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At least 19 recordsLinked to original sources
PubMed1989

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida.

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosome↗
PubMed1991

A mechanism for differential release of acrosomal enzymes during the acrosome reaction.

To study the organization of fertilization enzymes in the sperm acrosome, we isolated and characterized two physicochemically distinct acrosomal fractions of guinea-pig spermatozoa. A soluble fraction contained the 25,000-Mr acrosomal autoantigen, AA1, and most of the acrosomal hyaluronidase and dipeptidyl peptidase II activity. A particulate fraction, designated acrosomal matrix (AM), consisted of membraneless crescent-shaped structures, and contained most of the acrosomal proacrosin. The AM also contained a 28,000-Mr putative proacrosin-binding protein, and a very-high-Mr component which, on reduction, was dissociated into 48,000-Mr and 67,000-Mr subunits. Autoproteolytic dissolution of the AM correlated with proteolysis by acrosin of the 28,000-Mr and 48,000-Mr AM molecules. Components of both the AM and the soluble fraction were localized by immuno-electron microscopy to the electron-dense region of the guinea-pig sperm acrosome. We conclude that acrosomal molecules are segregated into soluble and matrix compartments. This segregation is a function of disulphide bonding and non-covalent interactions among the relatively few components of the AM. Association of acrosin with the AM may be the mechanism by which this enzyme's release from the spermatozoon during the acrosome reaction is delayed relative to the release of other acrosomal molecules.

Acrosin↗
PubMed1989

Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: population and single cell kinetics.

The acrosome reaction induced by the mouse egg's zona pellucida in mouse sperm has been shown to proceed in two stages as characterized empirically by sequential changes in patterns of chlortetracycline fluorescence on the sperm plasma membrane surfaces. The chlortetracycline fluorescence pattern characteristic of fully intact sperm is designated B; in sperm bound to structurally intact zonae that induce the acrosome reaction, the B pattern changes first to an intermediate pattern S and then to a terminal pattern AR characteristic of the completed acrosome reaction. In the same study, it was shown, using a 9-amino acridine fluorescent pH probe, that completion of the first stage was characterized by increase in H+ permeability such that the H+ gradient between sperm head and medium was dissipated. In this study, we show that the fluorescent pH probe 9-N-dodecylamino acridine and the intracellular Ca2+ fluorescent probe fura-2 are both localized to the anterior part of the sperm head encompassing the acrosomal compartment in intact sperm, and the fluorescence associated with each probe is lost as the first stage of the acrosome reaction is completed. Loss of the pH probe fluorescence, pattern N, corresponds to onset of H+ permeability, and loss of fura-2 fluorescence, pattern F, corresponds to onset of Ca2+ permeability. Localization of intracellular fura-2 fluorescence to the acrosomal compartment required extracellular Mn2+ to quench surface-bound fura-2 AM, the tetra-acetoxymethyl ester of fura-2 used to load the cells. Loss of acrosomal fura-2 fluorescence is due to quenching by tracer Mn2+ accompanying Ca2+. Onset of membrane permeability to both H+ and Ca2+, as seen by loss of patterns N and F, occurred in synchrony in populations of sperm bound to isolated, structurally intact zonae, with an overall time course of 210 min postbinding. The loss of pattern N in individual sperm cells bound to zonae was rapid, with a half time of 2.1 min. Concomitant with this rapid loss of pattern N was a shift in the amplitude of flagellar motion from large to small. The lag times to pattern N loss in 50 individual cells ranged from 30 to 140 min. The variable lag times determine the population kinetics; the rate of the endpoint reaction seen in the individual cells is rapid and constant. Dissipation of the H+ gradient with immediate loss of pattern N was readily achieved by addition of nigericin with no change in the time course of the onset of Ca2+ permeability of the membranes enclosing the acrosome.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1992

Interactions of human sperm acrosomal protein SP-10 with the acrosomal membranes.

The interaction of the human acrosomal protein SP-10 with the acrosomal membranes was analyzed by the ability of Triton X-114 (TX-114) and other agents to release SP-10 from the acrosome. Treatment of human sperm with TX-114 revealed a pool of SP-10 that was released by TX-114 and a pool of SP-10 that was TX-114-resistant. TX-114-resistant SP-10 was associated with the equatorial segment and with TX-114-resistant portions of the acrosomal matrix and the inner acrosomal membrane. Phase partitioning of TX-114-released and TX-114-resistant SP-10 pools showed that both were hydrophilic, indicating that these pools consist of proteins that are peripherally associated with, rather than integral to, the acrosomal membranes. Sequential treatments of human sperm with various agents showed that repeated washes with TX-114 or 1.5 M NaCl had little or no effect on TX-114-resistant SP-10, whereas treatment with a chaotropic salt (150 mM sodium thiocyanate) and buffers at pH extremes (pH 2.0 and 10.0) completely released this pool of SP-10 from the acrosome. Together the results suggest that SP-10 is a hydrophilic peripheral acrosomal membrane protein that may be associated with a TX-114-resistant "anchor."

Acrosome↗
PubMed1989

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction.

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1990

Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis.

The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.

Acrosome↗
PubMed1992

A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida-induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3-quinuclidinyl benzilate.

The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3-quinuclidinyl benzilate on the zona-induced acrosome reaction in mouse sperm.

Acrosome↗
PubMed1991

Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: characterization of an intermediate stage prior to the completion of the acrosome reaction.

The zona pellucida (ZP)-induced acrosome reaction in mouse sperm proceeds in two steps, identified by three sperm fluorescence patterns observed sequentially with the fluorescent probe chlortetracycline. Capacitated, acrosome-intact sperm displaying a B pattern proceed to an intermediate S pattern, and then progress from the S pattern to the fully acrosome-reacted AR pattern. Previously, it was not feasible to characterize the nature of the transient intermediate S pattern. Recently, it was demonstrated that sperm bind to the ZP of eggs treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and undergo a B to S transition, but do not complete the acrosome reaction. These cells accumulate in the S pattern and fail to undergo the S to AR transition (Endo, Y., Schultz, R. M., and Kopf, G. S. 1987a. Dev. Biol. 119, 119-209). The present study utilized ZP from TPA-treated eggs to assess the state of S pattern sperm. The kinetics of the B to S transition of sperm incubated with either structurally intact or solubilized ZP from untreated or TPA-treated eggs are identical. Addition of either solubilized ZP from untreated eggs or A-23187 to S pattern sperm bound to intact or solubilized ZP from TPA-treated eggs induces the S to AR transition, while ZP from TPA-treated or fertilized eggs does not. Loss of the transmembrane pH gradient in the anterior portion of the sperm head, monitored by the fluorescent pH probe 9-N-dodecyl aminoacridine, follows the B to S transition in sperm incubated with ZP from unfertilized eggs, but no loss is observed when the B to S transition is induced using ZP from TPA-treated eggs. Subsequent addition of solubilized ZP from untreated eggs or A-23187 results in the loss of the transmembrane pH gradient of these S pattern sperm. Addition of nigericin to S pattern sperm bound to ZP from TPA-treated eggs discharges the transmembrane pH gradient and causes the S to AR transition. In contrast, nigericin added to B pattern sperm discharges the pH gradient but does not induce a B to S transition. Electron microscopic evaluation of S pattern-arrested sperm using ZP from TPA-treated eggs reveals intact plasma and outer acrosomal membranes. These results suggest that ZP from TPA-treated and fertilized eggs are modified such that the ZP ligands inducing the S to AR transition are lost or are inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosome↗
PubMed1975

The organized distribution of acrosomal proteinase within the acrosomes of rabbit spermatozoa.

Acrosomal proteinase was found to be present in a highly organized distribution in the acrosomes of rabbit spermatozoa, using cytochemistry. This distribution consists of at least six linear loops of evenly spaced proteinase granules, which run diagonally across the flat side of the sperm head in a criss-crossing pattern. Two additional loops may be present, one encircling the tip of the spermatozoon in the region of the acrosome reaction, and the other at the posterior end of the acrosome. The results also seem to indicate that the enzyme granules are associated with the outer, and not the inner, acrosomal membrane.

Acrosin↗
PubMed1991

Evaluation of the acrosome reaction using monoclonal antibodies against different acrosomal antigens--comparison with the triple stain technique.

The acrosome reaction of human sperm was evaluated in vitro with the aid of there monoclonal antibodies TüS-1, TüS-19 and TüS-20, which react with antigens in the anterior part of the acrosome. Data were compared with results obtained using the triple stain technique. Seminal smears were performed prior to, and following incubation for 5 and 24 h in Hams-F10 containing 3% human serum albumin. Using the triple stain technique, 15.2 +/- 7.1% of sperm exhibited acrosome activation after 5 h and 16.8 +/- 8.4% after incubation for 24 h. The corresponding values obtained using antibody TüS-1 were 12.9 +/- 5.8 and 13.2 +/- 2.2%, with TüS-19 10.1 +/- 3.8 and 10.8 +/- 1.4% and with TüS-20, 9.0 +/- 3.4 and 10.4 +/- 2.9%. When the same sperm smears were stained firstly with rose Bengal, and secondly with TüS-1 both methods stained the same cells and the same region of the cells, thus indicating that both methods stain the same substrate. Staining of the acrosome with monoclonal antibodies gives clearer results than the triple stain technique, and could be used in preference in future studies dealing with diagnosis of the acrosome reaction in vitro.

Acrosome↗
PubMed1991

Stability of the acrosome of the brush-tailed possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) in vitro and after exposure to conditions and agents known to cause capacitation or acrosome reaction of eutherian spermatozoa.

Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33 degrees C, or room temperature when long-term sperm survival (greater than 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.

Acrosome↗
PubMed1975

An organized distribution of acrosomal proteinase in rabbit sperm acrosomes.

Acrosomal proteinase was found to be present in a highly organized distribution within the acrosomes of rabbit spermatozoa by cytochemistry. This distribution consists of at least six linear loops of evenly spaced proteinase granules, which run diagonally across the flat side of the sperm head in a crisscrossing pattern with the two most anterior loops surrounding the region of the acrosome reaction. This is the first description of an organized distribution of enzymes within a lysosome-like organelle.

Acrosome↗
PubMed1979

Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa.

A variety of treatment procedures was utilized to identify the origin and composition of the vesicles formed during the acrosome reaction of boar spermatozoa. Whether the acrosome reaction occurred spontaneously or was induced chemically the vesicles were hybrid vesicles composed of roughly equal proportions of plasma and outer acrosomal membranes.

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PubMed1992

The ionophore-induced acrosome reaction differs structurally from the spontaneous acrosome reaction.

The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-induced vesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation.

Acrosome↗
PubMedDate not supplied

Monoclonal antibodies against boar acrosomal antigens labelling undamaged acrosomes of spermatozoa in immunofluorescence test.

Monoclonal antibodies specific for acrosomal proteins were prepared and characterized. Two of these monoclonal antibodies, ACR.3 and ACR.11, reacted with the same molecular specificity of acrosomal antigens (18-20 kDa), antibody ACR.4 reacted with the 25-27 kDa antigens, and antibody ACR.2 reacted with several forms of boar acrosin (55, 53, 45, and 38 kDa). All monoclonal antibodies labelled the acrosomes of undamaged spermatozoa in immunofluorescence test. This test should be convenient as an immunological test of the sperm quality.

Acrosin↗
PubMed1992

Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin.

When western blots of human sperm proteins solubilized by acid extraction (presumably mainly acrosomal proteins) or by sodium dodecyl sulfate (SDS) were probed with biotin-conjugated Pisum sativum agglutinin (PSA), distinct sets of proteins were labelled in both preparations. When smears of human spermatozoa were treated with methanol either for 30 s or for 15 min and then exposed to FITC-conjugated PSA, the resulting fluorescence pattern essentially depended on the time of methanol treatment. With the longer treatment, fewer spermatozoa showed selective acrosomal labelling and more were labelled uniformly throughout, without a clear predilection for a single sperm region. With the shorter time of methanol treatment, the poorly topographically differentiated, whole-cell labelling was typical of dead spermatozoa as confirmed by a close correlation between the percentages of spermatozoa showing this type of labelling and of those stained supravitally with Hoechst 33258. The preferential whole-cell labelling of dead spermatozoa with PSA is considered to be due to increased availability of the nonacrosomal set of PSA-reactive sites in dead spermatozoa after a short treatment with methanol, whereas this treatment is probably not sufficient to expose most of these sites when applied to living spermatozoa. The simplicity of the staining protocol makes this method feasible in routine work in a number of clinical and research applications.

Acrosome↗