Contemporary Techniques for Freezing Mouse Spermatozoa

Mo Guan1, Debora Bogani1, Susan Marschall2, Marcello Raspa3, Toru Takeo4, Naomi Nakagata4, Rob Taft5, Martin Fray1

1 Mary Lyon Centre, Medical Research Council, 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), Rome, 4 Division of Reproductive Engineering, Center for Animal Resources & Development (CARD), University of Kumamoto, Kumamoto, 5 Division of Reproductive Technologies, Bar Harbor, Maine, The Jackson Laboratory
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140065
Online Posting Date:  September, 2014
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Abstract

Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L‐glutamine or monothioglycerol. Curr. Protoc. Mouse Biol. 4:85‐104 © 2014 by John Wiley & Sons, Inc.

Keywords: mouse; spermatozoa; cryoprotectant; freezing; epididymides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: The L‐Glutamine Sperm Freezing Method
  • Support Protocol 1: Preparation of Sperm Cryoprotective Agent Supplemented with 100 mM L‐Glutamine
  • Alternate Protocol 1: Freezing Sperm Using the Monothioglycerol (MTG) Method
  • Support Protocol 2: Preparation of Cryoproctective Agent Supplemented with Monothioglycerol
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The L‐Glutamine Sperm Freezing Method

  Materials
  • L‐glutamine‐supplemented cryoprotective agent (gCPA; protocol 2)
  • Liquid nitrogen (LN 2)
  • Male mice over 8 weeks of age
  • 70% ethanol
  • Modified HTF medium (see recipe)
  • TYH+MBCD sperm preincubation medium (see recipe).
  • Mineral oil (Sigma, cat. no. M8410)
  • 0.25‐ml plastic semen straws (Planer, cat. no. FZA201)
  • Paper tissue (Kimtech Science)
  • 35‐mm culture dishes (BD Falcon, cat. no. 351008)
  • Indelible marker pen or cryo‐fast labels (e.g., Brady labels)
  • Large LN 2 Dewar (e.g., Planer, cat. no. MVE‐XC 47‐11‐6)
  • Floating sperm‐freezing device (Fig.  ) prepared from 50‐ml syringe attached to a Perspex rod
  • Dissecting microscope (illuminated from above)
  • Dissecting instruments e.g., standard dissecting forceps, fine watchmakers forceps and scissors
  • Hot plate (37°C)
  • 1.0‐ml and 10‐ml syringes
  • Double‐impulse heat sealer or equivalent
  • Humidified 37°C, 5% CO 2 incubator
  • Small LN 2 Dewar
  • 16‐G metal rod
  • Wedge‐shaped 10‐µl pipet tips (e.g., Thistle Scientific, cat. no. AX‐T‐400‐L‐R‐S)
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ) and IVF (Guan et al., ) or Takeo et al. (2011)

Support Protocol 1: Preparation of Sperm Cryoprotective Agent Supplemented with 100 mM L‐Glutamine

  Materials
  • Ultrapure H 2O (e.g., embryo‐tested; Sigma)
  • L‐Glutamine (Sigma, cat. no. G8540)
  • D‐(+)‐raffinose pentahydrate (Sigma, cat. no. R7630)
  • Skim milk powder (BD Diagnostics, cat. no. 232100)
  • Ultrapure H 2O (e.g., Sigma embryo‐tested water)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Corning, cat. no. 430829)
  • 60°C water bath
  • Osmometer (e.g., Research Instruments)
  • 0.22‐µm syringe‐end disposable filter units (e.g., VWR, cat. no. 514‐7011)

Alternate Protocol 1: Freezing Sperm Using the Monothioglycerol (MTG) Method

  Additional Materials protocol 1)
  • Monothioglycerol‐supplemented cryoprotective agent (mCPA; protocol 4)
  • Freezing chamber (e.g., Styrofoam box or Thermosafe chests; Fisher Scientific, cat. no. 03‐530‐41; also see Fig.  )
  • 2.5‐cm‐thick styrofoam raft (Fig.  )
  • 140‐mm flat cassettes for holding straws (Hunter Scientific, cat. no. 16980/0601)

Support Protocol 2: Preparation of Cryoproctective Agent Supplemented with Monothioglycerol

  Materials
  • Ultrapure H 2O (e.g., Sigma embryo tested water)
  • D‐(+)‐raffinose (Sigma, cat. no. R7630)
  • Skim milk powder (BD Diagnostics, cat. no. 232100)
  • Monothioglycerol (Sigma, cat. no. M6145)
  • 60°C water bath
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Corning, cat. no. 430829)
  • 0.45‐µm syringe‐end filter (e.g., Millipore)
  • Osmometer (e.g., Research Instruments)
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Figures

Videos

Literature Cited

Literature Cited
  Bath, M.L. 2010. Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione. PloS One 5:e9387.
  Brown, S.D.M. and Moore, M.W. 2012. The International Mouse Phenotyping Consortium: Past and future perspectives on mouse phenotyping. Mamm. Genome 23:632‐640.
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Glenister, P.H. and Lyon, M.F. 1986. Long‐term storage of eight‐cell mouse embryos at −196 degrees C. J. In Vitro Fert. Embryo Transf. 3:20‐27.
  Glenister, P.H. and Thornton, C.E. 2000. Cryoconservation—archiving for the future. Mamm. Genome 11:565‐571.
  Guan, M., Bogani, D., Marschall, S., Raspa, M., Takeo, T., Nakagata, N. and Fray, M. 2014. In vitro fertilization in mice using the MBCD‐GSH protocol. Curr. Protoc. Mouse Biol. 4:67‐83.
  Liu, L., Nutter, L.M., Law, N., and McKerlie, C. 2009. Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice. J. Am. Assoc. Lab. Animal Sci. 48:39‐43.
  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. and Takeshima, T. 1993. Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains. Jikken Dobutsu 42:317‐320.
  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. Mamm. Genome 20:664‐673.
  Polge, C. 1951. Functional survival of fowl spermatozoa after freezing at −79 degrees C. Nature 167:949‐950.
  Scavizzi, F. and Raspa, M. 2006. Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically. Lab. Animals 40:70‐79.
  Stringfellow D.A.S.S. 1998. Manual of the International Embryo Transfer Society, 3rd ed. IETS, Savoy, Illinois.
  Sztein, J.M., Farley, J.S., and Mobraaten, L.E. 2000. In vitro fertilization with cryopreserved inbred mouse sperm. Biol. Reprod. 63:1774‐1780.
  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.
  Takeo, T., Hoshii, T., Kondo, Y., Toyodome, H., Arima, H., Yamamura, K., Irie, T., and Nakagata, N. 2008. Methyl‐beta‐cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol. Reprod. 78:546‐551.
Internet Resources
  http://www.infrafrontier.eu
  The European Mouse Mutant Archive (Europe), which hosts numerous Web‐based source materials of use to 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 cryobiologists.
  http://www.brc.riken.jp/inf/en/index.shtml
  The RIKEN BioResource Center, which hosts numerous Web‐based source materials of use to cryobiologists.
  http://jax.org
  The Jackson Laboratory (USA), which hosts numerous Web‐based source materials of use to cryobiologists.
  https://www.mmrrc.org/
  The Web site for Mutant Mouse Regional Resource Centre (USA), which hosts numerous Web‐based source materials of use to cryobiologists.
  http://findmice.org
  A searchable Web site for the International Mouse Strain Resource (IMSR), which links to mouse resources around the world.
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