Phorbol myristate acetate induces ruffling of the acrosome of human sperm

OBJECTIVE: To determine the effect of phorbol myristate acetate (PMA) on human acrosome morphology and the acrosome reaction. DESIGN: Controlled experiments on sperm and unfertilized oocytes from volunteers. SETTING: Academic research and teaching tertiary hospital. PATIENT(S): Sperm samples were from normospermic men and unfertilized oocytes from IVF patients. MAIN OUTCOME MEASURE(S): Acrosome morphology was assessed by using transmission and scanning electron microscopy. The acrosome reaction was assessed by using fluorescein-labeled Pisum sativum agglutinin. RESULT(S): PMA induced acrosome ruffling, indicated by a marked wavy appearance. A significant correlation was found between PMA-induced ruffling and PMA enhancement of the zona pellucida-induced acrosome reaction. Protein kinase C inhibitors bisindolylmalemide I and sangivamycin had no effect on PMA-induced acrosomal ruffling, but actin polymerization inhibitors cytochalasin B and cytochalasin D significantly decreased PMA-induced acrosomal ruffling. In contrast, bisindolylmalemide I, sangivamycin, cytochalasin B, and cytochalasin D significantly decreased both the zona pellucida-induced acrosome reaction and the PMA enhancement of the zona pellucida-induced acrosome reaction. CONCLUSION(S): PMA-induced acrosomal ruffling involves actin polymerization, possibly independent of conventional protein kinase C. Acrosomal ruffling is involved in the PMA augmentation of the zona pellucida-induced acrosome reaction.     

Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status

OBJECTIVE: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss. DESIGN: Prospective randomized study. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan. PATIENT(S): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program. INTERVENTION(S): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF. MAIN OUTCOME MEASURE(S): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red-conjugated antimouse immunoglobulin G antiserum. RESULT(S): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time. CONCLUSION(S): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss.     

Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients.

OBJECTIVE: To compare the function of a novel nongenomic progesterone (P) receptor on the human sperm surface (mediating the P-induced acrosome reaction) in spermatozoa from fertile donors and from infertile patients. To examine the possible implication of defective P receptor function as an etiologic factor in unexplained male infertility. DESIGN: Progesterone binding and P effects were assessed in sperm from infertile patients and compared with corresponding parameters for sperm from healthy donors. SETTING: Private hospital, medical research center, and a university-based andrological laboratory. PATIENTS, PARTICIPANTS: Sperm samples were from infertile patients (no pathology detected in their wives) attending our infertility clinic and from healthy sperm donors. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Progesterone binding sites were visualized with a fluorescein-labeled protein-P conjugate. Indo 1-AM (a fluorescent indicator of intracellular free Ca2+) was used to measure P-induced Ca2+ influx. Progesterone-induced acrosome reaction was monitored after acrosomal staining with Pisum sativum agglutinin. RESULTS: Among 8 patient sperm samples with normal spermiogram values (of 53 examined), 5 showed a reduced percentage of P-binding spermatozoa and an abnormal response to the hormone in terms of Ca2+ influx and the acrosome reaction. CONCLUSIONS: Defective function of a sperm surface P receptor is described in some cases of male infertility. The observed fluorescence microscopic patterns of hormone binding may be used to further investigate receptor activity in unexplained male infertility.     

Selective expression of a progesterone receptor on the human sperm surface.

OBJECTIVE: To visualize progesterone (P) binding sites on the sperm surface, examine the relationship between hormone binding and hormone action (acrosome reaction), and determine the size of the hormone-responsive sperm subpopulation. DESIGN: Kinetic analysis of P binding was combined with the assessment of the hormone effect using a fluorescent acrosomal marker. SETTING: Private hospital, medical research center, and a university-based andrological laboratory. PATIENTS, PARTICIPANTS: Sperm samples were from healthy volunteers with normal spermiogram values. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Progesterone binding was analyzed by fluorescence microscopy and flow cytometry using P coupled to fluorescein isothiocyanate-labeled bovine serum albumin. Tetramethylrhodamine isothiocyanate-labeled Pisum sativum agglutinin was used as acrosomal marker in double-labeling experiments. RESULTS: After in vitro capacitation, only few spermatozoa (approximately 10%) were able to bind P to the cell surface, but most of these cells subsequently generated the acrosome reaction in response to hormone binding. CONCLUSIONS: The expression of P receptor sites on the human sperm surface is a major factor controlling the P-induced acrosome reaction. Further studies are warranted to explore if defective expression of the receptor can compromise fertility.     

Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody

A monoclonal antibody, T-6, useful for detecting acrosome-reacted sperm based on an immunofluorescent assay, was employed to evaluate acrosomal status of human sperm that were tightly bound to hemisected human zonae pellucidae (hemizona assay). Over 90% of the bound sperm evaluated exhibited immunofluorescent patterns indicative of acrosome reaction. This staining method for evaluating the acrosomal status of sperm bound to the zona pellucida may enable definition of a group of male infertility patients heretofore not recognized.     

Comparative flow cytometric analysis of the human sperm acrosome reaction using CD46 antibody and lectins

Purpose: Our purpose was to determine the most suitable marker for the human sperm acrosome reaction, based on detection of CD46 antibody binding compared with lectin binding. Methods: Flow cytometric analysis of CD46 antibody versus lectins (PNA, PSA, and Con A) was used to quantify the acrosome reaction of human sperm. Results: Neither PSA nor Con A was able to detect significant changes in the spontaneous and ionophore-induced acrosome reactions compared to CD46 antibody. However, PNA was found to exhibit a binding pattern similar to that observed with CD46 and could be used to quantify measurable changes in acrosomal response to ionophore, albeit of a lower magnitude than the responses detected by CD46. Conclusions: We conclude that PNA binds to the inner acrosomal membrane of acrosome-reacted sperm and is suitable for use as a marker of the acrosome reaction by flow cytometry. Data are presented which clarify the assessment of the acrosome reaction when CD46 and lectins are used.     

Influence of antisperm antibodies on the acrosome reaction as determined by flow cytometry

OBJECTIVE: To evaluate the influence of antisperm antibodies on the acrosome reaction (AR). DESIGN: Clinical study. SETTING: University of Marburg, Department of Andrology, Clinical Training Center of the European Academy of Andrology. PATIENT(S): Spermatozoa from a pool of healthy donors were incubated with 30 seminal plasma samples from infertile men containing antisperm antibodies; they were compared to a control group of 10 samples without antisperm antibodies and five samples with buffer only. INTERVENTION(S): The spontaneous acrosome reaction (SAR) and the induced acrosome reaction (IAR) by calcium ionophore A23187 were observed and determined by means of a flow cytometer. Flow cytometric double-staining estimates of acrosomal integrity were determined by using a monoclonal antibody (TUS 19), marked with a secondary fluorescein isothiocyanate-labeled antibody. Cell viability was determined by counterstaining with propidium iodide (PI). MAIN OUTCOME MEASURE(S): Number of acrosome reacted spermatozoa. RESULT(S): The spermatozoa treated with antisperm antibodies showed significantly higher SAR and IAR responses than the control group. CONCLUSION(S): Some of the antisperm antibodies from individual patients are able to enhance the acrosome reaction in donor sperm, but none of them appeared to inhibit acrosome reaction.     

Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability.

OBJECTIVE: To determine the sensitivity and functional significance of the fluorescein isothiocyanate concanavalin A (FITC-ConA) staining method of assessment of acrosomal status. DESIGN: Treatments were assessed for their ability to induce human sperm acrosomal loss. Penetration of zona-free human eggs by treated spermatozoa was subsequently determined. SETTING: Zona-free human eggs were obtained from the Infertility Medical Centre, Richmond, Victoria, Australia. PATIENTS, PARTICIPANTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosomal loss of human spermatozoa was determined at 4 hours and 10 hours of treatment incubation. Penetration of zona-free human eggs was assessed 16 hours after reinsemination. RESULTS: Human spermatozoa incubated in a strontium- or lanthanum-based medium, or T6 + 10% maternal human serum (HS) supplemented with 12 mM 8-bromo cyclic guanosine 3,5'-monophosphate and 10 mM imidazole for a 4-hour period before transfer to fresh T6 + 10% HS for a further 6 hours, demonstrated a significant increase (P less than 0.05) in acrosomal loss compared with T6 + 10% HS for a total 10-hour incubation. This increase in acrosomal loss with test treatments correlated with an increase in the development of pronuclei of zona-free human eggs (r = +0.98). CONCLUSIONS: The FITC-ConA staining procedure therefore reflects biological function as assessed by the penetration of zona-free human eggs and consequently provides a further research tool for the investigation of the human sperm acrosome reaction.     

Light and electron microscopic immunolocalization of sperm proteins identified by monoclonal antibodies from the World Health Organization Task Force on Sperm Antigens.

Sperm antigens recognized by monoclonal antibodies (mAbs), S19, S69, S71, S72 and S77 submitted to the World Health Organization (WHO)-Sponsored Sperm Monoclonal Antibody Workshops were immunolocalized by light (LM) and transmission electron microscopy (TEM). S19 was surface reactive while mAbs S69, S71, S72 and S77 recognized internal antigens. Indirect immunofluorescence staining of permeabilized sperm with the mAbs revealed that S69 recognized an internal tail antigen, while S71, S72 and S77 recognized acrosomal proteins. Preservation of immunoreactivity after fixation in various combinations of glutaraldehyde, paraformaldehyde and tannic acid was evaluated for mAbs S69 to S77 using immunofluorescence microscopy. The epitopes recognized by these mAbs were adversely affected by these fixatives; therefore, pre-embedding immunogold staining was employed, prior to fixation, osmication, dehydration and embedding. Using this approach, the antigen recognized by mAb S19 was found associated with the plasmalemma of the head and tail of intact sperm. Monoclonal antibody S69 localized to the fibrous sheath. The mAbs S71, S72 and S77, which required sperm permeabilization to expose their acrosomal locus by LM, did not immunoreact with the plasmalemma at the TEM level. Ultrastructural examination of acrosome-reacted sperm revealed the localization of S71 and S77 on the inner and outer acrosomal membranes and with acrosomal matrix. The S72 antigen was associated with the inner and outer acrosomal membranes.     

Effect of incubating human sperm at room temperature on capacitation-related events

OBJECTIVE: To determine the effect of human sperm incubation at room temperature (20 degrees C) upon capacitation-related events. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF) was collected from women undergoing assisted reproductive treatment. INTERVENTION(S): Spermatozoa were incubated for up to 18 hours at 20 degrees C and/or 37 degrees C. MAIN OUTCOME MEASURE(S): Protein tyrosine phosphorylation patterns, development of hyperactivated motility, and induction of acrosome reaction (AR) in response to hFF. RESULT(S): Spermatozoa incubated for 18 hours at 20 degrees C showed an array of tyrosine phosphorylated proteins similar to noncapacitated cells. After incubation at 20 degrees C, the percentage of spermatozoa displaying hyperactivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared with cells kept the same time at 37 degrees C. Conversely, spermatozoa incubated overnight at 37 degrees C could respond to hFF, either at 37 degrees C or 20 degrees C. When preincubation at 20 degrees C was followed by sperm exposure to 37 degrees C, capacitation-related events could be activated. In capacitated cells (16 hours at 37 degrees C), 2-hour incubation at 20 degrees C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation. CONCLUSION(S): Human sperm incubation at room temperature does not allow capacitation, although it does not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when spermatozoa are exposed to 37 degrees C.     

Identification of sperm antigenic determinants with phylogenetically diverse and limited distribution using monoclonal antibodies

Mouse monoclonal antibodies capable of recognizing mouse sperm antigens were produced and the inter-species cross-reactivity of the antigenic determinants was examined. Of the seven clones independently established, it was found by indirect immunofluorescence staining that one was bound to the tail, one could recognize the head and five other antibodies reacted with the crescent-shaped anterior acrosome. Ultrastructural studies showed that the anti-head antibody recognized the sperm surface, and the anti-tail antibody reacted with the fibrous sheath. Five acrosomal antigenic determinants were found in the acrosome. These antibodies reacted with sperm from the male reproductive tract (testis, cauda epididymis, vas deferens), but not with somatic tissues. The anti-tail antibody reacted with sperm from four other mammalian species (musk shrew, rat, boar and human). The anti-head antibody and one of the anti-acrosome antibodies were bound only to mouse sperm. The other four acrosomal antigenic determinants showed various degrees of inter-species cross-reactivity. None of these antibodies reacted with chicken sperm.     

Differential diagnosis of immature germ cells in semen utilizing monoclonal antibody MHS-10 to the intra-acrosomal antigen SP-10

The monoclonal antibody (mAb) MHS-10 (IgG1) is a mouse antihuman sperm antibody which recognizes a polymorphic sperm protein, (SP-10), which has previously been localized within the acrosomal matrix and the acrosomal membranes. The SP-10 antigen has been shown to be sperm-specific and is not found in somatic tissues. It is stage specific, having been immunohistologically localized to Golgi phase spermatids and all subsequent phases of spermiogenesis. In the present study, acetone-dried smears from washed human semen containing significant numbers of round cells were probed with mAb MHS-10. Monoclonal antibody-labeled cells were visualized by a standard streptavidin-biotin immunoperoxidase method using a light microscope. The MHS-10 mAb immunoreacted with mature sperm and with a subset of round cells diagnosed as developing spermatids, which had been sloughed off from the testis at varying stages of acrosome formation. To rule out possible cross-reactivity of the mAb with leukocytes in semen, a leukocyte surface marker (anti-HLe-1) was used in conjunction with MHS-10. Round cell populations staining with MHS-10 did not stain with anti-HLe-1. The mAb MHS-10 is thus a promising probe for the identification and quantitation of immature germ cells in human semen.     

The fertilization antigen-1 does not have proteolytic/acrosin activity, but its monoclonal antibody inhibits sperm capacitation and acrosome reaction.

OBJECTIVE: To determine if human sperm surface fertilization antigen exhibits proteolytic or acrosin activity and to investigate the mechanism(s) whereby monoclonal antibody (mAb) to fertilization antigen inhibits human sperm penetration of zona-free hamster ova. DESIGN: Proteolytic and acrosin activities of human fertilization antigen were determined. Acrosomal status, acrosin activity, and motion characteristics were evaluated after incubation of human sperm with immunoaffinity-purified mAb to fertilization antigen. SETTING: Academic research environment. PARTICIPANTS: Fertile donors used as controls for infertile patients for fertility evaluation. INTERVENTIONS: Human spermatozoa were treated with mAb to fertilization antigen and induced to undergo acrosome reaction using calcium ionophore A23187. MAIN OUTCOME MEASURES: Proteolytic and acrosin activities of fertilization antigen. Sperm penetration assay, acrosomal status, and motion parameters. RESULTS: Fertilization antigen does not exhibit proteolytic or acrosin activity; however, its mAb completely blocks human sperm penetration of zona-free hamster ova. The mAb to fertilization antigen inhibits ionophore-induced acrosome reaction and blocks development of the hyperactivated state of human sperm cells. CONCLUSIONS: Monoclonal antibody to fertilization antigen blocks fertilization by inhibiting capacitation and acrosome reaction.     

Acrosin activity as a potential marker for sperm membrane characteristics in unexplained male infertility

OBJECTIVE: To evaluate sperm membrane system integrity in unexplained infertile male subjects with three consecutive conception failures on IUI even though semen clinical parameters were normal. DESIGN: Prospective study. SETTING: Medical biotechnology laboratory, School of Medical Science and Technology IIT Kharagpur, India. PATIENT(S): Twenty-nine patients with unexplained infertility, 17 normal proven-fertile healthy donors, and 21 infertile males with low motility but with other semen parameters remaining normal. INTERVENTION(S): Semen samples were collected from unexplained infertile patients as well as from healthy fertile donors after abstinence of 3-5 days and were analyzed according to World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Release of 5'-nucleotidase (plasma membrane marker), lactate dehydrogenase (mitochondrial marker) and free acrosin, proacrosin, and total acrosin (acrosomal membrane marker). RESULT(S): Plasma membrane integrity and respiratory activity of sperm cells were comparable in all three groups. The proacrosin-acrosin system was adversely affected in unexplained infertile subjects despite high sperm motility. CONCLUSION(S): Total acrosin activity may be considered as a sensitive biochemical marker for clinical evaluation of unexplained infertility in males.     

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa

OBJECTIVE: To develop a fast method for assessing acrosome status in human spermatozoa. DESIGN: Development of a new in vitro test to assess acrosome reaction in human spermatozoa. SETTING: Academic medical institution. PATIENT(S): Normozoospermic subjects. INTERVENTION(S): Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. MAIN OUTCOME MEASURE(S): Percentage of intact acrosomes. RESULT(S): The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. CONCLUSION(S): The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.     

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.     

Evaluation of acrosomal status using MH61-beads test and its clinical application.

OBJECTIVE: To evaluate the acrosomal status of viable sperm by MH61-beads test and its clinical application. DESIGN: Acrosomal status was evaluated using immunobeads coated with MH61 monoclonal antibody, which is specific for acrosome-reacted sperm. When viable sperm was coincubated with MH61-beads for an appropriate length of time, the formation of a sperm-bead complex was observed with a phase-contrast microscope, and the number of sperm binding to MH61-beads increased with the progression of the acrosome reaction. Using this agglutination, we developed the MH61-beads test to assess sperm function. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital. PARTICIPANTS: Forty-three volunteers and 20 males in our in vitro fertilization (IVF) and embryo transfer program. MAIN OUTCOME MEASUREMENT: The results of MH61-beads test were compared with the percentage of acrosome-reacted sperm detected by Pisium satinum agglutinin staining on 30 volunteers, with the results of sperm penetration assay (SPA) in 43 volunteers, and with the outcome of IVF in 20 patients. RESULTS: The results of MH61-beads test showed good reproducibility and correlated with the results of SPA using zona-free hamster eggs and IVF. CONCLUSIONS: The MH61-beads test, the results of which reflect the acrosomal status of sperm, may provide useful information concerning the fertilizing ability of sperm.     

Effect of erythrocyte-sperm separation medium on nuclear,acrosomal, and membrane maturity parameters in human sperm

Purpose

The purpose of this study is to investigate whether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation.

Methods

Semen samples from normozoospermic (n?=?36) and oligozoospermic (n?=?9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation.

Results

No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p?>?0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation.

Conclusion

ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

     

The sperm proteasome during sperm capacitation and fertilization

The 26S proteasome is a multi-subunit protease specifically targeting ubiquitinated proteins. A consensus has emerged from studies by multiple laboratories on the role of sperm-borne proteasomes in human, mouse, pig, bovine, ascidian and echinoderm fertilization. Major findings from the studies in various mammalian and non-mammalian fertilization systems are (1) proteasomes are present in the mammalian sperm acrosome and on the acrosomal surface; (2) ubiquitinated proteins are present on the mammalian, ascidian and echinoderm egg coat; (3) proteasomal proteolytic and ubiquitin-deconjugating (deubiquitinating) activities can be detected in viable, motile mammalian spermatozoa; (4) proteasomes remain associated with the sperm head following ZP-induced acrosomal exocytosis; (5) inhibition of ubiquitination and proteasomal proteolysis blocks fertilization in mammals, ascidians and echinoderms; (6) inhibition of proteasomal proteolysis alters the course of mammalian sperm capacitation and acrosomal exocytosis induced by sperm binding to the egg coat, zona pellucida (ZP); (7) depletion of the sperm surface-associated ATP blocks porcine and echinoderm fertilization, most likely by affecting the integrity of sperm proteasomes, of which several subunits are ATPases; (8) inhibition of proteasomal proteolysis blocks sperm–ZP penetration, but does not alter the rate of sperm–ZP binding in mammals, and (9) experimental modification of sperm-associated deubiquitinating activities shifts the balance of monospermic fertilization to polyspermic fertilization in vitro. Altogether, these studies provide evidence for the involvement of the 26S proteasome in multiple steps of animal and human fertilization, offering a novel model of sperm–egg coat interactions, and identifying a range of potential new sperm quality markers and contraceptive targets.     

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody OBF13

The variable reactivity of OBF13 monoclonal antibody to mouse sperm was studied using a fluorescein activated cell sorter. Sperm from cauda epididymis were incubated with ionophore A23187, subjected to indirect immunostaining and analyzed by a cell sorter. Three peaks showing different fluorescence intensities were observed. These peaks contained (i) not stained (N), (ii) acrosomal cap or anterior region stained (A) and (iii) head stained (H) sperm, respectively. H type sperm showed more intense integral fluorescence than A type sperm. It was also noted that the H type were observed when cauda epididymal sperm were incubated with A23187, but not among non-incubated, or A23187 treated caput epididymal sperm. When sperm were pre-categorized as "dead" or "alive" by propidium iodide staining, no A type were observed in the "live" population. These results suggest that the sperm exhibiting the OBF13 antigen in the acrosome region lost their viability before they accomplished a "true" acrosome reaction.