1 Department of Anatomy, Toho University School of Medicine, Ota-ku, Tokyo and 2 Tsukuba Primate Center, National Institute of Infectious Diseases, Tsukuba, Ibaraki, Japan
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Abstract |
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Key words: acrosome reaction/cryopreservation/cynomolgus monkey/electron microscopy/spermatozoa
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Introduction |
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Transmission electron microscopy (TEM) revealed that membranes of cryopreserved spermatozoa undergo structural alterations, such as swelling or vesiculation, that resemble the process of the acrosome reaction (Jones, 1973; Krogenaes et al., 1994
). However, the substantial number of dead spermatozoa resulting from the cryopreservation process makes it difficult to distinguish between acrosome-reacted live spermatozoa (true reaction) and damaged dead spermatozoa (false reaction). Therefore, the exact condition of the acrosome in cryopreserved spermatozoa remains unclear. When spermatozoa are cooled to a low temperature before freezing, membrane changes that resemble capacitation without significant morphological change occur before the acrosome reaction is induced (Zhao and Buhr, 1995
; Fuller and Whittingham, 1997
). In mammals, the acrosome reaction is associated with distinct morphological changes (e.g. swelling of the acrosome, fusion of the plasma and outer acrosomal membranes, vesiculation, and exocytosis of the acrosomal contents) that result in disintegration of the plasma and outer acrosomal membranes. To determine further whether such morphological changes would occur in the acrosome reactions of cryopreserved live spermatozoa, the spermatozoa used in this study were prepared using Percoll density gradient centrifugation. By using TEM and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), the acrosomal status and motility rates (% motile spermatozoa) were then assessed and compared between cryopreserved and fresh spermatozoa that were either not incubated, or were incubated for 2 h if cryopreserved, and for 2 or 8 h if fresh.
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Materials and methods |
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Cryopreservation of spermatozoa
The composition of the semen diluent and the process of cryopreservation were identical to those described previously (Sankai et al., 1994). In brief, 1 volume of the TYH suspension of washed spermatozoa was diluted in 9 volumes of TTE medium [Tes (Wako, Osaka, Japan), Tris-buffer, egg yolk base] at room temperature. The medium was prepared by adding 10% glycerol to TTE medium (TTE-G).
The sperm suspension was placed in test tubes, which were then placed in a water bath, the temperature of which was slowly decreased from room temperature to 5°C over a 2 h period. Equal volumes of 5°C TTE-G medium were then added at intervals of 510 min. Equilibrium of glycerol was assumed after this dilution was carried out for 30 min (i.e. 5% glycerol). The spermatozoa were transferred to cryostraws, which were sealed with straw powder, cooled at 5 cm above the surface of liquid nitrogen for 10 min, and then stored within the liquid nitrogen. Straws containing frozen spermatozoa were then thawed by placing them in a water bath at 37°C for 1 min. The thawed sperm suspension (total volume 1.25 ml) was then diluted with 2 ml TYH medium, layered over 90% Percoll, and centrifuged for 10 min at 800 g. The resulting pelleted, highly motile spermatozoa were used for the non-incubated and incubated sperm samples for TEM and FITC-PSA observations.
Survival assay of spermatozoa
The survival rate of the cryopreserved spermatozoa was determined by measuring the motility (% motile spermatozoa) among non-incubated and incubated spermatozoa. For fresh and cryopreserved non-incubated spermatozoa immediately after Percoll centrifugation, this survival rate was measured by placing 10 µl of sperm suspension under a coverslip and observing with a light microscope. For the incubated spermatozoa, the survival rate was measured directly in the dish, without Percoll centrifugation. Percoll centrifugation was used to remove substances such as cell debris and dead spermatozoa from the sperm suspensions.
The sperm pellets were divided into three aliquots and either resuspended directly in fixation solutions (i.e. not exposed to a capacitation-inducing substance, such as caffeine or dibutyryl cyclic AMP) for TEM and FITC-PSA analysis of non-incubated spermatozoa, or were resuspended in incubation medium to induce capacitation. The medium was TYH medium supplemented with 1 mmol/l caffeine and 1 mmol/l dibutyryl cyclic AMP, and the suspensions were incubated at 37°C in a humidified CO2-enriched (5%: 95%) atmosphere.
Acrosomal status determined by TEM observation
The fixative solution for TEM consisted of 0.2% tannic acid (Merck, Darmstadt, Germany), 4% paraformaldehyde and 3% glutaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.3). Fixation was performed by first adding 12 ml of fixative solution to 0.050.2 ml of the pelleted cryopreserved and fresh sperm samples. The spermatozoa were then immediately fixed by using microwave irradiation (MWI) in two 10 s pulses within 2 min at 4°C. The advantage of MWI is that cells can be fixed rapidly. After irradiation, each specimen was treated for 1 h at room temperature in the fixative solution. The spermatozoa were postfixed with Dalton's chrome-osmium fixative (pH 7.3) (Takeuchi and Takeuchi, 1985), dehydrated in ethanol, and embedded in Spurr resin. Thin sections were stained with uranyl acetate and lead citrate.
Acrosomal status and viability of spermatozoa determined by using FITC-PSA and propidium iodide
Sperm viability was determined by counting damaged spermatozoa, defined as spermatozoa with nuclei stained by propidium iodide (PI) (Wako Pure Chemical Industries Ltd., Osaka, Japan). After the spermatozoa had been washed, 1 ml samples of fresh and cryopreserved, non-incubated and incubated sperm samples were added to a 20 µl (0.27 mg/ml) solution of PI at room temperature and stained for 10 min.
After the samples had been stained with PI and washed, acrosomal status was determined as described previously (Cross et al., 1989; Mendoza et al., 1992
).
Air-drying from the standard procedure was excluded, the spermatozoa being maintained in a liquid phase on the basis of results from preliminary studies. It has been reported (Haas et al., 1988) that air-drying of spermatozoa resulted in the disappearance of the plasma membrane from over the outer and inner acrosomal membranes of the spermatozoan head. Briefly, the non-incubated and incubated spermatozoa were washed in protein-free Dulbecco's phosphate-buffered saline (DPBS) to remove protein from the solutions. The suspension was then immediately added to 1 ml of 95% ethanol fixation solution at 4°C. After washing, the spermatozoa were labelled with FITC-PSA (50 µg/ml; Vector Laboratories, Burlinghame, USA). After staining, the spermatozoa were mounted in 1,4-diazabicyclo-(2,2,2)octane (DABCO) to inhibit photo-bleaching of the fluorescence. The specimens were observed through a confocal laser-scanning microscope (Bio-Rad MRC-600, UK).
Classification of acrosomal status
Acrosomal status of the sperm specimens was determined using TEM and FITC-PSA observations. Spermatozoa were classified into five categories by TEM analysis: (i) intact spermatozoa, i.e. those possessing a complete plasma membrane. In cryopreserved sperm samples, this included spermatozoa with plasma membranes slightly separated from the outer acrosomal membrane, but with no alteration of the outer acrosomal membrane. These spermatozoa have also been suggested to be normal (Hofmo and Berg, 1989); (ii) swollen spermatozoa, i.e. those with an intact plasma membrane, a large acrosome, occasional ruffling or invagination of the outer acrosomal membrane, and an intact equatorial segment; (iii) vesiculated spermatozoa, i.e. those with a complete plasma membrane, sites of plasma and outer acrosomal membrane fusion, invagination of the outer acrosomal membrane, vesicles within the matrix of the acrosome, and an intact equatorial segment; (iv) fully acrosome-reacted spermatozoa, i.e. those without a plasma membrane, outer acrosomal membrane, or acrosomal matrix of the anterior acrosome, but with an intact equatorial segment and (v) dead spermatozoa, i.e. those identified by ruptured plasma membranes.
Acrosomal status was then classified into five categories using FITC-PSA analysis: (a) acrosome-intact spermatozoa, i.e. anterior heads have bound FITC-PSA uniformly; (b) acrosome-modified spermatozoa, i.e. anterior heads have bound FITC-PSA irregularly and in patches; (c) acrosome-reacted spermatozoa, i.e. anterior acrosome with no bound FITC, but the equatorial segment has bound FITC-PSA; (d) completely acrosome-reacted spermatozoa, i.e. no FITC-PSA has bound over the anterior portion and equatorial segment of the head; and (e) dead spermatozoa, i.e. nuclei stained intensely red by PI.
Statistical analysis
All data are expressed as mean ± SD. Differences between normally distributed data were analysed using Student's t-test, whilst the non-parametric Welch's test was used in the case of non-normally distributed data. Statistical significance was defined as P < 0.05. Calculations were performed using the Stat123/win statistics package (Shinko-Koeki Isyosyuppan, Tokyo, Japan).
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Results |
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Membrane fusion was sometimes observed among fresh spermatozoa (Figure 7A,B), and the rate of swollen spermatozoa increased remarkably from 0% before incubation to 37% following 2 h of incubation. Among cryopreserved spermatozoa, 2 h of incubation caused an increase in the number of vesicles within the swollen acrosomes, and some vesicles were dark (Figure 5A,B
). The dark vesicles resembled those of fresh spermatozoa that had been incubated for 2 or 8 h (Figures 8 and 9
). The vesiculated spermatozoa still possessed a discernible plasma membrane and outer acrosomal membrane by TEM (Figure 5A,B
). FITC staining of cryopreserved spermatozoa that had been incubated for 2 h revealed a significantly lowered (P < 0.01) rate of acrosome-reacted spermatozoa, and a significantly increased rate of completely acrosome-reacted spermatozoa.
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TEM showed no significant difference in the rate of acrosome-intact spermatozoa among fresh spermatozoa incubated for 8 h and cryopreserved spermatozoa incubated for 02 h. However, FITC staining revealed a significant difference (P < 0.03; Table III) in the rate of acrosome-intact fresh spermatozoa incubated for 8 h (68.1%) compared with cryopreserved spermatozoa incubated for 2 h (54.9%).
Incubation of fresh spermatozoa for 8 h significantly increased (P < 0.03; Table I) the rate of swollen spermatozoa in comparison with fresh spermatozoa incubated for 2 h. Many dark vesicles (Figures 8 and 9
) were present under the still-separated plasma and outer acrosomal membranes, and numerous swollen acrosomes contained a dark acrosomal matrix near the nucleus (Figure 9
). There was no significant difference in the rate of acrosome-reacted fresh spermatozoa between cryopreserved 2 h and fresh 8 h of incubation as determined by FITC staining (Table III
). The incidence of completely acrosome-reacted spermatozoa was highest in cryopreserved spermatozoa after 2 h of incubation and significantly lower in both fresh spermatozoa incubated for 8 h and non-incubated cryopreserved spermatozoa (Table II
).
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Discussion |
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Previously, it was shown that the concentration of intracellular Ca2+ increased during cooling of the spermatozoa from 4°C to 0°C (Zhao and Buhr, 1995), and further suggested that capacitation may be induced as a result of the extender's ability to eliminate phospholipids that are loosely attached to the plasma membrane (Quinn et al., 1980
). An increase in phospholipids, including arachidonic acid, and a decrease in sphingomyelin (SM) content occur both in cryopreserved spermatozoa (Buhr et al., 1994
) and acrosome-reacting fresh spermatozoa (Nikolopoulou et al., 1986
; Roldan and Fragio, 1994
). Such decreases in both SM invagination (Ikezawa et al., 1980
) and vesiculation of the membrane also occur as a result of treatment with erythrocyte sphingomyelinase (Bernheimer et al., 1974
). Moreover, the increase in arachidonic acid may directly induce the initiation of the acrosome reaction in fresh, incubated spermatozoa (Mack et al., 1992
). This similarity in the alteration of phospholipids in cryopreserved and fresh spermatozoa suggests that a similar change in lipid membrane fluidity must occur. Moreover, these findings also suggested that membrane alteration (fluidity change), sperm swelling and membrane fusion (due to signal transduction) in cryopreserved spermatozoa might have occurred due to mechanisms of phospholipid reorientation and pump dysfunction.
It is generally accepted that in mammalian spermatozoa, hybrid vesicles form after the fusion of the plasma and outer acrosomal membranes (Yanagimachi, 1981). In contrast, a false acrosome reaction is not associated with fusion of the plasma membrane and outer acrosomal membrane. Previous reports on ultrastructure may have inadvertently included many false acrosome reactions among cryodamaged dead spermatozoa (Krogenaes et al., 1994
). In the present samples, TEM indicated a larger proportion of dead spermatozoa than did PI staining. In addition, FITC-PSA staining of cryopreserved spermatozoa indicated acrosome reactions in 20.1%, while TEM indicated 0%. Cell death is judged by different criteria in the two methods; with TEM, alterations in the cell membranes are judged.
It was found that during the initial stage of the acrosome reaction, sites of plasma and outer acrosomal membrane fusion were frequently observed in fresh spermatozoa that had been incubated, and also in cryopreserved spermatozoa that had not been incubated, though typical hybrid vesicles were only seldom observed in fresh spermatozoa. However, invagination of the outer acrosomal membrane was similar to that reported for both human (Nagae et al., 1986; Tesarik et al., 1988
) and boar (Jones, 1973
) spermatozoa. Furthermore, healthy baby monkeys have been born using this IVF protocol with fresh spermatozoa (Sankai, 2000
).
Morphological evidence revealed an increased incidence of acrosomal vesiculation (Jones and Martin, 1973; Murdoch and Jones, 1978
) and alteration (Quinn et al., 1969
; Pursel et al., 1972
; Woolley and Richardson, 1978
; Holt and North, 1984
) after spermatozoa were diluted and cooled from 30°C to 0°C before freezing, with or without fixation (Deleeuw et al., 1990
). Spermatozoa that lose their acrosomes during cryopreservation remain viable (McLaughlin et al., 1993
); therefore, the current results suggest that acrosomal vesiculation can occur before or during cryopreservation.
The current results regarding acrosomal membrane alteration are consistent with previous results for both cynomolgus monkey (Tollner et al., 1990) and human spermatozoa (Esteves et al., 1998
). In monkeys (Tollner et al., 1990
) the FITC-PSA method was used to examine cryopreserved live monkey spermatozoa, and showed 31% to be intact, 35% acrosome-altered and 4.4% acrosome-reacted.
TEM observations of the current cryopreserved samples revealed that 21.2% of the spermatozoa had intact acrosomes, compared with 61.6% as revealed by FITC staining. In contrast, 28.8% of these spermatozoa were swollen when observed by TEM. Therefore, spermatozoa that were classified as intact by FITC-PSA analysis most likely included those that were classified as swollen by TEM. The latter technique shows that the outer acrosomal membrane and acrosomal contents are present in these spermatozoa, and therefore they stain well with FITC-PSA. Certainly, the proportion of acrosome-intact spermatozoa revealed that fresh and cryopreserved spermatozoa differ in their rates of the acrosome reaction after pentoxifylline treatment (Esteves et al., 1998).
One question which remains is whether the resemblance to acrosome reaction observed in cryopreserved spermatozoa contributed to the true acrosome reaction. Although 80% of the spermatozoa survived after 2 h of incubation, plasma membrane swelling was increased, as was the number of vesicles (including electron-dense vesicles) in the spermatozoa. These changes are similar to those observed in fresh spermatozoa undergoing the acrosome reaction after 8 h of incubation, and suggest that incubation may hasten the initiation of the signal transduction system in cryopreserved vesiculated spermatozoa and/or swelling spermatozoa revealed by TEM.
When cryopreserved spermatozoa were incubated for 2 h, there was a decrease in the rate of acrosome-reacting spermatozoa, accompanied by a significant increase in the rate of completely acrosome-reacted spermatozoa. These results clearly showed that the spermatozoa remained alive during the 2 h incubation, and that the acrosome reaction process progressed during incubation. However, it seems unlikely that the completely acrosome-reacted spermatozoa (lost equatorial segment spermatozoa) could pass through the zona pellucida. Furthermore, the rate of dead spermatozoa increased drastically when cryopreserved spermatozoa were incubated for more than 2 h (data not shown). In a previous study (Sankai et al., 1994) it was found that the cryopreserved spermatozoa which had been incubated for 2 h were hyperactivated. Here, we report that such spermatozoa were capable of fusion with zona-free cynomolgus monkey oocytes within 1530 min (data not shown). The fertilization of zona-free oocytes by these spermatozoa was not accelerated by a zona-induced acrosome reaction, whereas fresh spermatozoa incubated for only 2 h could not penetrate such oocytes (Sankai et al., 1994
). In the current study, only 4.3% (TEM) of fresh spermatozoa incubated for 2 h were vesiculated, which might explain this low rate of fertilization. Highly motile spermatozoa most likely experience a true reaction and become capable of fertilization. Furthermore, acrosome-reacted cynomolgus monkey spermatozoa can bind to the zona pellucida (Vandevoort et al., 1997
), unlike mouse spermatozoa (Bleil and Wassarman, 1983
).
It has been found (Critser et al., 1987) that the rate at which cryopreserved human spermatozoa penetrate zona pellucida-free hamster oocytes was highest (~55%) immediately after thawing. Others (Salamon and Maxwell, 1995
) found that cryopreserved ram spermatozoa displayed decreased viability and fertility when required to pass through the cervix and the female genital tract, but possessed a high oocyte fertilization rate (8595%) after intrauterine or tubal deposition (spermatozoa inseminated near the fertilization site) by laparoscopy. These results confirm that the acrosome reaction has already begun in cryopreserved spermatozoa, which explains why they require a shorter incubation period for in-vitro fertilization than do fresh spermatozoa. Thus, the fertility and viability of cryopreserved spermatozoa are reduced.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed at: Department of Anatomy, Toho University School of Medicine, 5-21-16 Omori Nishi, Ota-ku, Tokyo 143-8540, Japan. E-mail: okada2da{at}med.toho-u.ac.jp
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References |
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Submitted on January 3, 2001; accepted on July 9, 2001.