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BOR - Papers in Press, published online ahead of print November 28, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.065359
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BIOLOGY OF REPRODUCTION 78, 546–551 (2008)
DOI: 10.1095/biolreprod.107.065359
© 2008 by the Society for the Study of Reproduction, Inc.

Reproductive Technology; 

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

Toru Takeo 3, Takayuki Hoshii 5, Yuki Kondo 3, Hiroshi Toyodome 4, Hidetoshi Arima 4, Ken-ichi Yamamura 5, Tetsumi Irie 3, and Naomi Nakagata 2 6

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

INTRODUCTION

The cryopreservation of sperm provides a much simpler and more economical alternative to the freezing of embryos for the storage of genetically engineered strains of mice in resource facilities and research laboratories [1, 2]. 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 [3]. However, rates are remarkably low in frozen/thawed sperm of C57BL/6 mice, the main strain used not only for the production of transgenic mice but also as a backcross for targeted mutant mice [3–5].

Previously, we demonstrated that the low fertility of frozen/thawed C57BL/6 mouse sperm is related to cellular injury [6]. The mechanical aids for low-motile or immotile sperm, such as partial zona pellucida dissection, zona drilling, and intracytoplasmic sperm injection, have been exploited to better fertilize eggs produced with frozen/thawed C57BL/6 mouse sperm [7–10]. However, these procedures require special techniques and equipment.

In our observations, a large number of frozen/thawed C57BL/6 mouse sperm have reduced motility, but a small portion have normal motility [6]. The motile sperm exhibit a normal ability to bind the zona pellucida (ZP), but remarkably little ability to penetrate the ZP and low fertility. This would suggest that the frozen/thawed C57BL/6 sperm have not completely undergone capacitation. Therefore, it may be necessary to induce capacitation in preincubation medium to achieve a high fertilization rate for frozen/thawed C57BL/6 sperm.

One of the primary mechanisms of capacitation in mammalian sperm is cholesterol reduction in the plasma membrane by cholesterol acceptors, such as bovine serum albumin (BSA) and MBCD [11–14]. The β-cyclodextrin is a cyclic heptasaccharide consisting of {alpha}-(1–4)-glucopyranose units with a hydrophilic outer surface and a lipophilic cavity at its center, which is capable of forming inclusion complexes with many lipophilic agents by taking up a molecule [15]. MBCD strongly interacts with cholesterol to form a complex, resulting in a stimulation of cholesterol efflux and a disruption of lipid rafts on the plasma membrane [16–18]. In mice, the number of capacitated sperm was increased by preincubation with MBCD at 0.75 mM for 90 min, resulting in ability to fertilize in vitro in the absence of BSA [14].

Several observations suggest that MBCD is capable of activating fertility and improving cryosurvival of frozen/thawed sperm in other mammalian species [19–23]. MBCD increases the rate of capacitation, number of sperm bound to ZP, and sensitivity of the acrosome reaction, resulting in a high fertilization rate in vitro. However, there has been no report of the effects of MBCD on the fertilizing ability of frozen/thawed mouse sperm.

In this study, we examined the effects of MBCD on the fertilizing capacity of frozen/thawed C57BL/6 mouse sperm in vitro.

MATERIALS AND METHODS

Animals

C57BL/6J mice were purchased from CLEA Japan Inc. (Tokyo, Japan) and used as donors of sperm and oocytes. Female and male donors were an 8 wk and 12 wk old, respectively. Mice used as recipients for the transfer of two-cell embryos were from the ICR strain and were 8–16 wk old. All animals were kept under a 12L:12D cycle (lights on: 0700 to 1900 h) at a constant temperature of 22°C ± 1°C with free access to food and water. All animal experiments were carried out with the approval of The Animal Care and Use Committee of the Kumamoto University School of Medicine.

Media

Modified Krebs-Ringer bicarbonate solution (TYH) without BSA was used as a medium for sperm preincubation [24]. The medium contains MBCD (Sigma) at a concentration of 0.25, 0.50, 0.75, 1.0, 1.5, or 2.0 mM with 1.0 mg/ml polyvinylalchol (PVA; cold water soluble; Sigma) [14]. Human tubal fluid medium (HTF) and modified Whitten medium (mWM) were used for in vitro fertilization and culture of two-cell embryos to the blastocyst stage [25, 26].

Sperm Freezing and Thawing

The cryopreservation of sperm was performed as previously described [1]. After the male mice were killed by cervical dislocation, two-tailed caudal epididymides were taken from one male. The five portions in tissues were cut by microspring scissors in 100 µl of a cryopreservation solution consisting of 18% (w/v) raffinose pentahydrate and 3% (w/v) skim milk (R18S3) in a four-well multidish. The sperm were dispersed from the organs at room temperature for 3 min. After the epididymal tissues were removed, the sperm suspension at a concentration of 10 to 14 x 107 cells/ml was divided into eight aliquots of 10 µl. All specimens were put into a 0.25-ml plastic straw (IMV, Paris, France), and the straws were heat sealed. The straws then were cooled by putting them into the neck (liquid nitrogen gas layer) of a container for 10 min, and they were plunged directly into liquid nitrogen, where they were stored. After 5 days, the samples were removed from the liquid nitrogen and thawed in a water bath at 37°C for 10 min.

In Vitro Fertilization

Mature female mice were superovulated by an intraperitoneal injection of 7.5 IU eCG (TEIZO Pharmaceutical, Tokyo, Japan) followed by 7.5 IU hCG (TEIZO Pharmaceutical) 48 h later. Fourteen to fifteen hours after the injection, the mice were killed by cervical dislocation, and their oviducts were removed. The four to five cumulus-oocyte complexes (COCs) obtained from ampulla of the fallopian tube were introduced in a 90-µl drop of HTF medium covered with paraffin oil.

The 10-µl aliquot of thawed suspension was added to the 90-µl drop of preincubation medium covered with paraffin oil. The thawed sperm were preincubated in TYH (BSA free) with 0–2.0 mM MBCD for 60 min (experiment 1) or 1.0 mM MBCD for 5–120 min (experiment 2) at 37°C with 5% CO2 in air. After incubation, using a wedge-shaped pipette tip (0.5–10 µl; Quality Scientific Plastics), a 10-µl aliquot of sperm suspension was collected from the peripheral part containing motile sperm in the drop (Fig. 1). The sperm suspension was transferred to the insemination drop and incubated with COCs in HTF medium (final motile sperm concentration = 200–400/µl). At 8 h after insemination, fertilized eggs were examined by acetolacmoid staining for the formation of a male and female pronucleus and extrusion of a second polar body.


Figure 1
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  		FIG. 1. Collection of thawed sperm in preincubation medium. During preincubation, nonmotile sperm and aggregated sperm assembled near the center of the drop. Single motile sperm were swimming at the periphery of the drop. The pipette's tip was inserted at the edge of the drop (A) and an aliquot of 10 µl of sperm suspension was collected from the periphery in the direction of the arrow (B).

Embryo Culture and Transfer

After IVF, the fertilized oocytes that had developed to the two-cell stages at 24 h after insemination were divided into two groups: one group was further cultured in mWM for 72 h, and number of blastocysts stage embryos was recorded, whereas the other was transferred to the oviducts of pseudopregnant females on the day a vaginal plug was found (Day 1 of pseudopregnancy), and the recipients gave birth (experiment 3). When the offspring reached 8 wk of age, three pairs of females and males were randomly selected and mated within the same litter and observed regarding conception and delivery.

Cholesterol Quantification

Fresh or frozen/thawed sperm suspension were preincubated in preincubation medium (TYH with 4.0 mg/ml BSA or 0, 0.25, 1.0 mM MBCD containing 1.0 mg/ml PVA) at 37°C for 120 min. The sperm suspension was centrifuged (500 x g, 5 min) to collect the supernatant (released cholesterol) and pellet (cellular cholesterol). Cholesterol was extracted by the methods described by Bligh and Dyer [27]. After evaporation of the solvents, the amount of cholesterol was quantified with colorimetric enzyme assay kits from Cholesterol E-test Wako (Wako Pure Chemical Industries, Osaka, Japan) according to the protocol described by the manufacturer. The percentage of cholesterol efflux was calculated as released cholesterol / (released cholesterol + cellular cholesterol) x 100.

Cholesterol Visualization

After the preincubation described above, the aliquots of the sperm suspension were mixed with filipin (100 µg/ml) in TYH (BSA free) containing 1.0 mg/ml PVA for 30 min at room temperature in the dark. Filipin binds to cholesterol on the membrane and can be visualized by fluorescence (excitation: 365 nm; emission: 420 nm). The sperm were washed twice by centrifugation in phosphate-buffered saline. The stained sperm either were viewed by fluorescence microscopy (Biozero; Keyence Co.) or quantified based on the fluorescence intensity of filipin by FACS (Becton Dickinson, San Jose, CA) [28]. The relative mean fluorescence intensity was determined by dividing the fluorescence intensity of filipin-stained cells treated with BSA or MBCD by that without additives at predetermined intervals.

Statistical Analysis

The statistical analysis was performed using Prism version 3.0 (GraphPad, San Diego). Data are given as the mean ± SD. Comparison of the differences between the means for each treatment was done using ANOVA after arcsine transformation of the percentage data. Differences between the means were considered to be significant when P < 0.05 was achieved.

RESULTS

Experiment 1: Effects of MBCD on In Vitro Fertilization

Table 1 shows the dose-dependent effects of MBCD on the fertilization rate of frozen/thawed sperm in vitro. The proportion of pronuclear oocytes was significantly increased by MBCD at concentrations of 0.5–1.5 mM. The fertilization rate reached a maximum at 1.0 mM MBCD (68% ± 3.6%), whereas a high dose of 2.0 mM MBCD reduced the fertilizing ability of the sperm. Therefore, we used a dose of 1.0 mM in subsequent experiments to investigate the effects of MBCD.


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  		TABLE 1. Dose-dependent effects of MBCD on the fertilizing capacity of frozen/thawed mouse sperm in vitro.

Experiment 2: Time-Dependent Effects of MBCD on In Vitro Fertilization

The fertilizing ability of frozen/thawed sperm was enhanced by MBCD in a time-dependent manner (Table 2). The fertilization rate reached a maximum 30 or 60 min after thawing with 1.0 mM MBCD (64%–62%), whereas 4.0 mg/ml BSA gradually increased the rate for at least 120 min. Combination of 1.0 mM MBCD with 4.0 mg/ml BSA did not result in further improvement of fertilization rate in comparison with solo use of 1.0 mM MBCD (MBCD: 60.8% ± 8.5% vs. MBCD + BSA: 62.3% ± 4.5%, for 30 min).


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  		TABLE 2. Time-dependent effects of additives of BSA or MBCD on the fertilizing capacity of frozen/thawed mouse sperm in vitro.a

Experiment 3: In Vitro and In Vivo Development of Oocytes Derived from MBCD-Treated Sperm

The rate at which two-cell embryos derived from frozen/thawed sperm treated with MBCD developed to blastocysts or live young is shown in Table 3. The fertilization rate with MBCD was about three times as high as that with BSA (MBCD: 67% ± 8.4% vs. BSA: 18% ± 12.0%). In vitro, 83% of two-cell embryos developed into blastocysts in the MBCD-treated group. In vivo, 52% of transferred embryos were born in the MBCD-treated group. There was no significant difference in the developmental ability of two-cell embryos between the MBCD- and BSA-treated groups. Furthermore, we examined the reproductive ability of the offspring obtained. All females in each group became pregnant and delivered live young (average number of young born, MBCD: 7.0 ± 1.0 vs. BSA: 7.3 ± 4.0).


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  		TABLE 3. In vitro and in vivo development of 2-cell embryos produced with MBCD-treated sperm.

Experiment 4: MBCD Stimulated Cholesterol Efflux from Frozen/Thawed Sperm

The time course of cholesterol's efflux from frozen/thawed sperm with the addition of BSA or MBCD is shown in Figure 2. The efflux with BSA or MBCD occurred in a time-dependent manner. However, the effect of MBCD was greater and took less time. Moreover, the amount of cholesterol released was drastically increased and saturated at 30 min by MBCD.


Figure 2
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  		FIG. 2. Time-dependent effects of MBCD on cholesterol efflux from frozen/thawed sperm. A) Quantification of the amount of cholesterol released from sperm in medium with 4.0 mg/ml BSA (open circles) or 1.0 mg/ml PVA in the absence (closed circles) or presence of 0.25 (closed triangles) or 1.0 (closed squares) mM MBCD. All values are presented as the mean ± SD (n = 3). *P < 0.05 compared with control (4.0 mg/ml BSA) at each point.

Experiment 5: MBCD Removed Cholesterol from the Plasma Membrane of Frozen/Thawed Sperm

The distribution of cholesterol in the membrane was visualized using filipin (Fig. 3). Sperm labeled with filipin gave off strong fluorescence in the acrosomal region and weak fluorescence in elsewhere at 0 min (Fig. 3B). On the other hand, MBCD greatly reduced the amount of blue light in the entire area at 30 min (Fig. 3D). BSA gradually decreased the relative mean fluorescence intensity in a time-dependent manner, whereas MBCD drastically reduced the fluorescence in a short time (Fig. 3G).


Figure 3
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  		FIG. 3. Visualization of membrane cholesterol stained by filipin. A–D) Localization of filipin-labeled cholesterol in frozen/thawed sperm pretreated (B) or posttreated with MBCD (D). E–G) Quantification of fluorescence intensity of filipin-stained frozen/thawed sperm with 4 mg/ml BSA (E) or 1.0 mM MBCD (F) by FACS. The relative mean fluorescence intensities of filipin-stained frozen/thawed sperm treated with BSA (black circles) or MBCD (black triangles) are depicted in G. All values are presented as mean ± SD (n = 3). *P < 0.05 compared with control (4.0 mg/ml BSA) at each point. Bars = 5 µm (A–D).

Experiment 6: A Decreased Ability of Frozen/Thawed C57BL/6 Sperm To Release Cholesterol Compared with Fresh Sperm

In the presence of 4.0 mg/ml BSA, a smaller amount of cholesterol was released from frozen/thawed sperm compared with that from fresh sperm, especially for 120 min (Fig. 4).


Figure 4
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  		FIG. 4. Comparison between fresh and frozen/thawed sperm on efflux of cholesterol effects by BSA. A) Quantification of the amount of cholesterol released from fresh (black bars) or frozen/thawed (white bars) sperm in medium with 4.0 mg/ml BSA. All values are presented as mean ± SD (n = 3). *P < 0.05 compared with control (fresh sperm) at each point.

DISCUSSION

In the present study, we have shown that MBCD improves the fertilizing ability of frozen/thawed C57BL/6 mouse sperm in vitro. MBCD is capable of achieving a high fertilization rate, about three times that with BSA. The onset of cholesterol efflux from the frozen/thawed sperm treated with MBCD was rapid, and the effect was stronger than that of BSA. In addition, our results suggest that the increase in the fertilization rate of frozen/thawed sperm is associated with efflux of cholesterol from the cells.

There are many reports of MBCD promoting capacitation in mammalian species [13, 14, 28–32]. However, the mechanism of MBCD-mediated efflux of cholesterol and subsequent capacitation is not fully understood. MBCD-mediated efflux of cholesterol stimulated protein tyrosine phosphorylation during capacitation [13]. It is assumed that a loss of cholesterol induces a change in the stability and distribution of lipid rafts [33]. Lipid rafts are thought to function in signal transduction at the cell surface in response to intracellular and extracellular stimuli [33–35]. A change in the architecture of lipid rafts may regulate an influx of Ca2+ ion coupled with the release of intracellular Ca2+, leading to the opening of store-operated channels (SOCs) in the plasma membrane, such as Drosophila transient receptor potential (trp) in mouse sperm [36].

Our previous study demonstrated that frozen/thawed C57BL/6 mouse sperm induced functional disorders, such as decreased numbers of motile sperm and injured cellular membranes [6]. Furthermore, we found a decreased ability of frozen/thawed C57BL/6 sperm to release cholesterol compared with fresh sperm (Fig. 4). However, the mechanism responsible for the reduction in the efflux of cholesterol in frozen/thawed sperm has not been elucidated.

In somatic cells, cholesterol efflux is mediated by transmembrane proteins, like scavenger receptor BI and/or ATP-binding cassette transporter proteins, that give cellular cholesterol to cholesterol acceptors, such as high-density lipoprotein [37, 38]. ABCA17 protein exists in the anterior head of the sperm and may regulate the lipid composition of sperm [42]. In our study, it is likely that the functioning of these proteins or systems is disturbed during freeze/thawing process, resulting in the decreased ability to efflux cholesterol.

In the previous study, Choi and Toyoda showed cholesterol efflux after exposing fresh sperm to MBCD elevated fertilization rates, but lower compared with those obtained after exposure to BSA (MBCD, 51% vs. BSA, 63%) [14]. The results conflict with those in the present study. This discrepancy may be due to differences in the experimental conditions, such as the preincubation time and fresh vs. frozen/thawed sperm, between the two studies. Choi and Toyoda preincubated fresh sperm with 0.75 mM MBCD for 90 min, in which the fertilization rate of MBCD was lower than that of 4.0 mg/ml BSA. In the present study, the preincubation of frozen/thawed sperm with 1.0 mM MBCD for 120 min resulted in a lower fertilization rate than that with 4.0 mg/ml BSA. On the other hand, MBCD showed the maximum fertilization rate at 30 min, which was higher than that obtained after exposure to BSA for the same incubation period (Table 2).

Cholesterol efflux reached a plateau after exposure to 1.0 mM MBCD for 30 min, whereas BSA-induced efflux of cholesterol was slower, but BSA reached approximately the same level within a reasonable time period of 120 min (Fig. 2). Based on similar level of cholesterol efflux, similar fertilization rates are to be expected after exposing sperm to 1.0 mM MBCD (30 min) and 4 mg/ml BSA (120 min). However, there was more than 2-fold difference in fertilization rates between the two groups. This might be explained by the difference in the preincubation time between the two groups. With increasing preincubation time, frozen/thawed sperm tended to decrease in motility and form aggregates and/or cell debris in the medium, possibly leading to the reduction of fertilization rate with BSA.

Parinaud et al. demonstrated that hydroxypropyl-β-cyclodextrin (HPBCD) enhanced the ability of sperm binding to ZP in humans [38]. However, HPBCD induced spontaneous acrosome reaction after 4-h incubation. Spontaneous acrosome reaction induced with HPBCD might give rise to the low rate of fertilization. Considering the potential ability of MBCD to induce spontaneous acrosome reaction, we chose here shorter preincubation time with MBCD.

Recently, Bergeron et al. have reported that casein, a major component of whole milk and skim milk, protects sperm function during sperm storage [41]. Casein inhibits the loss of cholesterol from sperm membrane interacting with bull seminal plasma (BSP protein), which can stimulate capacitation by inducing cholesterol efflux. In the cryopreservation of mouse sperm, skim milk is used as a cryoprotectant in R18S3 medium [1]. This indicates that a constituent of skim milk such as casein may play a role in reducing the loss of cholesterol from sperm, acting as a cryoprotectant during sperm cryopreservation but as an inhibitor of cholesterol efflux in IVF. It will be necessary to investigate the relationship between inhibitory factors of cholesterol efflux and the fertilizing ability of frozen/thawed sperm.

It has been reported to be possible to improve the fertility of frozen/thawed C57BL/6 sperm by removing nonmotile sperm or cell debris. A study performed by Szczygiel et al. using a column of Sephadex G25 suggested that separation of motile populations of sperm prior to freezing improved the fertilization rate of frozen/thawed C57BL/6 sperm (nonseparated 16% versus separated 40%) [42].

The "swim-up" method is known to be a useful technique for separating motile sperm from nonmotile ones in human fertility clinics. In contrast to human sperm, it is difficult to collect adequate amount of motile sperm from frozen/thawed mouse sperm by the swim-up method, because of the presence of an overwhelmingly large number of nonmotile sperm and cell debris.

On the other hand, Bath et al. showed that when nonmotile sperm and cell debris were removed from frozen/thawed suspensions of C57BL/6 sperm with "swim-out" technique, the remainder exhibited a high fertilization rate (38%–88%) [43]. However, these methods are not practical because they need to perform complicated procedures by which the motile sperm are strictly separated from nonmotile sperm. On the other hand, the procedure described in the present study is simple. The sperm suspension was collected from peripheral part mainly containing motile sperm in the drop, which suspension may contain nonmotile sperm to some extent. In the present procedure, we could not obtain high fertilization rates even in the presence of BSA. Therefore, MBCD is necessary for developing a simple IVF method for frozen/thawed sperm achieving a high fertilization rate.

We used a different approach, focusing on the ability of frozen/thawed C57BL/6 sperm to release cholesterol. Our results show that MBCD activates the fertilizing ability of frozen/thawed C57BL/6 sperm, resulting in a high fertilization rate (63.7% ± 11.0%; Table 2).

We suggest that MBCD drastically improves the capacity of frozen/thawed C57BL/6 sperm to release cholesterol compared with BSA, thereby enhancing ability to fertilize in vitro. Moreover, the obtained offspring derived from the frozen/thawed sperm treated with MBCD had a normal reproductive ability. Therefore, MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm with a high fertilization rate.

ACKNOWLEDGMENTS

We wish to thank Dr. Toyoda (Obihiro University of Agriculture and Veterinary Medicine), Dr. Irikura (Department of Clinical Chemistry and Informatics, Kumamoto University), and Dr. Kaneko (Division of Reproductive Engineering, Kumamoto University) for helpful discussions and technical help. We are grateful to T. Kondo, Y. Haruguchi, K. Fukumoto, H. Machida, Y. Nakagawa, and M. Koga for excellent technical assistance and the Gene Technology Center in Kumamoto University for its important contributions to the experiments.

FOOTNOTES

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. Back

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

Received: 7 September 2007.

First decision: 26 September 2007.

Accepted: 19 November 2007.

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Y. Seita, S. Sugio, J. Ito, and N. Kashiwazaki
Generation of Live Rats Produced by In Vitro Fertilization Using Cryopreserved Spermatozoa
Biol Reprod, March 1, 2009; 80(3): 503 - 510.
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