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Male Reproductive Tract |
Kyudo Company Limited,3 Kumamoto 861-0104, Japan
Center for Animal Resources and Development,4 Kumamoto University, Kumamoto 860-0811, Japan
Department of Materials and Life Science,5 Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan
Department of Clinical Chemistry and Informatics,6 Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 860-8555, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, in vitro fertilization, male reproductive tract, sperm, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In 1990, Yokoyama et al. [9] and Tada et al. [10] reported the successful freezing of mouse sperm using a solution containing glycerol and raffinose. Okuyama et al. [11] then found that mouse sperm can be frozen in a solution containing raffinose and skim milk without glycerol. We were also subsequently successful in the cryopreservation of mouse spermatozoa, including transgenic strains (luciferase transgenic mouse), using an improved method [12, 13]. These results indicated that slow dilution after thawing prevents the sharp change in osmolarity and viscosity between the cryopreservation solution and diluent. Moreover, Thornton et al. [14] have demonstrated that it is possible to establish efficient, comprehensive, and extensive archives, and that potentially large numbers of offspring (>7000) can be derived from the frozen spermatozoa of a single mutant male mouse.
However, in general, high fertilization rates are not always obtained for the frozen spermatozoa of all mouse strains [15]. Notably, the fertilization rate of frozen C57BL/ 6 spermatozoa remains very low, although the rate can be increased by in vitro fertilization with partial zona pellucida dissection or the intracytoplasmic sperm injection technique [16, 17]. C57BL/6 is a major inbred strain, and its genetic background is well known. Furthermore, this strain is used not only for the production of transgenic mice [18], but also as a backcross for targeted mutant mice. Therefore, it is necessary to establish a cryopreservation method for C57BL/6 mouse spermatozoa that can maintain high fertilizing ability after thawing. In this study, C57BL/6 frozen-thawed mouse spermatozoa were examined ultrastructurally for any damage that could account for their low fertilizing ability.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inbred male (12- to 20-week-old) and female (8- to 12-week-old) C57BL/6J, BALB/cA, and DBA/2N mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Homozygous transgenic male (12- to 20-week-old) mice expressing the enhanced green fluorescent protein (EGFP) gene under the acrosin promoter on C57BL/6J background, acr3-EGFP [19, 20], were provided from the mouse embryo bank of Mitsubishi Kagaku Institute of Life Sciences (Machida-shi, Tokyo, Japan). All mice were kept according to the Guidelines for Animal Experiments of Kumamoto University and the Guide for the Care and Use of Laboratory Animals. They were maintained on a constant 12D:12L cycle with standard mouse chow and water available ad libitum.
Sperm Freezing and Thawing
Spermatozoa were obtained from C57BL/6J, BALB/cA, DBA/2N, and acr3-EGFP male mice (5 males/strain). After the male mice were killed humanely, one caudae epididymis was removed and placed into an 18% raffinose/3% skim milk solution. Spermatozoa from other caudae epididymides were used as a noncryopreserved control (fresh). Sperm cryopreservation and thawing were performed as described previously [15]. Briefly, 0.25-ml plastic straws (IMV, Paris, France) with 10-µl sperm aliquots collected at room temperature were frozen by exposure to liquid nitrogen vapor for 15 min before storage under liquid nitrogen. After 5 days, the samples were thawed in a water bath at 37°C for 10–15 min.
The thawed sperm suspension was incubated for 1.5 h with 5% CO2 in air at 37°C in a 200-µl drop of human tubal fluid (HTF) medium [21] prepared in our laboratory and covered with paraffin oil (NACALAI TESQUE Inc., Kyoto, Japan). Thawed C57BL/6J sperm samples were evaluated using five experiments: in vitro fertilization (IVF), motility analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zona-free assay. The DBA/2N sperm samples were evaluated using four experiments: IVF, motility analysis, SEM, and TEM. The BALB/cA sperm samples were evaluated using three experiments: IVF, motility analysis, and SEM. The acr3-EGFP sperm sample was evaluated using an acrosomal status assay.
In Vitro Fertilization
Inbred female mice were superovulated using an injection (i.p.) of 5 IU of eCG (Sigma Chemical Company, St. Louis, MO) followed by 5 IU of hCG (Sigma) 48 h later. Fourteen to fifteen h after the hCG injection, the females were killed and their oviducts were removed. The oocyte-cumulus complexes were isolated in a 200-µl drop of HTF medium covered with paraffin oil.
After the spermatozoa in the frozen plastic straw had thawed, the thawed sperm suspension was added to a 200-µl drop of HTF medium for IVF. The average concentration of these sperm was 8000 cells/µl. Five microliters of sperm suspension was added to the IVF medium (HTF) containing the oocyte-cumulus complexes (final sperm concentration = 200/µl). The IVF medium was placed in a sealed, modular incubator chamber gassed with 5% CO2 in air and maintained at 37°C for 8 h. The oocytes were then washed to eliminate excess sperm and were mounted in toto on a slide stained with lacmoid (whole-mount staining). The whole-mount staining samples were examined to assess fertilization. When totals of fertilized egg and unfertilized egg were less than 80, the data were not accepted.
Motility Analysis
The concentrations and motility rates of the fresh control and the frozen-thawed samples were determined using a C-IMAGING C-MEN computerized semen analyzer (Compix Inc., Lake Oswego, OR). The average number of cells counted per sample was approximately 2000. All counts were performed at 37°C. Motility was defined as linear direction at a speed of 50 µm/sec [22].
Scanning Electron Microscopy
Mouse spermatozoa (fresh or frozen-thawed) were incubated in HTF for 20 min at 37°C, washed twice with HTF, and fixed in 2.5% glutaraldehyde (EM Sciences, Fort Washington, PA) for 4 h at 4°C. The sperm samples were washed with PBS (IATRON Laboratories Inc., Tokyo, Japan) and incubated overnight at 4°C. The samples were then fixed in 2% osmic acid (EM Sciences); dehydrated sequentially in 50%, 70%, 80%, 90%, 95%, and 100% ethanol; critical point-dried in a critical point dryer; coated with palladium gold; and examined with a scanning electron microscope (S-800, Hitachi High-Technologies Co., Tokyo, Japan). When fewer than 80 sperm were visible in a fixed sample, the data were not used.
In Vitro Fertilization Using Zona-Free Oocytes
The oocyte-cumulus complexes were obtained from the oviducts of superovulated C57BL/6J female mice. Cumulus cells were removed by incubating the complexes for 3 min in HTF medium containing 0.1% hyaluronidase (type IV; Sigma). After washing in fresh HTF medium, the zona pellucida was dissolved by treating the oocyte for 30 sec to 1 min with acid Tyrode solution (pH 2.5). Finally, zona-free oocytes were washed in HTF medium 3 times and were used for in vitro fertilization. To assess fertilization, the zona-free oocytes were examined using lacmoid staining.
Transmission Electron Microscopy
The fresh and frozen-thawed spermatozoa were prefixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (PB, pH 7.4) for 2 h at 4°C and postfixed in 2% osmium tetroxide in PB for 2 h at 4°C. This was followed by dehydration and embedding in Epon 812 (TAAB Laboratories Equipment Ltd., Aldermaston, England). To select the optimal areas for this study, semithin sections were stained with toluidine blue. Ultrathin sections stained with uranyl acetate and lead citrate were examined via TEM (JEM-1230; JEOL, Tokyo, Japan).
Acrosomal Contents Status Assay Using acr3-EGFP Transgenic Mice
To assess the acrosomal contents, spermatozoa obtained from male acr3-EGFP transgenic mice were used. Immediately after collection from one caudae epididymis, the spermatozoa (fresh controls) were fixed for 5 min in 4% paraformaldehyde/PBS at room temperature [19]. Spermatozoa of other caudae epididymides were cryopreserved and then fixed after thawing (frozen-thawed samples). To determine whether acrosomal contents were present, samples (fresh and frozen-thawed) were observed under a fluorescent microscope.
Statistical Analyses
Normality of all variables was assessed through the use of the Kolmogorov-Smirnov test. Variables that were not normally distributed were arcsine transformed to approximate normality. Differences between in vitro fertilization rates before and after freezing were assessed with the paired t-test. The paired t-test was also used to analyze the difference in motility percentage of fresh and frozen sperm. The relation between fertility and the motility rate or cellular injury of spermatozoa was investigated by means of Pearson correlation coefficient. A significance level of 0.05 was used for all statistical tests, and two-tailed tests were applied. All statistical analyses were performed with Statview 5.0-J (SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fertilizing rate using fresh control and frozen-thawed sperm from three strains is shown in Figure 1. The fertilizing ability of frozen C57BL/6J and BALB/cA spermatozoa was less than that of fresh spermatozoa. In DBA/ 2N mice, the fertilizing ability of fresh and frozen-thawed sperm was identical, whereas all other strains had a significantly reduced rate.
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Sperm Motility after Freezing and Thawing
In order to elucidate the cause of decreased fertilizing ability in cryopreserved mouse spermatozoa, the sperm motility of frozen-thawed samples was examined. Figure 2 summarizes the motility of frozen-thawed sperm. Although the motility rates of frozen spermatozoa were lower than fresh spermatozoa, there was no significant difference among the motility rates of frozen spermatozoa in the three strains (P > 0.05).
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Scanning Electron Microscopy
The cell surface of frozen-thawed mouse spermatozoa was studied morphologically (Fig. 3, A–F). Fresh sperm showed few abnormalities, whereas frozen/thawed sperm lacked the rostral tip of the head (Fig. 3B); had disrupted acrosomes (Fig. 3, B, E, and F, arrowheads); lacked part of the mitochondrial sheath (Fig. 3C, arrowhead); showed a swollen flagellar base (Fig. 3C, arrow); and had coiled flagella (Fig. 3D, arrowhead). In the DBA/2N strain, abnormal cells were observed, but at a low rate (Fig. 4, 10.2%). As shown in Figure 4, the ratio of abnormal cells in other strains was higher than in DBA/2N. Notably, in C57BL/6J, almost all the sperm had suffered cellular injury.
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Cellular injury induced by freezing and thawing was mainly localized to the sperm head (81.2%). The middle piece and the tail of sperm were also damaged, although not severely. Furthermore, the dorsal anterior plasma membrane of the sperm head had notable defects, whereas the equatorial region and posterior head were normal after cryotreatment (Fig. 3, B, E, and F, arrowheads).
Transmission Electron Microscopy
To identify further causes of low fertility in frozen-thawed sperm, the ultrastructure was studied using TEM (Fig. 5, A–H). In the C57BL/6J strain, the plasma membrane changes in the acrosomal region were much more pronounced, whereas fresh sperm and DBA/2N spermatozoa did not show these defects (Fig. 5, A–D). Notably, both fresh controls and frozen DBA/2N spermatozoa had hydrolytic enzymes (Fig. 5, A–C, indicating high electron density in the acrosome), but frozen C57BL/6J sperm had no acrosomal contents (Fig. 5D, indicating low electron density in the acrosome).
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Furthermore, the mitochondria in the middle piece of frozen C57BL/6J spermatozoa had extremely variable and abnormal morphology compared with fresh sperm, whereas frozen DBA/2N spermatozoa had normal mitochondria (Fig. 5, E–H). The characteristic findings were mitochondria with an increased relative area of the matrix; thickening of the membrane, in particular the outer membranes; and swelling with loss of cristae (Fig. 5H, arrowheads).
Observation of Acrosomal Contents Using acr3-EGFP Transgenic Mice
In order to confirm the results of electron microscopy, the acrosomal content status using acr-3 EGFP transgenic mice sperm was examined (Fig. 6, A–D). As shown in Figure 6, A and B, in the case of fresh acr3-EGFP transgenic mouse spermatozoa, most sperm heads had acrosomal contents, as shown by green fluorescence (14.7%, the ratio of the sperm that does not have the acrosome contents). The profile of the fluorescence due to EGFP was identical to that of the acrosomal marker protein acrosin, indicating that EGFP was localized in the acrosome of acr3-EGFP mice spermatozoa.
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However, in frozen acr-3 EGFP spermatozoa, EGFP-negative cells were observed with high frequency (Fig. 6, C and D, 50.9%, the ratio of the sperm that do not have the acrosome contents). The EGFP-negative cells indicated that the acrosomal contents had leaked out during cryotreatment. These results demonstrate that most frozen sperm in C57BL/6J background mice had no acrosomal contents.
In Vitro Fertilization Using Zona-Free Oocytes
To determine the effects of the freezing injury, sperm penetration of zona-free oocytes was examined. When frozen-thawed C57BL/6J sperm were used to inseminate zona-free oocytes, the fertilization rate was higher than for intact oocytes (56.5% vs. 17.0%; Table 1). In addition, when frozen-thawed C57BL/6J sperm were used to inseminate intact oocytes, the fertilization rate was low, but cryopreserved C57BL/6J sperm had the ability to bind to the zona pellucida but not to penetrate it.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we found that the fertilization rate and sperm motility are not related in mouse spermatozoa (Figs. 1 and 2). Furthermore, in the case of frozen C57BL/6J spermatozoa, the percentage of damaged spermatozoa was 83.7% in total (Fig. 4). On the other hand, over 90% of frozen DBA/2N spermatozoa were intact. These observations suggest that cryopreservation-induced cellular injury is a potential cause of low fertilization. Quinn et al. [27] reported that freezing caused profound changes in the appearance of the acrosome in the majority of ram spermatozoa. In agreement with these results, we observed an abnormal acrosome in frozen C57BL/6J spermatozoa (Figs. 5 and 6). We also found that cryoinjury was localized to the dorsal anterior plasma membrane of the sperm head (Fig. 5). The proteins required for acrosome reaction are expressed in the rostral head region [28, 29].
Quinn et al. [27] also observed that in the midpieces changes occurred in the matrix of the mitochondria making up the mitochondrial sheath, the matrix appeared lighter in frozen spermatozoa than fresh spermatozoa, and loss of protein from the midpieces was confirmed histochemically. Imai et al. [30] reported that infertile human males with phospholipid hydroperoxidase glutathione peroxidase (PHGPx) defective spermatozoa accounted for about 10% of the total number of infertile males examined and for 35% of infertile males with oligoasthenozoospermia. The mitochondria in the midpiece of PHGPx-negative human spermatozoa have abnormal morphology: swollen, with loss of cristae. This phenotype is very similar to the mitochondrial cellular injury of frozen C57BL/6J spermatozoa (Fig. 5). Additionally, although there was no significant difference among the motilities of cryopreserved sperm of DBA/2N, BALB/cA, and C57BL/6J after thawing (Fig. 2), in this study the rate of spermatozoa with high progressive motility was lower for C57BL/6J than for DBA/2N and BALB/ cA under visual examination (data not shown). Thus, it appears that a defect in the mitochondria of frozen spermatozoa may be closely linked to lost fertilizing ability and high progressive motility.
Moreover, in the fluorescent study we found that the acrosome contents were missing from frozen acr3-EGFP mouse (C57BL/6J background) spermatozoa. The acrosome contents are vital proteins for passage through the zona pellucida surrounding an oocyte at fertilization, especially acrosin. The EGFP indicator expressed the same region as acrosin in the acrosome of frozen acr3-EGFP spermatozoa lost during cryotreatment (Fig. 6). In agreement with the results above, Müller et al. [31] reported that the plasma membrane of the acrosome was changed and the acrosomal contents were reduced in frozen ram spermatozoa. We demonstrated previously that the fertilization rate of C57BL/6J frozen spermatozoa could be increased by in vitro fertilization with oocytes subjected to partial dissection of the zona pellucida [16]. In this study, a relatively high fertilization rate was obtained when frozen C57BL/6J spermatozoa were used to inseminate zona-free oocytes. This knowledge and these results suggest that frozen C57BL/6J spermatozoa lost the ability to penetrate the zona pellucida as the result of decreased acrosome contents.
In conclusion, this study strongly suggests that the low fertilizing ability of frozen C57BL/6J spermatozoa resulted from injury to the head and tail caused by freezing and thawing. The acrosome of frozen C57BL/6J spermatozoa was damaged and their contents lost during cryotreatment. As a result, frozen C57BL/6J spermatozoa could not induce an acrosome reaction and could not penetrate the zona pellucida of the egg. Frozen C57BL/6J spermatozoa also lost essential motility for fertilization because of the damage to the mitochondria. Thus, the fertilizing ability of mouse spermatozoa was lost during cryotreatment.
This study provides new and important information to modify the cryopreservation method. However, from the results of these experiments, it is difficult to demonstrate how the cellular injury in spermatozoa, especially C57BL/ 6J spermatozoa, occurred after freezing and thawing, and further investigation is necessary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Naomi Nakagata, 2-2-1 Honjo, Kumamoto 860-0811, Japan. FAX: +81 96 373 6560; nakagata{at}gpo.kumamoto-u.ac.jp
Received: 18 October 2003.
First decision: 28 October 2003.
Accepted: 14 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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