In Vitro Fertilization in Mice Using the MBCD‐GSH Protocol

Mo Guan1, Debora Bogani1, Susan Marschall2, Marcello Raspa3, Toru Takeo4, Naomi Nakagata4, Martin Fray1

1 Mary Lyon Centre, Medical Research Council, Harwell Science and Innovation Campus, Oxfordshire, 2 Institute of Experimental Genetics, Helmholtz Zentrum Muenchen—German Research Center for Environmental Health (GmbH), Neuherberg, 3 Consiglio Nazionale delle Ricerche (IBCN) CNR‐Campus International Development (EMMA‐INFRAFRONTIER‐IMPC), A. Buzzati‐Traverso Campus, Rome, 4 University of Kumamoto, Kumamoto
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140059
Online Posting Date:  June, 2014
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Abstract

Historically, timed mating of either naturally cycling or superovulated females has been the mainstay of pre‐implantation embryo production. However, rising cage costs and the rapid expansion of biomedical research programs has necessitated the development of high‐throughput approaches to mouse embryo production. In vitro fertilization (IVF) represents one such versatile tool offering many advantages to busy mouse facilities in terms of efficient use of space and resources. For example, strains can be taken off the shelf, frozen quickly as sperm, and recovered at a later date, small colonies can be rapidly expanded to meet demand, and IVF can be used to rescue strains that fail to breed or where the founder male is ill or has died suddenly. This article describes an IVF protocol currently used by many reproductive technologists to assist mouse biology programs. Curr. Protoc. Mouse Biol. 4:67‐83 © 2014 by John Wiley & Sons, Inc.

Keywords: cryopreservation; methyl‐beta‐cyclodextrin; mouse; oocytes; spermatozoa; IVF

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: MBCD‐GSH In Vitro Fertilization Adapted for Using Frozen/Thawed Sperm
  • Alternate Protocol 1: MBCD‐GSH In Vitro fertilization Protocol for Freshly Harvested Sperm
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: MBCD‐GSH In Vitro Fertilization Adapted for Using Frozen/Thawed Sperm

  Materials
  • Female mice for oocyte harvesting (between 3 and 4 weeks old)
  • Pregnant mare serum gonadotrophin (PMS) (e.g., National Hormone and Pituitary Programme)
  • Human chorionic gonadotrophin (hCG, e.g., Intervet)
  • TYH with 0.75 mM methyl‐beta‐cyclodextrin (MBCD) sperm pre‐incubation medium (see recipe)
  • Mineral oil (Sigma, cat. no. M8410)
  • Reduced glutathione (Sigma, cat. no. G4251)
  • IVF culture medium (e.g., high Ca2+ human tubal fluid, HTF, see recipe)
  • Liquid nitrogen (N 2)
  • Frozen sperm in gCPA (Takeo and Nakagata, , )
  • M2 medium (Sigma, cat. no. M7167)
  • KSOM (e.g., Millipore/Chemicon, cat. no. MR‐020P‐5F) plus AA
  • Embryo‐tested water (e.g., Sigma, cat. no. W1503)
  • 10‐μl, 200‐μl, and 1.0‐ml pipets plus tips (e.g., Gilson)
  • 60‐mm culture dishes (Falcon, cat. no. 351016)
  • 35‐mm culture dishes (Falcon, cat. no. 351008)
  • 37°C, 5% CO 2 humidified incubator
  • 10‐ml graduated pipets (e.g., Fisher)
  • Analytical balance
  • 1.5‐ml microcentrifuge tubes
  • 0.22‐μm syringe‐end filters
  • 37°C water bath
  • Paper tissues
  • 16‐G metal rod
  • Dissecting instruments (e.g., standard dissecting forceps, fine watchmakers forceps, and scissors)
  • Dissecting microscope (100 to 400× magnification)
  • 10‐μl wedge‐shaped pipet tip (Thistle Scientific, cat. no. AX‐T‐400‐L‐R‐S)
  • Aspirator assembly (Sigma, cat. no. A5177)
NOTE: The most critical component of a successful IVF system is the culture medium, which must be capable of supporting sperm capacitation, fertilization, embryo development, and overcoming any risk of the two‐cell block to embryo development. A number of excellent commercial preparations are available, such as human tubal fluid (Lonza Group, cat. no. BE02‐036F) or Cook medium (Cook, cat. no. K‐RVFE‐50). Alternatively, defined media can be produced in‐house according to published protocols. The following protocol assumes use of a modified, high Ca2+ human tubal fluid described by Takeo and Nakagata ( ). See the recipe in Reagents and Solutions.

Alternate Protocol 1: MBCD‐GSH In Vitro fertilization Protocol for Freshly Harvested Sperm

  Additional Materials (also see protocol 1Basic Protocol)
  • Male mice (at least 8 weeks of age)
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Figures

Videos

Literature Cited

Literature Cited
  Bath, M.L. 2011. Optimized cryopreservation of mouse sperm based on fertilization rate. J. Reprod. Devel. 57:92‐98.
  Brinster, R.L. and Biggers, J.D. 1965. In‐vitro fertilization of mouse ova within the explanted fallopian tube. J. Reprod. Fertil. 10:277‐279.
  Donahue, L.R., Hrabe de Angelis, M., Hagn, M., Franklin, C., Lloyd, K.C., Magnuson, T., McKerlie, C., Nakagata, N., Obata, Y., Read, S., Wurst, W., Horlein, A., and Davisson, M.T. 2012. Centralized mouse repositories. Mammalian genome. Offic. J. Internat. Mammal. Genome Soc. 23:559‐571.
  Liu, L., Nutter, L.M., Law, N., and McKerlie, C. 2009. Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice. JAALAS 48:39‐43.
  Nakagata, N. and Takeshima, T. 1993. Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains. Jikken dobutsu. Exp. Animals 42:317‐320.
  Nakagata, N., Okamoto, M., Ueda, O., and Suzuki, H. 1997. Positive effect of partial zona‐pellucida dissection on the in vitro fertilizing capacity of cryopreserved C57BL/6J transgenic mouse spermatozoa of low motility. Biol. Reprod. 57:1050‐1055.
  Nakagata, N., Takeo, T., Fukumoto, K., Kondo, T., Haruguchi, Y., Takeshita, Y., Nakamuta, Y., Matsunaga, H., Tsuchiyama, S., Ishizuka, Y., and Araki, K. 2013. Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization. Cryobiology 67:188‐192.
  Nakagata, N., Takeo, T., Fukumoto, K., Haruguchi, Y., Kondo, T., Takeshita, Y., Nakamuta, Y., Umeno, T., and Tsuchiyama, S. 2014. Rescue in vitro fertilization method for legacy stock of frozen mouse sperm. J. Reprod. Devel. 60:167‐170.
  Ostermeier, G.C., Wiles, M.V., Farley, J.S., and Taft, R.A. 2008. Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PloS one 3:e2792.
  Peters, D.D., Lepikhov, K., Rodenacker, K., Marschall, S., Boersma, A., Hutzler, P., Scherb, H., Walter, J., and de Angelis, M.H. 2009. Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes. Mammalian genome. Offic. J. Internat. Mammal. Genome Soc. 20:664‐673.
  Takeo, T. and Nakagata, N. 2010. Combination medium of cryoprotective agents containing L‐glutamine and methyl‐{beta}‐cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm. Lab. Animals 44:132‐137.
  Takeo, T. and Nakagata, N. 2011. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl‐beta‐cyclodextrin. Biol. Reprod. 85:1066‐1072.
Internet Resources
  http://www.infrafrontier.eu
  The European Mouse Mutant Archive (Europe), which hosts numerous Web‐based source materials of use to IVF technicians and cryobiologists.
  http://card.medic.kumamoto‐u.ac.jp/card/english/index.html
  The Center for Animal Resources and Development (Japan), which hosts numerous Web‐based source materials of use to IVF technicians and cryobiologists.
  http://www.brc.riken.jp/inf/en/index.shtml
  The RIKEN BioResource Center, which hosts numerous Web‐based source materials of use to IVF technicians and cryobiologists.
  http://jax.org
  The Jackson Laboratory (U.S.A.), which hosts numerous Web‐based source materials of use to IVF technicians and cryobiologists.
  https://www.mmrrc.org/
  The website for Mutant Mouse Regional Resource Centre (U.S.A.), which hosts numerous Web‐based source materials of use to IVF technicians and cryobiologists.
  http://findmice.org
  A searchable website for the International Mouse Strain Resource (IMSR), which links to mouse resources around the world.
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