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,รพ 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) 435-0714.

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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