Human Reproduction Vol.19, No.1 pp. 114±117, 2004
DOI: 10.1093/humrep/deg511
Does ICSI require acrosomal disruption? An ultrastructural study Takumi Takeuchi, Liliana T.Colombero, Queenie V.Neri, Zev Rosenwaks and Gianpiero D.Palermo1 The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA 1
To whom correspondence should be addressed. E-mail:
[email protected]
BACKGROUND: Aggressive immobilization of sperm prior to ICSI signi®cantly improves fertilization rates, but the mechanism of this effect is not yet clear. This study was performed in order to assess the characteristics of mechanically immobilized human sperm by transmission electron microscope (TEM). METHODS: Sperm obtained from ejaculated semen samples from three different donors were immobilized in a standard manner for ICSI. They were then injected into the perivitelline space of mouse oocytes in order to be able to locate them by TEM. Intact motile sperm injected subzonally served as controls (n = 160). Finally, the `carrier' oocytes were ®xed and processed for TEM. RESULTS: A total of 300 sperm were mechanically immobilized and inserted into the perivitelline space of mouse oocytes. Ultrathin sections revealed consistent alterations in the acrosomal region including disruption of the plasma membrane, and disruption, vesiculation or even loss of the acrosome. Thus, all of the sperm assessed had undergone some disorganization of the head, in contrast to a majority of control sperm. CONCLUSIONS: Immobilization of sperm for ICSI by compressing and rolling the sperm tails induces a variable disruption and sometimes loss of the acrosome. This could well be a reason for the higher success rates when ICSI is performed using immobilized sperm. Key words: acrosome reaction/human sperm/ICSI/mechanical immobilization/transmission electron microscopy
Introduction In order to fertilize, mammalian sperm ®rst need to undergo capacitation in the female tract (Austin, 1951; Chang, 1951). Capacitation involves modi®cations in the sperm plasma membrane, which lead to hyperactivation and permit the acrosome reaction. The latter involves multiple fusions between the outer acrosome membrane and the overlying sperm plasma membrane, enabling the soluble contents of the acrosome to leak out through the fenestrated membranes (Barros et al., 1967), and coincidentally it prepares the surface over the equatorial segment for its fusogenic role (Yanagimachi, 1994). ICSI bypasses the events involved in physiological sperm penetration of the oocyte, and requires no speci®c pretreatment of sperm other than immobilization (Palermo et al., 1992, 1995; Vanderzwalmen et al., 1996). However, aggressive immobilization by compressing the tail prior to injection signi®cantly improves ICSI fertilization rates (Fishel et al., 1995; Gerris et al., 1995; Van den Bergh et al., 1995). Although the mechanism of this bene®cial effect is not yet clear, there is indirect evidence that such immobilization causes changes in the sperm permeability (Dozortsev et al., 1995a), and that it may possibly induce changes leading to 114
acrosomal disruption (Fishel et al., 1995; Palermo et al., 1996). Nevertheless, there has been no report on the ultrastructural state of sperm immobilized for ICSI. In this study, we have analysed membrane integrity and acrosomal characteristics of immobilized sperm using transmission electron microscopy (TEM). Materials and methods Sperm preparation and immobilization procedure Ejaculated semen samples obtained from consenting men with normozoospermia (under the Internal Review Board No. 0696-389) were processed by density gradient centrifugation as routinely performed for ICSI (Palermo et al., 1995). Semen parameters from each individual donor are summarized in Table I. Immediately before immobilization, 1 ml of the ®nal sperm suspension (~13106/ml) was placed in a 4 ml droplet of 10% polyvinylpyrrolidone (w/v) (PVP; Sigma Chemicals, USA) in human tubal ¯uid (HTF) medium buffered with HEPES medium (H-HTF; Irvine Scienti®c, USA) in the centre of a Petri dish (Palermo et al., 1995). For immobilization, motile sperm viewed under an inverted microscope at 3400 magni®cation (Olympus IX-70; New York/New Jersey Scienti®c Inc., USA) were positioned perpendicularly to an ICSI pipette. This was used to gently compress and roll the principal piece of the sperm tail in a repetitive
Human Reproduction vol. 19 no. 1 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
Ultrastructural study of immobilized sperm Table I. Semen characteristics of individual donors Semen parameters
Semen volume (ml) Sperm concentration (3106/ml) Sperm motility (%) Sperm morphologya (%) aKruger's
Donors A
B
C
2.3 58 53 6
3.6 95 61 8
3.3 70 76 9
strict criteria (Kruger et al., 1986).
manner until it became permanently kinked or convoluted (Palermo et al., 1996). Subzonal insertion of sperm into mouse oocytes Mouse oocytes were collected from B6D2F1 females and the subzonal insertion procedure was carried out as previously described (Palermo et al., 1991). To allow their study in the TEM, ~60 immobilized sperm were aspirated into an ICSI needle and injected together into the perivitelline space of oocytes, which acted as containers for these sperm (Figure 1). Motile (control) sperm were injected subzonally into oocytes in the same manner. The time required for sperm immobilization and oocyte injection ranged between 10 and 15 min. Sample processing for TEM Immediately after subzonal injection, each injected oocyte was ®xed for 1 h in 2% glutaraldehyde (Electron Microscopy Sciences, USA) in 0.1 mol/l cacodylate buffer at room temperature and overnight at 4°C. They were then washed in buffer, exposed to 2% osmium tetroxide (Electron Microscopy Sciences) for 1 h, dehydrated through increasing concentrations of ethanol up to 100%, and embedded in Spurr's resin (Electron Microscopy Sciences). Thick plastic sections were cut and stained with Toluidine Blue in borate buffer. When sperm were found, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and studied in a JEOL 100S TEM.
Results A total of 300 sperm from three different individuals were mechanically immobilized and inserted subzonally into ®ve mouse oocytes (Figure 1, ~60 sperm per oocyte). In addition, 160 motile sperm were injected similarly under the zona pellucida of mouse oocytes as controls. We evaluated 22 immobilized sperm originating from donor A, 11 from B, and 19 from C, while 7, 9 and 10 control sperm were examined respectively for each of the donors. Thus, a total of 52 immobilized and 26 intact sperm was studied in the TEM. Immobilized sperm all had alterations in the peri-acrosomal plasma membrane ranging from localized vesiculation to a complete disruption that allowed leakage of the soluble acrosomal content. While no changes in the chromatin were observed, some sperm even exhibited a complete loss of the acrosome. Each spermatozoon fell into one of four ultrastructural categories: (A) acrosome-intact (Figure 2a); (B) swelling of the acrosomal matrix (Figure 2b); (C) variable disruption of sperm head membranes and/or vesicle formation within the acrosomal matrix (Figure 2c); (D) loss of the carapace and content of the acrosome (Figure 2d). Since the proportions of each group were similar among the three different individuals, the data were combined (Table II).
Figure 1. A mouse oocyte containing injected immobilized sperm within the perivitelline space (original magni®cation 3600).
Table II. Effect of mechanical sperm immobilization on the status of the acrosome Acrosomal status
(A) Acrosome-intact sperm (B) Swelling of the acrosomal matrix (C) Disruption of the acrosomal complex and/or vesiculation within the acrosomal matrix (D) Acrosomal loss Total a,b c2,
No. of sperm (%) Non-immobilized
Immobilized
19 (73.1)a 6 (23.1)b
Nonea 7 (13.5)b
1 (3.8)b
36 (69.2)b
0b 26
9 (17.3)b 52
232, 1 df: effect of immobilization on acrosomal changes, P < 0.001.
No immobilized sperm had an intact acrosome: 86.5% had undergone disruption of the acrosome or it had disappeared completely (group C + D), and 13.5% were in the initial stages of the disruption (group B). In contrast, the majority of nonimmobilized cells (73.1%) were intact, with most of the remainder (23.1%; group B) showing some morphological change (Table II). Discussion Immobilization of sperm immediately before the ICSI procedure is one key to its consistent success (Fishel et al., 1995; Gerris et al., 1995; Van den Bergh et al., 1995; Palermo et al., 1996), but why is not clear. Most evidence suggests that this might depend on changes in the sperm plasma membrane (Dozortsev et al., 1995a), and our ®ndings support this view. Sperm membrane permeabilization may help to expose the sperm nucleus to the ooplasm, facilitating male pronucleus formation (Maleszewski, 1990; Yanagimachi, 1998). Removal of the sperm plasma membrane also seems to be necessary for oocyte activation. Indeed, calcium oscillations and subsequent 115
T.Takeuchi et al.
Figure 2. (a) Intact human sperm head. (b) Human sperm head showing local disruption of the plasma membrane and a scalloping of the acrosome. (c) Irregular swelling and decondensation of the acrosomal matrix and within which small circular vesicles are evident. (d) Human sperm heads that have lost their acrosomes (TEM 319 000).
oocyte activation appear to be induced after intracytoplasmic injection by a spermatozoon-associated oocyte activating factor(s) (Dozortsev et al., 1995b; Palermo et al., 1997), which can exert its effect only after damage or removal of this membrane (Swann et al., 1994). While calcium oscillations are certainly responsible for oocyte activation, these oscillations are believed to be induced by a sperm cytosolic factor(s) (Swann, 1990; Palermo et al., 1997). Although the oscillogenic molecule is presumably a soluble polypeptide released into the ooplasm at the time of gamete fusion, its location in the sperm head has not yet been clearly de®ned (Parrington et al., 1996; Wolny et al., 1999). However, it is considered to reside in the perinuclear theca (PT) (Kimura et a., 1998). It has been suggested that exposure of the PT to the ooplasm and its dissociation are linked to the beginning of calcium oscillations (Sutovsky et al., 1997; Kimura et al., 1998), although this is not always a requirement (Knott et al., 2003). Sperm of globozoospermic patients, which typically lack a PT and are considered unable to fertilize oocytes without assisted activation (Battaglia et al., 1997; Rybouchkin et al., 1997) have on occasion been able to do so (Lundin et al., 1994; Liu et al., 1995). The fact that mechanically immobilized sperm often displayed some modi®cation of the PT (Figure 2b and c) suggests that exposure of 116
the PT might be a contributing factor in the relative success of immobilized sperm during ICSI (Knott et al., 2003). The status of the acrosome after ICSI has been the subject of debate (Lacham-Kaplan and Trounson, 1995; Sathananthan et al., 1997). The present study demonstrates that immobilization elicits changes in the plasma membrane and acrosome, since these were always disrupted to varying degrees in immobilized sperm, in contrast to the control population. Since acrosomal disruption can sometimes occur spontaneously, it was not surprising to ®nd this in some 25% of the sperm in the control group as well (Lee et al., 1997). One problem in trying to perform ultrastructural studies on a small number of sperm is ®nding them in the TEM. Recently Cohen et al. (1997) used empty zonae pellucidae for cryopreservation of single human sperm, and we have followed this novel approach here, using oocytes as `carriers' to aid in localization of the treated sperm cells in the TEM. While sperm immobilization is usefully performed with the ICSI needle, immobilization by piezo-pulses (Huang et al., 1996) has also been practised, and some investigators have obtained identical fertilization rates using sperm exposed to a non-contact diode laser (Montag et al., 2000; Ebner et al., 2001). These authors noted that use of the diode laser reduces micromanipulation time, that the magni®cation of the laser
Ultrastructural study of immobilized sperm
objective allows for better evaluation of sperm morphology, and that its use eliminates the need for a viscous medium (e.g. PVP), which could be potentially toxic for the gametes. However, not only are laser and piezo systems more costly but the classical technique used here may be performed by any ICSI-trained embryologist with no extra equipment. In conclusion, the present study demonstrates that mechanical sperm immobilization induces changes in the acrosome and sperm head plasma membrane, providing a likely explanation for the higher success rates obtained when ICSI is performed using immobilized sperm. Acknowledgements We thank the clinical and scienti®c staff of The Center for Reproductive Medicine and Infertility. We are grateful to Professor J.Michael Bedford for his scienti®c support and Dr W.G.Breed for performing the electron microscopy.
References Austin CR (1951) Observation on the penetration of sperm into the mammalian egg. Aust J Sci Res [B] 4,581±596. Barros C, Bedford, JM, Franklin, LE and Austin, CR (1967) Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell Biol 34,C1±C5. Battaglia DE, Koehler JK, Klein NA and Tucker MJ (1997) Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm. Fertil Steril 68,118±122. Chang MC (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168,697±698. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW and Scott RT (1997) Cryopreservation of single human spermatozoa. Hum Reprod 12,994±1001. Dozortsev D, Rybouchkin A, De Sutter P and Dhont M (1995a) Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum Reprod 10,2960±2964. Dozortsev D, Rybouchkin A, De Sutter P, Qian C and Dhont M (1995b) Human oocyte activation following intracytoplasmic sperm injection: the role of the sperm cell. Hum Reprod 10,403±407. Ebner T, Yama C, Moser M, Sommergruber M, Hartl J and Tews G (2001) Laser assisted immobilization of spermatozoa prior to intracytoplasmic sperm injection in humans. Hum Reprod 16,2628±2631. Fishel S, Lisi F, Rinaldi L, Green S, Hunter A, Dowell K and Thornton S (1995) Systematic examination of immobilizing spermatozoa before intracytoplasmic sperm injection in the human. Hum Reprod 10,497±500. Gerris S, Mangelschots K, Van Royen E, Joostens M, Eestermans W and Ryckaert G (1995) ICSI and severe male-factor infertility: breaking the tail prior to injection. Hum Reprod 10,484±486. Huang T, Kimura Y and Yanagimachi R (1996) The use of piezo micromanipulation for intracytoplasmic sperm injection of human oocytes. J Assist Reprod Genet 13,320±328. Kimura Y, Yanagimachi R, Kuretake S, Bortkiewicz H, Perry ACF and Yanagimachi H (1998) Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol Reprod 58,1407±1415. Knott JG, Kurokawa M and Fissore RA (2003) Release of the Ca2+ oscillationinducing sperm factor during mouse fertilization. Dev Biol 260,536±547. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA and Smith K (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 46,1118±1123. Lacham-Kaplan O and Trounson A (1995) Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction. Hum Reprod 10,2642±2649. Lee DR, Lee JE, Yoon HS and Roh SI (1997) Induction of acrosome reaction in human spermatozoa accelerates the time of pronuclear formation of hamster oocytes after intracytoplasmic sperm injection. Fertil Steril 67,315± 320.
Liu J, Nagy Z, Joris H, Tournaye H, Devroey P and Van Steirteghem A (1995) Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia. Hum Reprod 10,626±629. Lundin K, Sjogren A, Nilsson L and Hamberger L (1994) Fertilization and pregnancy after intracytoplasmic microinjection of acrosomeless spermatozoa. Fertil Steril 62,1266±1267. Maleszewski M (1990) Decondensation of mouse sperm chromatin in cell-free extracts; a micromethod. Mol Reprod Dev 27,244±248. Montag M, Rink K, DelacreÂtaz G and van der Ven H (2000) Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa. Hum Reprod 15,846±852. Palermo G and Van Steirteghem A (1991) Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs. Mol Reprod Dev 30,339±345. Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 340,17±18. Palermo G, Cohen J, Alikani M, Adler A and Rosenwaks Z (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 63,1231±1240. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F and Rosenwaks Z (1996) Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum Reprod 11,1023±1029. Palermo GD, Avrech OM, Colombero LT, Wu H, Wolny YM, Fissore RA and Rosenwaks Z (1997) Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes. Mol Hum Reprod 3,367±374. Parrington J, Swann K, Shevchenko VI, Sesay AK and Lai FA (1996) Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379,364±368. Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P and Dhont M (1997) Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with de®cient oocyte activation capacity. Fertil Steril 68,1144± 1147. Sathananthan AH, Szell A, Ng SC, Kausche A, Lacham-Kaplan O and Trounson A (1997) Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)? Reprod Fertil Dev 9,703±709. Sutovsky P, Oko R, Hewitson L and Schatten G (1997) The removal of the sperm perinuclear theca and its association with the bovine oocyte surface during fertilization. Dev Biol 188,75±84. Swann K (1990) A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs. Development 110,1295± 1302. Swann K, Homa S and Carroll J (1994) An inside job: the results of injecting whole sperm into eggs supports one view of signal transduction at fertilization. Hum Reprod 9,978±980. Van den Bergh M, Bertrand E, Biramane J and Englert Y (1995) Importance of breaking a spermatozoon's tail before intracytoplasmic injection: a prospective randomized trial. Hum Reprod 10,2819±2820. Vanderzwalmen P, Bertin G, Lejeune B, Nijs B, Vandamme B and Schoysman R (1996) Two essential steps for a successful intracytoplasmic sperm injection: injection of immobilized spermatozoa after rupture of the oolema. Hum Reprod 11,540±547. Wolny YM, Fissore RA, Wu H, Reis MM, Colombero LT, ErguÈn B, Rosenwaks Z and Palermo GD (1999) Human glucosamin-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes. Mol Reprod Dev 52,277±287. Yanagimachi R (1994) Mammalian fertilization. In Knobil E and Neil JD (eds), The Physiology of Reproduction. Raven Press, New York, pp. 189± 317. Yanagimachi R (1998) Intracytoplasmic sperm injection experiments using the mouse as a model. Hum Reprod 13 (Suppl. 1),87±98. Submitted on May 16, 2003; resubmitted on June 28, 2003; accepted on September 16, 2003
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