Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis)

Akiko Okada1,4, Hiroaki Igarashi1, Masaru Kuroda1, Keiji Terao2, Yasuhiro Yoshikawa2,3 and Tadashi Sankai2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear. METHODS: Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa. RESULTS: Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted. CONCLUSIONS: Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.

Key words: acrosome reaction/cryopreservation/cynomolgus monkey/electron microscopy/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Successful cryopreservation of monkey spermatozoa has been limited, despite the need for reproductive technologies to assist in maintaining the genetic resources of non-human primates. Studies suggest that the successful cryopreservation of mammalian spermatozoa accelerates both capacitation (Watson, 1995Go; Gillan et al., 1997Go) and the acrosome reaction (McLaughlin et al., 1993Go). It has also previously been reported that cryopreserved cynomolgus monkey spermatozoa were able to fertilize oocytes after a shorter incubation period than was required for fresh spermatozoa (Sankai et al., 1994Go). However, different results were reported: in one study (McLaughlin et al., 1993Go) accelerated acrosome reaction was reported for a live human sperm population, whereas others (Cross and Hanks, 1991Go) reported near-100% intact acrosomes.

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, 1973Go; Krogenaes et al., 1994Go). 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, 1995Go; Fuller and Whittingham, 1997Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection and processing of semen
Four adult male cynomolgus monkeys (age 9–12 years) which had been bred and maintained at the Tsukuba Primate Center, National Institute of Infectious Diseases (Japan), were used in these studies. Fresh semen was collected from each monkey (anaesthetized with ketamine hydrochloride) by using electrode stimulation to the rectum. The mean volume of ejaculate was about 0.1 ml. Because fresh semen from cynomolgus monkeys tends to coagulate quickly, the semen was placed immediately after ejaculation into a centrifuge tube containing 2 ml of Tyrode's solution modified according to Toyoda, Yokoyama and Hoshi [TYH medium (119.37 mmol/l NaCl, 4.78 mmol/l KCl, 12.6 mmol/l CaCl2•2H2O, 1.19 mmol/l MgSO4•7H2O, 1.19 mmol/l KH2PO4, 25.07 mmol/l NaHCO3, 5.56 mmol/l glucose, 1.0 mmol/l pyruvic acid Na salt, 5 mg/ml bovine serum albumin (BSA), and penicillin-G Na salt, streptomycin sulphate] (Toyoda et al., 1971Go) and kept at 37°C for up to 30 min until liquefaction occurred. The liquefied sperm suspension was then gently layered onto 2 ml of 90% iso-osmotic Percoll (Pharmacia, Uppsala, Sweden). This Percoll was prepared by mixing 1 volume of 9% NaCl (w/v) solution with 9 volumes of Percoll. The sample was centrifuged at 800 g for 10 min, after which the spermatozoa with high motility were recovered from the bottom of the tube. Only fresh spermatozoa and cryopreserved spermatozoa with high motility were used in this study.

Cryopreservation of spermatozoa
The composition of the semen diluent and the process of cryopreservation were identical to those described previously (Sankai et al., 1994Go). 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 5–10 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 0.05–0.2 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, 1985Go), 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., 1989Go; Mendoza et al., 1992Go).

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., 1988Go) 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, 1989Go); (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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The acrosomal status of non-incubated and incubated fresh and cryopreserved spermatozoa is indicated in Table IGo (TEM) and Tables II and IIIGoGo (FITC-PSA and PI staining).


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Table I. Acrosomal alteration in fresh and cryopreserved cynomolgus monkey spermatozoa with and without incubation, as determined by transmission electron microscopy
 

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Table II. Acrosomal status of fresh and cryopreserved sperm determined by FITC-PSA and PI staining
 

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Table III. Statistical analysis of FITC-PSA staining
 
Non-incubated spermatozoa
More than 90% of the cryopreserved and fresh spermatozoa survived and were vigorously motile. Therefore, compared with fresh spermatozoa and incubated spermatozoa, there was no reduction in the number of motile spermatozoa recovered after 90% Percoll centrifugation. The incidence of dead spermatozoa among the cryopreserved spermatozoa revealed by TEM observation (13.6%) was higher than that revealed by FITC staining (0.3%). Almost every intact fresh spermatozoon possessed plasma and outer acrosomal membranes, and had an acrosomal matrix that was firm and clear by both TEM (see Figure 6Go) and FITC observations (see Figure 10Go).



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Figure 6. Non-incubated, fresh cynomolgus monkey spermatozoa. The plasma membrane (PL), outer acrosomal membrane (OAM) and acrosomal matrix are intact. Scale bar = 0.5 µm.

 


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Figure 10. (TOP) Non-incubated fresh spermatozoa. Left: FITC-PSA staining; right: PI staining. Acrosome-intact spermatozoa are intensely stained by FITC-PSA, but there is no staining by PI. These six spermatozoa are classified as live, acrosome-intact. Scalebar = 5 µm.

 
More than 90% of the fresh spermatozoa had intact acrosomes as determined by both TEM and FITC observation. However, in cryopreserved spermatozoa, the incidence of intact acrosomes (Figure 1Go) was significantly lower; 21.2% by TEM and 61.6% by FITC staining (Tables I and IIGoGo).



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Figure 1. Non-incubated cryopreserved cynomolgus monkey spermatozoa. The acrosomal matrix, the smooth plasma membrane (PL) and outer acrosomal membrane (OAM) are intact. Scale bar = 0.5 µm

 
Only 8.1% of the fresh spermatozoa had altered acrosomes (modified spermatozoa + acrosome-reacted spermatozoa) by FITC observation, and none by TEM. In contrast, high rates (28.8%) of swollen spermatozoa (Figure 2Go) and 36.4% vesiculated spermatozoa were observed among cryopreserved spermatozoa by TEM. These vesiculated spermatozoa exhibited several pale vesicles (Figures 3 and 4GoGo), and no dark vesicles.



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Figure 2. Non-incubated cryopreserved cynomolgus monkey spermatozoa. The anterior portion of the acrosome has begun to swell and the outer acrosomal membrane (OAM) is irregular, but the equatorial region remains intact (arrow). Scale bar = 0.5 µm.

 


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Figure 3. Non-incubated cryopreserved cynomolgus monkey spermatozoa. (A) The anterior portion of acrosome has two vesicles within the clear acrosomal matrix. The equatorial segment is intact (arrow). (B) An enlargement of the boxed anterior portion of the acrosome shown in (A). Fusion (*) of the smooth plasma membrane (PL) and outer acrosomal membrane (OAM) is evident. Two vesicles with clear contents are indicated (arrowheads). Both deeply invaginated and shallowly invaginated regions of the OAM are obvious (arrows). Scale bar = 0.1 µm.

 


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Figure 4. Non-incubated cryopreserved cynomolgus monkey spermatozoa. (A) Many clear vesicles are evident within the more greatly swollen acrosome. Only a few sites of fusion between the smooth plasma membrane (PL) and outer acrosomal membrane (OAM) are clear, but the equatorial segment is intact (arrow). (B) An enlargement of the boxed anterior portion of the acrosome shown in (A). Four clear vesicles (arrowheads) are shown. Fusions (*) between the PL and OAM and a dense acrosomal matrix near the nuclear membrane are evident. Scale bar = 0.1 µm.

 
Occasionally, TEM observations of the non-incubated cryopreserved spermatozoa with intact equatorial segments revealed both invagination of the outer acrosomal membrane and fusion sites (Figure 3A,B and Figure 4A,BGoGo). Additionally, FITC staining revealed modified spermatozoa, acrosome-reacted spermatozoa (Figure 11Go) and completely acrosome-reacted spermatozoa totalling 38.1% (Table IIGo). This was comparable with the 36.4% of vesiculated spermatozoa revealed by TEM (Table IGo). However, unlike FITC staining, TEM did not reveal the existence of acrosome-reacted spermatozoa.



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Figure 11. (MIDDLE) Non-incubated cryopreserved spermatozoa. Left: FITC-PSA staining; right: PI staining. There are five acrosome-modified spermatozoa showing intense staining and irregular FITC-PSA brightness on the head, but there is no PI staining. There is one acrosome-reacted, live spermatozoon. The anterior acrosome lacks FITC-PSA staining; only the equatorial segment has intense FITC-PSA staining (arrow). Scale bar = 5 µm.

 
Spermatozoa incubated for 2 h
More than 80% of the recovered cryopreserved and fresh spermatozoa survived after 2 h of incubation. The merged FITC-PSA/PI-stained image of a 2 h-incubated cryopreserved spermatozoon shows yellowish staining of the nucleus, and indicates an acrosomally intact, dead spermatozoon (Figure 12Go). PI staining revealed a significant increase in the rate of dead cryopreserved spermatozoa, from 0.3% before incubation to 9.1% after incubation. However, TEM showed no significant difference between the rate of dead cryopreserved spermatozoa at 0 h (13.6%) and 2 h (15.0%).



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Figure 12. (BOTTOM) Cryopreserved spermatozoa incubated for2 h. Merged images of FITC-PSA and PI stained specimen. There are seven acrosome-intact spermatozoa with intense FITC-PSA on the sperm head. One sperm nucleus appears yellow (arrowhead), which results from the merged colour of the green FITC staining and the faintly red nucleus stained by PI. This staining pattern indicates a damaged spermatozoon with an intact acrosome. Another live, acrosome-modified spermatozoon shows irregular and moderate FITC-PSA staining (arrow). Scale bar = 5 µm.

 
According to TEM studies, the incidence of intact acrosomes among fresh spermatozoa was significantly lower (P < 0.001) after 2 h of incubation. In the cryopreserved spermatozoa, however, TEM did not reveal a significantly lower rate of intact acrosomes after 2 h of incubation (Table IGo).

Membrane fusion was sometimes observed among fresh spermatozoa (Figure 7A,BGo), 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,BGo). The dark vesicles resembled those of fresh spermatozoa that had been incubated for 2 or 8 h (Figures 8 and 9GoGo). The vesiculated spermatozoa still possessed a discernible plasma membrane and outer acrosomal membrane by TEM (Figure 5A,BGo). 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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Figure 7. Fresh cynomolgus monkey spermatozoa incubated for 2 h. (A) A few points of fusion are visible (*). Some areas with invagination of the outer acrosomal membrane (OAM) are visible. The acrosome is nearly normal in size. (B) Enlargement of the boxed anterior portion of the acrosome shown in (A). PL and OAM fusion (*), as well as the invagination of the OAM (arrow), are evident. Scale bar = 0.1 µm.

 


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Figure 5. Cryopreserved spermatozoa after 2 h of incubation. (A) Separate plasma membrane (PL) and outer acrosomal membrane (OAM) and dark, clear vesicles are shown. The equatorial segment is intact (arrow). Scale bar = 0.5 µm. (B) An enlargement of the boxed anterior portion of the acrosome shown in (A). A clear vesicle (arrowhead) and two dark vesicles (arrows) are shown. Scale bar = 0.1 µm.

 


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Figure 8. Fresh cynomolgus monkey spermatozoa incubated for 2 h. The dark (arrows) and clear vesicles are obvious. Scale bar = 0.5 µm.

 


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Figure 9. Fresh cynomolgus monkey spermatozoa incubated for 8 h. (A) Several vesicles (arrowheads) are visible within the swelling acrosome. (B) Enlargement of the boxed anterior portion of the acrosome shown in (A). Separate plasma membrane (PL) and outer acrosomal membrane (OAM) are still visible. The vesicles (arrowheads) and a dense acrosomal matrix (right side *) are shown. Scale bar = 0.1 µm.

 
Fresh spermatozoa incubated for 8 h
Although the survival rate for fresh spermatozoa incubated for 8 h was low (>70%) compared with that for non-incubated spermatozoa, some spermatozoa showed hyperactive movement. The rate of dead spermatozoa increased during the first 2 h of incubation as revealed by TEM, and did not significantly increase further between 2 and 8 h. Although it was a little higher when monitored by TEM, the value for dead spermatozoa at 8 h was similar for both PI staining and TEM.

TEM showed no significant difference in the rate of acrosome-intact spermatozoa among fresh spermatozoa incubated for 8 h and cryopreserved spermatozoa incubated for 0–2 h. However, FITC staining revealed a significant difference (P < 0.03; Table IIIGo) 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 IGo) the rate of swollen spermatozoa in comparison with fresh spermatozoa incubated for 2 h. Many dark vesicles (Figures 8 and 9GoGo) were present under the still-separated plasma and outer acrosomal membranes, and numerous swollen acrosomes contained a dark acrosomal matrix near the nucleus (Figure 9Go). 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 IIIGo). 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 IIGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It has previously been reported that a 2 h incubation of cryopreserved cynomolgus monkey spermatozoa were capable of fertilizing oocytes, which then developed to blastocyst stage, whereas fresh spermatozoa were incapable of fertilization (Sankai et al., 1994Go). The current results include evidence that alteration of the acrosomal membranes occurred in 38.1% (FITC-PSA) or 36.4% (TEM) of non-incubated cryopreserved live spermatozoa, in contrast with either 0% (TEM) or only 8.1% (FITC-PSA) of non-incubated fresh spermatozoa. This raises the question as to why membrane alterations that resemble acrosome reactions occur in cryopreserved spermatozoa.

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, 1995Go), 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., 1980Go). An increase in phospholipids, including arachidonic acid, and a decrease in sphingomyelin (SM) content occur both in cryopreserved spermatozoa (Buhr et al., 1994Go) and acrosome-reacting fresh spermatozoa (Nikolopoulou et al., 1986Go; Roldan and Fragio, 1994Go). Such decreases in both SM invagination (Ikezawa et al., 1980Go) and vesiculation of the membrane also occur as a result of treatment with erythrocyte sphingomyelinase (Bernheimer et al., 1974Go). Moreover, the increase in arachidonic acid may directly induce the initiation of the acrosome reaction in fresh, incubated spermatozoa (Mack et al., 1992Go). 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, 1981Go). 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., 1994Go). 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., 1986Go; Tesarik et al., 1988Go) and boar (Jones, 1973Go) spermatozoa. Furthermore, healthy baby monkeys have been born using this IVF protocol with fresh spermatozoa (Sankai, 2000Go).

Morphological evidence revealed an increased incidence of acrosomal vesiculation (Jones and Martin, 1973Go; Murdoch and Jones, 1978Go) and alteration (Quinn et al., 1969Go; Pursel et al., 1972Go; Woolley and Richardson, 1978Go; Holt and North, 1984Go) after spermatozoa were diluted and cooled from 30°C to 0°C before freezing, with or without fixation (Deleeuw et al., 1990Go). Spermatozoa that lose their acrosomes during cryopreservation remain viable (McLaughlin et al., 1993Go); 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., 1990Go) and human spermatozoa (Esteves et al., 1998Go). In monkeys (Tollner et al., 1990Go) 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., 1998Go).

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., 1994Go) 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 15–30 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., 1994Go). 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., 1997Go), unlike mouse spermatozoa (Bleil and Wassarman, 1983Go).

It has been found (Critser et al., 1987Go) that the rate at which cryopreserved human spermatozoa penetrate zona pellucida-free hamster oocytes was highest (~55%) immediately after thawing. Others (Salamon and Maxwell, 1995Go) 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 (85–95%) 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Drs F.Cho and T.Yoshida of the Tsukuba Primate Center for their invaluable advice, and Mr H.Tsuchiya of the Tsukuba Primate Center and Mr K.Murakami of Toho University for their technical assistance. They also thank Dr S.Nagae of Toho University and Dr H.Nagashima of the University of Meiji for their reading and valuable advice on the manuscript. This study was supported in part by a grant from the Ministry of Health and Welfare of Japan and by special coordination funds for promoting Science and Technology.


    Notes
 
3 Present address: Department of Medical Science, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,Tokyo 113-8657, Japan Back

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 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 3, 2001; accepted on July 9, 2001.