Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.

Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca²⁺-dependent sialic acid-binding protein (SABP) of humanendometrium, which specifically bound to human sperm head plasmamembrane in vitro, was found to increase the percentage motilityand acrosome-reacted pattern of uncapacitated spermatozoa. Theprotein was synthesized in the endometrium and secreted intothe uterine fluid. This intra-uterine factor, which is apparentlyadvantageous in vitro in inducing human sperm capacitation,may play a significant role in promoting the postrelease maturationof ejaculated spermatozoa by enhancing ⁴⁵Ca uptake into spermatozoaby a pathway which is insensitive to calcium-channel blockers.However, the ⁴⁵Ca uptake could be enhanced on exposure to thedivalent cation ionophore A23187 and inhibited in the presenceof the calmodulin inhibitor trifluoperazine. The SABP also inducesan increase in intracellular Ca²⁺ in spermatozoa, as seen byFURA-2 AM studies. Furthermore, overlay studies show human SABPto be a Ca²⁺-binding protein. The data presented here suggestthat SABP induces invitro sperm capacitation and the subsequentacrosome reaction by increasing intracellular Ca²⁺ concentration.     

Acrosome reactivity in spermatozoa of different morphology in response to stimulation with follicular fluid

The ability of the morphologically abnormal spermatozoon toundergo the processes necessary for fertilization is unknown;one of the essential processes is the acrosome reaction. Inorder to assess this, spermatozoa from 10 known fertile donorswere incubated with either follicular fluid or Earle's mediumcontaining 3 mg/ml bovine serum albumin at 37°C for 6 h.The spermatozoa were then stained with 2% Trypan Blue priorto being fixed in gluteraldehyde and stained for the presenceof the acrosomal cap using the triple stain. Fifty live spermatozoain each of four morphological categories (normal head, largehead, small head, abnormal neck or tail) were examined and thenumber of acrosome-reacted spermatozoa determined. There wasa significant difference between the morphological groups inthe baseline number of acrosome-reacted spermatozoa, determinedby examining sperm samples incubated in Earle's medium; however,the number of spermatozoa undergoing the acrosome reaction inresponse to stimulation with follicular fluid (i.e. the numberof spermatozoa acrosome-reacted in follicular fluid minus thenumber acrosome-reacted in Earle's medium) was similar for allmorphological groups. This suggests that abnormal sperm morphologydid not affect the response of spermatozoa to activation ofthe acrosome reaction by exogenous stimuli.     

Andrology: Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation

In this study, human spermatozoa obtained from donors (n = 15)with normal semen characteristics were cryopreserved in humansperm preservation medium, supplemented with the phosphodiesteraseinhibitor pentoxifylline at concentrations of 0, 1, 3 and 10mM. The effect of pentoxifylline on cryopreserved spermatozoawas determined by monitoring changes in sperm motility and acrosomemorphology by labelling the spermatozoa with fluorescein-conjugatedconcanavalin A lectin. Cryoprotectant supplemented with 1 mMpentoxifylline was found to improve post-thaw progressive motilityfrom 15.3 ± 2.4 (control) to 23.1 ± 3.8% (P <0.01), and total motility from 27.4 ± 3.3 (control) to38.2 ± 3.9% (P < 0.05) without reducing the percentageof spermatozoa with normal acrosomal regions, and so appearsuseful for cryopreservation purposes. The beneficial effectsof 1 mM pentoxifylline on sperm motility were shown to be maintainedpost-thaw over a 6 h time course. Cryoprotectant supplementedwith 3 mM pentoxifylline was found to improve only post-thawprogressive motility, from 15.3 ± 2.4 (control) to 20.7± 3.0% (P < 0.05). However, cryopreservation in thepresence of 10 mM pentoxifylline was found to have a significantly(P < 0.01) detrimental effect on acrosome morphology post-thaw,reducing it from 29.0 ± 2.0 (control) to 21.0 ±2.4% without affecting sperm motility. This suggests that assessmentof the acrosomal region may indicate subtle deleterious effectsof cryoprotectant supplements that cannot be determined frompost-thaw motility assessments alone. These findings differfrom previous studies in that a lower concentration of pentoxifylline(1 mM) was found to be optimal for cryopreservation purposes.     

Andrology: The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa

The usefulness of sub-zonal injection (SZI) for the treatmentof severe male factor infertility has been restricted by lowand unpredictable fertilization rates and the high risk of polyspermyafter the injection of multiple spermatozoa. In this prospectivestudy, we have evaluated whether sperm morphology and the percentageof acrosome-reacted spermatozoa at the time of injection canbe used to predict SZI fertilization outcomes. Populations ofmotile spermatozoa equivalent to those injected were collectedfrom the medium/oil interface immediately after SZI of eachcohort of oocytes. Morphology was assessed using the World HealthOrganization 1987 criteria and the acrosomal status of spermatozoawas determined after staining with rhodamine-conjugated Pisumsativum agglutinin. A fertilization index (FI) was calculatedto express the actual fertilizing potential of the spermatozoainjected. In all, 67 patients underwent 72 SZI cycles. The overallfertilization and polyspermy rates were 36 and 47% respectively,and a clinical pregnancy rate per transfer of 22% was achieved.Linear regression analysis demonstrated a statistically significantrelationship between morphology and the FI (r = 0.506, P <0.0001). Patients with <10% normal morphology always hada FI < 10%, and this was reflected by low fertilization andpolyspermy rates and the high number (32%) of cycles with completefailure of fertilization in this group. In patients with >10% normal morphology, there were two patterns: low (10% FI)or high (>10% FI) fertility. This was evident in the fertilization(23 and 85%, respectively) and polyspermy (25 and 68%, respectively)rates of these two patient sub-groups. While the percentageof acrosome-reacted spermatozoa at the time of injection wasweakly correlated with the FI (r = 0.292, P < 0.05), it couldnot be used to predict differences in fertilization potentialbetween patient sub-groups. We conclude that sperm morphologyand acrosomal status at the time of injection are of limiteduse in predicting SZI fertilization outcomes, although patientswith poor morphology ( 10% normal) have lower fertilizationand polyspermy rates.     

Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C

To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.     

Selection and characterization of human acrosome reacted spermatozoa

In order to study the characteristics of human acrosome reactedspermatozoa, we developed a method to select them. Spermatozoa,obtained from 20 fertile volunteers, were selected by usingGB24 antibody fixed on magnetic immunobeads, after acrosomereaction induced by 50% follicular fluid. Bead-bound spermatozoawere then detached using sheep anti-mouse IGG F(ab')2 antibody.This method allowed recovery of 170 ± 48x10³ spermatozoa(n = 20), free of GB24 antibody, as assessed by incubation withFITC-rabbit anti-mouse antibody. The percentage of acrosomereacted spermatozoa in the selected population was 88 ±3% versus 32 ± 6% in the whole sperm population. Concerningsperm morphology, the percentage of head abnormalities was lowered(15 ± 3% versus 20 ± 3%). The motility of selectedspermatozoa was dramatically reduced (7 ± 3% versus 53± 7% in the whole population) despite no difference inviability (84 ± 3% versus 80 ± 4%). However, theviability after an 18 h incubation was very low (1 ±0.5% versus 46 ± 5%). These results show that acrosomereaction occurs in the most morphologically normal spermatozoaand is followed by a loss in motility and a decrease in longevity.     

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro

It is known that only acrosome-reacted spermatozoa can fusewith the oolemma during normal fertilization with zona pellucida-intactoocytes. The aim of this study was to determine if the oolemmaof human zona pellucida-free oocytes selectively binds spermatozoawith normal morphology and a reacted acrosome. Oocytes thatfailed to fertilize in vitro because of severe sperm defectswere used. The zona pellucida was removed with acidic (pH 2–3)saline. Sperm samples were obtained from normal fertile donorsand normozoospermic men. Motile spermatozoa were selected witha swim-up technique and 2x10⁶/ml incubated with oocytes. Theresults from 23 experiments showed that at 2 h there was a significantlyhigher mean percentage of acrosomereacted spermatozoa boundto the zona pellucida (mean ±SD, 42±22) than inthe insemination medium (27 ± 12). In contrast, all spermatozoabound to the oolemma at 2h were acrosome reacted. Furthermore,each fresh zona pellucida had>100 spermatozoa bound comparedwith an average of 28 (range 4–81) spermatozoa bound perzona pellucida-free oocyte. There was no significant differencein the zona pellucida-induced acrosome reaction between fresh(45 ± 21)and salt-stored (35 ± 22) zonae pellucidae.The percentage with normal morphology was significantly higherfor spermatozoa bound to the zona pellucida (84 ± 13)and oolemma of zona pellucida-free oocytes (71 ± 25)than for spermatozoa in insemination medium (39 ± 11)(P<0.01). Extending the time of incubation of spermatozoawith zona pellucida-intact oocytes increased the proportionof spermatozoa undergoing the acrosome reaction (n=6, 2 h, 41± 23; 3 h, 53 ± 31; 4 h, 61 ± 34). However,there was a large variation in the percentage of acrosome reactionsamong spermatozoa bound to the zona pellucida between individualsperm samples. In conclusion, only acrosome-reacted spermatozoacan bind to the oolemma of zona pellucida-free oocytes. Bothfresh and salt-stored human zonae pellucidae are equally effectivein inducing the acrosome reaction. Both the zona pellucida andthe oolemma of zona pellucida-free oocytes selectively bindspermatozoa with normal morphology     

Effect of platelet-activating factor on motility and acrosome reaction of human spermatozoa

The effect of platelet-activating factor (PAF) on motility parametersand induction of the acrosome reaction in human spermatozoawas investigated in 36 unselected men with different degreesof initial sperm motility. The characteristics of sperm movementwere assessed by computer-assisted sperm analysis (Hamilton-ThornMotility Analyser) and the percentage of acrosome-reacted spermatozoawas evaluated after 1 h incubation with PAF (10 nM) and stainingwith fluorescent peanut lectin. We found that short-term (4h max) incubation with PAF significantly enhanced total andprogressive sperm motility as well as acrosome reaction. Anincrease of sperm motility in response to PAF was present in16 out of the 25 subjects studied (defined as responders) andwas inversely correlated with basal motility. In the 11 samples(six responders and five non-responders) where the incubationwith PAF was prolonged overnight, an increase of sperm motilitywas present in all the subjects studied. Similarly, an increasein numbers of acrosome reactions in response to 10 nM PAF waspresent in 20 out of the 26 subjects examined, and was inhibitedby the PAF receptor antagonist L659 989. Our results indicatea possible physiological role for PAF in fertilization and suggesta potential use of PAF in in-vitro fertilization techniquesin cases of reduced sperm motility.     

Variable kinematics of capacitating human spermatozoa

Human spermatozoa were prepared by swim-up fromsemen into in-vitrofertilization (IVF) culture medium, and their movement recordedby videomicrography using NTSC video to give 60 images/s onfreeze-frame playback. Trajectoriesin which spermatozoa appearedto switch fromone type of motility pattern to another (fromnon-hyperactivatedto transitional or star-spin hyperactivated),were reconstructed manually and the movement characteristicsdetermined for short consecutive sequences along eachtrack.The spermatozoa were able to switch between allof these motilitypatterns, apparently at random. The seobservations illustratedthat hyperactivated motility is areversible state in human spermatozoa,with phase changesfrom hyperactivated to non-hyperactivatedmotility patternsalong trajectories. It was not necessary forspermatozoato pass through the transitional hyperactivated motilityphasewhen switching from non-hyperactivated to starspinhyperactivatedmotility or vice versa.     

Induction of acrosome reaction in human spermatozoa used for subzonal insemination.

Human spermatozoa were injected into the perivitelline space of oocytes from 43 couples (44 cycles) in whom fertilization had failed in conventional in-vitro fertilization (IVF). The spermatozoa were treated to enhance the percentage of acrosome-free spermatozoa either by incubation for 24 h in T6 medium with 50% follicular fluid (v/v) or by incubation for 24 h in T6 medium followed by electroporation and incubation for a few hours in T6 medium with 3.5 mM pentoxifylline. After these two procedures, the mean percentage of acrosome-free spermatozoa increased to 35.5 and 53.9% respectively. Up to three spermatozoa were injected into the perivitelline space of metaphase II oocytes; few oocytes were damaged during the injection procedure. The overall fertilization rate was 30.9% of the 433 oocytes that were intact after subzonal insemination. Only 3% of the injected oocytes had more than two pronuclei. The cleavage rate of the fertilized oocytes was 80%. There was no difference in the fertilization and cleavage rates between the two sperm treatment procedures. One, two or three embryos were replaced in 34 cycles and seven patients became pregnant. In three of the four ongoing pregnancies, prenatal diagnosis by amniocentesis indicated a normal karyotype.     

Andrology: Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry

Acrosomal status and viability were evaluated simultaneouslyon human spermatozoa using flow cytometry. Samples were dividedinto three aliquots and randomly assigned to one of three treatments:(i) cryopreservation; (ii) 10 µM calcium ionophore [A23187in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control).Acrosomal status was evaluated using monoclonal antibodies recognizingMH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouseimmunoglobulin (IgG) was used as a second antibody. Sperm viabilitywas assessed using Hoechst 33258 (H258) exclusion. The followingfactors were analysed: (i) the specificity of the monoclonalantibodies for the human acrosome; (ii) the relative effectivenessof flow cytometry and direct fluorescent microscopy scoringand (iii) the acrosomal status and viability of the control,ionophore-treated, and cryopreserved spermatozoa. Across alltreatments, the MH61 and CD46 monoclonal antibodies resultedin acrosomal status values (acrosome-reacted/viable spermatozoa)which were not significantly different (P > 0.05): control,1.0 ± 0.3% and 1.5 ± 0.6% (mean ± SEM);A23187, 42.8 ± 3.5% and 38.1 ± 3.5%; cryopreserved,8.2 ± 2.0% and 9.9 ± 1.3%; respectively. However,acrosomal status among treatments differed significantly (P< 0.01). Flow cytometric and direct fluorescent microscopyassessments were significantly correlated (r² = 0.96, P <0.01). These results indicate that flow cytometry, using anacrosome-specific monoclonal antibody and a supravital dye,provides an objective and efficient method to evaluate humansperm acrosomal and viability status simultaneously.     

Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa

The in-vitro effects of several concentrations of Ureaplasmaurealyticum on the motility, membrane integrity and morphologyof washed spermatozoa from healthy donors were studied. A significantreduction in sperm motility and signs of membrane alteration,directly related to U.urealyticum concentration and contacttime were observed. Scanning electron microscopy examinationshowed masses of U.urealyticum attached to the head and middlepiece of some of deformed spermatozoa. It is suggested thatU.urealyticum is involved in sperm changes leading to male infertility,particularly when there is heavy U.urealyticum colonizationor specific infections with this microorganism.     

Tryptase inhibits motility of human spermatozoa mainly by activation of the mitogen-activated protein kinase pathway

BACKGROUND: We previously localized protease-activated receptor 2 (PAR-2) on human spermatozoa and demonstrated that activation of PAR-2 by the mast cell (MC) product tryptase inhibits sperm motility. Importantly, tryptase-secreting MCs are encountered in the male and female genital tract, implying that MC-spermatozoa interactions may be as yet unrecognized factors affecting sperm fertilizing ability. In order to elucidate how tryptase via activation of PAR-2 acts in human spermatozoa, we studied intracellular signal transduction events. METHODS AND RESULTS: Impairment of sperm motility by tryptase was not dependent on the presence of extracellular Ca2+ and tryptase did not alter intracellular Ca2+ levels. Pre-incubation with pertussis toxin (PTX) failed to prevent tryptase effects on sperm motility. Western blot analyses revealed that tryptase increased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2, an effect which was blocked by the MAPK pathway inhibitor PD98059. Pre-treatment of spermatozoa with this inhibitor also blocked the inhibtion of sperm motility evoked by tryptase. CONCLUSIONS: These results indicate that tryptase acts via the ERK1/2 pathway to inhibit motility of human spermatozoa.     

Computer-assisted measurement of hyperactivation in human spermatozoa: differences between European and American versions of the Hamilton-Thorn motility analyser

Spermatozoa from healthy donors of proven fertility were analysedfor percentage hyperactivation, mean curvilinear velocity (VCL),mean progressive velocity (VSL), amplitude of lateral head displacement(ALH) and percentage linearity (LIN) using two versions of theHamilton-Thorn motility analyser (HTMA) running at differentframe rates. The sort fraction criteria developed using a machineutilizing 30 Hz gave comparable figures for the percentage spermatozoadisplaying motility patterns concomitant with hyperactivationwhen compared to that developed for a machine using a framerate of 25 Hz. Statistically significant differences were observedwhen criteria developed using 30 Hz machines were transferredto those using 25 Hz. The frame rate dependency of VSL and VCLare thought to account for these differences; values for ALHand LIN were largely unaffected by frame rate. These resultshave implications for workers using the European version ofthe HTMA (at 25 Hz) with sort criteria, containing frame rate-dependentvariables, which have been developed by American colleaguesusing the machines of a higher frame rate. More appropriatesort criteria for workers using European (25 Hz) machines aresuggested.     

Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear. METHODS: Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa. RESULTS: Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted. CONCLUSIONS: Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.     

A comparison of three methods for detecting the acrosome reaction in human spermatozoa

This study was designed to compare three different fluorescentprobes to assay the acrosome reaction in human spermatozoa:chlortetracycline (CTC), mannosylated bovine serum albumin (BSA)labelled with fluorescein (MAF), and quinacrine (QN)- Normalhuman sperm ejaculates were washed and allowed to swim up for30–60 min. Samples were examined under epifluorescencefor the percentage of the acrosome reacted spermatozoa, as detectedby the three probes. There was no significant difference betweensamples of fresh, uncapadtated spermatozoa evaluated with CTC,MAF or QN; all gave <10% reacted. Following capacitationfor 3 h, the percentage of spontaneously reacted spermatozoawas higher than in fresh spermatozoa; CTC and MAF gave the samepercentage (12%), while QN indicated a higher percentage (18%)of reacted spermatozoa (P < 0.001). Following exposure toionophore A23187 at 1 h, the percentage of acrosome reactionsincreased to a mean of 31% as detected with CTC or MAF; themean percentage (45%) was significantly higher with QN (P <0.0001). Further incubation up to 2 h with A23187 did not changethese percentages. These results suggest that the QN probe detectsthe onset stage of the acrosome reaction, whereas the CTC andMAF probes detect the later stages in which the acrosomal capis lost. Use of the two types of probe provides a means forfiner resolution of the time course of the acrosome reactionin the human spermatozoa.