Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Modification of human sperm function by human follicular fluid: a review

This review assesses whether human follicular fluid (hFF) is able to modify human sperm function in vitro. Addition of hFF has been found to stimulate the motility of washed human spermatozoa, to increase the percentage of hyper-activated spermatozoa, to induce the acrosome reaction, to attract spermatozoa to the site of fertilization and to facilitate penetration of the oocyte by spermatozoa. It is possible that hFF could provide a favourable environment around the oocyte for fertilization by spermatozoa. Inclusion of hFF in gamete transfer medium may also improve the success rate of assisted reproduction technology. Purification of individual components in hFF which modify different aspects of sperm function awaits further investigation.     

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P 相似文献   

Hemizona assay (HZA) demonstrates effects of characterized mouse antihuman sperm antibodies on sperm zona binding.

Characterized antihuman sperm monoclonal antibodies from mice were evaluated using the hemizona assay (HZA) to determine whether sperm:zona binding was effected. The seven monoclonal antibodies were characterized using human sperm in agglutination, immobilization, and penetration assays. Semen was provided by four fertile men and used in the HZA to determine if the presence of a monoclonal antibody would affect tight binding of the sperm to the zona pellucida. Pre-incubation of MA-14 for 1 h with the sperm induced a 33-54% reduction of the number of tightly bound sperm. This antibody reacts to an antigen located on the acrosome and midpiece. Experiments in which there was no pre-incubation of the antibody with sperm, resulted in no significant reduction in the number of sperm bound in the HZA. These findings suggest that an anti-human sperm antibody produced in mice can modulate sperm:zona binding. Reduction in zona binding could indicate a cause of immune-related infertility and this test may be useful in selecting an antigen for contraceptive vaccine development.     

Extracellular signal-regulated kinase activation involved in human sperm-zona pellucida binding

In a previous study involving the inhibition the mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), we found that the very specific MAPK kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced acrosome reaction. As an intact acrosome on the spermatozoa is a prerequisite in ensuring tight binding to the ZP, we investigated the zona binding potential of spermatozoa after PD098059 treatment of sperm, followed by exposure to solubilised human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa, exposed to solubilised ZP, bound significantly more to the ZP, as compared to control spermatozoa also exposed to solubilised ZP (26.5 +/- 3.7 vs. 13.8 +/- 2.8, P < 0.05). No significant differences in binding to the ZP were observed between PD098059 treated and untreated sperm populations after A23187 exposure. These results can be interpreted to support the idea that the ZP-induced AR is the physiologically relevant exocytotic event, as it is the ZP-induced AR, and not the spontaneous (culture medium) or A23187 induced AR, that appears to be mediated through an ERK-mediated signal transduction process.     

Assessment of sperm function in fertile and infertile men

Summary. The sperm function of fertile men (control), infertility patients (experimental), and men with varicocele were compared. The bioassays used were the follicular fluid-induced acrosome reaction, the binding to the zona pellucida, and the penetration of zona-free hamster oocytes. The percentage (mean ± SEM) of reacted spermatozoa was 35 ± 3 in the control, 22±1 in the experimental, and 22 ± 3 in the varicocele. The minimum value of acrosome reaction in control men was 20%. The mean number of zona-bound spermatozoa was 250 ± 30 in the control, 160 ± 28 in the experimental, and 196 ± 44 in the varicocele. The minimum number of zona bound spermatozoa in control men was 50. The mean number of hamster oocytes penetrated was 50 ± 8 in the control, 19 ± 3% in the experimental, and 10 ± 3 in the varicocele. The minimum number of oocytes penetrated in control men was 6%. In the experimental group, 22 men had a normal sperm function, 58 had 1 or 2 bioassays below the minimum (relative dysfunction), and 10 had all bioassay below the minimum (abnormal sperm function). The results of these bioassays could help to reclassify the infertile men in several subgroups.     

Relationship between semen characteristics, a-glucosidase and the capacity of spermatozoa to bind to the human zona pellucida

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind sperniatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of α-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low α-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.     

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution.

The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.     

Binding of 17-beta-estradiol to the outer surface and nucleus of human spermatozoa.

The binding of 3H-17-beta-estradiol to human ejaculated spermatozoa and to its subcellular structures was studied. The binding kinetics of the labeled steroid to whole spermatozoa followed a parabolic pattern. Scatchard-type plots showed the presence of high-affinity binding sites (1.56 +/- 0.23 X 10(4) per sperm cell) with an apparent Kd of 6.6 X 10(-10) M. In competition experiments testosterone was partially effective in decreasing 17-beta-estradiol binding, whereas progesterone and 17-alpha-estradiol were ineffective. Study of membrane fractions obtained from estradiol-labeled spermatozoa showed that under saturating conditions 75-84% of the bound steroid was bound to sperm membranes. Nuclear fractions obtained from estradiol-labeled spermatozoa showed only 10% of the total bound radioactivity. When isolated sperm nuclei were incubated in the presence of the purified receptor-17-beta-estradiol complex obtained from the high speed supernatant of human uterus almost no transfer of radioactivity to the nuclei was observed.     

The influence of a mineral oil overlay on the zona pellucida binding potential of human spermatozoa

Summary.  The objective of the study was to evaluate the effect of mineral oil on zona pellucida binding potential of human spermatozoa. The study compared zona binding using micro volume droplets under mineral oil as apposed to micro droplets in cryopreservation straws. Spermatozoa from eight proven fertile sperm donors were used. One hundred and fifty five matched hemizonae in 50 μl, 100 μl and 200 μl insemination sperm droplets were co-incubated; (i) under mineral oil and (ii) 0.5 ml plastic cryopreservation straws. The results were analysed to determine the number of the zona bound spermatozoa during each experiment. Microvolumes with an oil overlay had a decrease in sperm bound per hemizona of 38% (mean±SD; 563±415 vs. 921±597), 51% (mean±SD; 392±359 vs. 800±566 sperm) and 18% (mean±SD; 502±369 vs. 618±445) in 200 μl, 100 μl and 50 μl respectively, compared to microvolumes in cryopreservation straws. It was concluded that mineral oil may have some detrimental factors which interfere with zona binding of spermatozoa.     

Human follicular fluid stimulates motility and velocity of washed human sperm in vitro

Human follicular fluid collected by laparoscopic oocyte pick-up during IVF was studied with a computer-assisted semen analyser to evaluate the effect of hFF on human sperm motility and velocity. Freshly ejaculated human sperm were washed with phosphate buffered saline and mixed with either PBS or hFF. At various incubation periods of time, hFF increased both sperm motility and velocity as compared with control (P less than 0.01). After incubation of sperm with hFF at 37 degrees C and 5% CO2 in air for 0, 1, 3, 6, and 12 h, the amplitude increase of motility were 49%, 77%, 330%, 2020%, and 3340% when individual control motility was considered to be 100%. The amplitude increase of curvilinear velocity were 43%, 51%, 67%, 152%, 278%, respectively. Comparison of the motility and velocity of the sperm treated with hFF between 0 and 12 h, showed that hFF preserved both motility and velocity in vitro (P less than 0.01). The stimulatory effect of hFF was retained after boiling at 100 degrees C for 30 min, or after being filtered through Amicon membrane cones, but it disappeared if the hFF had been pre-treated with chymotrypsin. However, hFF did not stimulate the motility and velocity of unwashed sperm in freshly ejaculated human semen. A non-dialyzable and heat-stable factor(s) with a molecular weight below 50,000 in hFF may improve and maintain the motility and velocity of washed human sperm. Whether this factor could be used to improve pregnancy rate in assisted reproduction awaits further investigation.     

Functional aspects of human sperm binding to the zona pellucida using the hemizona assay

The hemizona assay (HZA) has facilitated investigations of sperm function in relation to zona pellucida binding. In this study, the authors examined: 1) the association between hyperactivated sperm motility and HZA binding; 2) the binding kinetics and efficiency of sperm from subfertile men; and 3) the influence of sperm freezing and thawing on binding capacity. For each HZA, a nonviable human oocyte was cut into equal zona hemispheres. The mean number of bound sperm and the incidence of hyperactivation were significantly greater for samples of sperm from fertile men compared with sperm from subfertile men (P less than 0.05). Subfertile sperm had a binding curve that paralleled the curve for fertile sperm, although the magnitude of binding was markedly reduced. Freezing and thawing of sperm from fertile samples impaired their capacity to bind to the zona pellucida. The HZA binding efficiency was reduced by 30%, although the binding curves for fresh versus frozen samples remained parallel.     

Human follicular fluid stimulates the motility of washed human sperm

Human follicular fluid (hFF) was collected by laparoscopic oocyte pickup during IVF to evaluate the effect of hFF on human sperm motility with a transmembrane migration method. Freshly ejaculated human sperm were washed with phosphate buffered saline (PBS) and mixed with either PBS or hFF. Amplitude of motility increases were 38% and 72% in washed fertile sperm and washed asthenozoospermic sperm when individual control motility was considered to be 100%. The stimulatory effect of hFF was lost when preheated at 100 degrees C for 30 minutes. hFF collected from mature follicles stimulated sperm motility better than that collected from intermediate or immature follicles. hFF did not stimulate the motility of unwashed sperm in freshly ejaculated human semen. A heat labile factor(s) in hFF may stimulate the motility of washed human sperm. Whether this factor could be used to improve the success rate of IVF and artificial insemination awaits further investigation.     

Sperm binding to the human zona pellucida and calcium influx in response to GnRH and progesterone

In this study the effect of the sequential exposure of spermatozoa to progesterone and gonadotrophin-releasing hormone (GnRH) upon zona binding and the intracellular free Ca2+ concentration was evaluated. Sperm aliquots were treated as follows: (a) 0.7 micro mol l(-1) progesterone or 0.1% DMSO (progesterone solvent) followed by 50 nmol l(-1) of GnRH; (b) 50 nmol l(-1) of GnRH or distilled water (GnRH solvent) followed by 0.7 micro mol l(-1) of progesterone. Additional aliquots were incubated with DMSO or distilled water (controls) and with 0.7 micro mol l(-1) of progesterone or 50 nmol l(-1) of GnRH. All treatments were for 5 min. Motile spermatozoa were incubated in modified Tyrode's medium, at 37 degrees C, 5% CO2, 10 x 10(6) spermatozoa ml(-1), for 4.5 h. Intracellular Ca2+ concentration and sperm-zona binding was evaluated using fura 2 and the hemizona assay, respectively. GnRH and progesterone increased sperm-zona binding and the Ca2+ concentration. Regarding zona binding, the effect of GnRH was significantly greater when the spermatozoa had been previously treated with progesterone (progesterone-->GnRH=185 +/- 116 zona-bound spermatozoa versus DMSO-->GnRH=99 +/- 15, P < 0.001). On the other hand, previous treatment with GnRH did not modify their subsequent response to progesterone (GnRH-->progesterone= 114 +/- 19 zona-bound spermatozoa versus distilled water-->progesterone=108 +/- 22, NS). The results regarding intracellular Ca2+ showed a similar pattern. These findings suggest a priming effect of progesterone upon a GnRH-induced increase in sperm-zona binding and intracellular Ca2+.     

Changes in sperm phosphotyrosine proteins by human follicular fluid in mice

The effects of follicular fluid on the acrosome reaction (AR) and phosphorylation of tyrosine residues of the sperm proteins were examined in mouse epididymal sperm. Human follicular fluid (hFF) increased AR in the capacitated sperm. Genistein, a receptor tyrosine kinase (RTK) inhibitor, inhibited spontaneous AR. When the genistein was primed, hFF-induced AR was attenuated but the A23187-induced AR was not, suggesting that potentiation of AR by hFF attributed to the activation of RTK upstream the mobilization of Ca2+. Phosphotyrosine proteins of Mr 27 to 116 kDa were markedly increased in capacitated sperm but this increase was abrogated by genistein. hFF increased tyrosine phosphorylation of Mr 56 kDa protein with genistein sensitive manner, suggesting that 56 kDa phosphotyrosine protein might be involved in capacitation and AR by follicular fluid.     

Teratospermia in domestic cats compromises penetration of zona-free hamster ova and cat zonae pellucidae

The ability of spermatozoa to bind and penetrate zona-free hamster ova and the zonae pellucidae of domestic cat oocytes in vitro was compared between normospermic (greater than 60% structurally normal spermatozoa per ejaculate) and teratospermic (less than 40% normal spermatozoa per ejaculate) domestic cats. The effects of culture media (Biggers, Whitten, Whittingham [BWW] versus modified Krebs Ringer bicarbonate [mKRB]) and simple dilution (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU) on penetration also were examined. High percentages of structurally normal spermatozoa were bound to zona-pellucida-free hamster ova regardless of the morphological forms in the inseminant. Mean percent normal spermatozoa bound to ova in DR, NS, and SU sperm aliquots from teratospermic male cats were not different (P greater than 0.05) from similarly treated normospermic aliquots. However, the percent penetration of hamster ova by normospermic ejaculates (10.5%) was superior (P less than 0.05) to that of teratospermic ejaculates (2.8%). Although swim-up processing improved percent sperm motility, progressive motility, and normal morphology in teratospermic ejaculates (P less than 0.05), no difference was observed in ovum penetration among the DR-treated, NS-treated, and SU-treated spermatozoa (P greater than 0.05). Culture medium had no effect on sperm binding in the hamster assay, but ovum penetration rate by spermatozoa in the normospermic ejaculates was enhanced (P less than 0.05) using mKRB (13.5%) when compared with BWW (7.6%) medium. Spermatozoa from teratospermic cats were capable of binding and penetrating cat zonae; however, sperm-zona interaction (defined as percent of oocytes with spermatozoa binding to or penetrating into the zona) was different (P less than 0.05) between normospermic (65.3%) and teratospermic (24.2%) cats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications of surface carbohydrates on bovine spermatozoa mediated by oviductal fluid: a flow cytometric study using lectins

The objective of the present study was to characterize and quantify changes in exposed saccharide residues of bovine sperm during capacitation in oviductal fluid (ODF) using flow cytometry (FC). Bovine sperm were incubated with 0% or 50% non-luteal ODF for 30 min or 3.5 h. After incubation, sperm were labelled with 11 fluorescein isothiocyanate-labelled lectins and evaluated for lectin binding with FC. Furthermore, inhibiting sugars were used to determine specificity of lectin binding to oligosaccharides on the sperm surface. After 30 min incubation, there was a 91% decrease in fluorescence intensity of labelled sperm incubated in WGA, a 76% decline for Con A, 75% decline for BS-I and a 36% decline for DBA. These differences remained approximately the same over the 3.5-h incubation. Interestingly, although there was no reduction in UEA-I binding at 30 min, a significant reduction (23%) was observed at 3.5 h. Con A fluorescence was mostly inhibited with either alpha-d-glucose or alpha-d-mannose (86% and 90% respectively). BS-I fluorescence was reduced after prior incubation of the control samples with N-acetyl-galactosamine and galactose by 74% and 80% respectively. After prior incubation with N-acetyl-galactosamine DBA fluorescence reduced by 18% in the control samples. With UEA-I no fluorescence reduction was observed after prior incubation with l-fucose. We have demonstrated that capacitation of bovine sperm in ODF is accompanied by a quantitative reduction in individual lectin binding sites. These modifications may be crucial to the subsequent signalling events involved with sperm-zona binding, zona penetration or interaction with the oolema.     

Use of microbeads for the detection of binding sites on the human zona pellucida: a scanning electron microscopy (SEM) assay

Summary. One prerequisite for fertilization is the specific binding of spermatozoa to the zona pellucida. However, the factors and mechanisms involved in this gamete contact are not well understood. Gamete recognition and binding are species‐specific and are controlled by oligosaccharides of the zona and their corresponding carbohydrates on the spermatozoon. By using a specific lectin we developed a technique to detect those oligosaccharides on the human zona pellucida that might be involved in the binding process. Microbeads (Ø = 2.8 µm), used as artificial spermatozoa, were coated with lectin Con A and cultured together with 75 unfertilized oocytes (group A) remaining after intracytoplasmic sperm injection. Con A binds specifically to α‐d ‐mannose and α‐d ‐glucose. As a control, 75 unfertilized oocytes after intracytoplasmic sperm injection (group B) were also cultured together with Con A‐covered microbeads, but in a medium containing a binding inhibiting sugar (α‐methyl‐mannopyrasosid). The number and distribution of the microbeads on human oocytes of both groups were analysed on scanning electron microscopy images. Beads on oocytes of group A had binding patterns similar to those of spermatozoa. They were distributed in an extremely heterogeneous way with various numbers of bound beads both on individual and different oocytes. Most of the group A oocytes (85%) had more than 50 beads bound to the zona, in contrast to the control oocytes of group B, where 68% had less than 10 bound beads. The use of an inhibiting sugar abolished the binding capacity of the microbeads nearly completely. This technique is a powerful tool for the detection of binding sites on the zona pellucida, i.e. those sugars that are responsible for contact between spermatozoa and the zona pellucida.     

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.     

Assisted reproductive technologies may obviate apparent immunologic infertility.

Spermatozoal autoantibodies have been associated with reduced fertilization by natural coital methods. Nine subfertile men were evaluated who repeatedly tested positive for spermatozoal autoantibodies as characterized by direct immunobead test. Using the hemizona assay, we determined whether tight binding of spermatozoa to the zona pellucida was reduced in these test males as compared to a fertile male whose semen had been cryopreserved and thawed immediately prior to testing. The average number of spermatozoa tightly bound to the zona pellucida from the subfertile male was significantly reduced compared to the fertile male (mean +/- SD, P less than or equal to 0.01) 19.5 +/- 8 versus 77.1 +/- 49. Seven of the nine couples eventually had successful fertilization using intrauterine insemination or gamete intrafallopian tube transfer and one couple conceived with natural coital insemination. Our findings, albeit limited, suggest that greater caution should be used in implicating associations of spermatozoal autoantibodies with absolute infertility, because novel assisted reproductive technologies often may obviate conventional encumbrances on opportunities for pregnancy.