) invite applications from investigators willing to participate in a multisite National Cooperative Program on Mouse Sperm Cryopreservation utilizing the cooperative agreement (U01) mechanism. The specific goal of this cooperative Program will be to establish a facile, reliable and standardized protocol for mouse sperm cryopreservation and mouse strain reconstitution so that frozen sperm banks can be used as a major, cost-effective, form of repository for the thousands of genetically altered mouse strains that will be produced in the Mouse Genome Project and related efforts. Presently, the cryopreservation and strain reconstitution technology for mouse sperm is only adequate to allow the sperm of some outbred and hybrid mouse strains to be frozen, thawed and competent to fertilize eggs in vitro (IVF) that yield live and healthy offspring after embryo transfer. This strain-specificity may be circumvented by using a combination of frozen or freeze-dried sperm with intracytoplasmic sperm injection (ICSI), in which frozen or freeze-dried sperm or sperm heads are directly injected into the egg cytoplasm. Thus, to address the specific goal of this Program, protocols must be submitted that will utilize sperm cryopreservation or freeze-drying followed by fertilization through IVF and ICSI, culminating in the production of live and healthy offspring at an efficient rate. In addition, the cooperative group is expected to examine long-term outcomes of these protocols such as adult phenotypes, including fertility, pathology and behavior. Ultimately, the resultant strains must be pathogen-free. The protocol developed through this Program will become an essential part of a larger effort on the structural and functional genomics of mice that is designed to map and sequence the mouse genome as well as to produce and characterize thousands of genetically altered strains of mice. These mice will be used for biomedical and behavioral research on the full range of public health topics. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS-led national activity for setting priority areas. This Request for Applications (RFA), Mouse Sperm Cryopreservation, is related to the priority area of family planning. Potential applicants may obtain a copy of "Healthy People 2000" at http://www.crisny.org/health/us/health7.html. ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic for-profit and non-profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. Racial/ethnic minority individuals, persons with disabilities, and women are encouraged to apply as Principal Investigators. MECHANISM OF SUPPORT This RFA will use the National Institutes of Health (NIH) cooperative agreement (U01) award mechanism. The major difference between a cooperative agreement and a research grant is that there will be substantial programmatic/scientific involvement of the NICHD staff Research Coordinator above and beyond the levels required by the traditional program management of grants. Specifically, an NICHD staff member will cooperate with the Principal Investigators as a partner in the research and serve as Research Coordinator. The Research Coordinator will assist the recipient(s) in a cooperative way but without assuming direction, prime responsibility or a dominant role in the activity. A cooperative agreement is an assistance mechanism whose purpose is to support, stimulate and expedite the recipient"s activities through a partnership role. All awardees in this Program will agree to accept the participatory and cooperative nature of the group process. Details of the responsibilities, relationships, and governance of the activities to be funded under the cooperative agreements to be awarded for this Program are discussed later in this document under Terms and Conditions of the Award. The anticipated award date is August 1, 1999. Because the nature and scope of the research proposed in response to this RFA may vary, it is anticipated that the size of the awards will vary. Numbers of awards and levels of support will depend upon the receipt of a sufficient number of applications of high scientific and technical merit. Although the Program is provided for in the financial plans of the NICHD and the NCRR, awards pursuant to this RFA are contingent upon the availability of funds for this purpose. This RFA is a one-time solicitation for the purpose of the present competition. At this time, the NICHD and NCRR have not determined whether or how this Program will be continued beyond the present solicitation. Any future determination in this regard will be publicly announced. FUNDS AVAILABLE NICHD and NCRR intend to commit approximately $2 million (including direct and indirect costs) for the first year of the entire cooperative program, with NICHD contributing $1.5 million and NCRR, $500,000. It is also anticipated that up to four awards will be made with an award period of three years. RESEARCH OBJECTIVES Background Stunning technology has developed over the last eighteen years that enables scientists to make precise additions, deletions or alterations to the genetic material of mammals in order to study the influence of genes on developmental processes, and on the function of specific adult tissues in normal and pathological states as well as the construction of animal models of specific human diseases. These genetic changes have been maintained in vivo by propagating the genetically altered organisms in breeding colonies or preserving them as banks of frozen embryos. The mouse has been the ideal species, because of its well-characterized genetic makeup, short generation time and ease of use in the laboratory. However, maintaining live mice in breeding colonies and freezing embryos is expensive, labor-intensive, and/or technically demanding. An NIH conference on "Priority Setting for Mouse Genomics and Genetics Resources, was held in March, 1998. A major conclusion of the conference was that the cryopreservation of mouse sperm should be the primary mode of storing and disseminating the thousands of genetically altered mice that will be produced as a result of the Mouse Genome Project and related efforts. This is a scientifically attractive and cost-effective strategy. However, the viability of mouse sperm after cryopreservation is poor compared with that of other mammalian species, such as the bovine, and to a degree, the human. For this reason, it was suggested at the conference that there should be an immediate emphasis on technology development for improving the cryopreservation of mouse sperm combined with facile methods for mouse strain reconstitution. A panel was convened by NICHD and NCRR in June, 1998 to discuss the state of the art of mouse sperm cryopreservation, the use of ICSI and further technology development that might be needed in order to enhance the utility of this approach for mouse germ plasm preservation. The panel concluded that indeed the strain-specificities exist, that more studies need to be done on the genetic factors that influence freezing and strain reconstitution success, and that efforts need to be made to streamline protocols for sperm freezing followed by ICSI. The panel also noted that sperm freezing per se is not difficult, the recovery is the problem. Thus, for the outbred and hybrid strains in which some success has been achieved using sperm cryopreservation and IVF, there is no need to use ICSI. Indeed, some transgenic and knockout strains are now preserved only in the form of frozen sperm. For inbred strains, however, and perhaps for the thousands of new strains that will be produced through ethylnitrosourea (ENU) mutagenesis in the Mouse Genome Project, it is anticipated that there will many cases in which conventional technology will not work. For these, the combination of sperm cryopreservation or freeze-drying followed by ICSI would seem to be the most direct method for establishing sperm banks, circumventing numerous strain- specific barriers to normal fertilization. While the use of ICSI in human clinics has been successful and widespread, it has only recently been demonstrated in the mouse. Long-term studies have not been done in either the human or the mouse on such topics as fertility, pathology, behavior or other phenotypic characteristics after ICSI. This point must be underlined since the selection process for which humans and which mouse strains are selected for ICSI will often be based upon sperm immotility or failure to successfully fertilize eggs in vitro. Sperm immotility or IVF failure after freezing may be due to gene or chromosome damage. This possibility of damage reinforces the need for intensive follow-up of offspring produced through ICSI. Fortuitously, the last ten years of intensive research has provided vast improvements in the in vitro fertilization of mouse eggs and the culture of preimplantation embryos for many mammalian species, including the mouse. These advances in the efficiency of fertilization and embryo culture, then, provide an important part of the protocol such that the emphasis of this Program can be focussed upon the freezing or freeze-drying of sperm, improvement of the ICSI technology and then improving the success rates of subsequent embryo development after embryo transfer into live and healthy offspring, with long-term studies of the phenotypes obtained, including fertility, pathology and behavior. The eventual benefit to the field and to the scientific community is anticipated to be the optimization and standardization of the procedures necessary to generate a mouse sperm repository for preservation of genetically altered animals. Objectives and scope The overriding goal of this cooperative program will be to establish a facile, reliable and standardized protocol for mouse sperm cryopreservation and mouse strain reconstitution that will be useful for all strains, including those produced by ENU mutagenesis, transgenesis, gene targeting, knockouts, etc. This will necessarily include testing the competence of cryopreserved or freeze-dried sperm to fertilize eggs successfully using IVF and ICSI and to produce live and healthy offspring. The improved efficiency and standardization of the ICSI technology will be an important feature of this Program. Likewise, the long-term follow-ups of mice produced through frozen or freeze-dried sperm followed by ICSI will be very important since ICSI, as well as mouse sperm freezing, is quite new so there is little data on potential harmful effects. Thus, the effects of the freezing-ICSI protocol must also be monitored carefully at the gene and chromosome levels. Strains selected for study must include several genetically altered strains, including those which are produced largely through ENU mutagenesis, but also through transgenesis, gene targeting, knockouts, etc. One of the strains studied in the Program should be the 129 SvEvtac strain since it is favorable for gene targeting and many knockout mice and embryonic stem cell lines have been made with that strain owing to the stability of cell lines and the ease of maintaining inbred lines. Frozen sperm should first be tested for the effects of these genetic alterations on the recovery of viable and fully competent sperm. Those strains would then be tested using conventional IVF for a satisfactory level of recovery. For those strains in which recovery is unsatisfactory, then ICSI would be used. Ultimately, genetic effects upon sperm function discovered in this way will provide valuable tools for the study of the genetic control of sperm physiology, but that is beyond the scope of this Program. Also, the resultant cryopreserved strains must be pathogen- free. Since there is concern about the transmission of pathogens through sperm, screening procedures for pathogens should be included in the experimental protocols if possible. Newborns can be examined in order to assure the faithful passage of the desired genotype. Furthermore, offspring must be examined for adult phenotypes, fertility, pathology and behavior. This cooperative program will include the following special features: o The protocols will be approved, developed and/or modified by the Steering Committee through a consensus process (see SPECIAL REQUIREMENTS: 1. The Steering Committee), o All sites must have the capability to conduct fertilization experiments using IVF and/or ICSI, o All investigators must focus clearly on the establishment of a facile, reliable and standardized protocol for mouse sperm cryopreservation or freeze- drying followed by IVF and/or ICSI. The production of live and healthy offspring is a critical goal, some will study the longer term phenotypic effects, such as fertility, adult pathology, and behavior, o Approaches will be determined and prioritized, o Detailed experimental designs will be established in order to maximize chances of success and to facilitate statistical analysis, satisfactory levels of recovery will be determined, o Progress reports will be transmitted to the cooperating sites and NICHD, o Results will be analyzed and disseminated in a timely manner, o Meetings will be held at least twice a year to discuss progress and future plans. SPECIAL REQUIREMENTS The minimal requirements for applicants are as follows (See also REVIEW CONSIDERATIONS below): o Competent, experienced principal investigators who are committed to this problem and who are willing to cooperate with the other Principal Investigators and the NICHD Research Coordinator, o Access to properly managed animal colonies with breeding capabilities, o Access to genetically altered mice as described above, o Demonstrated capability and experience to produce and evaluate sufficient numbers of fertilizations, embryos, fetuses, live offspring or other endpoints for sound statistical analysis, o Experience with IVF and/or ICSI followed by preimplantation embryo culture and embryo transfer, o Excellent technical resources necessary for conduct of the experiments, and o Evidence of departmental and institutional support and commitment. Terms and Conditions of Award Cooperative agreements are assistance mechanisms subject to the same administrative requirements as grants. The special Terms and Conditions of Award are in addition to, not in lieu of, otherwise applicable OMB administrative guidelines, HHS, PHS, and NIH grant regulations, policies and procedures, with particular emphasis on HHS regulations at 42 CFR Part 74 and 92. The NIH Information for Management and Planning Analysis and Coordination (IMPAC) system and indirect cost award procedures will apply to cooperative agreement awards in the same manner as for grants. Business management aspects of these awards will be administered by the NICHD Grants Management Branch in accordance with HHS, PHS, and NIH grant administration requirements. The following terms and conditions of the cooperative agreement award, and details of the arbitration procedures pertaining to the scope and nature of the interaction between the NICHD and the participating awardees will be incorporated into the Notice of Grant Award and provided to the Principal Investigator as well as to the institutional official at the time of award. These procedures will be in addition to the customary programmatic and financial negotiations which occur in the administration of grants. 1. The Steering Committee The planning and implementation of the study will be done by a Steering Committee consisting of the Principal Investigators of each participating site and one NICHD staff member from the Reproductive Sciences Branch who will serve as Research Coordinator. One staff member from NCRR will serve as an ex officio member of the Steering Committee. Protocols, approaches and experimental designs will be approved, prioritized, developed and/or modified by the Steering Committee through a consensus process. These may be based upon protocols, approaches and designs approved during the initial review process but may include new protocols. The Steering Committee will meet at least twice a year. The purpose of these meetings will be to share scientific information, assess scientific progress, identify new research opportunities, exploit opportunities for collaboration within the Program and with outside scientists when appropriate, ensure the rapid dissemination of the findings, establish priorities that will facilitate the translation of basic research findings to the practical goals of this work, and to conduct other business of the Program. The Steering Committee will select a Chair for the meetings from among the Principal Investigators. 2. Awardee Responsibilities and Rights The primary responsibilities of the awardees are: o Determine experimental approaches o Design protocols o Conduct experiments o Analyze and interpret the results o Present results and plans at Steering Committee meetings o Publish results o Modify, delete or add protocols o Accept and participate in the cooperative nature of the group process. Awardees will retain custody of and primary rights to their data developed under the award, subject to Government rights of access consistent with current HHS, PHS, and NIH policies. 3. The degree of programmatic/scientific assistance by the NICHD staff Research Coordinator includes: o Participation in the development of optimal approaches and protocol designs (and adjustments of protocols and approaches when needed). The Research Coordinator will assist and facilitate the process, rather than directing it. o Assistance and review of all phases of the study to assure consistency of protocol compliance, to improve and strengthen cooperation between the sites, and to help redirect efforts, if necessary. In the event of disagreement among participants, the Research Coordinator will assist in forming an arbitration panel acceptable to participants (see below). o Participation in data analyses, interpretation and publication of study results. o Identification, jointly with awardees, of the need to modify or terminate a site if technical performance requirements are not met. 4. Arbitration When agreement between an awardee and NIH staff cannot be reached on scientific and/or programmatic issues that may arise after the award, an arbitration panel will be formed. The panel will consist of one person selected by the Principal Investigators, one person selected by NICHD staff, and a third person selected by these two members. The decision of the arbitration panel, by majority vote, will be binding. This special arbitration procedure in no way affects the right of an awardee to appeal an adverse action in accordance with PHS regulations at 42 CFR Part 50, Subpart D, and HHS regulations at 45 CFR Part 16. LETTER OF INTENT Prospective applicants are asked to submit, by December 4, 1998, a letter of intent that includes a descriptive title of the proposed research, the name, address, and telephone number of the Principal Investigator, the identities of other key personnel and participating institutions, and the number and title of the RFA in response to which the application may be submitted. Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, the information that it contains allows NICHD staff to estimate the potential review workload and avoid conflict of interest in the review. The letter of intent is to be sent to Dr. Richard J. Tasca at the address listed under INQUIRIES. APPLICATION PROCEDURES The research grant application form PHS 398 (rev. 5/95) is to be used in applying for these cooperative agreements. These forms are available at most institutional offices of sponsored research and from the Division of Extramural Outreach and Information Resources, National Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda MD 20892-7910, Email: grantsinfo@nih.gov, telephone: (301) 710-0267. Content of Applications Applications in response to this RFA should include: o A description of the capabilities of the site to meet or exceed the minimal requirements (see SPECIAL REQUIREMENTS above). o A proposed three-year research plan that should be the applicant"s perception of the study and of his/her cooperative role. This plan should demonstrate the applicant"s knowledge, ingenuity, practicality, and commitment to improving sperm cryopreservation and strain reconstitution. Budget The instructions for budget estimates provided with the Research Grant application form (PHS 398) should be followed. Eligible indirect costs will be awarded in the same manner as for research project grants (R01). Budgets will be reviewed on the basis of appropriateness for the work proposed. Allowable costs and policies governing the research grants programs of the NIH will prevail. In planning the budget section of the application each applicant should submit budget estimates for all three years including estimates of staffing needs. Since the final protocol(s) for this study will not be exactly known at the time of submission of the application, the budget request should be based on the plan proposed by the applicant and should reflect the scope of the project. The requested budget should not exceed approximately $500,000 total costs (direct plus indirect) per year. Include estimates for staffing needs, although it is expected that some modification will be needed once the final research protocol(s) have been developed. The budget must also include estimates of travel expenses for two meetings of two days each of the Steering Committee per year. In the first year, however, support should be requested for the principal investigators to travel to as many as two meetings in the first six months as required for initial planning activities including joint identification and specification of research topics, the development of protocols and the presentation of new results. The first planning meeting will be held in Bethesda, Maryland in August, 1999. The RFA label available in the PHS 398 (rev. 5/95) application form must be affixed to the bottom of the face page of the application. Failure to use this label could delay processing of the application such that it may not reach the review committee in time for review. In addition, the RFA title, Mouse Sperm Cryopreservation, and number, HD-98-012, must be typed on line 2 of the face page of the application form and the YES box must be checked. Submit a signed, typewritten original of the application, including the Checklist, and three signed photocopies, in one package to: CENTER FOR SCIENTIFIC REVIEW NATIONAL INSTITUTES OF HEALTH 6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710 BETHESDA, MD 20892-7710 BETHESDA, MD 20817 (for express/courier service) At the time of submission, two additional copies of the application must be sent to: Scott Andres, Ph.D. Division of Scientific Review National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 5E03 BETHESDA, MD 20892-7510 ROCKVILLE, MD 20852 (for express/courier service) Applications must be received by January 12, 1998. If an application is received after that date, it will be returned to the applicant without review. The Center for Scientific Review (CSR) will not accept any application in response to this RFA that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of substantial revisions of applications already reviewed, but such applications must include an introduction addressing the previous critique. REVIEW CONSIDERATIONS Upon receipt, applications will be reviewed for completeness by the CSR and for responsiveness to the RFA by NICHD staff. Incomplete and/or non- responsive applications will be returned to the applicant without further consideration. Applications that do not meet the minimum requirements (see SPECIAL REQUIREMENTS above) of this RFA will be judged non-responsive and will be returned to the applicant. Applications that are complete and responsive to the RFA will be evaluated for scientific and technical merit by an appropriate peer review group convened by NICHD, in accordance with the review criteria stated below. As part of the initial merit review, a process may be used by the initial review group in which applications will receive a written critique and undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of the applications under review, will be discussed, assigned a priority score, and receive a second level review by the National Advisory Child Health and Human Development Council (NACHHD) and by the National Advisory Research Resources Council (NARRC). Review Criteria The goals of NIH-supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. The reviewers will comment on the following aspects of the application in their written critiques in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals. Each of these criteria will be addressed and considered by the reviewers in assigning the overall score weighting them as appropriate for each application. Note that the application does not need to be strong in all categories to be judged likely to have a major scientific impact and thus deserve a high priority score. For example, an investigator may propose to carry out important work that by its nature is not innovative but is essential to move a field forward. 1. Qualifications, experience and commitment of key personnel o Scientific and administrative abilities of the Principal Investigator and other team members, as evidenced by pertinent publications. o Knowledge and experience in areas relevant to the conduct of the experiments proposed. o Commitment of time for the proposed study and stated willingness to work and collaborate with other sites and the NIH in the manner summarized in this RFA. 2. Protocols and Procedures o Appropriateness of the application to the objectives of the study as outlined in this RFA. o Scientific and technical merit of the proposal, including feasibility and approach. o Demonstration of cost-effectiveness through improved mouse sperm cryopreservation and strain reconstitution. 3. Facilities and Management o Adequacy of administrative and technical capabilities. o Adequacy of animal facilities and appropriateness of animal care management. o Institutional assurance to provide support to the study in such areas as fiscal administration, personnel management, space allocation, procurement, planning and budgeting. 4. Budgeting o Appropriateness of budget. 5. Animal Welfare, Biohazards. o The initial review group will also examine the provisions for the protection of animal subjects and the safety of the research environment. Schedule Letter of Intent Receipt Date: December 4, 1998 Application Receipt Date: January 12, 1999 NACHHD Council Review: June 1999 Earliest Award Date: August 1, 1999 AWARD CRITERIA The anticipated date of award is August 1, 1999. Applications recommended for funding by the NACHHD Council will be considered for award based upon scientific and technical merit as determined by peer review, program balance, including in this instance, sufficient compatibility of features to enhance the likelihood of a successful collaborative program, and availability of funds. INQUIRIES Inquiries concerning this RFA are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding scientific program issues and address the letter of intent to: Richard J. Tasca, Ph.D. Center for Population Research National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8B01 - MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 496-6515 FAX: (301) 496-0962 Email: rt34g@nih.gov Direct inquiries regarding fiscal matters to: Melinda Nelson Grants Management Branch National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8B17 - MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 496-5481 FAX: (301) 402-0915 Email: mn23z@nih.gov AUTHORITY AND REGULATIONS This Program is described in the Catalog of Federal Domestic Assistance No. 93.864, Population Research. Awards are made under authorization of the Public Health Service Act, Title IV,Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants policies and Federal Regulations 42 CFR 52 and 45 CFR Part 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. The PHS strongly encourages all grant and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people.

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