The fatty acid composition of phospholipids of spermatozoa from infertile patients

The lipid composition of the sperm membrane has a significant effect upon the functional characteristics of spermatozoa. In the present study we investigated the fatty acid (FA) composition of subpopulations of spermatozoa separated on a discontinuous Percoll gradient (47:90%) and the FA composition of phospholipids (PL) of sperm heads and tails in both normal and abnormal semen samples. In normozoospermic samples, polyunsaturated fatty acids (PUFA) represented 34.0 +/- 1.3 (mean +/- SE, mole %) and 25.6 +/- 1.2% of total FA of PL of the 47 and 90% Percoll fractions respectively. Docosahexaenoic acid (22:6omega3, DHA) contributed to more than 60% of total PUFA. DHA was significantly lower in both the 47% (P < 0.05) and the 90% (P < 0.01) Percoll fractions of oligozoospermic samples and in the 90% Percoll layer of asthenozoospermic samples (P < 0.01), compared with normozoospermic samples. The omega6/omega3 ratio was significantly increased in both Percoll fractions of samples with oligozoospermia (47%, P < 0.001 and 90%, P < 0.001) or with asthenozoospermia (47%, P < 0.05 and 90%, P < 0.001) compared with normozoospermic samples. The oxidative potential index (OPI) of spermatozoa recovered from the 47% Percoll layer was significantly higher (P < 0.0001) than of those recovered from the 90% Percoll. Mean melting point (MMP), an index of membrane fluidity, was significantly lower in head than in tails (P < 0.01) of spermatozoa, and also in both the 47% (P < 0.01) and 90% (P < 0.001) Percoll fractions of normozoospermic samples in comparison with oligozoospermic samples. The MMP was significantly higher (P < 0.05) in samples of patients with idiopathic oligo/asthenozoospermia, varicocele, and male accessory gland infection (MAGI). These differences in FA composition of PL in subpopulations of human spermatozoa, and in their heads and tails may be related to sperm maturity and to differences in physiological function.      

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.     

Andrology: An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percollcentrifugation was assessed in an in-vitro fertilization programmeon 71 semen samples of a well-defined quality [total numberof type A (WHO criteria) motile spermatozoa]: category I (n= 21) with > 100 x 10⁶, II (n = 31) with 15–100 x 10⁶,III (n = 11) with 5–15 x 10⁶ and IV (n = 8) with <5 x 10⁶ type A motile spermatozoa. Oocytes were inseminated4 h after oocyte retrieval, alternately with spermatozoa derivedfrom swim-up and Percoll preparation. Both selection proceduresresulted in a significantly higher (P < 0.001) percentagemotility as compared to fresh semen. For low-quality samples(III and IV), however, swim-up was more effective in selectinghighly motile (P = 0.004) and morphologically normal spermatozoa(P < 0.05). For high-quality samples, this difference mighthave been masked by introducing a swim-up step to remove Percollparticles. Regardless of the initial sperm quality, the meanfertilization rate was significantly higher (P = 0.003) whenPercoll-treated spermatozoa were used for insemination (51.3versus 37.8%). For semen of groups I and II, no difference infertilization capacity was observed according to the sperm preparationmethod. Despite the lower percentage motility and normal morphologyfor the Percoll compared to the swim-up treatment in groupsIII and IV, fertilizing capacity was significantly (P < 0.001)in favour of this selection method (65.3 versus 26.5% in groupIII, 47.6 versus 11.6% in group IV). Based on these results,it may be concluded that a subgroup of patients exhibiting poorsemen quality can benefit from Percoll semen preparation interms of improved fertilizing capacity.     

Sperm selection by PD-10 sephadex columns: comparison with SpermPrep filtration and Percoll centrifugation

The efficacy of a disposable, prepacked column (PD-10) containingSephadex G-25, to select motile spermatozoa, was compared withanother column for sperm filtration (SpermPrep) and centrifugationthrough Percoll gradients. Aliquots of washed sperm suspensionswere processed by the three techniques. The number of motilecells and the proportion of total spermatozoa selected was similarfor all methods. Recovery of spermatozoa showing optimal movementwas 145.9 ± 30% (mean ± SEM) with PD-10 columnsand 131.9 ± 32% with Percoll, both significantly higherthan SpermPrep (71.9 ± 11%; P < 0.05). The straightline velocity of motile cells was lower in samples processedby SpermPrep (29.3 ± 2 µm/s) compared to both PD-10(34.7 ± 1 µm/s) and Percoll (34.9 ± 2 µm/s;P = 0.07). When whole semen was processed, total sperm recoverywith PD-10 was 61.7 ± 8% versus 47.7 ± 7% withPercoll (P < 0.001). Percoll centrifugation improved thepercentage of morphologically normal spermatozoa more than PD-10.Similar proportions of motile spermatozoa and cells with optimalmotility were obtained by both methods. We conclude that PD-10filtration columns can be used to prepare semen in the laboratoryas a practical alternative to other methods.     

Genetics: Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence int-situ hybridization

The aim of this study was to evaluate the efficacy of enrichingX-bearing human spermatozoa using 12-step (25–80%) discontinuousPercoll gradients. X- and Y-bearing spermatozoa were simultaneouslyidentified in neat semen (controls) and in 80% Percoll fractionsfrom the same samples using double-label fluorescence in-situhybridization and chromosome-specific DNA probes. Hybridizationand labelling efficiencies of 95–99% were obtained inall samples. The mean ratio of X- to Y-bearing spermatozoa inthe controls was 49.0: 48.2, whereas there was a significantenrichment (P < 0.0001) of X-bearing spermatozoa in the Percollfractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratiovaried from 1.1–1.5 in individual Percoll samples. Therewere no significant differences in the proportions of aneuploidspermatozoa (XX, YY, XY) between the control and Percoll fractions.We conclude that 12-step discontinuous Percoll gradients enrichX-bearing spermatozoa, but the degree of enrichment is insufficientfor use in preconceptional sex selection.     

Regulators of sperm function: Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa

The involvement of protein kinases in platelet activating factor(PAF)-induced acrosome reaction of human spermatozoa was investigatedusing specific inhibitors of protein kinase A (PKA), proteinkinase C (PKC) and protein tyrosine kinase (PTK). PAF (10^–9–10^–11M) treatment of spermatozoa enhanced the acrosome reaction ina dose-dependent manner (32 ± 4% at 10^–9 M, 28±4%at 10^–10 M and 24 ± 3% at 10^–11 M respectively).When spermatozoa were preincubated with PKA, PKC and PTK inhibitor(KT5720, calphostin C and genistein) for 15 min prior to additionof PAF, there was a significantly reduced acrosome reactioninduced by PAF, but complete inhibition was not observed. Onthe other hand, combined use of three inhibitors completelyinhibited PAF-induced acrosome reaction to levels of non-treatedsamples. These results suggest that the induction of the acrosomereaction by PAF treatment may involve the activation of PKA,PKC and PTK signalling pathways, and that interaction betweenthese pathways may regulate complex mechanisms of PAF-inducedacrosome reaction. acrosome reaction/human spermatozoa/platelet activating factor/protein kinase     

Fertilization and early embryology: A simple technique to quantify human sperm--zona pellucida binding assays

A simplified method for assessing the degree of spermzona pellucidabinding was developed. The zonae pellucidae of salt-stored,failed-fertilized human oocytes were each inseminated with between1 x 10⁵ and 1 x 10⁶ motile spermatozoa/ml, prepared by a directswim-up method from 11 individuals with normal sperm counts,as defined by the World Health Organization. Following 4 h ofincubation at 37°C in humidified air, the zonae pellucidaewere ‘washed’ by vigorous pipetting to remove anyloosely attached spermatozoa. The zonae were then placed individuallyin microwells and dissolved by exposure to acidified (pH <2.0) medium to form a fluid monolayer. The slides were sealedand the number of spermatozoa in the monolayer counted by eachof three observers within 24 h. There was good agreement inthe counts between the different observers, with a mean coefficientof variation of only 7.4% and a range of 1.8–16.7%. Itwas notable that the highest coefficients of variation occurredat the extremes of sperm numbers and that the results were stableovernight. The method is also able to identify observer biaswithin this variation, indicating the potential for improvementsin assay performance. The technique reported has the advantageover current sperm-zona pellucida binding assays of allowingthe determination of the precise number of spermatozoa boundto a zona pellucida while producing a slide which remains stableovernight.     

Andrology: Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity

Endogenous nitric oxide (NO) is an important functional mediatorin several physiological systems, including the reproductivesystem. However, when generated in excessive amounts for longperiods, mainly during immunological reactions, NO is cytotoxicand cytostatic for invading microbes, as well as for the cellsgenerating it and the tissues present around it. Since infertilityassociated with urogenital tract infection in males and femalesis also accompanied by reduced sperm motility and viability,it is possible that reduced fertility in these patients is dueto NO-induced sperm toxicity. We therefore evaluated the directeffects of NO, chemically derived from S-nitroso-N-acetylpenicillamine(SNAP, 0.012–0.6 mM) and sodium nitroprusside (SNP, 0.25–2.5mM), on the motility and viability of human spermatozoa. Furthermore,we tested whether inhibition of NO synthesis prevents spermmotility and viability by incubating washed total cells presentin the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester(L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoawith SNAP or SNP decreased forward progressive sperm motilityand straight line velocity, and also increased the percentageof immotile spermatozoa in a concentration-dependent manner.Furthermore, the percentage of immotile spermatozoa positivelycorrelated with the percentage of dead spermatozoa. In contrastto freshly prepared SNAP, SNAP preincubated for 48 h had noeffect on the motility and viability of the spermatozoa. Furthermore,as compared to untreated controls, a significantly higher percentageof forward progressive sperm motility as well as viability (P< 0.05) was maintained in washed semen incubated with L-NAME(0.15 mM). Seminal plasma concentrations of nitrite-nitrate(stabile metabolites of NO/10⁶ spermatozoa correlated positively(P < 0.05) with the percentage of immotile spermatozoa. Ourresults suggest that NO can cause sperm toxicity as well asinhibit sperm motility. In conclusion, excessive NO synthesisin response to infection and inflammation could be an importantfactor contributing to functional change of the spermatozoa,leading to their dysfunction and to infertility.     

Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa

Human oocytes can be fertilized with high rates of success underin-vitro conditions even if only low numbers of spermatozoaare used. A culture system has been developed in which fertilizationis performed in haematocrit capillary tubes (length 75 mm; i.d.0.8–0.9 mm). Oocytes were fertilized in 5–10µlof different sperm suspensions containing a total of 500, 1000,2000 and 4000 spermatozoa per oocyte (0.1–0.4 x 10⁶ spermatozoa/ml).Oocytes were obtained from 10 patients participating in an in-vitrofertilization programme; of these, 32 oocytes were fertilizedin capillary tubes and 32 oocytes were cultured using standardmethods (1 ml culture medium in tissue culture tubes; 0.1–0.2x 10⁶ spermatozoa/ml). The overall fertilization rate of oocytescultured in tissue culture tubes was 78% (25/32) and the fertilizationrates in capillary tubes using 4000, 2000, 1000 or 500 spermatozoaper oocyte were 71% (5/7), 86% (6/7), 60% (6/10)and 50% (4/8),respectively. The fertilization rate of mature oocytes was highercompared with immature oocytes when fertilization was performedin culture tubes (83 and 63%) or in capillary tubes (74 and44%). Fertilization in capillary tubes using a 10µl ofoocyte and spermatozoa suspension compared to 5µl seemedto provide better culture conditions, resulting in higher fertilizationand cleavage rates. These preliminary results indicate thatfertilization of human oocytes under in-vitrio conditions canbe achieved even with very low numbers of spermatozoa     

Evidence for {gamma}-aminobutyric acid specific binding sites on human spermatozoa

The possible presence of -aminobutyric acid (GABA) specificbinding sites on human spermatozoa was investigated. Swim-uppreparations of human spermatozoa were incubated with radiolabelledGABA in the presence of unlabelled GABA, alternatively displacersof GABA_A/B receptors and GABA transport proteins. The resultsindicate that GABA specific binding sites are present on thesurface of human spermatozoa, and that these binding sites possiblyindicate the presence of GABA transport proteins. Furthermore,GABA at different concentrations was added to swim-up preparationsof human spermatozoa. Possible effects of GABA on sperm motiltty,hyperactivation and acrosome reaction were explored. No significantdifferences were observed between treated groups and controlsconcerning motiltty parameters and hyperactivation. Incubationwith GABA did not cause any increase in spontaneous acrosomereaction. However, spermatozoa treated with the calcium ionophoreA-23187 showed a small but significantly increased ability toundergo the acrosome reaction following preincubation in 10^–4M GABA (P < 0.05). -aminobutyric acid (GABA)/spermatozoa/sperm motility/transport proteins     

Molecular Human Reproduction: Evidence for {gamma}-aminobutyric acid specific binding sites on human spermatozoa

The possible presence of -aminobutyric acid (GABA) specificbinding sites on human spermatozoa was investigated. Swim-uppreparations of human spermatozoa were incubated with radiolabelledGABA in the presence of unlabelled GABA, alternatively displacersof GABA_A/B receptors and GABA transport proteins. The resultsindicate that GABA specific binding sites are present on thesurface of human spermatozoa, and that these binding sites possiblyindicate the presence of GABA transport proteins. Furthermore,GABA at different concentrations was added to swim-up preparationsof human spermatozoa. Possible effects of GABA on sperm motility,hyperactivation and acrosome reaction were explored. No significantdifferences were observed between treated groups and controlsconcerning motility parameters and hyperactivation. Incubationwith GABA did not cause any increase in spontaneous acrosomereaction. However, spermatozoa treated with the calcium ionophoreA-23187 showed a small but significantly increased ability toundergo the acrosome reaction following preincubation in 10–⁴M GABA (P < 0.05).     

Analysis of the flow cytometer stain Hoechst 33342 on human spermatozoa

Several procedures exist for processing sperm cells for sexpreselection. Flow cytometric separation using the fluorochromestain Hoechst 33342, chemically known as bisbenzimide, is themost promising. The objective of this study was to determinethe effect of bisbenzimide on spermatozoa assessed by meansof the sperm survival test and to analyse the p-globin genein sperm DNA after exposure to increasing concentrations ofbisbenzimide. Donor (n = 16) sperm specimens were pooled andwashed in a discontinuous Percoll gradient 95: 47%, dividedand incubated in tubes containing bisbenzimide at concentrations0 (control), 0.9, 9, 90, 900 and 9000 µM at 25°C andscanned on a computer-aided sperm motility analyser at 0, 1,4 and 24 h. Spermatozoa were also incubated in a known mutagen,ethidium bromide, as positive control. After 24 h of incubation,the treated sperm cells were processed through DNA extractionand polymerase chain reaction (PCR) performed with primers targetingthe p-globin gene. The amplified DNA products were analysedfor evidence of mutation in 5% polyacrylamide gel electrophoresisand 20: 80 denaturing gradient gel electrophoresis (DGGE) andfurther confirmed in 30: 40 DGGE. The results showed completecessation of motility in sperm incubated in the presence of900 µM or higher concentrations of bisbenzimide. The beatcross frequency sperm parameter was significantly differentat the 90 µM or higher concentration of bisbenzimide comparedwith the control. At concentrations <900 µM bisbenzimide,there were no differences in the remaining sperm kinematic parameters(percentage rapid progressive, percentage total progressive,sperm velocities, linearity, straightness, amplitude of lateralhead displacement and percentage hyperactive motility). PCRand DGGE analyses of spermatozoa treated with bisbenzimide showedno evidence of mutation in the representative region of thef3-globin gene at concentrations <900 µM. The datasuggest an inhibitory effect of bisbenzimide on human spermmotility at 900 µM or higher concentrations of bisbenzimide.The decrease in sperm motility and rapid progression were notdue to changes in pH. Point mutation in the representative regionof the p-globin gene in human spermatozoa was detected onlyat high concentrations (     

Relationships between motility parameters, morphology and acrosomal status of human spermatozoa

A total of 130 semen samples were examined for motility (bycomputer-assisted sperm analysis), morphology and acrosomalstatus. A high positive correlation was found between percentagesof normal forms and progressive motility in the whole semen(r = 0.539, P < 0.0001) as well as in the Percoll fraction(r = 0.702, P < 0.0001). Among the specific abnormalities,acrosome defects were most highly correlated with progressivemotility (r = –0.492, P < 0.0001, in the Percoll fraction).The percentage of total spontaneously acrosome-reacted spermatozoain the Percoll fraction was negatively correlated with the progressivemotility (r = –0.499, P < 0.0001) and with the percentageof normal forms (r = –0.430, P < 0.0001). Surprisingly,the percentage of total spontaneously acrosome-reacted spermatozoawas poorly linked with head abnormalities but displayed significantpositive correlations with the percentages of bent tails (r= 0359, P < 0.0001) and of coiled tails (r = 0371, P <0.0001). These data suggest that sperm defects are often linkedtogether, reflecting spermiogenesis and/or epididymal dysfunctions.     

Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa

Recombinant human ZP3 (rhuZP3) generated by Chinese hamsterovary cells transfected with a plasmid containing human ZP3cDNA was used to study the acrosome reaction (AR) and intracellularcalcium fluxes in capacitated human spermatozoa. Conditionedmedium containing rhuZP3 significantly induced the AR (P <0.005)in 59.4 ± 4.7% of spermatozoa (control = 8.5 ±3.1%) and caused complete acrosomal loss in a further 17.2 ±3.8% of cells (control = 3.7 ± 0.7%; mean ± SEM,n = 5). Sperm motility was not affected and acrosomal exocytosisin response to rhuZP3 was also shown to be time-dependent. Basalconcentrations of sperm intracellular calcium were measured(82 ± 7 nM; mean ± SEM, n = 9). A transient increasein intracellular calcium (typically up to 400–450 nM)occurred within 1 min of rhuZP3 addition and was followed bysustained lower values of calcium (200–400 nM). Theseresponses were dependent on the amount of rhuZP3. This is thefirst report of zona protein-induced changes in intracellularcalcium levels in human spermatozoa. The results support thepremise that ZP3 is an aganist of the human sperm AR and thatrhuZP3 generated in a eukaryotic cell is effective in this respect. acrosome reaction/calcium/human/spermatozoa/ZP3     

Andrology: Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction

This study was designed to test the effects of pentoxifyllineand progesterone upon capacitation of fresh human spermatozoa.Capacitation and acrosomal integrity were assessed using thefluorescent probe chlortetracycline on spermatozoa co-stainedwith a supravital fluorescent dye, Hoechst 33258. Hyperactivatedmotility was measured using computer-assisted movement analysis.After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent‘B’ pattern, characteristic of capacitated, acrosome-intactcells, increased significantly (P < 0.01), though no increasein ‘AR’ pattern, characteristic of acrosome-reactedcells, was detected. There was a significant increase in hyperactivemotility (P < 0.001). Exposure to progesterone (1µg/ml;60 min) resulted in a significant increase in ‘B’pattern (P < 0.05) and ‘AR’ pattern (P < 0.005),though no effect on the expression of hyperactivation was detected.No effect upon hyperactivation was detected on exposure of freshor cryopreserved spermatozoa to a physiological range of progesteroneconcentrations (0.1–1000 ng/ml). Sequential exposure topentoxifylline then progesterone resulted in a significant increasein ‘B’ pattern, acrosome loss and hyperactivation.Sperm viability was not affected in any treatment group. Theseobservations suggest that pentoxifylline and progesterone affectcapacitation through independent mechanisms. Stimulation ofboth capacitation and acrosome reaction resulted from sequentialexposure to pentoxifylline and progesterone. This may have implicationsfor sperm handling for assisted reproductive techniques.     

Standardization and quality control of sperm concentration and sperm motility counts in semen analysis

The paper reports a study of standardization and quality controlof sperm concentration counts and visual motility assessmentsin human semen analyses performed for infertility investigationsand from internal quality control procedures. Sperm concentrationdeterminations were performed in Improved Neubauer haemocytometerson volumetric dilutions made using a positive displacement pipettorfor sampling the liquefied semen. In addition to a standard1+19 dilution a second dilution of either 1+9, 1+19 or 1+49was made according to whether the estimated sperm concentrationwas <20, 20–100 or > 100 x l0⁶/ml respectively.The duplicate determinations of sperm concentration were highlysignificantly correlated (P << 0.001) with <5% variability.Parallel visual sperm motility assessments were made by twopairs of technicians and showed highly significant correlations(P << 0.001) between technicians in the determinationof the percentages of motile and progressive spermatozoa aswell as the subjective rating of sperm progressivity. When thesevalues were incorporated into a calculated motility index whichgave added weight to the progressive spermatozoa and to theirquality of progression the correlations between techniciansremained highly significant (P << 0.001) with averagedifferences of the order of 1.0%. Therefore, provided that sufficientattention is paid to technician training, regular standardizationchecks and the use of only proven reliable procedures, quantitativelyaccurate values for sperm concentration and motility can beobtained in routine semen analyses.     

Uterine flushing: a method to recover spermatozoa and leukocytes

The process of sperm transport from the cervix, where a leukocyticreaction is initiated, through the uterus to gain access tothe site of fertilization is very poorly understood. This preliminarystudy was designed to utilize a uterine flushing technique todetermine firstly, the number of spermatozoa that can be recoveredfrom the uterine cavity at 4 h post-insemination, around thetime of ovulation, and secondly, to establish whether the spermatozoainitiate a leukocytic response while present. Uterine flushingwas carried out in 10 potentially fertile women at 4 h post-inseminationwith donor semen, 24–36 h after the onset of the luteinizinghormone (LH) surge. The flush fluid was analysed for the numbersof spermatozoa and leukocytes present. In 8/10 women spermatozoawere retrieved from the uterus, in consistently low numbers(median 46, range 3–415). In 5/5 women leukocytes wererecovered (median, 2.75 x 10⁸/l, range 2.0 x 10⁸–12.7x 10⁸/l) from an origin other than peripheral blood contamination.These results suggest firstly that the flushing technique wasa consistent method for retrieving spermatozoa and leukocytesfrom the uterine cavity, secondly that only low numbers of spermatozoacan be retrieved on flushing and thirdly that the leukocyticreaction to spermatozoa extends to the uterine cavity.     

Characterization of human zona pellucida glycoproteins

The human egg may only be fertilized by one spermatozoon toprevent polyploidy. In most mammals, the primary block to polyspermyoccurs at the zona pellucida (ZP). Little is known of the humanZP and the changes occurring following fertilization to preventpolyploidy. Using antibodies directed against synthetic peptidespredicted from the human ZP2 and ZP3 cDNA, we identified ZP3as a 53–60 kDa glycoprotein and ZP2 as a 90–110 kDaglycoprotein in prophase-I oocytes. Characterization of theZP from metaphase II arrested eggs (inseminated–unfertilizedand fertilized–uncleaved), shows no visible modificationof ZP3, but demonstrates that ZP2 undergoes limited proteolysisin the amino terminal domain, to a 60–73 kDa species, denotedZP2_p, which remains linked to the proteolysed fragments by intramoleculardisulphide bonds. A lack of ZP2 proteolytic activity in acrosomalsupernatants is consistent with an oocyte origin for the protease.The ZP2-specific protease may be released during cortical granuleexocytosis which occurs during meiotic maturation and followingsperm–egg fusion as part of the block to polyspermy. Sincemouse ZP2 acts as a secondary sperm receptor, it is possiblethat intact ZP2 binds a secondary egg binding protein, whereascleaved ZP2 does not, suggesting a possible mechanism for theblock to polyspermy. glycosylation/human zona pellucida/proteolysis/ZP2/ZP3 ⁴ Current address: Centre for Immunology, St Vincent's Hospitaland University of New South Wales, Sydney, 2010, Australia ⁵ To whom correspondence should be addressed     

The paradoxical effects of pentoxifylline on the binding of permatozoa to the human zona pellucioda

In the presence of pentoxifylline, human spermatozoa are inducedto increase certain motion characteristics; however, the roleof this drug in fertilization remains equivocal. In this study,the influence of pentoxifylline on one aspect of fertilization,that is spermzona binding, has been examined. Results from afluorescence label competitive zona binding (CZB) test showedthat spermatozoa exposed to a pentoxifylline challenge of between0.1 and 5 mM, which was curtailed after 1 h by washing, hada decreased (P < 0.01) ability to bind to intact zona comparedwith control spermatozoa. The washing procedures also removed(decrease P < 0.01 compared with peak values) some of theenhanced motion characteristics induced by pentoxifylline. Theseresults were in contrast with those obtained using experimentalconditions that maintained an increased curvilinear velocity(VCL) and lateral head displacement (ALH) (increase P < 0.001above baseline controls) in the continued presence of pentoxifylline.Using a hemizona binding (HZB) assay, 3 mM pentoxifylline increased(P < 0.001) spermzona binding almost 20% above zona bindingwith unexposed control spermatozoa. It was concluded that, inthe presence of pentoxifylline, there is increased sperm bindingto the zona pelludda; however, if the drug is removed by washing,the sperm binding to the zona is decreased in concert with theremoval of the enhanced motion characteristics. The applicationof zona solubilization by acidic conditions in a microchamberenabled the precise determination of sperm numbers in both ofthe spermzona binding assays, and the results demonstrated thata wide variation in sperm numbers was observed in each test,with 63–580 spermatozoa bound in the CZB assay and 56–1340spermatozoa bound on a hemizona.     

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