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: gdpalerm{at}med.cornell.edu
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Abstract |
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Key words: acrosome reaction/human sperm/ICSI/mechanical immobilization/transmission electron microscopy
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Introduction |
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ICSI bypasses the events involved in physiological sperm penetration of the oocyte, and requires no specific 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 significantly improves ICSI fertilization rates (Fishel et al., 1995
; Gerris et al., 1995
; Van den Bergh et al., 1995
). Although the mechanism of this beneficial 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 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).
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Materials and methods |
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Results |
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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).
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Discussion |
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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 defined (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 modification of the PT (Figure 2b and c) suggests that exposure of 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 find 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 finding 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 magnification of the laser 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.
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Acknowledgements |
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References |
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Submitted on May 16, 2003; resubmitted on June 28, 2003; accepted on September 16, 2003.