Abstract
In the last few years sperm cryopreservation was rapidly established as the technique to efficiently manage production, preservation, and transportation of genetically engineered mice. However, occasionally, the reduced fertility of the frozen-thawed mouse sperm can make it difficult to revitalize the mouse by in vitro fertilization (IVF). In particular, the frozen-thawed sperm of C57BL/6 mice, widely used as the background of choice for genetically engineered strains, show very low fertility after freezing and thawing. To overcome this problem, we have developed a new protocol for sperm cryopreservation and IVF with frozen-thawed C57BL/6 sperm as well as other mouse strains. This protocol has the following three modifications: (1) addition of l-glutamine to the sperm cryoprotectant, (2) addition of methyl-β-cyclodextrin to the sperm preincubation medium, and (3) addition of reduced glutathione to the fertilization medium. These modifications greatly enhanced the fertility of frozen-thawed C57BL/6 sperm, resulting in a stable fertilization rate >80% in IVF. Our results indicate that this robust protocol for sperm cryopreservation may improve the archiving and distributing system for genetically engineered mice.
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References
Yokoyama M, Akiba H, Katsuki M, Nomura T (1990) Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Jikken Dobutsu 39:125–128
Tada N, Sato M, Yamanoi J, Mizorogi T, Kasai K, Ogawa S (1990) Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. J Reprod Fertil 89:511–516
Okuyama M, Isogai S, Saga M, Hamada H, Ogawa S (1990) In vitro fertilization (IVF) and artificial insemination (AI) by cryopreserved spermatozoa in mouse. J Fertil Implant 7:116–119
Takeshima T, Nakagata N, Ogawa S (1991) Cryopreservation of mouse spermatozoa. Jikken Dobutsu 40:493–497
Nakagata N, Takeshima T (1992) High fertilizing ability of mouse spermatozoa diluted slowly after cryoprservation. Theriogenology 37:1283–1291
Nakagata N (1992) Production of normal young following insemination of frozen-thawed mouse spermatozoa into fallopian tubes of pseudopregnant females. Jikken Dobutsu 41:519–522
Nakagata N, Matsumoto K, Anzai M, Takahashi A, Takahashi Y, Matsuzaki Y, Miyata K (1992) Cryopreservation of spermatozoa of a transgenic mouse. Jikken Dobutsu 41:537–540
Nakagata N, Takeshima T (1993) Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains. Jikken Dobutsu 42:317–320
Nakagata N (1993) Production of normal young following transfer of mouse embryos obtained by in vitro fertilization between cryopreserved gametes. J Reprod Fertil 99:77–80
Nakagata N, Ueda S, Yamanouchi K, Okamoto M, Matsuda Y, Tsuchiya K, Nishimura M, Oda S, Koyasu K, Azuma S, Toyoda Y (1995) Cryopreservation of wild mouse spermatozoa. Theriogenology 43:635–643
Nakagata N, Okamoto M, Ueda O, 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 (2000) Cryopreservation of mouse spermatozoa. Mamm Genome 11:572–576
Davisson M (2006) FIMRe: Federation of International Mouse Resources: global networking of resource centers. Mamm Genome 17:363–364
Donahue L, Angelis M, Hagn M, Franklin C, Lloyd K, Magnuson T, McKerlie C, Nakagata N, Obata Y, Read S, Wurst W, Hörlein A et al (2012) Centralized mouse repositories. Mamm Genome 23(0):559–571
Nishizono H, Shioda M, Takeo T, Irie T, Nakagata N (2004) Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury. Biol Reprod 71:973–978
Songsasen N, Leibo SP (1997) Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 35:255–269
Sztein JM, Farley JS, Mobraaten LE (2000) In vitro fertilization with cryopreserved inbred mouse sperm. Biol Reprod 63:1774–1780
Ostermeier GC, Wiles MV, Farley JS, Taft RA (2008) Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PLoS One 3:e2792
Fray MD (2009) Biological methods for archiving and maintaining mutant laboratory mice. Part I: Conserving mutant strains. Methods Mol Biol 561:301–319
Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, Irie T, 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
Takeo T, 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 Anim 44(2):132–137
Takeo T, Nakagata N (2011) Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod 85(5):1066–1072
Nakagata N, Takeo T, Fukumoto K et al (2014) Rescue in vitro fertilization method for legacy stock of frozen mouse sperm. J Reprod Dev 60:168–171
Renard P, Grizard G, Griveau JF, Sion B, Boucher D, Le Lannou D (1996) Improvement of motility and fertilization potential of postthaw human sperm using glutamine. Cryobiology 33:311–319
Trimeche A, Yvon JM, Vidament M, Palmer E, Magistrini M (1999) Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. Theriogenology 52:181–191
Kundu CN, Das K, Majumder GC (2001) Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system. Cryobiology 42:21–27
Khlifaoui M, Battut I, Bruyas JF, Chatagnon G, Trimeche A, Tainturier D (2005) Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level. Theriogenology 63:138–149
Al Ahmad MZ, Chatagnon G, Amirat-Briand L, Moussa M, Tainturier D, Anton M, Fieni F (2008) Use of glutamine and low density lipoproteins isolated from egg yolk to improve buck semen freezing. Reprod Domest Anim 43:429–436
Amirat-Briand L, Bencharif D, Vera-Munoz O, Bel Hadj Ali H, Destrumelle S, Desherces S, Schmidt E, Anton M, Tainturier D (2009) Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: preliminary results. Theriogenology 71:1209–1214
Ohtani Y, Irie T, Uekama K, Fukunaga K, Pitha J (1989) Differential effects of alpha-, beta- and gamma-cyclodextrins on human erythrocytes. Eur J Biochem 186:17–22
Choi YH, Toyoda Y (1998) Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod 59:1328–1333
Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG, Kopf GS (1999) Cholesterol efflux-mediated signal transduction in mammalian sperm: beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J Biol Chem 274:3235–3242
Bath ML (2010) Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione. PLoS One 5(2):e9387
Nakagata N, Yamamura K (2009) Current activities of CARD as an international core center for mouse resources. Exp Anim 58:343–350
Eppig JT, Strivens M (1999) Finding a mouse: the International Mouse Strain Resource (IMSR). Trends Genet 15:81–82
Wadman M (2012) Lab-animal flights squeezed. Nature 489:344–345
Quinn P, Kerin JF, Warnes GM (1985) Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 44:493–498
Nakagata N (2011) Cryopreservation of mouse spermatozoa and in vitro fertilization. Methods Mol Biol 693:57–73
Toyoda Y, Yokoyama M, Hoshi T (1971) Studies on the fertilization of mouse eggs in vitro: I. In vitro fertilization of eggs by fresh epididymal sperm. Jpn J Anim Reprod 16:147–151
Takeo T, Nakagata N (2015) Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS One 10:e0128330
Acknowledgments
The authors thank our CARD research group members Kiyoko Fukumoto, Yukie Haruguchi, Tomoko Kondo, Yuko Nakamuta, Yumi Takeshita, Hiroko Matsunaga, Manami Nishimura, Tomoko Umeno, Eri Ishida, Fumi Takahashi, Mari Iwamoto, Eri Kohagura Tomoko Matsumoto and Shuuji Tsuchiyama, my students: Aki Tsutsumi, Taichi Omaru, Kazuhito Sakoh, Yuta Ishizuka, Satohiro Nakao, Hidetaka Yoshimoto, Yuki Sakai, and Yuka Horikoshi, Ayumi Mukunoki for their technical assistance and helpful discussions.
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Takeo, T., Sztein, J., Nakagata, N. (2019). The CARD Method for Mouse Sperm Cryopreservation and In Vitro Fertilization Using Frozen-Thawed Sperm. In: Liu, C., Du, Y. (eds) Microinjection. Methods in Molecular Biology, vol 1874. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8831-0_14
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DOI: https://doi.org/10.1007/978-1-4939-8831-0_14
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