Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa.

Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red). The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.     

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics

Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender with 4% glycerol. Spermatozoal quality was assessed at four different points: at less than 15 min after collecting and before processing (Step 1); after centrifugation and just before freezing (Step 2); immediately after thawing less than 3 h after freezing (Step 3); and immediately after thawing 10 to 20 d after freezing (Step 4). Acrosome-specific monoclonal antibody detected differences (P <0.05) among steps and ejaculates within stallions. All parameters of spermatozoal motion, including the percentage of motile sperm, percentage of progressively motile sperm, curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and radius of the average path for circularly swimming sperm, differed (P <0.05) among steps, and most of these parameters differed among ejaculates within a stallion and among stallions. For Steps 2 and 3, 62 and 37% of the sperm were motile, and 56 and 23% of all motile sperm had a curvilinear velocity of >100 mum/sec. Most damage to sperm occurred as a result of freezing-thawing, whereas centrifugation of sperm caused only minor damage.     

Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa.

The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.     

Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.     

Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose Cl-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1% Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine     

A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion sperm and is associated with spermatozoal function.

Sperm hyaluronidase has been implicated in sperm penetration of the extracellular matrix of the cumulus oophorus and may play a crucial role in gamete interaction and fertility in mammals. The objectives of this study were to characterize the enzyme activity of equine sperm hyaluronidase and to investigate its cellular distribution. Zymography of stallion sperm plasma membrane extracts was used to identify hyaluronidase activity in protein bands. Affinity-purified polyclonal IgG raised against equine sperm hyaluronidase was used to label fresh and capacitated stallion sperm, followed by indirect immunofluorescence. Equine sperm plasma membrane extracts displayed 3 major protein bands with potent hyaluronidase activity of approximately 54, 59, and 83 kDa. Under reducing conditions, a single protein band was observed at 62 kDa, although the reduced sample exhibited no enzyme activity. The polyclonal IgG labeled the postacrosomal region of stallion sperm and was redistributed over the acrosomal region during in vitro capacitation in a significant percentage of sperm cells. These studies suggest that a specific protein localized to the equine sperm head displays hyaluronidase activity, gets redistributed over the acrosomal region during capacitation, and may be important in fertility in this species.     

Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose C1-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1% Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine or be used as a tool in pest/rodent management.     

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.     

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody A-1

We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A-1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Changes in lectin binding to bovine sperm during heparin-induced capacitation

Changes in the plasma membrane of bovine sperm during heparin-induced capacitation were detected by the binding of fluorescent labeled lectins to unfixed sperm. Of the seven lectins evaluated, only binding of wheat germ agglutinin (WGA) changed with capacitation. Sperm were classified into one of 5 patterns (p1–p5) based on staining with WGA, presence or absence of propidium iodide (PI) staining (dead or alive), and acrosomal integrity (acrosome intact or reacted). The major changes associated with capacitation occurred in p1 and p2. Sperm in p1 exhibited diffuse WGA binding over the anterior sperm head, were alive, and had intact acrosomes. In p2, sperm were also acrosome intact and alive, but lacked WGA binding. When sperm were incubated under capacitating conditions with heparin, there was a decrease over time in the percentage of sperm classified in p1 (p < 0.05) and an increase in the percentage of sperm in p2 (p < 0.05). When capacitation by heparin was delayed by the inclusion of glucose in the culture medium, the same heparin-dependent changes in the percentage of sperm in p1 and p2 were delayed (p < 0.05). When capacitation by heparin was inhibited by including protamine in the culture medium, the percentage of sperm in p1 or p2 was not different from sperm incubated without heparin. Heparin-induced capacitation was associated with a loss of WGA binding to the bovine sperm head. © 1996 Wiley-Liss, Inc.     

Assessment of boar sperm viability using a combination of two fluorophores

A combination of the fluorophore probes, calcein acetylmethyl ester (CAM) and ethidium homodimer (EH), were used to assess viability of ejaculated boar spermatozoa. Both CAM and EH have been used as indicators of biosynthetic activity and membrane integrity in monolayer cell cultures, with CAM shown to permeate and undergo enzymatic cleavage in viable monolayer cells giving the cell a green fluorescence, and EH penetrating only membrane damaged cells giving cells a red fluorescence. To determine if these fluorophores can be used to assess boar sperm viability, ejaculates from 10 boars were divided into 3 test groups (cytotoxic-treated, swim-up and washed), utilizing a split-ejaculate technique; each group consisted of both a probe-treated and control sample. Sample viability was ascertained in the control groups by visual estimation of the percentage motile spermatozoa, whereas the number of spermatozoa showing green (CAM = viable) or red (EH = non-viable) fluorescence were quantitated for each of the probe-treated groups using a fluorsecein or rhodamine filter, respectively. All spermatozoa exposed to the combined probes had an uptake of one or both fluorophores. The cytotoxic-treated group exhibited 0% gross motility, with 100% of the sperm heads showing red fluorescence. In the swim-up group, no difference was detected (P > 0.05) between control gross motility and the percentage of completely green fluorescing spermatozoa (85% vs. 86.6%, respectively). In the washed group, a significant difference (P = 0.039) was detected between gross motility estimates and the percentage of calcein-green fluorescent spermatozoa (57% vs. 60%, respectively). This study demonstrated that 1) CAM fluoresces only viable sperm, giving off a green fluorescence, 2) EH fluoresces in only non-viable sperm, giving off a red fluorescence, 3) visual estimation of motile sperm can approximate a semen sample's viability, but is not as precise as fluorophore determination, and 4) sperm incubation with the fluorophore combination CAM and EH provided an accurate technique for the objective assessment of boar sperm viability via their distinct fluorescent patterns in boar sperm.     

A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis

Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (P相似文献   

Behavior of bull spermatozoa in bovine uterine tube epithelial cell co-culture: an in vitro model for studying the cell interactions of reproduction

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.     

Antibody-induced changes on rabbit sperm surface inhibit gamete interaction

Interaction of specific ligands with cell surface molecules may induce reorganization of surface components. A monoclonal antibody (B-12) against sperm surface antigens of 40kDa size induced molecules on the plasma membrane overlying the acrosome of rabbit sperm to cluster in small aggregates at 0°C (patching). At an elevated temperature of 37°C these clusters of antigen antibody complexes collected into a large aggregate on one pole of the cell forming a cap (capping). This cap-like structure showed a reduction in size over a period of time and eventually disappeared from the sperm surface. Inhibition of capping by sodium azide indicated that it is an energy-dependent process. Patching of antigens did not require energy. Involvement of sperm head cytoskeleton in the process of capping was evident from potentiation of cap formation by cytoskeleton disrupting agents like cytochalasin B and D. Patching of antigen antibody complexes was not affected by either of the agents. The loss of antigen antibody complexes from sperm surface was mainly due to shedding of the complexes in the surrounding media. Sperm with patches of antigen antibody complexes did not adhere to oocytes. Sperm from the group where a majority of the sperm were denuded of the antigen antibody complexes also did not bind to oocytes. © 1994 Wiley-Liss, Inc.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider,Aquarius remigis

Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi‐aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of flavin adenine dinucleotide (FAD), which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization. J. Cell. Physiol. 226: 999–1006, 2011. © 2010 Wiley‐Liss, Inc.     

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.     

Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation

We previously targeted EGFP (a mutant of green fluorescent protein) to the lumen of the mouse sperm acrosome and reported the time course of EGFP release during the acrosome reaction. In the study reported here, we estimated the pH within the mouse sperm acrosome utilizing the pH-dependent nature of EGFP fluorescence. The average intra-acrosomal pH was estimated to be 5.3 +/- 0.1 immediately after sperm preparation, gradually increasing to 6.2 +/- 0.3 during 120 min of incubation in TYH media suitable for capacitation. Spontaneous acrosome reactions were noted to increase concomitantly with acrosomal alkalinization during incubation. We also demonstrated that acrosomal antigens detected by monoclonal antibodies MN7 and MC41 did not dissolve following the acrosome reaction in pH 5.3 media, but dissolved at pH 6.2. These data suggest that acrosomal alkalinization during incubation conducive for sperm capacitation may function to alter acrosomal contents and prepare them for release during the acrosome reaction.     

Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida

Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor‐mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model. J. Cell. Physiol. 220: 611–620, 2009. © 2009 Wiley‐Liss, Inc.