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

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.

Chromosome analysis of mouse zygotes produced by intracytoplasmic injection of spermatozoa exposed to acrosome reaction inducing agents methyl-β-cyclodextrin and calcium ionophore A23187

“…Type A, intact acrosome; type B, condensed acrosome; type C, collapsed acrosome; and type D, elimination of the acrosome MβCD-treated spermatozoa and ICSI zygotes derived from MβCD-treated spermatozoa. These findings demonstrate that MβCD has no detrimental effect on sperm chromatin, and cytogenetically supports the previous finding that IVF embryos derived from MβCD-treated spermatozoa show normal developmental ability [15]. In contrast, the increased incidence of structural chromosome aberrations was found in ICSI zygotes derived from acrosome-intact spermatozoa after incubation in H-TYH without MβCD.…”

“…It has been demonstrated that methyl-β-cyclodextrin (MβCD) allows mouse spermatozoa to capacitate by promotion of cholesterol efflux from the plasma membrane [13][14][15]. Calcium ionophore A23187 is commonly used to induce the acrosome reaction in mammalian spermatozoa.…”

Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

“…However, there is a possibility that a medium containing no Ca 2+ has some other effect on the fertility of the frozen sperm. Takeo et al [23] reported that MBCD improves the fertility of frozen C57BL/6 sperm by stimulating cholesterol efflux. It will be important to investigate whether or not SP in a medium containing no Ca 2+ stimulates cholesterol efflux from the cell, and whether or not MBCD affects acrosome loss.…”

“…When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23]. Successful IVF with fresh sperm depends on the media used.…”

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

“…Mechanical damage to the sperm membrane occurs most often in spermatozoa from the C57BL/6 (B6) strain, one of the standard inbred stains of genetically engineered mice. This has prompted several technical improvements for cryopreservation of B6 spermatozoa by modifying the composition of the cryopreservation solution or the sperm preincubation medium [16][17][18][19].Little attention has been given to the containers used for freezing spermatozoa because the plastic straws currently used are generally satisfactory. However, these straws are used specifically for sperm freezing and are less familiar than conventional cryotubes in most laboratories.…”