Analysis of sperm function in globozoospermia: implications for the mechanism of sperm-zona interaction

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1 FERTILITY AND STERILITY Copyright 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Analysis of sperm function in globozoospermia: implications for the mechanism of sperm-zona interaction R. John Aitken, Ph.D.*t Lorraine Kerr, B.Sc.* Virginia Bolton, Ph.D.:j: Timothy Hargreave, F.R.C.S. Medical Research Council (MRC) Reproductive Biology Unit, Centre for Reproductive Biology and Department of Surgery and Urology, Western General Hospital, Edinburgh, and King's College School of Medicine and Dentistry, University of London, London, United Kingdom The globozoospermic condition has provided a unique opportunity to determine how the abnormal mitochondrial organization and acrosomalloss associated with this syndrome, influence sperm function. Despite the abnormal midpiece architecture, the movement characteristics of the spermatozoa, in terms of the curvilinear, path, and progressive velocities, amplitude of head displacement, and hyperactivation were all within the normal range. Similarly, the behavior of the spermatozoa on Percoll gradients was normal, although the capacity of the isolated fractions to generate reactive oxygen species was negligible. Of particular significance was the fact that the globozoospermic spermatozoa were incapable of sperm -oocyte fusion or binding the human zona pellucida, even after an intracellular calcium signal had been generated with the ionophore A The sudden induction of sperm -zona interaction could, however, be achieved by the use of a ferrous ion promoter system to induce limited lipoperoxidation. This result demonstrates that the enhancing effect of peroxidation on sperm-zona adhesion involves a direct action on the properties of the sperm-plasma membrane, rather than an indirect consequence of acrosomal damage and acrosin leakage. Such findings emphasize the value of specific teratozoospermic conditions, such as globozoospermia, in dissecting the mechanisms that regulate human sperm function. Fertil Steril54:701, 1990 In a number of recent studies, constructive use has been made of specific teratozoospermic conditions to elucidate the mechanisms involved in the regulation of human sperm function. 1 2 For example, spermatozoa from patients exhibiting Kartagener's syndrome, Received December 4, 1989; revised and accepted May 23, * MRC Reproductive Biology Unit, Centre for Reproductive Biology. t Reprint requests: R. John Aitken, Ph.D., MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom. :j: Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, University of London. Department of Surgery and Urology, Western General Hospital. characterized by a lack of motility due to complete absence of dynein arms from the axoneme, have been used to examine the role of sperm movement in the control of sperm-oocyte fusion. 1 Similarly, the spermatozoa from patients exhibiting the round-headed sperm syndrome or globozoospermia, 2 characterized by a disorganization of the midpiece and the complete absence of an acrosomal vesicle, have been used to investigate the significance of the acrosome as a site of acrosin and phospholipase A 2 activity and as a component of the mechanisms involved in the processes of sperm-oocyte fusion and nuclear decondensation.2 In this study, we have extended these analyses of sperm function in globozoospermia, with particular emphasis on the mechanisms involved in sperm-zona recognition. Aitken et al. Globozoospermia 701

2 MATERIALS AND METHODS The conventional semen profile was constructed using the World Health Organization (WHO) protocol3 after incubation at 37oC for 30 minutes to induce liquefaction. All of the globozoospermic samples were produced by a single 38-year-old, infertile patient exhibiting this syndrome; details of the semen profiles are provided in Results. The movement characteristics of the spermatozoa were determined at 37 C using a computerized digital image analysis system (Hamilton Thorn Research, Danvers, MA) with the following parameter settings: Minimum contrast 12, low velocity gate 10 J.Lm/s, low size gate 0.4, high size gate 1.6, low intensity gate 0.5, and high intensity gate 2.0. Complete and accurate capture of the sperm tracks was verified using the Hamilton Thorn's playback facility. The proportion of hyperactivated cells was determined from video recordings made of the spermatozoa over a 5-hour incubation period at 37oC in the medium of Biggers, Whitten, and Whittingham (BWW) 1 on a Panasonic NV-L25 HQ (Matsushita Electric Industrial Co., Osaka, Japan) four-head, video recorder. Hyperactivated cells were identified using the criteria laid down by Robertson et al. 4 and included both the star-spin and transitional trajectories. The morphology of the spermatozoa was examined by both phase contrast microscopy and on stained preparations (Testsimplets, Boehringer Mannheim, West Germany), whereas the absence of an acrosome was confirmed by staining the outer acrosomal membrane with a fluorescein conjugated peanut (Arachis hypogea) lectin. 5 Preparation of the spermatozoa for functional assays was achieved using a two-step Percoll gradient comprised of 3 ml of 80% Percoll overlaid with 3 ml of 40% Percoll in a sterile conical-based centrifuge tube. Isotonic Percoll (100%) was prepared by adding 10 ml of lox concentrated medium 199 to 90 ml of Percoll followed by supplementation with 300 mg bovine serum albumin, 3 mg sodium pyruvate, and 0.37 ml sodium lactate syrup. 6 The cells were fractionated by centrifugation for 20 minutes at 500 X g after which the 80% pellet was recovered, washed once with 5 ml medium BWW, 7 and finally resuspended at a concentration of 10 X 10 6 /ml. The zona-free hamster oocyte penetration test was carried out as described previously, 8 except that the cells were stimulated by a 3-hour incubation with the free acid of A23187 before washing and resuspension in fresh medium BWW containing zona-free hamster oocytes. After an additional 3 hours of incubation, the hamster oocytes were removed, washed to remove loosely adherent spermatozoa, and finally compressed to a depth of about 30 J.Lm before assessing the levels of sperm-oocyte fusion by phase contrast microscopy. Sperm-zona interaction was assessed using saltstored (1.5 M MgS0 4 :1% dextran) unfertilized, human ova derived from a human in vitro fertilization (IVF) program. Three separate experiments were performed to investigate the conditions under which globozoospermic spermatozoa could be induced to interact with human zonae pellucidae. In the first experiment, the spermatozoa from the 80% Percoll fraction were pre incubated for 3 hours, at a concentration of 10 X 10 6 /ml in medium BWW, with a parallel incubation containing spermatozoa from a normal fertile donor, serving as the control. The second experiment was identical except that the spermatozoa from both the globozoospermic patient and fertile donor were preincubated for 3 hours in the presence of2.5 J.LM A In the third experiment, the influence of lipid peroxidation on sperm-zona interaction was investigated as previously described, 9 the propagation of the peroxidation chain reaction being achieved by exposure to a ferrous ion promotor system (200 J.LM ferrous sulfate and 1 mm sodium ascorbate) for the last hour of a 3-hour preincubation period. In all three experiments, the spermatozoa were pelleted at the end of the preincubation period by centrifugation for 5 minutes at 500 X g and resuspended at 10 X 10 6 /ml in fresh medium BWW before dissemination as 50 ul drops under liquid paraffin and the introduction of 7 to 12 salt-stored human zonae pellucidae. After an additional 2 hours, the zonae pellucidae were washed three times to remove loosely adherent cells and photographed at X250 under phase contrast illumination to record the number of spermatozoa adherent to the zona surface. Although the zonae pellucidae used in this assay had previously been exposed to spermatozoa during a failed IVF attempt, the presence of residual spermatozoa on the stored zonae could not have influenced the outcome of the assay since the globozoospermic cells were easily identified by their characteristic round-headed morphology (Fig. 1), and there was no detectable binding in the nonperoxidized globozoospermic controls. The small standard errors observed in the quantification of sperm-zona binding in the normozoospermic con- 702 Aitken et al. Globozoospermia Fertility and Sterility

3 pernatant collected and assayed for activity using the method of Polakoski et al. 12 One milli-international Unit (miu) of activity was defined as that amount of enzyme that causes the hydrolysis of one nanomole a-n-benzoyl-l-arginine ethyl ester in 1 minute at 25 C. Proacrosin activation was performed as described by Goodpasture et al. 11 by a 20-minute incubation at ph 8.0. The influence of acrosin on sperm -oocyte fusion was investigated by incubating globozoospermic spermatozoa for 3 hours with a combination of neutralized acrosin and 1.25 JIM A23187, before washing the cells with BWW and adding the zona-free hamster oocytes. RESULTS Figure 1 Spermatozoa from a patient with globozoospermia photographed by (A) phase contrast microscopy and (B) after staining (Testsimplets, Boehringer Mannheim, West Germany). Note the complete absence of acrosomes and the consistently abnormal midpiece morphology. trois also suggested that previous exposure of the zonae pellucidae to spermatozoa did not induce significant variation in their potential for sperm binding. Reactive oxygen species production was measured by chemiluminescence using 100 JIM luminol (5- amino- 2,3- dihydro -1,4- phthalazinedione) as previously described. 10 The potential ability of the globozoospermic spermatozoa to generate superoxide anion was investigated using nicotinamide-adenine dinucleotide phosphate (NADPH) as the electron donor. Duplicate 25 JIL aliquots of spermatozoa, at a concentration of 20 X 106 /ml, were air dried into the wells of a 96-well microtiter plate. Into each well was pipetted 50 JIL NADPH (2.5 mg/ml), 50 JIL ofnitroblue tetrazolium (2.45 mg/ml), and 100 JIL Trizma base (Sigma Chemical Company, St. Louis, MO), ph 7.0 at 37 C. Duplicate control wells incorporated 50 JIL superoxide dismutase (100 Jig/mL) and included only 50 JIL Trizma base to maintain a constant volume. The plates were then incubated for 4.5 hours and finally read on a microtiter plate reader, using a 540-nm filter. Proacrosin/ acrosin extraction was performed as described by Goodpasture et aly In brief, five human ejaculates were layered over 1 M sucrose and centrifuged at 6000 X g for 15 minutes. The sperm pellet was then resuspended in 10% glycerol, adjusted to ph 2.8 with 1 M HCl, allowed to acid extract overnight at 4 ac for 30 minutes, and the su The conventional semen profile for the patient exhibiting globozoospermia gave a mean ± (SE) sperm concentration of 88.6 ± 14.7 X 106 /ml (range 54 to 134 X 106 /ml) and a mean± SE percentage motility of 58.8% ± 5.2% (range 44% to 70%) after five ejaculates had been analyzed. The morphology was typical of the globozoospermic syndrome 2 (Fig. 1) and affected 100% of the cellsthe uniform absence of an acrosomal vesicle being indicated on stained preparations and confirmed by the complete failure of fluorescein-conjugated Arachis hypogea lectin to bind to the mature spermatozoa. After fractionation on the two-step Percoll gradient, the cells partitioned into high (base of the 80% layer) and low (40%:80% interface) density populations with a minority of the recovered spermatozoa (32% and 46% for the two ejaculates analyzed) being located in the latter. Fractionation of 33 samples from normal fertile donors on the same form of gradient produced a similar division of the cells into high and low density populations, with 39% (loth to 90th percentile range of 25.6% to 51.8%) of the spermatozoa being located at the 40%:80% interface. The spermatozoa from the globozoospermic patient were also typical of the normal population in that once they had been washed free of seminal plasma and incubated in vitro, there was a marked increase in mean path velocity, (35 to 64 Jim/s) progressive velocity (31 to 53 Jim/s), curvilinear velocity (43 to 88 Jim/s), and amplitude oflateral sperm head displacement (2.9 to 5.3 Jim). These cells were also characterized by the appearance of hyperactivated motility, the incidence of which gradually increased over 5 hours, to affect 21% of the sperm population (Fig. 2). Aitken et al. Globozoospermia 703

4 Figure 2 Direct plots of sperm tracks from a globozoospermic patient after 5 hours capacitation at 37 c. Videotape of the spermatozoa was scanned at 25 frames per second with a Hamilton Thorn image analyzer and the plots subsequently generated using a Hewlett Packard Printer Plotter, (Hewlett Packard, Martinez, CA). Arrow indicates hyperactivated cell. The spermatozoa of this patient exhibited an extremely weak chemiluminescent signal in the presence of luminol (Fig. 3), suggesting that the spermatozoa were either producing low levels of reactive oxygen species or that an intracellular factor, such as peroxidase, which is normally involved in the oxidation of luminol, is absent from these cells. To investigate this possibility, horse radish peroxidase (2 MM) was added to the incubation medium to promote the oxidation of luminol by hydrogen peroxide released into the extracellular space Under these conditions, a weak chemiluminescent signal was obtained from the globozoospermic spermatozoa recovered from the 40%:80% Percoll interface. However, the spermatozoa recovered from the base of the 80% fraction exhibited no response to peroxidase treatment, indicating an extremely low level of extracellular hydrogen peroxide release. (Fig. 3) Furthermore, even when the 12myristate, 13-acetate phorbol ester (PMA) was added to these fractions, there was no stimulation of reactive oxygen species production (Fig. 3). That the plasma membranes of these globozoospermic spermatozoa possessed the potential for superoxide formation was confirmed by exposing cells, permeabilized by air drying, to NADPH in the presence of nitro blue tetrazolium. The superoxide-dismutase inhibitable generation of formazan observed under these conditions indicated that these spermatozoa could generate 0 2- when provided with an appropriate source of electrons. The intensity of this activity (11.5 nmol 704 Aitken et al. Globozoospermia /106 spermatozoa per hour for cells from the 40%:80% Percoll interface and 10"8 nmol 0 2-./106 spermatozoa/hour for spermatozoa at the base of the 80% fraction) was of the same order that we have obtained using identical techniques with spermatozoa from normal fertile donors, which gave median values (loth to 90th percentile) of 18.5 (13.7 to 28.9) and 11.9 (8.5 to 22.1) nmol 0 2-./106 spermatozoa per hour for cells taken from the 40%: 80% Percoll interface and the base of the 80% fraction respectively (n = 30). The zona-free hamster oocyte penetration assay was carried out using spermatozoa pelleted in the 80% Percoll fraction. At doses of 2.5 MM and 5.0 MM A23187, the globozoospermic spermatozoa were 60% and 68% motile, respectively, but no penetrations were observed in the 29 oocytes examined. In contrast, a parallel control sample from a normal, fertile donor gave penetration rates of 100% (mean of 10.4 spermatozoa penetrating each oocyte; n = 11) and 81.8% (mean of 2.8 spermatozoa penetrating each oocyte; n = 11) with the same two doses of ionophore. Addition of 8.4 miu of acrosin as well as A23187 to the incubation medium did not improve the fertilizing potential of the globozoospermic spermatozoa, which still failed to bind to, or fuse with, zona-free hamster oocytes (n = 25). The cells from the globozoospermic patient also failed to exhibit any capacity for adhesion to the PMA Peroxidase 400. u., 0 U; 300 E 1 :I 0 ~..,.. u <: u 200 <: e :I...I ~----,...---~----, Time (mlns) Figure 3 Chemiluminescent signals obtained from spermatozoa collected either from the 40%:80% Percoll interface (e) or from the base of the 80% fraction (0) in the presence ofluminol. PMA = 12 myristate, 13-acetate phorbol ester. Fertility and Sterility

5 Figure 4 (A), Absence of sperm binding to the human zo~a pellucida when incubated with 10 X 106 /ml globozoosperm1c spermatozoa and (B), stimulation of sperm binding to th_e zona pellucida when a ferrous ion promoter has been used to mduce limited lipid peroxidation in these cells. 8 surface of the human zona pellucida. Hence, in contrast to the control incubations containing normal fertile spermatozoa, in which a mean ± SE of ± 2.9 spermatozoa/104 Jim 2 of zona surface (n = number of zonae = 8) was observed, there was no detectable attachment of globozoospermic spermatozoa to the human zona pellucida (n = 7). Even when the spermatozoa were stimulated with 2.5 JIM A23187, no significant binding of globozoospermic spermatozoa to the zona surface was observed (n = 8). In contrast, when, in the absence of A23187, such spermatozoa were exposed to a ferrous ion promoter system to induce lipid peroxidation, 8 the motility of the cells was unchanged, but large numbers of globozoospermic cells suddenly became bound to the zona surface (Fig. 4), giving a mean value of 50.6 ± 3.8 spermatozoa/104 Jlm 2 (n = 12). Replication of this experiment with a second ejaculate from the same globozoospermic patient 4 months later gave the same result. Hence, in contrast to the absence of zona binding observed with these spermatozoa under control conditions (n = 6), a mean of 56.6 ± 1.3 spermatozoa/104 Jim 2 (n = 7) was recorded after peroxidation of the spermatozoa with a ferrous ion promoter. DISCUSSION Globozoospermia is a rare, but well-characterized condition that has been exploited in this study to shed light on the mechanisms regulating human sperm function. In several respects, the ejaculates of globozoospermic patients appear to be normal, at least with regard to the concentration of spermatozoa and their motility. Detailed analysis of the movement characteristics of the spermatozoa by computerized digital image analysis also indicated that all of the individual components of sperm movement in this patient were typical of those found in the normal fertile population, including the capacity to exhibit hyperactivated movement after capacitation. 15 This result is of interest because the midpiece of these spermatozoa is highly abnormal particularly with respect to the mitochondria, which are either absent or severely disorganized.2 The spermatozoa from the globozoospermic patient were also found to partition normally5 on Percoli gradients with approximately 60% of the recovered cells progressing to the base of the 80% fraction and the remainder, characterized by a low level of motility, arresting at the 40%:80% interface. The cells from both regions of the gradient were characterized by a low capacity for reactive oxygen species production, despite the demonstrable potential of the sperm plasma membrane to generate superoxide anion. Such low levels of reactive oxygen species generation have, in previous studies, been positively associated with the functional competence of human spermatozoa, particularly with respect to sperm-oocyte fusion. 10' 16 However, the spermatozoa from the globozoospermic patient differed from the normal population in that they failed to exhibit sperm-oocyte fusion even when stimulated with the calcium ionophore A Previous studies have also shown that the spermatozoa from globozoospermic patients do not spontaneously fuse with zona-free hamster oocytes Although it is possible that these cells contain unrecognized defects in addition to the absence of an acrosome, such studies suggest that either the acrosome reaction itself is necessary for sperm -oocyte fusion or that some process concomitant with the acrosome reaction, such as calcium entry, is involved in triggering the fusion process. In this context, it has been proposed that calcium entry at the moment of the acrosome reaction induces fusogenic changes in the sperm plasma membrane through the activation of phospholipase A2.19 Activation of this enzyme is then thought to lead to the generation of membrane-perturbing lysophospholipids, which are responsible for the inaitken et al. Globozoospermia 705

6 duction of the acrosome reaction and the genesis of a fusogenic equatorial segment capable of initiating sperm-oocyte fusion. The fact that globozoospermic spermatozoa, which exhibit a normal capacity for phosphoiisase A2 activity,2 do not respond to the calcium influx induced by A23187 indicates that neither the activity of this enzyme nor elevated levels of cytoplasmic calcium are sufficient stimulus, per se, for initiating fusion with the oocyte. It is therefore possible that some additional component, residing in the acrosome, is essential for fusion to occur. It is already known that only acrosome-reacted spermatozoa can attach to the surface of the oocyte by means of the plasma membrane overlying the postacrosomal region of the sperm head.19 The specific binding of acrosin to this area of the sperm head after the acrosome reaction20 may be significant in view of the fact that the acrosin molecule exhibits fucose-binding properties,21 and fucose oligomers can inhibit the binding of normal human spermatozoa to the surface of zona-free hamster oocytes.22 It therefore seems reasonable to propose that even though globozoospermic spermatozoa are competent to exhibit phospholipase A2 activity, without acrosin to mediate sperm-oocyte adhesion, they are unable to fuse with the vitelline membrane even in the presence of A A preliminary attempt was made to examine this hypothesis by incubating the globozoospermic spermatozoa in the presence of acrosin extracts prepared from normal functional spermatozoa. The absence of a significant effect of exogenous acrosin on the process of sperm-oocyte fusion is of some clinical interest in that it would appear to exclude this approach as a straightforward form of therapy for such patients. It is possible that larger amounts of acrosin or more purified preparations might have brought about a biological response, although there would be obvious logistical problems in preparing larger amounts of this material for therapeutic purposes. The lack of an effect on sperm-oocyte fusion may mean that such spermatozoa do not possess the appropriate surface properties (receptors?) to bind acrosin on the external surface of the plasma membrane in the manner that is normally observed in functional cells. 20 The spermatozoa from the globozoospermic patient also failed to bind to the zona pellucida, supporting the notion that the acrosome reaction, and in particular acrosin, may be directly involved in sperm-zona interaction.20 The above-mentioned capacity of acrosin to bind fucose residues21 and the ability of fucose oligomers23 to block sperm-zona interaction in the human are both consistent with this hypothesis. The possible role of trypsin-like proteases in mediating sperm-zona interaction is also consistent with an involvement of acrosin in this process. 24 Furthermore, ultrastructural studies indicate that during the earliest stages of the acrosome reaction, acrosin becomes exteriorized and bound to the plasma membrane overlying the aerosomal region,20 where it would be in an ideal position to mediate sperm-zona adhesion. The binding of spermatozoa to the zona surface, however, is a multi-faceted process, and, apart from acrosin, there may be additional mechanisms involved in mediating sperm-egg recognition In a recent study, 8 limited peroxidation of human spermatozoa in the presence of a ferrous ion promotor was also shown to significantly enhance the ability of these cells to bind to the zona pellucida. On the basis of these results, it has been suggested that peroxidative changes in the sperm plasma membrane, induced by reactive oxygen species generated in response to the calcium influx that accompanies the acrosome reaction, may constitute an additional mechanism by which sperm-zona adhesion is mediated. However, the question of whether the binding ofperoxidized human spermatozoa to the zona pellucida reflects on underlying physiological mechanism is still unresolved. For instance, because lipid peroxidation is known to selectively damage the plasma membrane overlying the acrosome,25 it is possible that the enhancement of sperm-zona interaction, induced by a ferrous ion promoter system, is simply a reflection of acrosin leakage from acrosomes that have been damaged during the peroxidation process. The experiments carried out in this study on acrosome less spermatozoa indicate that this cannot be the case. Exposure of spermatozoa from the globozoospermic patient to a ferrous ion promoter system resulted in the sudden acquisition of a capacity to bind to the zona pellucida via mechanisms that must reflect a direct effect of peroxidation on the properties of the plasma membrane. It is difficult to imagine how such an unequivocal result, which is of both clinical and scientific interest, could have been obtained without recourse to the globozoospermic condition. REFERENCES 1. Aitken RJ, Ross A, Lees MM: Analysis of sperm function in Kartogener's syndrome. Fertil Steril40:696, Aitken et al. Globozoospermia Fertility and Sterility

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