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.     

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions.Design: Laboratory experimental study.Setting: University gynecology unit.Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics.Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential).Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion.Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF.Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.     

Biochemical and functional characterization of the human zona pellucida

A successful interaction between spermatozoa and the zona pellucida is critical for fertilization. This biological step reflects multiple sperm functions, including the acquisition and completion of capacitation, recognition and binding to specific zona pellucida receptors, and induction of the physiological acrosome reaction. The recognition of carbohydrate sequences by complimentary receptors has been demonstrated in gamete interaction in different animal species. It has been proposed that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human spermatozoa to the homologous zona pellucida) and advanced methods of carbohydrate analysis have been used to test this hypothesis. Compelling evidence exists to demonstrate that oligosaccharide recognition is also required for specific, tight human gamete binding. The induction of the acrosome reaction using the physiological inducers, i.e. the zona pellucida and progesterone, was also examined. It has also been demonstrated that there is a priming effect of the steroid on the acrosome reaction inducing capacity of the zona pellucida. These studies may allow for a better understanding of human gamete interaction in physiological and pathological situations.     

The role of acrosin in sperm penetration through human cervical mucus.

Enzymes have been implicated in facilitating cervical mucus penetration by spermatozoa. One of these enzymes in the neutral proteinase acrosin, which is associated with the sperm acrosome. To determine the validity of this hypothesis, human spermatozoa were incubated with the following acrosin inhibitors: p-aminobenzamidine (AB), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and p-nitropheyl-p'-guanidino benzoate (NPGB). An in vitro slide test system was developed which allowed inhibitor-treated and control spermatozoa to be evaluated against the same human cervical mucus sample. At inhibitor concentrations far exceeding those necessary for the inhibition of human acrosin, there was no effect on spermatozoal penetration into or through the mucus. These findings indicate that, in man, acrosin activity is neither necessary nor facilitory to sperm penetration of cervical mucus. Evidence is also presented that demonstrates the superiority of the newly developed double-interface slide test, especially for comparative purposes, over the tests currently in use.     

High progesterone concentrations induce acrosome reaction with a low cytotoxic effect.

OBJECTIVE: To determine the optimal conditions to obtain live acrosome-reacted spermatozoa for micromanipulation. DESIGN: Experiments were performed to determine time and dose-dependent effects of calcium ionophore A23187 or steroids on acrosome reaction of fertile donor sperm. The percentages of total reacted and live reacted spermatozoa were assessed with the peanut agglutinin lectin procedure. RESULTS: Incubation with 1 mmol/L progesterone (P) induced 48% +/- 17% acrosome reaction after 6 hours. Motility and viability remained high (49% +/- 3% and 70% +/- 2%, respectively) and thus the percentage of live reacted spermatozoa was 27% +/- 5%. Incubation with A23187 (5 mumol/L for 30 minutes) gave similar results for the percentage of live reacted spermatozoa (26% +/- 4%) but with a lower motility and viability (25% +/- 7% and 53% +/- 2%, respectively; P less than 0.05). CONCLUSIONS: These results show that high concentration of P is an effective way to induce acrosome reaction in preparation for micromanipulation.     

Blocking effect of sperm immobilizing antibodies on sperm penetration of human zonae pellucidae

To investigate the mechanism of the blocking effect of sperm immobilizing antibodies on human fertilization, an in vitro zona penetration test was carried out using media containing the IgG fraction extracted from sperm immobilizing antibody-negative or-positive serum. The sperm penetration rate of the test was 100% (6/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-negative serum, whereas it was only 17% (1/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-positive serum. Electron microscopic observation of the sperm immobilizing antibody-negative and-positive serum-treated spermatozoa showed that the number of acrosome-reacted spermatozoa was significantly greater in the sperm immobilizing antibody-negative serum than in the antibody-positive serum. Therefore, it appears that one of the blocking mechanisms of the spermatozoal penetration of the zona pellucida by sperm immobilizing antibodies may be due to inhibition of the acrosome reaction in the spermatozoa.     

The effects of calmodulin and it's antagonist, W-7, on the acrosome reaction and fertilization of human spermatozoa

The acrosome reaction of mammalian spermatozoa has been shown to be dependent upon an influx of Ca2+ following capacitation. Recently it was shown that calmodulin which activates various enzymatic activities in a calcium-dependent manner is contained in the acrosomal portion of sperm obtained from diverse species including human. Therefore calmodulin appears to be the primary target for Ca2+-dependent regulatory process involved in the acrosome reaction. This report examines the effects of calmodulin and it's antagonist, W-7, on the acrosome reaction in human spermatozoa and the fertilization with zona free hamster eggs. The results are as follows: 1) In the experiment on fertilization in mBWW medium with 30nM of calmodulin, there was no significant difference between the calmodulin and the control. (2) In fertilization in mBWW medium with 2.5 to 25 microM of W-7, there were no significant changes in the fertilization rate, but there was a significant decrease in the fertilization rate when the concentration of W-7 was elevated up to 50 microM. (3) When fertilization in mBWW was performed using spermatozoa pretreated with W-7, the fertilization rates were more markedly and promptly elevated with insemination times than the control. (4) When the eggs and spermatozoa were pretreated with W-7 before insemination and then placed in mBWW medium, the fertilization rate was markedly decreased. (5) Triple stain method and transmission electron microscopy showed that a large proportion of spermatozoa had undergone the acrosome reaction in mBWW medium containing W-7 with normal manner. From the results given above, it appears that calmodulin plays an important role in the acrosome reaction and fertilization in human spermatozoa.     

Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

OBJECTIVE: To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. DESIGN: The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. SETTING: Samples were collected from patients referred to university hospital infertility clinics. MATERIALS: Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. RESULTS: During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). CONCLUSIONS: Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.     

Acrosome reaction of human sperm used for in vitro fertilization

The acrosomal reaction of human spermatozoa was assessed, according to a triple-stain technique, at the end of a 17-hour capacitation period in the search for a predictive test of sperm efficiency in an in vitro fertilization program. A 400% increase in the acrosomal reaction rate of the living spermatozoa was observed (4.2% +/- 3.2% before incubation versus 16% +/- 11% after incubation). No correlation could be evidenced between the abilities for spermatozoa to react and to fertilize mature human oocytes in vitro, whatever the initial semen quality. The acrosome reaction rate was not enhanced by the presence of the cumulus oocyte complex, by the selection of motile spermatozoa, or by the increase in albumin concentration of the culture medium.     

Effects of human follicular fluid on the capacitation and motility of human spermatozoa

OBJECTIVE: To investigate the capacitation and motility kinetics of spermatozoa treated with human follicular fluid (FF). DESIGN: Controlled, experimental laboratory study.Setting: University-based gynecology unit. PATIENT(s): Human FF was collected from women undergoing assisted reproductive treatment. Semen samples were obtained from men visiting subfertility clinics. INTERVENTION(s): Spermatozoa were incubated with human FF under various experimental conditions. Spermatozoa incubated with Earle's balanced salt solution were used as the control. MAIN OUTCOME MEASURE(s): Chlortetracycline staining patterns and sperm motility parameters. RESULT(s): The rate of capacitation in the human FF-treated spermatozoa was significantly higher than that in the control spermatozoa after 1 hour and 3 hours of treatment. The percentage of acrosome-reacted spermatozoa also was significantly higher after human FF treatment than after control treatment. These effects of human FF were dose-dependent. Human FF-treated spermatozoa maintained their velocities at the zero-hour level for 5 hours, whereas the velocities of the control spermatozoa decreased significantly after 1 hour. Human FF treatment significantly increased the beat cross-frequency above the rate at zero hour for 5 hours. The hyperactivation of the human FF-treated spermatozoa remained stable for 3 hours, whereas that of the control spermatozoa decreased significantly after 1 hour of incubation. Significantly more human FF-treated spermatozoa underwent hyperactivation than did control spermatozoa after 1 hour and 3 hours of treatment. The effects of human FF on beat cross-frequency and hyperactivation were dose-dependent. CONCLUSION(s): Human FF promotes capacitation and the acrosome reaction within a short period. It also stimulates or maintains various sperm motility parameters.     

Induced acrosome reactions as fertility predictor

Purpose This study was conducted to determine the fertility predictive value of acrosome reaction rates and indices (induced minus control) of human spermatozoa. By comparing these outcomes with in vitro fertilization success. The effect of oocyte-cumulus complex exposure on the induction of the acrosome reaction was also analyzed. Patients attending our assisted reproduction unit for infertility treatment were included in the study. Acrosome reactions were determined on ethanolpermeabilized smears using FITC-conjugated Pisum sativumagglutinin. The acrosome reaction inducing agent used was calcium ionophore A23187 (10 M/ml).Results Poor correlations were found between all the acrosome reaction rates and indices and in vitro fertilization. The presence of oocyte-cumulus complexes had no effect on the spontaneous acrosome reactions, but had a significant effect on the inducibility of the acrosome reaction. Exposure to oocyte-cumulus complexes resulted in the mean percentage sperm induced to be 7.8% (SE= 3.1%) higher compared to the control samples.Conclusions Acrosome reaction rates and indices were therefore found to have no significant value in the prediction of male fertility andlor in vitro fertilization success. This study did, however, show that exposure to oocyte-cumulus complexes significantly increases the inducible sperm population.     

Antiestrogenic effect of clomiphene citrate: correlation with serum estradiol concentrations

The antiestrogenic effect of clomiphene citrate (CC) on cervical mucus was evaluated in women receiving 150 mg CC daily for 5 days. Daily cervical mucus scores and serum estradiol (E2) concentrations were determined in control (n = 25) and CC (n = 24) cycles. E2 concentrations were significantly higher in the CC-treated women (mean +/- standard error of the mean, 1254 +/- 102 pg/ml versus 337 +/- 18 pg/ml, P less than 0.0001). Despite supraphysiologic E2 concentrations, however, cervical mucus scores were significantly reduced in the CC-treated group (P less than 0.01). These data indicate that CC exerts a direct suppressive effect on cervical mucus despite markedly increased E2 concentrations.     

Induction of the human sperm acrosome reaction by human oocytes

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P greater than 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P greater than 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.     

In vitro penetration of human sperm into bovine cervical mucus: effects of sperm washing and exposure to low temperature

Standardized bovine cervical mucus penetration by human sperm in vitro provides information for evaluating male fertility. Normal semen specimens from 20 sperm donors and 17 infertile men were tested for cervical mucus penetration. Rigorous control of test temperature was necessary to guarantee the reliability and reproducibility of cervical mucus penetration. Sperm washing was found to significantly improve cervical mucus penetration for infertile men, from 18 +/- 2.2 to 27 +/- 3.4 mm, P less than .025. Sperm washing for normal donors had no apparent effect on cervical mucus penetration (58 +/- 1.5 mm prewash, 55 +/- 1.7 mm postwash, P greater than .1). The authors conclude that: 1) temperature for cervical mucus penetration testing is critical to reliability, and 2) cervical mucus penetration is a useful screening tool for in vitro procedures proposed to improve sperm function.     

Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction.

OBJECTIVE: To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. MAIN OUTCOME MEASURES: HA and acrosome reaction. RESULTS: Sperm HA was significantly increased at 10 minutes by P both at 10(-7) M (9.27 +/- 1.59%; mean +/- SEM) and 10(-6) M (9.39 +/- 1.94%) when compared with untreated spermatozoa (5.62 +/- 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10(-6) M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10(-6) M to P at 10(-7) M (12.43 +/- 3.31%) or P at 10(-6) M (13.52 +/- 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. CONCLUSION: Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.     

The influence of various molecules on the occurrence of the human sperm acrosome reaction

The acrosome reaction is essential for fertilization, but the mechanism of the acrosome reaction of human spermatozoa is not clear at the present time. We studied the mechanism to analyze the cause of unexplained infertility, the appropriate timing of insemination, and the environment of spermatozoa prior to fertilization. For this study, we examined the effects of Ca++, Mg++, Kallikrein, Phospholipase A2, p-bromophenacyl bromide (Phospholipase A2 specific inhibitor), Lysophosphatidyl choline, Arachidonic acid, and Glyceryl monooleate using in vitro penetration assay employing zona- free hamster eggs. Results obtained were as follows. When human spermatozoa were incubated in mBWW with Ca++ or (and) Mg++ free medium, the acrosome reaction was inhibited. When human spermatozoa were incubated in mBWW with Kallikrein (1.0-4.0 KU ml), the acrosome reaction was promoted. When Phospholipase A2 was used at concentrations of 0.2 and 2.0 unit/ml, penetration rates showed the same tendency as in the control. But when p-bromophenacyl bromide was tested at concentrations of 1 X 10(-5) - 1 X 10(-3)M, penetration rates were inhibited when compared with the control. When human spermatozoa were incubated in medium containing Lysophosphatidyl choline (50 micrograms/ml), Arachidonic acid (5-50 micrograms/ml), and Glyceryl monoleate (300-400 micrograms/ml), the acrosome reaction was accelerated.     

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.     

Presence of prolactin in human cervical mucus.

The presence of prolactin in human cervical mucus was demonstrated by radioimmunoassay. The levels of prolactin in cervical mucus were significantly higher (260 ng/ml +/- 47.5 SE) than those in sera of normally menstruating women (30.28 ng/ml +/- 2.0 SE).     

How animal embryo research led to the first documented human IVF

Research studies using animal gametes led to the first documented human IVF in 1969. A key contribution was the development of a reliable culture medium for IVF of hamster oocytes, which was then used successfully with human gametes in the laboratory. This article describes how this was accomplished. Hamster IVF was obtained using a bicarbonate-buffered culture medium based on Tyrode's solution. A dose-response experiment showed that IVF in this species was highly dependent on the pH of the culture medium. At pH 7.2 or less, very few oocytes were penetrated and spermatozoa did not undergo acrosome reactions. Over the pH range 7.3-7.5, 40% of oocytes were fertilized in vitro, while at pH 7.6 or higher, more than 75% of oocytes were fertilized and most spermatozoa exhibited acrosome reactions. There was no apparent effect of pH on sperm motility. These experiments showed that in the hamster, fertilization in vitro is highly pH dependent, most likely through pH effects on the sperm acrosome reaction and sperm:zona binding. The data led to formulation of a culture medium, Tyrode-B, that consistently supported high levels of IVF of hamster oocytes. When this medium was used for human gametes, IVF was observed and documented for the first time. Spermatozoa penetrating through the zona pellucida and sperm components within the ooplasm were detected, and some oocytes exhibited two pronuclei. These observations suggested that human IVF might be useful for alleviation of infertility.