Influence of sperm immobilization on onset of Ca 2 oscillations after ICSI

Human Reproduction Vol.16, No.1 pp. 148 152, 2001 Influence of sperm immobilization on onset of Ca 2 oscillations after ICSI K.Yanagida 1,3, H.Katayose 1, S.Hirata 2, H.Yazawa 1, S.Hayashi 1 and A.Sato 1 1 Department of Obstetrics and Gynecology, Fukushima Medical University, Fukushima, Japan and 2 Department of Obstetrics and Gynecology, Yamanashi Medical University, Yamanashi, Japan 3 To whom correspondence should be addressed at: 1 Hikarigaoka Fukushima, Fukushima 960-1295, Japan. E-mail: kyana@fmu.ac.jp 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 Ca 2 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 Ca 2 oscillations was observed at 4.8 to 80.4 min after ICSI. Ca 2 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 Ca 2 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 Ca 2 oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports. Key words: Ca 2 oscillations/icsi/piezo/spermatozoa oocyte interaction/sperm immobilization onset time of Ca 2 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 Ca 2 oscillations induced in oocytes. The correlation between the method of sperm immobilization and the oocyte-activating ability was examined, and the influence of sperm immobiliza- tion on clinical performance evaluated. Introduction 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, Ca 2 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 Ca 2 oscillations in the oocyte occur within 5 h of ICSI. However, a previous report indicated that in human oocytes, Ca 2 oscillations develop between 2 and 12 h [mean ( SEM) 6.2 1.9 h] after ICSI (Tesarik et al., 1994). This Materials and methods 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 148 European Society of Human Reproduction and Embryology

Sperm immobilization and Ca 2 oscillations swim-up method using human tubal fluid (HTF) medium (Irvine to penetrate deeply into the ooplasm without piezo driving, and when Scientific, Santa Ana, CA, USA), supplemented with 6% plasmanate the oolemma was extended sufficiently it was punctured by a single cutter (PPF, Bayer Pharmaceutical Co., Osaka, Japan). When the piezo pulse. No ooplasm was aspirated into the pipette when the swim-up method was not effective, sperm suspensions were prepared spermatozoa were injected. by centrifugal washing (250 g for 10 min) with HEPES-buffered Measurement of Ca 2 oscillations HTF (mhtf). Just before ICSI, the oocytes were incubated in HTF supplemented Eleven metaphase II oocytes were provided by three patients, none with 44 µmol/l fluo-3 acetoxymethyl ester (Fluo-3/AM; Molecular of whom had undergone ICSI because of failures in sperm collection. Probes Inc., Eugene, OR, USA; dissolved in dimethyl sulphoxide) Thirty-eight metaphase I oocytes taken from 26 patients treated by for 45 min and washed three times. The washed oocytes were ICSI were further incubated; 21 metaphase I oocytes subsequently then put in mhtf in a microinjection chamber. A spermatozoon became metaphase II and were used for the experiment. Ovarian immobilized by one of the methods described above was injected stimulation and oocyte collection were performed as described previ- into an oocyte by ICSI; immediately afterwards, changes in Ca 2 ously (Yanagida et al., 1999). The oocytes were collected trans- concentration in the oocyte ([Ca 2 ] i ) were measured using a Biovaginally 35 h after the administration of human chorionic Rad MRC-600 (Nippon Bio-Rad Lab., Tokyo, Japan) confocal laser gonadotrophin (HCG), and then incubated for ~5 h and pipetted in scanning microscope system. ICSI and calcium measurements were mhtf containing 0.025% hyaluronidase (Type VIII, Sigma Chemical performed using the same microscope. Measurements were started Co., St Louis, MO, USA) to remove cumulus cells. Oocytes were immediately after ICSI and continued for a maximum of 3 h at then evaluated for maturity. Metaphase II oocytes were further intervals of 10 20 s. incubated for 5 h in HTF until the experiment was commenced. Metaphase I oocytes were incubated in HTF for 16 24 h to obtain Clinical results by immobilization method metaphase II oocytes for the experiment. 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 Sperm immobilization and eosin staining immobilization: a pipetting group (130 treatment cycles); a squeezing The influence of sperm immobilization on the sperm plasma membrane group (76 treatment cycles); and a piezo group (159 treatment cycles). was examined by eosin staining. A small drop of sperm suspension Rates of fertilization, cleavage, and pregnancy for the three groups and 1% eosin Y (in mhtf; Eosin Y, Sigma), used to immobilize the were then compared. motile spermatozoa, was prepared in an ICSI chamber. Next, using a microinjector and a micromanipulator mounted on an inverted Statistical analysis microscope (IX-70, Olympus, Tokyo, Japan) and equipped with Analysis of variance (ANOVA) and Fisher s protected lasting signi- Hoffman modulation (Hoffman Modulation Contrast, Model EP, ficant difference (PLSD), Student s t-test and the χ 2 -test were used Olympus), a motile spermatozoon was carefully aspirated into the for statistical analysis where appropriate. A P value of 0.05 was injection pipette (outer diameter of tip 5 µm) from the suspension. considered statistically significant. The spermatozoon was immobilized in the drop of eosin Y, and the time from immobilization until the entire sperm head stained red (magnification, 300) was measured. The methods of sperm immobil- Results ization were the squeezing method, the pipetting method, and the piezo method. The squeezing method was achieved by squeezing the Influence of sperm immobilization on the sperm plasma upper one-third of the sperm tail against the chamber bottom with membrane the pipette tip. Two other methods were assessed by the use of three Motile spermatozoa were immobilized by the aforementioned to five pipettings without squeezing and piezo-pulse application to methods. Figure 1 shows the average time (in seconds) required the upper one-third of the sperm tail using a piezo micromanipulator for the entire sperm head to be stained red. Sperm staining (PMM-MB-A, Prime Tech Ltd, Tuchiura, Japan). Sperm immobilizatook (on average) 5.0 s in the piezo group, 42.2 s in the tion with the piezo micromanipulator was conducted as described squeezing group, and 220.7 s in the pipetting group. Significant previously (Yanagida et al., 1999). differences were observed between these three methods. When Fluorometric measurement of Ca 2 oscillations after ICSI motile spermatozoa were left without immobilization in the drop of eosin Y solution, motility disappeared 6 min after Method of ICSI The same chamber (Chambered coverglass, Nunc, Inc., Naperville, exposure and the sperm heads began to stain red. IL, USA) was used for ICSI and fluorometric measurement. At the centre of the chamber, 3 µl drops of mhtf, sperm suspension and Fluorimetric measurement of Ca 2 oscillations after ICSI 8% polyvinyl pyrrolidone (PVP, mol. wt 360 000; Sigma) solution in Initial increases in [Ca 2 ] i were recorded at a mean ( SD) D-PBS were placed in line, then covered with mineral oil (Sigma). of 28.3 19.4 min (range: 4.8 to 80.4 min) after ICSI. Figure 2 The chamber was mounted onto the stage of a Nikon Diaphoto shows the initial change of [Ca 2 ] i and Ca 2 oscillations (4.8 microscope equipped with the microinjection system and warmed min after ICSI) in one oocyte. Increases in [Ca 2 ] i immediately to 37 C. ICSI was conducted as described previously (Yanagida after ICSI were caused by the flow of extracellular Ca 2 into et al., 1999). the oocyte after puncture by the injection pipette; this reaction Sperm immobilization was conducted in an 8% PVP drop by the was completed in ~1 min. In this case, Ca 2 oscillations began three methods mentioned previously. For oocyte injection, immobilfrom 4.8 min after ICSI. Figure 3 shows the changes in [Ca 2 ized spermatozoa (or motile spermatozoa when used) were drawn ] i into an injection pipette, tail first, and injected into the oocyte by for 160 min after ICSI in a second oocyte; regular increases using a piezo micromanipulator. First, the pipette was allowed to in [Ca 2 ] i at intervals of ~11 min can be identified. penetrate only through the zona pellucida while piezo pulses (5 10 The time required for initial Ca 2 oscillations after ICSI by pulses, at ~0.5 Hz rate) were applied. The needle was then allowed pipetting, squeezing and piezo pulse application are shown in 149

K.Yanagida et al. Figure 1. Evaluation of damages to immobilized spermatozoa using eosin stain. Motile spermatozoa were immobilized using one of the three selected methods (A, pipetting; B, squeezing; C, piezo), and the time required for full staining of the sperm head was measured (original magnification, 300). Fisher s PLSD revealed significant differences between A and B (P 0.0001), between B and C (P 0.005), and between A and C (P 0.0001). Figure 3. Ca 2 oscillations developed after ICSI. Fresh oocyte. Motile spermatozoa were immobilized by applying piezo pulses and one spermatozoon was injected by ICSI. The x-axis shows elapsed time after ICSI, and the y-axis shows relative fluorometric intensity of [Ca 2 ] i. The oocyte continued Ca 2 oscillations for more than 3 h, and attained the 4-cell stage at 48 h after ICSI. Figure 4. Rates of fertilization and cleavage after ICSI for different sperm immobilization methods (A, pipetting; B, squeezing; C, piezo). Significant differences were identified between A and C, Figure 2. Initial development of Ca 2 oscillations after ICSI. Fresh and between B and C. No significant differences were observed oocyte. Motile spermatozoa were immobilized by applying piezo between three methods (χ 2 -test). pulses and one spermatozoon was injected by ICSI. Time 0 indicates the start of ICSI. The graph shows relative intensities, contains optical-system switching noise, and shows the increase in fluorometric intensity of [Ca 2 ] i due to the flow of extracellular oocytes measurable for Ca 2 oscillations was 69.0 min after Ca 2 into the oocyte after puncture by the injection needle. The ICSI. first increase in [Ca 2 ] i in the oocyte was observed 4.8 min after ICSI. Immobilization methods and clinical results Clinical results classified by immobilization method are shown Table I. With these methods, no significant differences in the in Table II, and indicate no significant differences among the time of initial Ca 2 oscillations were seen between fresh three groups in terms of average age, average number of oocytes and in-vitro-matured (IVM) oocytes. In fresh oocytes, oocytes treated by ICSI, and average number of embryos Ca 2 oscillations developed significantly earlier with the piezo transferred. group than with the pipetting group (P 0.05); this pattern The rates of fertilization and cleavage are shown in Figure 4. was not observed for IVM oocytes. In the case of a combination The piezo group showed a fertilization rate of 78.3%, significantly of fresh and IVM oocytes, Ca 2 oscillations were observed higher than that of the pipetting and squeezing groups earlier in the piezo group than other groups. A total of 12 (P 0.001 and 0.01 respectively). No significant differences IVM oocytes was examined in the non-immobilized group; were found between the pipetting and squeezing groups; neither seven of the oocytes did not show Ca 2 oscillations within were there any significant differences between groups in 3 h, while three out of these seven had motile spermatozoa in terms of cleavage rate. In addition, there were no significant the oocytes and four did not show Ca 2 oscillations during differences in pregnancy rate among these three groups measurement. The average onset time of the remaining five (Figure 5). 150

Sperm immobilization and Ca 2 oscillations 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 Ca 2 oscillations in the piezo method Figure 5. Pregnancy rate (%) after ICSI for different sperm than the other two methods. We also observed eosin staining immobilization methods. No significant differences were found of the spermatozoa without immobilization. It seemed that the between the groups (χ 2 -test). reason for staining was dependent upon the cell toxicity of eosin Y. In addition, we observed Ca 2 oscillations in 42% Discussion (5/12) of oocytes injected with spermatozoa without immobilization. There are two key aspects of fertilization in ICSI, namely This may be related to the small diameter (5 µm) of sperm immobilization and secure injection of the spermatozoon the needle and the fact that motile spermatozoa suffered less into an oocyte (Dozortsev et al., 1995; Svalander et al., damage during the ICSI procedure. 1995; Vanderzwalmen et al., 1996). Injection of a motile Because damage was induced in the sperm plasma membrane spermatozoon without sperm immobilization leads to poor after immobilization, the sperm nucleus decondensing factor fertilization rates (Hoshi et al., 1995; Vanderzwalmen et al., of the oocyte can enter the spermatozoon and induce initial 1996). In such cases, spermatozoa with moving tails have been swelling of the head. As a result of this swelling, the sperm observed in the oocyte even within 2 h after ICSI treatment plasma membrane ruptures and sperm-associated oocyte activating (unpublished data), and spermatozoon oocyte interaction is factors are released into the ooplasm to induce oocyte considered to be obstructed by the normal sperm plasma activation (Dozortsev et al., 1997). In our earlier study, the membrane. The damage to the sperm membrane after immobilization onset of sperm head swelling began 30 min after ICSI, when may induce gradual disruption of other parts of the human ejaculated spermatozoa were injected into hamster sperm membrane. This situation would make it easier for oocytes after immobilization, and we observed the swelling sperm factors (Dale and DeFelice, 1990; Swann, 1990) to act with acetolacmoid stain (Yanagida et al., 1991). As for on ooplasmic factors related to signal transduction of oocyte testicular spermatozoa, the onset began from 15 min after ICSI activation. Neither the nature nor the mode of action of the because they have fewer S S bonds in protamine (unpublished sperm factor(s) have been identified definitively (Parrington data). Based on the results of this research, we observed the et al., 1996; Fissore et al., 1999), and it has not been clear onset of Ca 2 oscillations 4.8 min after ICSI. In these cases, whether sperm factors induce only oocyte activation, or oocyte swelling of the sperm head had not occurred at the time when activation and Ca 2 oscillations. It has been reported (Yanagida Ca 2 oscillations began. Hence, swelling of the sperm head is et al., 2000) that mouse elongated spermatids activated mouse not always necessary for the release of sperm factor. oocytes but did not induce normal Ca 2 oscillations. These In our study, the piezo method yielded significantly higher workers observed sporadic intracellular Ca 2 increases following fertilization rates than the other two methods, with greater elongated-spermatid injection. However, normal Ca 2 degrees of immobilization leading in turn to higher rates of oscillations could be observed following injection of two fertilization. It has been reported (Palermo et al., 1996) that elongated spermatids. On the basis of these results, it might aggressive sperm immobilization (achieved by permanently be considered that the sperm factor induced the initial increase crimping the sperm flagellum between the middle piece and in intracellular Ca 2 and also played the role of oscillator. the tail) improves fertilization rates. No significant differences Since fertilization by ICSI does not entail spermatozoon in cleavage rate and pregnancy rate were found among the oocyte fusion, the action of these sperm factors is important three immobilization methods in these studies, and we cannot for accomplishing fertilization. Sperm factor is reported to provide an explanation for this phenomenon. The investigation exist in the equatorial segment of the sperm head (Parrington period of this study was long, extending from April 1996 until et al., 1996), or in the perinuclear materials (Kuretake et al., June 1998. Thus, the difference may depend on the times 1996). when we performed ICSI. It was found that, when a motile spermatozoon was immobil- Ca 2 oscillations due to spermatozoon oocyte interaction ized, 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 were observed at 4.8 min after ICSI in the shortest onset case. Earlier reports cite initial Ca 2 oscillations in human oocytes occurring at between 2 and 12 h (average 6.2 h) after ICSI 151

K.Yanagida et al. (Tesarik et al., 1994). Our results differ considerably from Hoshi, K., Yanagida, K., Yazawa, H. et al. (1995) Intracytoplasmic sperm injection using immobilized or motile human spermatozoon. Fertil. Steril., those of the aforementioned report, and differences in the 63, 1241 1245. sperm immobilization methods used may be responsible for Kuretake, S., Kimura, Y., Hoshi, K. and Yanagimachi, R. (1996) Fertilization this discrepancy. With ICSI, extrusion of the second polar and development of mouse oocytes injected with isolated sperm heads. Biol. Reprod., 55, 789 795. body could be observed from 2 h after ICSI in about half Miyazaki, S. and Igusa, Y. (1981) Fertilization potential in golden hamster of the oocytes fertilized (Nagy et al., 1994). Hence, the eggs consists of recurring hyperpolarizations. Nature, 290, 702 704. spermatozoon oocyte interaction must be occurring within 2 Nagy, Z.P., Liu, J., Joris, H. et al. (1994) Time-course of oocyte activation, h after ICSI. We showed the average onset time of initial Ca 2 pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, 1743 1748. oscillations to be 28.3 19.4 min (range: 4.8 80.4 min), Nakano,Y., Shirakawa, H., Mitsuhashi, N. et al. (1997) Spatiotemporal while others (Nakano et al., 1997), using an isolated mouse dynamics of intracellular calcium in the mouse egg injected with a sperm head, reported Ca 2 oscillations to be induced within spermatozoon. Mol. Hum. Reprod., 3, 1087 1093. Palermo, G., Joris, H., Derde, M.P. et al. (1993) Sperm characteristics and 30 min in mouse oocytes treated by ICSI. outcome of human assisted fertilization by subzonal insemination and We conclude that differences in immobilization methods intracytoplasmic sperm injection. Fertil. 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