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Methyl-Beta-Cyclodextrin Improves Fertilizing Ability of C57BL/6 Mouse Sperm after Freezing and Thawing by Facilitating Cholesterol Efflux from the Cells¹

Department of Clinical Chemistry and Informatics,³ and Department of Physical Pharmaceutics,⁴ Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan Division of Developmental Genetics,⁵ Institute of Molecular Embryology and Genetics, and Division of Reproductive Engineering,⁶ Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan

ABSTRACT

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% ± 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.

fertilization, in vitro fertilization, methyl-beta-cyclodextrin, sperm, sperm cryopreservation

¹Supported in part by grants from New Industry Creative Type Technology Research and Development Promotion Program, from the Ministry of Economy, Trade, and Industry in Japan, and the Sasakawa Scientific Research Grant from the Japan Science Society.

Correspondence: ²FAX: 81 96 373 6560; e-mail: nakagata{at}gpo.kumamoto-u.ac.jp