The kinetics of the acrosome reaction of human spermatozoa and its correlation with in vitro fertilization.

OBJECTIVE: To study the reaction pattern of acrosome reaction in human semen and correlate it to the results of in vitro fertilization (IVF). DESIGN: The percentage of acrosome-reacted spermatozoa of 41 IVF semen samples was determined after 0, 2, 4, and 24 hours of incubation in human tubal fluid medium supplemented with 10% human pool serum. SETTING: St. Radboud Hospital, Catholic University of Nijmegen, The Netherlands. PATIENTS: Forty-one IVF couples. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosome reaction was determined using fluorescein isothiocyanate conjugated concanavalin A lectin. To avoid false-positive signals from dead spermatozoa, the sperm viability was determined. RESULTS: Three kinetic patterns of acrosome reaction could be distinguished: (1) normal reacting pattern (percentage of acrosome-reacted spermatozoa less than 10% at 2 hours and greater than 5% at 4 hours; 75% fertilization in IVF); (2) a quickly reacting pattern (percentage of acrosome-reacted spermatozoa greater than 10% at 2 hours; 22% fertilization in IVF); and (3) a nonreacting pattern (percentage of acrosome-reacted spermatozoa less than 5% at all time intervals studied; 15% fertilization in IVF). CONCLUSIONS: The timing of acrosome reaction and the percentage of acrosome-reacted spermatozoa are very important parameters in IVF.     

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.     

囊性纤维化跨膜转导调节因子(CFTR)表达率与人精子获能及顶体反应间相关性研究

目的:研究人精子囊性纤维化跨膜转导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)表达率与精子获能及顶体反应间的相关性。方法:通过间接免疫荧光染色法观察CFTR在人精子上的表达率,金霉素染色法评估精子获能及顶体反应。结果:CFTR定位在人精子赤道板上,随着CFTR表达率降低,精子获能率也随之降低,故CFTR表达率与人精子获能呈正相关(r=0.985,P0.01),而且精子顶体反应率也随之减少,因此CFTR表达率与人精子顶体反应呈正相关(r=0.979,P0.05)。结论:人精子CFTR表达率与精子获能及顶体反应呈正相关。     

High potassium concentration and the cumulus corona oocyte complex stimulate the fertilizing capacity of human spermatozoa

Progressively motile spermatozoa were incubated for 24 hours in culture media containing 4.7 or 25 mM K, in the presence or absence of hamster cumulus oophorus. The percentage of spermatozoa with progressive motility was significantly higher at 24 hours in the presence of cumulus corona oocyte complexes, irrespective of K concentration. A significant decrease in sperm mortality was observed with the association of 25 mM K and cumulus cells. A higher percentage of acrosome reaction was observed in spermatozoa incubated in 25 mM K when compared with 4.7 mM K, irrespective of time and the presence or absence of cumulus. The percentage of penetrated oocytes at 2 and 5 hours of incubation was higher when sperm had been incubated in 25 mM K than in 4.7 mM K. The presence of cumulus in the culture medium induced an additional significant increase in the percentage of penetrated oocyte. Although at 24 hours of incubation the percentage of acrosome reaction was higher than at 2 and 5 hours, the percentage of penetrated oocytes did not increase proportionally.     

Isolation of human spermatozoa membrane antigens binding sperm-immobilizing and sperm-agglutinating antibodies.

Peptides of human spermatozoa were dissolved with Hyamine 2389 and Triton X-100 and separated by chromatography on Biogel P4 columns into seven fractions. The antigenic activities of the separated sperm-membrane fractions were tested according to their capacity to inhibit sperm agglutination and sperm immobilization (immune inhibition test) in human sera of sterile patients. Sperm-agglutinating and sperm-immobilizing activity was tested by the microtray agglutination and microtray immobilization test. A titer reduction was achieved only in sperm-immobilizing sera. Four sperm antigenic fractions revealed in the majority of the repeatedly tested sperm-immobilizing sera an inhibition of the antigen-antibody reaction. No reaction was observed after exhaustive absorption of the tested seven antigen fractions with sperm-agglutinating sera. Therefore the conclusion can be drawn that sperm-agglutinating and sperm-immobilizing antibodies react with different sperm antigens. Normal human sera without sperm antibodies served as control. As no sperm agglutination or sperm immobilization was obtained after absorption of these control sera our antigen fractions do not produce sperm agglutination or sperm immobilization.     

NO对抗精子抗体阳性大鼠精子自发顶体反应的影响

目的 :探讨 NO对抗精子抗体 ( As Ab)阳性大鼠精子自发顶体反应的影响。方法 :采用主动免疫法建立 As Ab阳性大鼠动物模型 ;浅盘凝集实验和 ELISA法检测大鼠血清 As Ab;考马斯亮蓝染色法进行精子顶体染色 ,以观察大鼠自发精子顶体反应率。结果 :As Ab阳性大鼠精子自发 AR%下降 ,且内源性 NO含量、精子内 SOD和 Na+ -K+ ATP酶活性明显低于对照组 ;低剂量 NO( SNP1 0 - 9～ 1 0 - 8mol/L)可提高 As Ab阳性大鼠精子自发顶体反应率、SOD活性 ,但对 Na+ -K+ATP酶活性无影响 ( P>0 .0 5 )。高剂量 NO( SNP1 0 - 6 ～ 1 0 - 4mol/L)则进一步降低上述三项指标。结论 :As Ab阳性大鼠自发顶体反应率降低可能与精子内 NO生成减少、O2 - ·产生增多 ( SOD活性降低 )有关。低浓度 NO可以灭活过量的超氧化物而提高 As Ab阳性大鼠精子自发顶体反应率 ;但过高浓度 NO则损害精子的功能     

A comparative study of raw and prepared semen samples from two consecutive days.

OBJECTIVE: To study and compare raw and prepared semen samples from two consecutive days by conventional sperm parameters, various motility characteristics of spermatozoa determined by computer-aided sperm analysis and calcium ionophore-induced acrosome reaction. STUDY DESIGN: Semen samples of male partners in couples undergoing 81 cycles of double intrauterine insemination were studied. The first sample was produced after abstinence of 2-7 days and the second, 24 hours after the first. Both samples were processed by isolate sperm separation medium. RESULTS: Semen volume, sperm concentration and total motile spermatozoa were significantly reduced in day 2 raw and prepared samples, whereas normal morphology, motility characteristics and percentage of acrosome-reacted spermatozoa increased significantly in day 2 inseminated samples as compared to day 1. Oligospermic, asthenospermic and teratozoospermic samples showed a significant improvement in concentration, various motility characteristics and normal morphology of spermatozoa in day 2 samples as compared to day 1. CONCLUSION: Men with normal samples showed improvements in normal morphology and acrosome-reacted spermatozoa, whereas those with subnormal semen samples from day 1 showed a significant improvement in concentration, various spermatozoal velocities and normal morphology on day 2.     

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.     

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.     

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.     

Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay

Human spermatozoa were incubated in culture medium containing human serum albumin (HSA) to promote capacitation, which was monitored by a rapid chlortetracycline (CTC) fluorescence assay. Four CTC fluorescence patterns were readily distinguished, one of which appeared to be correlated with capacitated sperm. When capacitated sperm were treated with either ionophore A23187 or acid-solubilized mouse zonae pellucidae to induce the acrosome reaction, the CTC assay identified acrosome-reacted sperm by lack of fluorescence on the head. Fresh sperm would not undergo the induced acrosome reaction. The percentages of acrosome-reacted sperm identified by the CTC assay in induced and control populations were the same as those identified by the presently used indirect immunofluorescence and triple stain assays.     

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.     

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.     

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa

Background and Aims:   Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa.
Methods:   Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4 h in modified Beltsville thawing solution supplemented with 0, 20 or 40 ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of ¹⁴C-glucose were evaluated during 0–4 h of incubation.
Results:  The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly ( P  < 0.05) at 1–3 h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly ( P  < 0.05) when the spermatozoa were treated with 20 ng/mL relaxin. Viability was not significantly ( P  > 0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of ¹⁴C-glucose were increased in correlation with AR up to 4 h of incubation.
Conclusion:  We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5 : 215–220)     

Sperm immobilization antibodies in infertile male sera decrease the acrosome reaction: A possible mechanism for immunologic infertility

Objective: To study the effect of sperm-immobilizing antibodies from male sera on spontaneous and A23187-induced acrosome reactions (AR). Design: Swim-up spermatozoa obtained from three fertile donors were incubated with 13 sera with sperm-immobilizing antibodies obtained from infertile men and three control sera obtained from healthy fertile males. Sperm acrosomes were examined by staining with pisum sativum agglutinin labeled with fluorescein isothiocyanate (30 µg/ml; Sigma Chemical Co., St. Louis, MO) as spontaneous and A23187 (used at a final concentration of 10 µM; Sigma Chemical Co.) induced. Results: The incidence of spontaneous AR of spermatozoa incubated with antisperm antibody positive male sera (6.2±0.7) was significantly (P<0.001) lower than that of spermatozoa incubated with control sera (10.7±0.5). And the incidence of A23187-induced and -inducible (incidence of induced minus spontaneous) ARs of spermatozoa incubated with sperm antibody-positive male sera (12.4±1.9 and 6.2±1.9) was significantly lower (P<0.001) than that of spermatozoa incubated with control sera (31.0±0.5 and 20.3±0.9). Sperm-immobilizing antibody-positive sera decreased spontaneous, A23187-induced, and inducible ARs. Conclusions: Sperm-immobilizing antibodies from male sera interfere with fertilization by inhibiting the AR.     

Effect of fructose on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa

Aim:  The present study was carried out to investigate the effects of fructose supplementation in glucose containing mTALP medium on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa.
Methods:  Boar spermatozoa were preincubated, swum-up, resuspended and then incubated for 6 h in mTALP medium supplemented with 0, 0.5, or 1.0 mmol fructose in the presence of 5.0 mmol glucose. After completion of the specified incubation period, motility was determined subjectively on the basis of speed of progression and on the type of forward movement of spermatozoa; acrosome status was evaluated by applying a triple staining technique; and in vitro fertilization capability was assessed by acetic–orcein staining.
Results:  The combination of fructose and glucose (0.5 + 5.0 mmol) supplements in mTALP medium improved sperm motility significantly ( P <  0.05), more than glucose alone (5.0 mmol) at 2–6 h of incubation. Acrosome reaction (live spermatozoa) and the sperm penetration rate was increased significantly ( P <  0.05) when the spermatozoa were treated with the combination of fructose and glucose compared with glucose alone, but the incidence of polyspermy was not significantly different between the treatments.
Conclusion:  These results suggest that the combination of glucose and fructose as supplements in mTALP medium improve the progressive motility, acrosome reaction and fertilization capability of boar spermatozoa. (Reprod Med Biol 2006; 5: 255–261)     

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.     

Effect of swim-up sperm washing and subsequent capacitation on acrosome status and functional membrane integrity of normal sperm

OBJECTIVE: Sperm preparation techniques select sperm population with improved sperm motion characteristics. We sought to determine whether the swim-up technique selects spermatozoa with the ability to undergo hypoosmotic swelling, and how swim-up and subsequent capacitation affect the acrosome reaction rate. METHODS: Semen specimens from 15 normal donors were divided into unprocessed, swim-up, and capacitated groups, and sperm motion characteristics, ability to undergo hypoosmotic swelling, and acrosome reaction rate were measured. RESULTS: Sperm motility, viability, and all motion characteristics (except linearity) were significantly increased in both swim-up and capacitated specimens. The ability to respond to hypoosmotic swelling was significantly higher in the spermatozoa isolated by swim-up. The percentage of acrosome-reacted spermatozoa remained unchanged in both unprocessed and swim-up groups, but was significantly higher in the capacitated group. CONCLUSIONS: Swim-up isolates sperm with greater ability to undergo hypoosmotic swelling, but does not change the acrosome reaction rate. In vitro capacitation of spermatozoa selected by swim-up enhances the acrosome reaction rate.     

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.     

Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride

To test the specificity of sperm fractions solubilized by N-acetylpyridinium chloride, human spermatozoa were washed 3 times with phosphate-buffered saline and resuspended in 5 ml of the solubilizing solution. The mixture was ultrasonated for 5 minutes in ice water and then centrifuged for 20 minutes. The supernatant was used for specificity tests. Details of the techniques used are given. Using N-acetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions were extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in 2-dimensional immuno-electrophoresis. In runs against sperm-immobilizing sera, 9 sperm fractions were visualized. In runs against rabbit antihuman spermatozoa serum 11 fractions were found. Most of the antigenic fractions that were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed in many instances. Sperm as well as species specificities were found in some fractions. None of the fractions found could be detected on the surface membranes of intact human spermatozoa. Specific reactivity against a sperm-immobilizing serum was detected but no specific activity against a sperm-agglutinating serum was found. The fraction designated Gi is considered to be the "immobilizing" antigen. The origin of the antigens detectable on the surface membranes of intact spermatozoa was not determined.