An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.

The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10-40 micromol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.     

Reassessing the role of progesterone in fertilization--compartmentalized calcium signalling in human spermatozoa?

Progesterone is present at micromolar concentrations in the vicinity of the oocyte. Human spermatozoa generate a biphasic rise in intracellular calcium concentration ([Ca(2+)](i)) and undergo the acrosome reaction upon progesterone stimulation, suggesting that the hormone acts as a secondary inducer or 'primer' of the acrosome reaction in association with the zona pellucida. However, the sensitivity of human spermatozoa to progesterone is such that many cells may undergo the acrosome reaction prematurely, compromising their ability to fertilize. We have shown that exposing human spermatozoa to a progesterone gradient, simulating the stimulus encountered as sperm approach the oocyte, results in a novel response. A slow rise in [Ca(2+)](i) occurs, upon which, in many cells, [Ca(2+)](i) oscillations are superimposed. Cells showing this pattern of response do not undergo the acrosome reaction, but instead show an alternating pattern of flagellar activity associated with peaks and troughs of [Ca(2+)](i). A Ca(2+) store in the rear of the sperm head apparently generates this complex signal, functioning as an '[Ca(2+)](i) oscillator'. We propose that: (i) the acrosome reaction and flagellar beat are regulated by separate Ca(2+) stores; (ii) these stores are mobilized through different mechanisms by different agonists; and (iii) progesterone in vivo acts as a switch for the oscillator which regulates the flagellar beat mode.     

An evaluation of couples with failure of fertilization in vitro.

Attempts at in-vitro fertilization (IVF) may be used as a method of evaluating whether in a given couple, the inability of the sperm to fertilize the oocyte may be the cause of infertility. We evaluated all IVF patients in our practice who had at least one cycle with no fertilization to determine how often this was an isolated event or was repeated in multiple cycles; would poor semen quality be found as a frequent cause; and how well can a donor sperm or oocyte 'probe' uncover which of the two is the problem? Of 35 couples who used their own gametes exclusively, 30 (85.7%) had at least one cycle with zero fertilization; 42.5% of those failing to fertilize in cycle 1 and 35% of those failing in cycle 2 had a subnormal concentration of motile spermatozoa, morphology or hypo-osmotic swelling test scores. The pregnancy rate per cycle with both husband's and wife's gametes was only 2.3% (3/130), but was 8.3% for those using donor spermatozoa (3/36) and 18.2% (2/11) for donor oocytes. Thus, failing to fertilize in a given cycle does not necessarily predict failure to fertilize in a subsequent cycle, but does predict a poor fertility outcome unless donor gametes are used.     

Should ICSI be the treatment of choice for all cases of in-vitro conception?

The objective of this study was to examine different clinical scenarios of in-vitro conception, viz. fertilization with conventional IVF, IVF with high insemination concentration (HIC) and intracytoplasmic sperm injection (ICSI), and assess on a sibling oocyte comparison the hypothesis that ICSI should be performed in all cases requiring in-vitro conception. ICSI with husband's spermatozoa had a higher incidence of fertilization as compared with IVF or IVF with HIC with donor spermatozoa (if previous failure of fertilization had occurred) for unexplained infertility. Similarly, ICSI with husband's spermatozoa had as high an incidence of fertilization as IVF with donor spermatozoa for patients with severe oligozoospermia, asthenozoospermia and/or teratozoospermia, even when the spermatozoa were not selected for their morphology. Two studies were performed to assess ICSI in potential oocyte-related failure of IVF, viz. when fertilization occurred in >50% of oocytes for one group of patients, and in <50% of oocytes in a second group. In both of these studies a significant proportion of the oocytes that failed to fertilize with conventional IVF eventually fertilized after ICSI. The overall conclusion was that ICSI as a first option offers a higher incidence of fertilization, maximizes the number of embryos and minimizes the risk of complete failure of fertilization for all cases requiring in-vitro conception. However, among other concerns, current knowledge of ICSI as an outcome procedure does not provide the confidence to use this process in all cases of IVF for the time being.     

Localisation of clusterin in normal human sperm by immunogold electron microscopy

In the human male reproductive tract, two forms of clusterin have been detected: the conventional heterodimeric form and a novel acrosomal form. On human sperm the novel form of clusterin is present in the acrosomal region of acrosome intact sperm only. The aim of this study was to determine the site of localisation of the acrosomal form of clusterin using immunogold electron microscopy on normal human sperm. Using the E5 anticlusterin mAb and a preembedding technique, acrosomal clusterin was localised in the acrosomal contents. Immunogold particles were detected on ethanol fixed spermatozoa that were subjected to Triton X-100 permeabilisation treatment. These sperm had lost their plasmalemma and outer acrosomal membrane. Specific immunogold labeling was present over the surface mainly of the acrosomal contents exposed by the loss of the plasma-lemma and outer acrosomal membrane. Immunogold particles were also detected in the equatorial segment of the sperm. These data confirm that the acrosomal form of clusterin is associated with the contents of the acrosome.     

Correlation of Post Swim-Up Acrosome Index with in-vitro Fertilization Outcomes

Purpose

A prospective study was planned to determine the relationship between post swim-up acrosome index (AI) evaluation and fertilization outcomes in an in vitro fertilization (IVF) program.

Materials and Methods

Infertile couples who have applied to IVF were admitted into this study when the male partner''s sperm concentration was > 20×10⁶/mL and motility > 30%. Pre- and post swim-up semen quality parameters including concentration, motility, sperm morphology and AI were evaluated in a prospective, randomized and blinded fashion. The couples were divided prospectively into 2 groups. In group I (25 couples) 50 000 sperm per oocyte were used for insemination considering post swim-up acrosome index, and in group II (25 couples) 50 000 sperm per oocyte were used for insemination without considering post swim-up acrosome index.

Results

Pre- and post swim-up AI were 30.8 ± 3.4 and 17.8 ± 4.5 in group I, and 31.4 ± 3.6 and 16.3 ± 4.7 in group II (p > 0.05) respectively. The significant improvement in morphology and motility after double wash swim-up procedure has been observed. However, double wash swim-up procedure could not eliminate head and especially acrosomal defects which would directly effect fertilization capacity in conventional IVF program. In group I, 85.3% of oocytes were fertilized, with a 48% pregnancy rate; in group II, 71.0% of oocytes were fertilized, with a pregnancy rate of 20%. Fertilization and pregnancy rates were significantly different (p < 0.05) between the two groups.

Conclusion

We have concluded that it could be useful to consider post swim-up AI of sperm inseminated in conventional IVF cycles, which correlates with high fertilization and pregnancy rates.     

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction

We studied the effect of media composition on sperm capacitation,using Biggers—Whitten—Whittingham (BWW) medium,Ham's-F10 and a modified Tyrode's medium (HSM) supplementedwith bovine serum albumin (BSA) or fetal cord serum (FCS). Weevaluated the effect of chemical environment and protein supplementationon the sperm motion parameters of curvilinear velocity and linearity,and on the ability of incubated spermatozoa to undergo follicularfluid induced acrosome reaction. Neither chemical compositionnor protein supplementation of capacitation media greatly affectedmotion parameters after 2 h incubation. Furthermore, chemicalcomposition had only a small effect on the ability of spermatozoato undergo the acrosome reaction upon exposure to follicularfluid. A higher proportion of spermatozoa underwent acrosomereaction after incubation in HSM (8% control (C); 28% follicularfluid) than in BWW (8% C, 17% follicular fluid) or Ham‘sF-10 (6% C, 19% follicular fluid). By contrast, protein sourceproved critical in determining acrosome reaction inducibility.Spermatozoa incubated in BSA-supplemented media showed a 4-foldincrease in acrosomal discharge when exposed to follicular fluid(6% C, 22% follicular fluid) compared to controls while spermatozoaincubated in FCS were unable to undergo acrosome reaction (6%C, 6% follicular fluid). Simultaneous addition of FCS to BSAcapacitation medium blocked acrosome reaction inducibility andthe late addition of BSA, after sperm incubation in FCS, didnot facilitate acrosome reaction. We propose that an inhibitorof sperm capacitation is present in FCS and therefore, the selectionof optimum incubation conditions for spermatozoa may be of criticalimportance when evaluating or treating infertile patients.     

Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Sperm-zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had 相似文献   

Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon

This study was undertaken to determine the site of the acrosomereaction of spermatozoa penetrating into freshly inseminatedhuman oocytes. The inseminated oocytes were treated with ananti-acrosin monoclonal antibody and the bound antibody wasvisualized at the ultrastructural level with the use of a secondperoxidase-conjugated antibody. Quantitative analysis of serialthin sections cut throughout the specimens showed that the numberof spermatozoa within the zona pellucida (potentially fertilizingones) corresponded to the number of acrosin deposits associatedwith acrosomal ghosts on the zona pellucida surface. As it isknown that a large acrosin bundle is liberated from a spermatozoonat a well-defined point of the acrosome reaction, these findingsindicate that the acrosome reaction of the fertilizing spermatozoonmust be exactly synchronized with its penetration through theegg vestments, apparently by the action of specific acrosomereaction-promoting substances in the oocyte/cumulus complex.These results represent a theoretical basis for evaluation ofdirect and indirect laboratory tests for human sperm acrosomereaction. ‘Good’ sperm samples should display elevatedlevels of acrosome-reacted spermatozoa after the administrationof an appropriate stimulus and low levels of spontaneous acrosomereactions.     

The spontaneous acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (10 donors x 3 replicates) using Percoll density gradientsat 30–60 min post-ejaculation. Sperm suspensions wereincubated in a complex ’synthetic tubal fluid‘ culturemedium (STF) at 37°C under 5% CO₂ in air for up to 36 h.Parallel aliquots were incubated with 50 µM A23187 toinduce maximum acrosome loss (AR_max). Acrosome reactions wereassessed using both the triple-stain (TS) technique and fluorescentpeanut agglutinin (PNA) lectin-labelling. During incubation,the proportion of TS acrosome reacted spermatozoa increasedfrom 9.1 to 54.3% with AR_max being 68.3%. Spermatozoa showingintact acrosomes by PNA labelling decreased from 68.4 to 26.1%over 36 h of incubation (AR_max = 13.8%). Simultaneously, spermatozoashowing complete acrosomal loss (no PNA labelling) increasedfrom 8.1 to 27.0% (AR_max= 46.3%). Therefore, while only 23.5%of cells were actually undergoing acrosomal changes at the startof incubation, this had increased to 46.9% after 36 h (AR_max=40.7%). These experiments clearly show that even in selectedpopulations, not all human spermatozoa are capable of undergoingan acrosome reaction. However, the incidence of acrosomal changesafter 36 h of incubation did approach the AR_max. These levelsof spontaneous occurrence of the human sperm acrosome reactionwere higher than those reported in many other in-vitro incubationstudies: an improvement that may be attributable to the morephysiological nature of the STF culture medium     

The sperm acrosome: functional and clinical aspects

The acrosome of mammalian spermatozoa contains high amountsof acrosin, which is believed to be essential for gamete fusion,particularly for binding to, and penetration of, the zona pellucida.In addition, its activation from pro–acrosin seems tobe associated with the capacitation process. Furthermore, itmight facilitate cervical mucus penetration and intrauterinesperm migration by releasing kinins from kininogen, as wellas participating in the acrosome reaction and in chromatin decondensationin the oocyte. Considering these functions of the acrosome duringthe pro–cess of fertilization, the morphology and functionalintegrity of the acrosome in ejaculated human spermatozoa areof fundamental inportance in attachment, species–specificbind–ing and zona penetration preceding gamete fusion.It is therefore mandatory to focus on acrosomal membrane func–tionsand consider particularly the occurrence of acrosomal disturbancesin spermatozoa from men of barren marriages. For this purpose,biochemical and immunocytochemkal methods have been used toinvestigate acrosin activity and possible alterations of theacrosomal membrane system in dif–ferent groups of sub–andinfertile patients. Of particular interest is the ability ofspermatozoa to undergo the acrosome reaction, which can be studiedusing the triple–stain techni–que. Of all the spermatoIogkaJgroups investigated (normo–, astheno–,oligo–,terato–and polyzoospermia), polyzoosper–mk semen samples showedsevere acrosomal disturbances indicating functionally defectiveacrosomes which might hinder fertilization. The data indicatethe need for the introduction of acrosomal markers in clinicalandrology for proper diagnosis of acrosomal sperm defects.     

Andrology: High fertilization prediction by flow cytometric analysis of the CD46 antigen on the inner acrosomal membrane of spermatozoa

The study was set up to determine the relationship between thehuman sperm acrosome reaction and fertilization in couples undergoingroutine in-vitro fertilization (IVF) treatment. Prospectivedata analysis was carried out on all IVF patients during a 6month period. Exceptions were those patients having insufficientsperm concentration to allow both acrosome reaction determinationand insemination. The main outcome measures were the predictionof fertiliza tion in IVF patients using flow cytometric analysisof the spontaneous and ionophore-induced acrosome reaction [givingthe acrosomal response to ionophore challenge (ARIC) score]in the male partner's spermatozoa versus standard analyticalmethods of sperm motion parameters and morphology. Stepwiselogistic regression indicated only two independent factors predictiveof fertilization: ARIC score (x² = 109.6, P < 0.0001) andpost-Percoll % motility (x² 8.8, P < 0.003). Of patientswith an ARIC score of >10, 92% had >30% of oocytes fertilized;100% of patients with an ARIC score of <10 had <30% fertilizationof oocytes. The sensitivity and specificity of the assay systemwere 1.00 and 0.82 respectively. The results would indicatethat the ARIC test as measured by flow cytometric analysis ofCD46 binding is a sensitive and specific assay for use in theprediction of fertilization in IVF patients, thus enabling directchannelling of those patients with ARIC scores of <10 intothe more invasive micro-assisted fertilization schemes.     

Lectin binding as a biological test in vitro for the prediction of functional activity of human spermatozoa

Adequate acrosome reaction is one of the essential events thathas to occur in successful mammalian fertilization. The purposeof the present study was to assess the acrosome reaction inhuman spermatozoa by means of iodine-labelled lectins concanavalinA and peanut agglutinin ([¹²⁵I]Con A and [¹²⁵I]PNA). Six spermsamples in the control (fertile) group were compared with 24samples obtained from infertile patients. The acrosome reactionin both groups was induced in vitro by adding follicular fluid.Iodine-labelled lectins were bound to the sperm surface, andalteration in the binding capacity for [¹²⁵I]PNA and [¹²⁵I]ConA after induced acrosomal reaction was the main parameter forthe prediction of acrosome reaction and fertilizing ability.It is hoped that with the availability of this test, in-vivodata may be accumulated.     

Immunochemical localization and characterization of proteins from mouse cauda epididymis using human sperm specific monoclonal antibody

Monoclonal antibodies (mAbs) to sperm specific antigens were generated by fusion of mouse myeloma cells (SP2/0) with splenocytes of female BALB/c mice hyperimmunized with washed human spermatozoa. The resultant hybridomas producing antisperm antibodies were first screened by ELISA. Out of these, one of the mAbs (D2G4) which recognized target antigens restricted to the acrosomal cap was chosen for these studies. The mAb D2G4 was found to be an agglutinating antibody and was also found to cross-react with mouse epididymal spermatozoa in ELISA and indirect immunofluorescence. The origin of antigens reacting with monoclonal antibody D2G4 was investigated. When frozen sections of murine testis and various regions of epididymis were reacted with mAb D2G4, only the cauda epididymal region was stained. Western blot of proteins from the epididymal spermatozoa and fluid indicated the presence of two bands of mol. wt 45 and 26 kd. These bands were identical under reducing and non-reducing conditions. These observations suggest that the two proteins are structurally similar or at least have a common epitope. These data indicate that the proteins recognized by D2G4 are acquired by spermatozoa during their passage and storage in the cauda epididymis.     

In-vitro fertilization in cases with severe sperm defect: use of a swim-across technique and medium supplemented with follicular fluid.

The purpose of this study was to evaluate a new method of in-vitro fertilization (IVF) in patients with severe sperm defects. Unlike the conventional swim-up method, spermatozoa and oocytes are placed in opposite corners of the bottom of the incubation dish so that sperm swimming is horizontal instead of vertical. Another difference between the swim-across and swim-up techniques is that the incubation medium is supplemented with 20% follicular fluid. After a randomized series (protocol I) of 15 IVF attempts had demonstrated that swim-across was more effective than swim-up in terms of fertilization and cleavage, we began a second series (protocol II) using only swim-across. A total of 124 couples with motile sperm counts less than 1 x 10(6) spermatozoa/ml of semen were included in protocol II. Clinical parameters (age, tubal damage) and number of recovered oocytes were recorded and compared in patients who did (group A: n = 94) and did not (group B: n = 74) achieve fertilization. In group A the fertilization rate was 36.7% and, out of the 94 transfers that were made, there were 21 clinical pregnancies and 12 full-term pregnancies with 16 live births. The number of oocytes collected (12 versus 7.7, P less than 0.001) and the incidence of tubal damage (50% versus 24.3%, P less than 0.001) was significantly higher in group A than in group B. Using logistic regression analysis, we showed a significant correlation between fertilization and progressive motility, percentage normal spermatozoa, number of recovered oocytes and tubal damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Andrology: Comparison between fluorescent peanut agglutinin lectin and GB24 antibody techniques for the assessment of acrosomal status

In order to compare fluorescent peanut (Arachis hypogaea) agglutininlectin and GB24 antibody (specific for the inner acrosomal membrane)techniques for the assessment of acrosome reaction, both methodswere applied on semen specimens obtained from patients undergoingin-vitro fertilization (IVF). The acrosome status was evaluatedafter a 4 h incubation in B2 medium with and without calciumionophore A23187. Results obtained with both techniques werecompared and studied as a function of IVF outcome. The percentageof spontaneous acrosome-reacted spermatozoa was higher whenassessed by lectin than by GB24 (19 ± 2% versus 11 ±1%; P < 0.001). The difference between the two methods [lectinsminus GB24) was significantly higher in abnormal than in normalspermatozoa (10 ± 2% versus 4 ± 2%; P < 0.05),but did not significantly correlate with the percentage of acrosomeswith abnormal morphology (r = 0.28; NS). When studied in relationto the IVF results, the response to A23187 was higher in successesthan in failures (45 ± 2% versus 34 ± 4%; P <0.05) but there was no significant difference between methods.Thus the assessment of acrosome reaction is strongly influencedby the method used, particularly in abnormal spermatozoa. Sincethe results obtained with lectins were higher in abnormal spermatozoa,GB24 seems to be more effective for assessment of true acrosomereaction.     

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody

The study was designed in order to investigate the action ofprogesterone on the spontaneous and ionophore-induced humanspermatozoa acrosome reaction in vitro. The principle of theassay system is flow cytometric analysis of CD46 antibody bindingto the inner acrosomal membrane. The technique is a simple andobjective method of analysis, allowing fluorescent analysisof a large segment (5000 spermatozoa) of the spermatozoa populationunder investigation, with concomitant isolation of the livefraction of the spermatozoa population. Four concentrationsof progesterone (1, 25, 50, and 100µg/ml) were examinedfor their effects on spermatozoa capacitated for 4 and 24 h.In addition, motility parameters were examined by the CellSoft2000 automated semen analyser system. Analysis of variance revealedthat progesterone had no effect on either the spontaneous acrosomereaction or the ionophore-induced acrosome reaction at both4 h and 24 h of spermatozoa capacitation times. Further, noeffects on sperm motility parameters or on spermatozoa viabilitycould be attributed to progesterone. We therefore conclude thatprogesterone had no objectively measurable effects on eitherthe sperm acrosome reaction or sperm motility parameters, asmeasured in normal sperm populations.     

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     

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.     

gamma-Aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa

The sperm acrosome reaction takes place in response to progesterone and zona pellucida. Progesterone may act on more than one type of surface receptor, of which one is a gamma-aminobutyric acid (GABA) type A-like receptor. Although there is direct evidence of GABA initiation of mouse sperm acrosome reaction, there are conflicting results regarding GABA- induced exocytosis in human spermatozoa. We have examined whether GABA would initiate exocytosis in human spermatozoa using the chlortetracycline assay and a zona-free hamster oocyte test. Human spermatozoa preincubated for > or = 3 h in Biggers-Whitten-Whittingham medium with 0.35% bovine serum albumin underwent acrosome reactions in response to GABA, with maximal responses in spermatozoa preincubated for 9 h. The effect was concentration-dependent. Preincubated spermatozoa treated with GABA were able to fertilize a higher proportion of zona-free oocytes, with a higher number of spermatozoa penetrating each oocyte. Exposure of preincubated spermatozoa to GABA and progesterone together resulted in a higher proportion of acrosome reactions than when each agonist was used alone. The effect of GABA was mediated by the influx of extracellular Ca2+ because inclusion of EGTA or the Ca2+ channel antagonist La3+ prevented GABA-induced acrosome reactions. These results indicate that GABA can initiate exocytosis in capacitated human spermatozoa and open up possibilities for studies of signalling mechanisms activated upon occupancy of the GABAA receptor present on the sperm surface.