1 Department of Obstetrics and Gynecology, Fukushima Medical University, Fukushima, Japan and 2 Department of Obstetrics and Gynecology, Yamanashi Medical University, Yamanashi, Japan
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Abstract |
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Key words: Ca2+ oscillations/ICSI/piezo/spermatozoaoocyte interaction/sperm immobilization
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Introduction |
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Materials and methods |
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Preparation of gametes
Semen samples were taken from fertile volunteers and liquefied for about 30 min. Motile spermatozoa were collected principally by the swim-up method using human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA), supplemented with 6% plasmanate cutter (PPF, Bayer Pharmaceutical Co., Osaka, Japan). When the swim-up method was not effective, sperm suspensions were prepared by centrifugal washing (250 g for 10 min) with HEPES-buffered HTF (mHTF).
Eleven metaphase II oocytes were provided by three patients, none of whom had undergone ICSI because of failures in sperm collection. Thirty-eight metaphase I oocytes taken from 26 patients treated by ICSI were further incubated; 21 metaphase I oocytes subsequently became metaphase II and were used for the experiment. Ovarian stimulation and oocyte collection were performed as described previously (Yanagida et al., 1999). The oocytes were collected transvaginally 35 h after the administration of human chorionic gonadotrophin (HCG), and then incubated for ~5 h and pipetted in mHTF containing 0.025% hyaluronidase (Type VIII, Sigma Chemical Co., St Louis, MO, USA) to remove cumulus cells. Oocytes were then evaluated for maturity. Metaphase II oocytes were further incubated for 5 h in HTF until the experiment was commenced. Metaphase I oocytes were incubated in HTF for 1624 h to obtain metaphase II oocytes for the experiment.
Sperm immobilization and eosin staining
The influence of sperm immobilization on the sperm plasma membrane was examined by eosin staining. A small drop of sperm suspension and 1% eosin Y (in mHTF; Eosin Y, Sigma), used to immobilize the motile spermatozoa, was prepared in an ICSI chamber. Next, using a microinjector and a micromanipulator mounted on an inverted microscope (IX-70, Olympus, Tokyo, Japan) and equipped with Hoffman modulation (Hoffman Modulation Contrast, Model EP, Olympus), a motile spermatozoon was carefully aspirated into the injection pipette (outer diameter of tip = 5 µm) from the suspension. The spermatozoon was immobilized in the drop of eosin Y, and the time from immobilization until the entire sperm head stained red (magnification, x300) was measured. The methods of sperm immobilization were the squeezing method, the pipetting method, and the piezo method. The squeezing method was achieved by squeezing the upper one-third of the sperm tail against the chamber bottom with the pipette tip. Two other methods were assessed by the use of three to five pipettings without squeezing and piezo-pulse application to the upper one-third of the sperm tail using a piezo micromanipulator (PMM-MB-A, Prime Tech Ltd, Tuchiura, Japan). Sperm immobilization with the piezo micromanipulator was conducted as described previously (Yanagida et al., 1999).
Fluorometric measurement of Ca2+ oscillations after ICSI
Method of ICSI
The same chamber (Chambered coverglass, Nunc, Inc., Naperville, IL, USA) was used for ICSI and fluorometric measurement. At the centre of the chamber, 3 µl drops of mHTF, sperm suspension and 8% polyvinyl pyrrolidone (PVP, mol. wt 360 000; Sigma) solution in D-PBS were placed in line, then covered with mineral oil (Sigma). The chamber was mounted onto the stage of a Nikon Diaphoto microscope equipped with the microinjection system and warmed to 37°C. ICSI was conducted as described previously (Yanagida et al., 1999).
Sperm immobilization was conducted in an 8% PVP drop by the three methods mentioned previously. For oocyte injection, immobilized spermatozoa (or motile spermatozoa when used) were drawn into an injection pipette, tail first, and injected into the oocyte by using a piezo micromanipulator. First, the pipette was allowed to penetrate only through the zona pellucida while piezo pulses (510 pulses, at ~0.5 Hz rate) were applied. The needle was then allowed to penetrate deeply into the ooplasm without piezo driving, and when the oolemma was extended sufficiently it was punctured by a single piezo pulse. No ooplasm was aspirated into the pipette when the spermatozoa were injected.
Measurement of Ca2+ oscillations
Just before ICSI, the oocytes were incubated in HTF supplemented with 44 µmol/l fluo-3 acetoxymethyl ester (Fluo-3/AM; Molecular Probes Inc., Eugene, OR, USA; dissolved in dimethyl sulphoxide) for 45 min and washed three times. The washed oocytes were then put in mHTF in a microinjection chamber. A spermatozoon immobilized by one of the methods described above was injected into an oocyte by ICSI; immediately afterwards, changes in Ca2+ concentration in the oocyte ([Ca2+]i) were measured using a Bio-Rad MRC-600 (Nippon Bio-Rad Lab., Tokyo, Japan) confocal laser scanning microscope system. ICSI and calcium measurements were performed using the same microscope. Measurements were started immediately after ICSI and continued for a maximum of 3 h at intervals of 1020 s.
Clinical results by immobilization method
A total of 365 couples treated by ICSI in our institution between July 1996 and April 1998 was divided into three groups based on sperm immobilization: a pipetting group (130 treatment cycles); a squeezing group (76 treatment cycles); and a piezo group (159 treatment cycles). Rates of fertilization, cleavage, and pregnancy for the three groups were then compared.
Statistical analysis
Analysis of variance (ANOVA) and Fisher's protected lasting significant difference (PLSD), Student's t-test and the 2-test were used for statistical analysis where appropriate. A P value of < 0.05 was considered statistically significant.
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Results |
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Immobilization methods and clinical results
Clinical results classified by immobilization method are shown in Table II, and indicate no significant differences among the three groups in terms of average age, average number of oocytes treated by ICSI, and average number of embryos transferred.
The rates of fertilization and cleavage are shown in Figure 4. The piezo group showed a fertilization rate of 78.3%, significantly higher than that of the pipetting and squeezing groups (P < 0.001 and < 0.01 respectively). No significant differences were found between the pipetting and squeezing groups; neither were there any significant differences between groups in terms of cleavage rate. In addition, there were no significant differences in pregnancy rate among these three groups (Figure 5
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Discussion |
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It was found that, when a motile spermatozoon was immobilized, the sperm head was stained immediately by live stains such as eosin (Dozortsev et al., 1995) or the Live/Dead Sperm Fertilight Kit (Garner and Johnson, 1995
). This means that some low-molecular weight substances may enter the sperm head when the sperm membrane is damaged by immobilization. Therefore, the time of action of sperm factors may be related to the time taken to stain the sperm head with eosin after prior immobilization. Immobilization methods can be broadly classified as three types: pipetting, squeezing, and piezo application. However, no reports have yet assessed the degree of damage that each of these methods causes to the sperm plasma membrane. To evaluate this, the time required to achieve full eosin staining of the immobilized spermatozoan head was measured. The results indicated that the piezo method produces the earliest staining of the sperm head, but most likely causes the most severe damage to the sperm membrane, followed by squeezing and pipetting. Indeed, we observed earlier onset of oocyte Ca2+ oscillations in the piezo method than the other two methods. We also observed eosin staining of the spermatozoa without immobilization. It seemed that the reason for staining was dependent upon the cell toxicity of eosin Y. In addition, we observed Ca2+ oscillations in 42% (5/12) of oocytes injected with spermatozoa without immobilization. This may be related to the small diameter (5 µm) of the needle and the fact that motile spermatozoa suffered less damage during the ICSI procedure.
Because damage was induced in the sperm plasma membrane after immobilization, the sperm nucleus decondensing factor of the oocyte can enter the spermatozoon and induce initial swelling of the head. As a result of this swelling, the sperm plasma membrane ruptures and sperm-associated oocyte activating factors are released into the ooplasm to induce oocyte activation (Dozortsev et al., 1997). In our earlier study, the onset of sperm head swelling began 30 min after ICSI, when human ejaculated spermatozoa were injected into hamster oocytes after immobilization, and we observed the swelling with acetolacmoid stain (Yanagida et al., 1991
). As for testicular spermatozoa, the onset began from 15 min after ICSI because they have fewer SS bonds in protamine (unpublished data). Based on the results of this research, we observed the onset of Ca2+ oscillations 4.8 min after ICSI. In these cases, swelling of the sperm head had not occurred at the time when Ca2+ oscillations began. Hence, swelling of the sperm head is not always necessary for the release of sperm factor.
In our study, the piezo method yielded significantly higher fertilization rates than the other two methods, with greater degrees of immobilization leading in turn to higher rates of fertilization. It has been reported (Palermo et al., 1996) that aggressive sperm immobilization (achieved by permanently crimping the sperm flagellum between the middle piece and the tail) improves fertilization rates. No significant differences in cleavage rate and pregnancy rate were found among the three immobilization methods in these studies, and we cannot provide an explanation for this phenomenon. The investigation period of this study was long, extending from April 1996 until June 1998. Thus, the difference may depend on the times when we performed ICSI.
Ca2+ oscillations due to spermatozoonoocyte interaction were observed at 4.8 min after ICSI in the shortest onset case. Earlier reports cite initial Ca2+ oscillations in human oocytes occurring at between 2 and 12 h (average 6.2 h) after ICSI (Tesarik et al., 1994). Our results differ considerably from those of the aforementioned report, and differences in the sperm immobilization methods used may be responsible for this discrepancy. With ICSI, extrusion of the second polar body could be observed from 2 h after ICSI in about half of the oocytes fertilized (Nagy et al., 1994
). Hence, the spermatozoonoocyte interaction must be occurring within 2 h after ICSI. We showed the average onset time of initial Ca2+ oscillations to be 28.3 ± 19.4 min (range: 4.880.4 min), while others (Nakano et al., 1997
), using an isolated mouse sperm head, reported Ca2+ oscillations to be induced within 30 min in mouse oocytes treated by ICSI.
We conclude that differences in immobilization methods affect the timing of initial Ca2+ oscillations and that the sperm immobilization method may be important for the rapid release of sperm factors that initiate oocyte activation. The present study also showed that Ca2+ oscillations develop earlier in human oocytes treated by ICSI than has been indicated in previous reports.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 27, 2000; accepted on October 2, 2000.