Human Reproduction, Vol. 16, No. 1, 148-152,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Influence of sperm immobilization on onset of Ca2+ oscillations after ICSI
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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Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca2+ oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca2+ oscillations was observed at 4.8 to 80.4 min after ICSI. Ca2+ oscillations developed earlier with the piezo method (14.4 ± 6.4 min) than other methods (pipetting, 43.1 ± 20.2 min, P < 0.01; squeezing, 18.4 ± 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca2+ oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca2+ oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.
Key words: Ca2+ oscillations/ICSI/piezo/spermatozoa—oocyte interaction/sperm immobilization
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Introduction |
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Two mechanisms exist for oocyte activation: one due to the signals generated by spermatozoon–oocyte fusion mediated by receptors on the oocyte plasma membrane; the other due to signals from soluble sperm factors. In the past, it has been suggested that oocyte activation is related to signals from both receptors and sperm factors. Since spermatozoon–oocyte fusion is bypassed in intracytoplasmic sperm injection (ICSI), the signals from sperm factors are thought to be important for oocyte activation in the mechanism of fertilization by ICSI. Sperm immobilization before ICSI is considered necessary for efficient fertilization to occur (Svalander et al., 1995
; Vanderzwalmen et al., 1996). The sperm plasma membrane is disrupted after sperm immobilization, this damage favouring the liberation of the soluble sperm factor(s) that induce oocyte activation (Dozortsev et al., 1997). During the early stages of spermatozoon–oocyte interaction, Ca2+ oscillations in oocytes can be observed (Miyazaki and Igusa, 1981; Cuthbertson et al., 1981). Using the original ICSI technique, second polar bodies are usually observed within 5 h, and male and female pronuclei in the oocyte no sooner than 6 h (Nagy et al., 1994). Thus, it seems that Ca2+ oscillations in the oocyte occur within 5 h of ICSI. However, a previous report indicated that in human oocytes, Ca2+ oscillations develop between 2 and 12 h [mean (± SEM) 6.2 ± 1.9 h] after ICSI (Tesarik et al., 1994). This onset time of Ca2+ oscillations was considered to be too late for oocyte activation. The suggestion was made that these differences of timing might be related to differences in the sperm immobilization method used for the ICSI procedure. Reported methods of sperm immobilization include pipetting (Redgment et al., 1994; Gearon et al., 1995; Yanagida et al., 1999), squeezing, and the piezo method (Yanagida et al., 1999). The squeezing method has involved touching (Palermo et al., 1993; Tesarik et al., 1994; Silber, 1995), rubbing (Atiee et al., 1995; Vanderzwalmen et al., 1996), stroking (Sakkas et al., 1996) or pressing (Dozortsev et al., 1994; Payne et al., 1994; Silber et al., 1995). The degree of sperm plasma membrane disruption following immobilization was studied by observing eosin staining of the spermatozoa, as well as Ca2+ oscillations induced in oocytes. The correlation between the method of sperm immobilization and the oocyte-activating ability was examined, and the influence of sperm immobilization on clinical performance evaluated. |
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Human oocytes were taken from consenting patients with the approval of the Ethics Board at our institution.
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 16–24 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 (5–10 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 10–20 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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Influence of sperm immobilization on the sperm plasma membrane
Motile spermatozoa were immobilized by the aforementioned methods. Figure 1
shows the average time (in seconds) required for the entire sperm head to be stained red. Sperm staining took (on average) 5.0 s in the piezo group, 42.2 s in the squeezing group, and 220.7 s in the pipetting group. Significant differences were observed between these three methods. When motile spermatozoa were left without immobilization in the drop of eosin Y solution, motility disappeared 6 min after exposure and the sperm heads began to stain red.
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Fluorimetric measurement of Ca2+ oscillations after ICSI
Initial increases in [Ca2+]i were recorded at a mean (± SD) of 28.3 ± 19.4 min (range: 4.8 to 80.4 min) after ICSI. Figure 2
shows the initial change of [Ca2+]i and Ca2+ oscillations (4.8 min after ICSI) in one oocyte. Increases in [Ca2+]i immediately after ICSI were caused by the flow of extracellular Ca2+ into the oocyte after puncture by the injection pipette; this reaction was completed in ~1 min. In this case, Ca2+ oscillations began from 4.8 min after ICSI. Figure 3 shows the changes in [Ca2+]i for 160 min after ICSI in a second oocyte; regular increases in [Ca2+]i at intervals of ~11 min can be identified.
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The time required for initial Ca2+ oscillations after ICSI by pipetting, squeezing and piezo pulse application are shown in Table I. With these methods, no significant differences in the time of initial Ca2+ oscillations were seen between fresh oocytes and in-vitro-matured (IVM) oocytes. In fresh oocytes, Ca2+ oscillations developed significantly earlier with the piezo group than with the pipetting group (P < 0.05); this pattern was not observed for IVM oocytes. In the case of a combination of fresh and IVM oocytes, Ca2+ oscillations were observed earlier in the piezo group than other groups. A total of 12 IVM oocytes was examined in the non-immobilized group; seven of the oocytes did not show Ca2+ oscillations within 3 h, while three out of these seven had motile spermatozoa in the oocytes and four did not show Ca2+ oscillations during measurement. The average onset time of the remaining five oocytes measurable for Ca2+ oscillations was 69.0 min after ICSI.
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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There are two key aspects of fertilization in ICSI, namely sperm immobilization and secure injection of the spermatozoon into an oocyte (Dozortsev et al., 1995
; Svalander et al., 1995; Vanderzwalmen et al., 1996). Injection of a motile spermatozoon without sperm immobilization leads to poor fertilization rates (Hoshi et al., 1995; Vanderzwalmen et al., 1996). In such cases, spermatozoa with moving tails have been observed in the oocyte even within 2 h after ICSI treatment (unpublished data), and spermatozoon–oocyte interaction is considered to be obstructed by the normal sperm plasma membrane. The damage to the sperm membrane after immobilization may induce gradual disruption of other parts of the sperm membrane. This situation would make it easier for sperm factors (Dale and DeFelice, 1990; Swann, 1990) to act on ooplasmic factors related to signal transduction of oocyte activation. Neither the nature nor the mode of action of the sperm factor(s) have been identified definitively (Parrington et al., 1996; Fissore et al., 1999), and it has not been clear whether sperm factors induce only oocyte activation, or oocyte activation and Ca2+ oscillations. It has been reported (Yanagida et al., 2000) that mouse elongated spermatids activated mouse oocytes but did not induce normal Ca2+ oscillations. These workers observed sporadic intracellular Ca2+ increases following elongated-spermatid injection. However, normal Ca2+ oscillations could be observed following injection of two elongated spermatids. On the basis of these results, it might be considered that the sperm factor induced the initial increase in intracellular Ca2+ and also played the role of oscillator. Since fertilization by ICSI does not entail spermatozoon–oocyte fusion, the action of these sperm factors is important for accomplishing fertilization. Sperm factor is reported to exist in the equatorial segment of the sperm head (Parrington et al., 1996), or in the perinuclear materials (Kuretake et al., 1996).
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 S–S 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 spermatozoon–oocyte 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 spermatozoon–oocyte 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: 4.8–80.4 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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We thank Dr Rosario Aiko Orgaz Asanuma for her assistance in the preparation of the manuscript.
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Notes |
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3 To whom correspondence should be addressed at: 1 Hikarigaoka Fukushima, Fukushima 960-1295, Japan. E-mail: kyana{at}fmu.ac.jp
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References |
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Atiee, S.H., Pool, T.B. and Martin, J.E. (1995) A simple approach to intracytoplasmic sperm injection. Fertil. Steril., 63, 652–655.[Web of Science][Medline]
Cuthbertson, K.S.R., Whittingham, D.G. and Cobbold, P.H. (1981) Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature, 294, 754–757.[Medline]
Dale, B. and DeFelice, L.J. (1990) Soluble sperm factors, electrical events and egg activation. In Dale, B. (ed.), Mechanism of Fertilization. Springer-Verlag, Berlin, pp. 475–487.
Dozortsev, D., De Sutter, P. and Dhont, M. (1994) Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum. Reprod., 9, 2139–2144.
Dozortsev, D., Rybouchkin, A., De Sutter, P. et al. (1995) Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum. Reprod., 10, 2960–2964.
Dozortsev, D., Qian, C., Ermilov, A. et al. (1997) Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 min after injection as a result of the sperm-oocyte interaction. Hum. Reprod., 12, 2792–2796.
Fissore, R.A., Reis, M.M. and Palermo, G.D. (1999) Isolation of the Ca2+ releasing component(s) of mammalian sperm extracts: the search continues. Mol. Hum. Reprod., 5, 189–192.
Garner, D.L. and Johnson, L.A. (1995) Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod., 53, 276–284.[Abstract]
Gearon, C.M., Taylor, A.S. and Forman, R.G. (1995) Factors affecting activation and fertilization of human oocytes following intracytoplasmic sperm injection. Hum. Reprod., 10, 896–902.
Hoshi, K., Yanagida, K., Yazawa, H. et al. (1995) Intracytoplasmic sperm injection using immobilized or motile human spermatozoon. Fertil. Steril., 63, 1241–1245.[Web of Science][Medline]
Kuretake, S., Kimura, Y., Hoshi, K. and Yanagimachi, R. (1996) Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol. Reprod., 55, 789–795.[Abstract]
Miyazaki, S. and Igusa, Y. (1981) Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations. Nature, 290, 702–704.[Medline]
Nagy, Z.P., Liu, J., Joris, H. et al. (1994) Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, 1743–1748.
Nakano,Y., Shirakawa, H., Mitsuhashi, N. et al. (1997) Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon. Mol. Hum. Reprod., 3, 1087–1093.
Palermo, G., Joris, H., Derde, M.P. et al. (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil. Steril., 59, 826–835.[Web of Science][Medline]
Palermo, G.D., Schlegel, P.N., Colombero, L.T. et al. (1996) Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum. Reprod., 11, 1023–1029.
Parrington, J., Swann, K., Shevchenko, V.I. et al. (1996) Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature, 379, 364–368.[Medline]
Payne, D., Flaherty, S.P., Jeffrey, R. et al. (1994) Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection. Hum. Reprod., 9, 2051–2057.
Redgment, C.J., Yang, D., Tsirigotis, M. et al. (1994) Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro. Hum. Reprod., 9, 680–683.
Sakkas, D., Urner, F., Bianchi, P.G. et al. (1996) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum. Reprod., 11, 837–843.
Silber, S.J. (1995) Epididymal and testicular spermatozoa and intracytoplasmic sperm injection. Assist. Reprod. Rev., 6, 45–53.
Silber, S.J., Nagy, Z., Liu, J. et al. (1995) The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum. Reprod., 10, 2031–2043.
Svalander, P., Forsberg, A.S., Jakobsson, A.H. et al. (1995) Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility. Fertil. Steril., 63, 828–837.[Web of Science][Medline]
Swann, K. (1990) A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs. Development, 110, 1295–1302.
Tesarik, J., Sousa, M. and Testart, J. (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod., 9, 511–518.
Vanderzwalmen, P., Bertin, G., Lejeune, B. et al. (1996) Two essential steps for a successful intracytoplasmic sperm injection: injection of immobilized spermatozoa after rupture of the oolemma. Hum. Reprod., 11, 540–547.
Yanagida, K., Yanagimachi, R., Perreault, S.D. et al. (1991) Thermostability of sperm nuclei assessed by microinjection into hamster oocytes. Biol. Reprod., 44, 440–447.[Abstract]
Yanagida, K., Katayose, H., Yazawa, H. et al. (1998) The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans. Hum. Reprod., 14, 448–453.
Yanagida, K., Yazawa, H., Katayose, H. et al. (2000) Oocyte activation induced by spermatids and the spermatozoa. Int. J. Androl., 23 (Suppl. 2), 63–65.
Submitted on June 27, 2000; accepted on October 2, 2000.
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