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.     

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.     

In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry

The process of sperm chromatin decondensation occurs when a spermatozoon enters an ovum. Protamine disulphide bonds are reduced to SH and the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA which then combines with histones. Defective chromatin decondensation will thus prevent further development of the male pronucleus. In this study human sperm samples were incubated in vitro at 28 degrees C (using a medium in which the polyanion, heparin, substitutes for nucleoplasmin and beta- mercaptoethanol for egg glutathione) for 10, 20 and 30 min before stopping the reaction with formalin (to 3.6%). The DNA of the fixed cells was stained with Acridine Orange by a one-step method and subjected to flow cytometry and data analysis, in which a zone characteristic of condensed chromatin is outlined on red-green fluorescence contour plots. After 20 min of incubation 97% of the control spermatozoa that were in the mature window (WIN M) had decondensed and moved out of this region. Defects in sperm decondensation were seen in four semen samples of the 20 that were tested. In cases where spermatozoa fail to produce a fertilized egg the cause may lie with defective chromatin quality, including failure of the sperm chromatin to decondense. The method described here is a simple procedure for detecting sperm samples containing such defective cells.      

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.     

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     

Pentoxifylline potentiates ionophore (A23187) mediated acrosome reaction in human sperm: flow cytometric analysis using CD46 antibody

The study was designed to investigate the effects of pentoxifyllineon the acrosome reaction of human spermatozoa in vitro, andto determine whether the reaction is differently modulated aftersperm selection by multiple tube swim-up and Percoll buoyantdensity centrifugation. The acrosome reaction was induced invitro by using calcium ionophore (A23187) and was detected bymeasuring the fluorescence of FITC-conjugated goat anti-mouseimmunoglobulin bound to CD46 antibody (which binds to the CD46receptor site on the inner acrosomal membrane) by flow cytometry.Spermatozoa separated on Percoll displayed significantly lowerspontaneous acrosome reactions (P= 0.002) than did those separatedby the swim-up technique. Pentoxifylline did not, by itself,induse acrosome reaction, but after induction with ionophore,it significantly increased the reaction (P= 0.0003) and thisincrease was seen to be greater when Percoll separation wasused as compared to the swim-up technique (P= 0.0002). We thereforeconclude that percoll selection of motile spermatozoa togetherwith pentoxifylline treatment may be of value in assisted reproductivetechniques, as an increased ARIC score arouse after both treatments,and that flow cytometry allows a precise and rapid quantificationof the acrosome reaction.     

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.      

The incidence of spontaneous acrosome reaction in homogeneous populations of hyperactivated human spermatozoa.

The incidence of spontaneous acrosome reaction occurring in 1314 individually selected hyperactivated (HA) human spermatozoa was compared to that occurring in 8226 individually selected non-hyperactivated spermatozoa (non-HA) sampled over an incubation time course to allow for capacitation. Two-way analysis of variance showed a significant difference between HA and non-HA spermatozoa for the mean percent acrosome reacted (R), partially acrosome reacted (PR) and combined total (R+PR) (P < 0.001). One-way analysis showed that among the HA spermatozoa there were marked differences among the proportion showing R+PR at the various time points (P = 0.005). Using the same end point, there was no significant evidence of change with time for the non-HA spermatozoa. The overall data indicated that HA human spermatozoa have a greater propensity for spontaneous acrosomal loss than non-HA spermatozoa during incubation in synthetic culture media.     

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

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

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

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

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca(2+)](i), plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca(2+)](i) before and after freezing-thawing showed a significant reduction of basal [Ca(2+)](i) in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca(2+)](i) both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca(2+)](i) and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca(2+)](i) rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.     

Andrology: Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry

Inasmuch as caput epididymal and even testicular spermatozoaare now being used to generate pregnancies by direct injectioninto the oocyte, differences in the chromatin of spermatozoafrom proximal and distal locations in the epidldymis were studied.Acridine Orange staining was used to investigate chromatin structurein human spermatozoa which had left the testis and were undergoingmaturation in the epididymis. Measurement of green and red fluorescenceIntensities of human spermatozoa by flow cytometry demonstrateda decrease in binding of Acridine Orange to DNA as the spermatozoatraversed the epididymis. Using spermatozoa from the cauda epididymisas the standard, the percentages of spermatozoa from the efferentduct, proximal corpus epididymis, midcorpus epididymis, distalcorpus epididymis, proximal cauda epidldymis and distal caudaepididymis that had matured with regard to chromatin condensationwere 28 ± 5, 39 ± 3, 49 ± 5, 64 ±5, 69 ± 6 and 74 ± 4% respectively. It may beconcluded that eggs fertilized by ejaculated spermatozoa receivea more highly condensed form of chromatin than that receivedby eggs Inseminated with proximal epididymal or testicular spermatozoa.     

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     

Enhancement of motility and acrosome reaction in human spermatozoa: differential activation by type-specific phosphodiesterase inhibitors

Inhibition of sperm phosphodiesterase (PDE) has been shown to increase cAMP concentrations and stimulate motility and the acrosome reaction. While several PDE genes exist in mammals, little is known about the physiological role of PDE forms expressed in human spermatozoa. Using type-selective inhibitors, we identified two of the PDE forms expressed in human spermatozoa and studied their involvement in sperm function. Selective inhibitors of calcium-calmodulin-regulated PDE1 (8-methoxy- isobutyl-methylxanthine) and cAMP-specific PDE4 (RS-25344, Rolipram) were used to study PDE forms in human sperm extracts. 8-MeIBMX and Rolipram/RS-25344 inhibited sperm PDE activity by 35-40 and 25-30% respectively. Subcellular fractionation of the sperm homogenate suggests these pharmacologically distinct forms may be located in separate cellular regions. To evaluate the functional significance of different PDE forms, the effect of type-specific PDE inhibition on sperm motility and the acrosome reaction was examined. PDE4 inhibitors enhanced sperm motility over controls without affecting the acrosome reaction, while PDE1 inhibitors selectively stimulated the acrosome reaction. These data indicate at least two distinct PDE types exist in human spermatozoa. Our findings also support the hypothesis that PDE subtypes affect sperm function by regulating separate pools of cAMP and may ultimately offer novel treatments to infertile couples with abnormal semen parameters.      

Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate

Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post- thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline- treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in-vitro fertilization.      

Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization

Capacitation is defined as the series of transformations that spermatozoa normally undergo during their migration through the female genital tract, in order to reach and bind to the zona pellucida, undergo the acrosome reaction, and fertilize the egg. During this process, extensive changes occur in all sperm compartments (head and flagellum; membrane, cytosol, cytoskeleton), factors originating from epididymal fluid and seminal plasma are lost or redistributed and membrane lipids and proteins are reorganized; ion fluxes induce biochemical modifications and controlled amounts of reactive oxygen species are generated; spermatozoa develop hyperactivated motility; and complex signal transduction mechanisms are initiated. The main purpose of capacitation is to ensure that spermatozoa reach the eggs at the appropriate time and in the appropriate state to fertilize these eggs, by finely-controlling the rate of the changes necessary to prime spermatozoa and by activating all the mechanisms needed for the subsequent acrosome reaction. The reversibility of some of the mechanisms leading to sperm capacitation may therefore be a very important aspect of the fine regulation and perfect timing of this process.      

Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm

BACKGROUND: The aim of this study was to determine the relationship between human sperm hyperactivation (HA), sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction (AR) of ZP-bound sperm in vitro. METHODS: Sperm samples from 129 infertile men were studied. Motile sperm (2 x 10(6)) selected by Pure sperm were incubated with four oocytes in 1 ml human tubal fluid supplemented with 10% human serum. After 2-h incubation, the number of sperm bound to the ZP and the AR of ZP-bound sperm were examined. Velocities and HA of sperm in insemination medium were assessed by Hamilton-Thorn Sperm Analyzer. RESULTS: The HA was highly correlated with the ZP-induced AR in all the subjects (rho = 0.626, P < 0.001). In the 69 men with < or = 100 sperm bound/ZP, allowing accurate counts, HA was not significantly correlated with sperm-ZP binding. Men with <7% HA sperm were more likely to have very low ZP-induced AR. Only normal sperm morphology was significantly correlated with sperm-ZP binding (rho = 0.346 and 0.446 in semen and insemination medium, respectively, both P < 0.001). Sperm motility and velocities were significantly correlated with sperm morphology but not with either sperm-ZP binding or the ZP-induced AR. CONCLUSIONS: The correlation of HA with the ZP-induced AR of ZP-bound sperm suggests a mechanistic link between HA and the physiological AR in humans. Assessment of HA of capacitated sperm in vitro may be a useful clinical test for male infertility associated with defective ZP-induced AR that does not require the use of human oocytes.