Andrology: A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein

Neoglycoproteins with N-acetylglucosamine residues (BSA-GIcNAc)induced specifically the acrosome reaction (AR) in human spermatozoa.Our objective was to investigate the relationship between thisphenomenon and the in-vitro fertilization (IVF) rate. Spermsuspensions from IVF protocols were incubated with BSA-GlcNAc(t), using calcium ionophore (i) or medium alone (c) as positiveor negative controls. When the normalized AR percentage ratio(STIM) (%ARt-%ARc): (%ARi-%ARc) was compared with fertilizationrate in 31 couples from our IVF programme, a positive correlationwas found (r = 0.46, P < 0.01). The fertilization rate inpatients with STIM 0.2 was higher than in non-responders (STIM< 0.2); 72 ± 7% compared with 5 ± 3%. The overallpredictive value of this test for adequate fertilization rate(>30%) was 87%, sensitivity 91% and specificity 78%. Falsepositives were 9% and false negatives 22%. For successful fertilizationrates (>60%), the results were: overall predictive value,84%; sensitivity 100%; specificity 64%. False positives were23% and no false negatives were found. The results indicatedthat the induction of AR in human spermatozoa by GlcNAc-neoglycoproteinscould be used to predict their fertilizing ability in vitro.     

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.     

Fertilization and early embryology: Progesterone-induced acrosome reaction: potential role for sperm acrosome antigen-1 in fertilization

We have established a monoclonal antibody (mAb) AG7 defininga sperm acrosome antigen-1 (SAA-1) on spermatozoa from the humanand several mammalian species. MAb AG7 inhibits fertilizationof mouse eggs in vitro and in vivo. An important characteristicof mAb AG7 is its inhibition of the rise in intracellular calciuminduced by progesterone in human spermatozoa. Here we show that,following the acrosome reaction, SAA-1 is lost from the capof human spermatozoa but remains detectable in the equatorialregion. Acrosome reaction assays demonstrated a clear differencebetween progesterone- and A23187-induced acrosome reactions.For induction of the acrosome reaction with progesterone, aminimum capacitation time of 6 h was required. A23187 inducedthe acrosome reaction regardless of capacitation time. MAb AG7completely inhibited the progesterone-induced acrosome reaction,but not the A23187-induced acrosome reaction in human spermatozoa.Differences in the pattern of calcium flux induced by the twoagents might account for this phenomenon. The inhibition ofthe progesterone-induced acrosome reaction by mAb AG7 impliesa regulatory function of SAA-1 during the human sperm acrosomereaction.     

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit

This study was conducted to investigate the effect of plateletactivating factor (PAF) on the acrosome reaction and fertilizingcapacity of spermatozoa, and development of the resulting embryosin the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF,or high ionic strength medium (HIS) prior to subzonal sperminjection (SUZI) into 326 mature oocytes, or morphological assessmentof the acrosome reaction. The rates of fertilization and blastocystformation were compared among the three treatment groups. Acrosomereaction was assessed by fluorescein isothiocyanate-conjugatedPisum sativum agglutinin (FITC–PSA) staining and electronmicroscopy. PAF-treated spermatozoa fertilized the oocytes ata significantly higher rate (56.1%) than did lyso-PAF-(36.8%,P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa.The embryos produced by PAF-treated spermatozoa showed significantlyhigher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%,P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa.FITC-PSA staining demonstrated a significantly higher incidenceof acrosome reaction in PAF-treated spermatozoa (45.8%) thanin Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01)treated spermatozoa. Acrosome reaction of PAF-treated spermatozoawas also confirmed by electron microscopy. PAF treatment ofspermatozoa enhances fertilizing capacity for SUZI possiblyby augmenting the acrosome reaction. Enhanced embryonic developmentwas also found in the oocytes fertilized by SUZI of PAF-treatedspermatozoa.     

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.     

Fertilization and early embryology: Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro

The acrosome reaction was determined in aliquots from ejaculatesof 74 patients undergoing in-vitro fertilization at the Universityof Giessen, Germany, by means of the triple-stain technique.The percentage of acrosome-reacted spermatozoa after low-temperatureinduction of the acrosome reaction was not significantly relatedwith the fertilization rate (H test, P = 0.693, SJ test, P =0.366). However, all patients showing < 13.0% acrosome-reactedspermatozoa had poor fertilization rates. Highly significantdifferences between patients could be detected by correlatingthe inducibility of the acrosome reaction with the fertilizationrate (H test, P = 0.018; SJ test, P = 0.004); patients withhigh fertilization rates showed a corresponding high inducibilityof acrosome reactions. From our results, it is evident thatpercentages of acrosome-reacted spermatozoa < 13.0% or aninducibility of the acrosome reaction of <7.5% are indicativeof subfertility.     

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization

In this study we have investigated responsiveness to progesteronein spermatozoa from a group of unselected male partners of couplesundergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulatedintracellular Ca²⁺ ([Ca²⁺]_i) and percentage increase in acrosomereaction in the same sperm sample used for oocyte inseminations.[Ca²⁺]_i was measured with a fluorimetric method, while the acrosomereaction was assessed using a fluorescent probe (fluoresceinisothiocyanate-labelled peanut lectin). The average percentage[Ca²⁺]_i as well as the rate of increase in the frequency ofacrosome reaction following progesterone challenge were significantlylower (P < 0.005) in the group of patients with a fertilizationrate <50%. In addition, significant correlations betweenthe fertilization rate and the progesterone-stimulated [Ca²⁺]_iand acrosome reaction increases (r = 0.78 and r = 0.79 respectively)were observed. Furthermore, in cases of fertilization failure,no increase of [Ca²⁺]_i or acrosome reaction was observed inresponse to progesterone with the exception of one case. Ourresults indicate that [Ca²⁺]_i and acrosome reaction increasesin response to progesterone can be of value in the predictionof sperm fertilizing ability. As the two parameters were significantlycorrelated to each other (r = 0.86), the two assays have similarIVF predictive value and might be used interchangeably as adiagnostic tool in the assignment of male patients to the differentkinds of assisted fertilization techniques.     

Fertilization and early embryology: Intracytoplasmic sperm injection does not overcome an oocyte defect in previous fertilization failure with conventional in-vitro fertilization and normal spermatozoa

A cohort comprising a total of 447 oocyte aspirations due tomale factors (n±258) or to previous fertilization failureby IVF in the presence of normal sperm parameters (n±189)was studied. We found a significantly reduced implantation andpregnancy rate per transfer in the group with previously failedIVF attempts compared to the male factor group (P <0.001).No differences were found in age, number of oocytes retrieved,number of embryos transferred or quality of embryos scored atthe time of transfer. However, the fertilization and cleavagerates were found to be reduced in the group with previous failedIVF cycles. It is therefore concluded that previous fertilizationfailure despite normal sperm parameters in an 1VF cyde may notbe alleviated by the intracytoplasmlc sperm injec tion (ICSI)procedure. These patients might suffer from oocyte defects aswell.     

Andrology: Osmo-sensitivity of the human sperm acrosome reaction

The osmo-sensitivity of the human sperm acrosome reaction wasinvestigated by determining the effect of hyper- and hypo-osmolalconditions on the ionophore A23187- and dbcAMP-induced reactionof both capacitated and non-capacitated spermatozoa. Hyper-osmolalconditions inhibited the agonist-induced reactions of both typesof spermatozoa. Hypo-osmolal conditions caused a spontaneousloss of acrosomes from capacitated but not from non-capacitatedspermatozoa. The loss of acrosomes under hypo-osmolal conditionswas further enhanced by dbcAMP but not by ionophore A23187.Although significant decreases in sperm viability were occasionallyobserved at the high and low osmolalities, these decreases werenot consistent and could not account for the observed loss ofacrosomes. It is concluded that the human sperm acrosome reactionis osmo-sensitive. The acrosome reaction stimulated by ionophoreA23187 (raises intracellular Ca²⁺) and dbcAMP (activates proteinkinase A which causes protein phosphorylation) appears to involvewater entry downstream from the action of these agonists. Preincubationin albumin (capacitation) causes human spermatozoa to lose theiracrosomes under hypo-osmolal conditions. Finally, capacitationis not an essential prerequisite to the acrosome reaction aslong as agonists are used that by-pass certain membrane-relatedevents.     

Andrology: The effect of sperm preparation methods on the fertilization rate of oocytes micro-inseminated by subzonal sperm injection

Three methods were used to prepare spermatozoa for subzonalinjection into mature oocytes. In method A, the washed spermsuspension was incubated for 18 h in modified T6 culture medium.Method B consisted of incubating the sperm suspension for 6h in regular T6 culture medium. In method C, the sperm suspensionwas incubated for 6 h in regular T6 culture medium containing20% (v/v) follicular fluid. The percentages of acrosome-freespermatozoa and fertilization rates were compared for 42 treatmentcycles assigned randomly to the three sperm preparation methods.The sperm suspensions prepared by methods A and C each had significantlyhigher proportions of acrosome-free spermatozoa compared tosuspensions prepared by method B. The fertilization rates ofoocytes micro-injected with spermatozoa prepared by methodsA and C were significantly higher than for method B. Eight clinicalpregnancies resulted from 28 cycles in which embryo replacementoccurred. We conclude that the fertilization rate followingsubzonal sperm injection is related directly to the percentageof acrosome-free spermatozoa in the sperm suspension used formicro-injection     

Artificial induction of the acrosome reaction in human spermatozoa

This study investigated the use of human follicular fluid andpentoxifylline as inducers of the human sperm acrosome reactionin vitro. Motile sperm suspensions were prepared using a discontinuousPercoll gradient, preincubated for 3 h, divided into aliquotsand exposed to various concentrations of non-heart-inactivatedfollicular fluid for 1 and 24 h and pentoxifylline for 30 min.Detection of the acrosome reaction involved the combined useof a fluorescent vital stain, H33258 [GenBank] , and fluorescein isothiocyanate-conjugatedpeanut agglutinin (FITC-PNA). A short (1 h) exposure to follicularfluid at concentrations of 50% or more, did not compromise spermmotility and significantly increased the proportion of spermatozoahaving completed the acrosome reaction. Similarly, a 30 minexposure to pentoxifylline also significantly increased theproportion of spermatozoa having completed the acrosome reaction.     

The acrosome reaction in human sperm from men of proven fertility

Suspensions of motile sperm were prepared from semen samplesdonated by 40 men of recent, proven fertility and incubatedunder capacitating conditions for 24 h. At selected time-pointsaliquots were removed, assessed for motility, fixed and examinedwith the electron microscope to determine the rate of acrosomeloss. Data indicate that acrosome loss increases significantlywith time, but absolute values are relatively low. After 24h a mean of 15.4% sperm had initiated the acrosome reaction;this figure included 9.7% which had completed it. The proportionof cells at intermediate stages was similar throughout incubation({small tilde}5%), indicating that initiation of the acrosomereaction occurs at a fairly constant rate. In four samples motilitydeclined over 24 h and in six, contaminating cells were observed.In the majority of these 10, acrosome loss was higher than thatobserved in the remaining 30 samples. Additionally, the assessmentof <25 000 cells during this study made it possible to evaluatespecific ultrastructural features of the normal acrosome reactionin human sperm. Six stages were identified, with the intermediateones involving loss of acrosomal matrix material while outermembranes appear to retain their integrity; this contrasts sharplywith the current view of the generalized mammalian sperm acrosomereaction.     

Clinical importance of zona pellucida-induced acrosome reaction and its predictive value for IVF

The study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. ZIAR tests were performed using 0.25 zona pellucida/microliter co-incubated with spermatozoa for 60 min. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between stimulated and unstimulated (spontaneous) sperm populations. Results were compared with IVF rates of metaphase II oocytes. Interactive dot diagrams divided the patients into two groups, i.e. ZIAR <15% and ZIAR > 15%, with mean fertilization rates of 49 and 79% respectively. The sensitivity and specificity for ZIAR results versus fertilization were 93 and 100% respectively. The area under the curve was 99% and the 95% confidence interval did not include 0.5 which implies that the ZIAR test is able to predict fertilization failure among IVF patients. In conclusion, the ZIAR test has diagnostic potential since it can assist the clinician to identify couples that will benefit from intracytoplasmic sperm injection therapy.     

Andrology: Pentoxifylline-enhanced acrosome reaction correlates with fertilization in vitro

To evaluate the possible effect of pentoxifylline on the acrosomereaction (AR) and its correlation with in-vitro fertilization(IVF), sperm samples obtained from 51 patients who underwentIVF treatment were studied. Acrosome reactions were evaluatedas spontaneous, pentoxifylline-treated and calcium ionophore(A23187) induced, before and after treatment. The correlationof AR with fertilization in vitro in spermatozoa pre-treatedwith pentoxifylline was sought. In cases with failure or verylow fertilization rate (10%) in their previous trials, spermatozoaafter swim-up were treated before insemination. Spontaneousacrosome loss remained low even after treatment (mean ±SD: 8.18 ± 1.74%). Response to A23187 was enhanced significantly(P < 0.001) by pre-treatment with pentoxifylline in 33 controlcases (group A) in which fertilization in vitro was previouslysuccessful without this treatment. Patients with at least twoepisodes of failed fertilization were divided into two groups.In 11 cases (group B), the IVF rate was improved significantly(P < 0.001) by the treatment. This was not observed in sevencases (group C) in which the treatment induced no increase inIVF rate. We achieved nine (27.3%) pregnancies in group A andfive (45.4%) pregnancies in group B. This study demonstratedthat pentoxifylline enhanced A23187 induced the acrosome reactionand this effect was correlated with improvement in IVF rate.     

Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol

This study reports the subsequent embryo development of cryopreservedmature human oocytes following insemination or intracytoplasmicsperm injection (ICSI). Metaphase II oocytes were cryopreservedusing a slow freezingrapid thawing procedure employing the cryoprotectant1,2- propanediol. The study was conducted at two centres. Thenormal insemination of cryopreserved oocytes was undertakenin one centre, and ICSI of cryopreserved oocytes in the other.Both methods resulted in a 50% normal fertilization rate. Alow rate of abnormal fertilization was observed in the inseminatedgroup of oocytes (5%) compared with 21% for the ICSI oocytes;this was not significantly different. Embryo development wasassessed daily for 7 days. All normal fertilized cryopreservedoocytes in both groups cleaved on day 2, with a similar appearanceto in-vitro fertilization and ICSI embryos. In the normal inseminatedoocytes, there was a significant decrease in the number of embryoscleaving on day 3 (33%) compared with the development of ICSIoocytes, with a subsequent gradual reduction over days 4 and5 (22 and 11% respectively) resulting in one early blastocyston day 7 (11%). In contrast, all ICSI-generated embryos continuedto cleave on day 3, with a gradual reduction over subsequentdays (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day7, two of the blastocysts had started to hatch, resulting ina 66% hatching rate of blastocysts formed from ICSI of cryopreservedoocytes. This is the first study to show normal developmentto the hatching blastocyst stage following ICSI of cryopreservedhuman oocytes.     

Intracytoplasmic sperm injection in the mouse

Intracytoplasmic sperm injection (ICSI) into mouse oocytes involvesa very low survival rate. This study was designed to determinewhy ICSI frequently fails in mice. Metaphase II oocytes wereobtained from superovulated 4–6 week old F₁ hybrid mice.Spermatozoa were retrieved from the epididymis of 12–14week old F₁ hybrid mice. The spiked microinjection pipette usedto inject a spermatozoon into the ooplasm had outer and innerdiameters of 10 and 8 µm respectively. The oocytes usedin the first part of the study were not activated (group 1).Some oocytes were incubated with calcium ionophore for 5 min(group 2). The injected oocytes were evaluated 6, 20, 48 and72 h after injection. A total of 143 eggs in each group underwentICSI. In group 1, sperm heads escaped into the perivitellinespace. In all, 63 (47%) of the remaining oocytes were damagedduring the injection or had degenerated by the first evaluation.The survival rate was 53%, but fertilization did not occur.In group 2, 31 oocytes (22%) were damaged during microinjectionor soon degenerated. Two oocytes underwent accidental subzonalinsemination. Six oocytes were fertilized (4.2%) among the 78%of survivors. After injection, the sperm tail was found in thecytoplasm (27 and 31% in groups 1 and 2 respectively), the perivitellinespace (45% in both groups) or protruding through the zona pellucida(28 and 23% respectively). More oocytes degenerated when thetail remained in the cytoplasm, i.e. 78% in group 1 and 36%in group 2.     

Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

Human sperm capacitation and the acrosome reaction.

A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phospholipid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion.     

Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay

The effect of anti-sperm antibodies (ASA) on the rate of acrosome reactions (AR) during "in vitro" capacitation of human sperm used for the hamster egg penetration assay (HEPA) was assessed. Motile sperm suspensions from donors were exposed to several sera and seminal plasma with sperm head-directed ASA, then they were washed and capacitated "in vitro." After capacitation, the proportion of acrosome-reacted viable sperm was assessed by staining with Fluoresceinated Pisum Sativum Agglutinin and supravital stain Hoechst 33258. ASA of any immunoglobulin class did not significantly affect either the AR rate, or the hamster egg penetration rate. In conclusion, interference of ASA on spontaneous AR rate during "in vitro" capacitation can not be advocated as an explanation of the impairment of the interaction of human sperm with egg or its vestments, which have been reported in several studies.     

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (5 donors x 3 replicates) using Percoll gradients at 30–60 min post-ejaculation and preincubated in a complex ‘syntheitictubal fluid’ culture medium (STF) at 37° C under 5%CO₂ in air for 6h. Aliquots of these suspensions were then incubatedfor a further 2 h in STF containing 0, 5, 25, 50, 75 and 100%(v/v) pooled human follicular fluid (FF). Another aliquot wastreated with 10 µm A23187 in STF for 20min and then incubatedin fresh STF medium for a futher 2h to induce maximal acrosomeloss. Acrosome reactions were assessed using both the triple-staintechnique and fluorescent peanut agglutinin lectin-labelling.Sperm motility and movement characteristics were assessed fromvideorecordings using digital image analysis (CellSoft). Exposureto FF caused only relatively small proportions of the preincubatedspermatozoa to undergo acrosome reactions. The size of theseresponsive sub-populations was smaller than that capable ofresponding to a Ca²⁺ influx generated by A23187. Increased FFconcentrations induced a progressive loss of motility and trendsfor changes in movement characteristics that may have been relatedto reduced intracellular Ca²⁺. This interpretation of theseobervations is that while FF may act to stimulate or promotethe human sperm acrosome reaction it does not appear to be aspecific inducer of it. Consequently, a precise role for FFat the relatively low concentrations that would be expectedto be present in the tubal ampulla in the physiological regulationof human fertilization remains unproven