Summary. The morphology of spermatozoa bound to the zona pellucida (ZP) of 264 oocytes that had failed to fertilize in 58 in vitro fertilization procedures was studied by light microscopy. The percentage of spermatozoa with normal morphology bound to the ZP (mean 77, range 8\p=n-\100) was significantly higher than in the insemination medium (mean 42, range 2\p=n-\80). The abnormal spermatozoa bound to the ZP had small oval (47%), pyriform (46%), amorphous (5%) and tapering (2%) heads. Other abnormalities of morphology were not observed in ZP-bound spermatozoa. The mean rates of binding to the ZP of spermatozoa with normal morphology (44, 95% confidence limits 42\p=n-\46,number bound/ZP/105/ml inseminated) were much higher than for abnormal spermatozoa with small oval 6\m=.\5 (5\ m=. \ 5\ p=n-\ 7\ m=. \ 6), pyriform 5\ m=. \ 9 (4\ m=. \ 8\ p=n-\ 7\ m=. \ 3), amorphous 1\m=.\0 (0\ m=. \ 5\ p=n-\ 2\ m=. \ 0) and tapering 2\m=.\5 (1\ m=. \ 2\ p=n-\ 5\ m=. \ 3) heads. The morphologically abnormal forms not found on the ZP were infrequent in the insemination medium (tail defects, large oval, round, pin and duplicate heads and cytoplasmic droplets) but upper 95% confidence limits for standardized binding ratios of <60% indicated that these were unlikely to bind to the zona with rates approaching those of normal spermatozoa (standardized binding ratio 180%). A large number of uniformly normal spermatozoa bound to the ZP when the percentage normal morphology in the insemination medium was >40%. The proportions of abnormal spermatozoa on the ZP were significantly correlated with the proportions of abnormal spermatozoa in the insemination medium. Spermatozoon head dimensions were measured with a micrometer in samples from 14 patients. While there were no consistent changes in all samples, the means for head width and area were significantly larger and the ratio of the length to width was smaller for spermatozoa on the ZP than for those in the insemination medium. The head length and ratio of length to width of spermatozoa in samples with good morphology were significantly less than in samples with poor morphology. The percentage of spermatozoa with normal morphology, motility and the ratio of head length to width in the insemination medium were positively correlated with the percentage of spermatozoa with normal morphology bound to the ZP. Logistic regression analysis showed that the diagnosis of tubal infertility and the rate of binding of spermatozoa with normal morphology to the ZP were positively related to fertilization rates in vitro while the rate of binding of spermatozoa with pyriform heads was negatively related. In conclusion, human ZP are highly selective for spermatozoa with normal morphology. The frequency of binding of abnormal spermatozoa to the ZP was mainly dependent on semen quality. Detailed analysis of spermatozoa bound to the ZP should be useful for determining the range of morphology characteristics of spermatozoa with good or poor fertilizing potential.

Acrosome reactions induced by the calcium ionophore A23187 and zona pellucida (ZP) were studied. Sperm samples were obtained from fertile men or men with normal semen analysis and normal sperm-ZP binding. Oocytes were obtained, with the consent of the patients, after the failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were incubated with 10 microM A23187 for 1 h, four oocytes for 2 h or solubilized ZP (4 ZP/microliters) for 2 h. Spermatozoa bound to the ZP were dislodged and collected in a small volume of phosphate-buffered saline by aspirating the oocytes with a glass pipette with an inner diameter (120 microns) slightly smaller than the diameter of the oocyte. The acrosome status of the spermatozoa was determined using fluorescein-labelled Pisum sativum agglutinin. The proportion of spermatozoa undergoing the acrosome reaction on the ZP at 2 h varied over a wide range (5-99%), but the agreement between results for the same semen sample exposed to different groups of oocytes was good: the standard deviations of the differences being 9%. Pre-incubation of spermatozoa for 2 h did not increase the ZP-induced acrosome reaction. Re-incubation of ZP with the same sperm suspension for 2 h after removing ZP-bound spermatozoa from the first 2 h incubation produced a significantly lower ZP-induced acrosome reaction in the second incubation (22 +/- 16%) than in the first incubation (30 +/- 14%; P < 0.001, n = 20). There was no significant difference in the ZP-induced acrosome reaction with oocytes with ZP which had or had not been penetrated by spermatozoa during the in-vitro fertilization insemination. Pre-incubation of spermatozoa with solubilized ZP blocked sperm-ZP binding. However, the acrosome reaction induced by solubilized ZP (4 ZP/microliters) was significantly lower than the acrosome reaction induced by intact ZP (10 +/- 5 and 30 +/- 13% respectively, n = 11, P < 0.001), but there was a high correlation (Spearman r = 0.822, P < 0.01) between the results. On the other hand, although the average of the acrosome reaction was similar for A23187 (42%) and for ZP (43%), there was no significant correlation between the results for the two stimuli (n = 60). In conclusion, a useful method for assessing the ZP-induced acrosome reaction has been developed using oocytes which failed to fertilize in vitro. The lack of a relationship between the result of the chemical (A23187) and physiological (ZP) stimuli for the acrosome reaction in the same subjects questions the biological basis of using A23187 for tests of sperm function. Solubilized human ZP in a concentration that blocks sperm-ZP binding is a less efficient inducer of the acrosome reaction than is intact ZP. It is possible that the three-dimensional structure of the ZP is important for induction of the acrosome reaction or that spermatozoa which bind to the ZP are more likely to acrosome react. Assessment of the physiological acrosome reaction for diagnosis of sperm defects which interfere with the fertilization process should be c...

Acrosin was measured in the semen used for sperm preparation for in-vitro fertilization (IVF) in 118 patients. Acrosin levels correlated with the proportion of spermatozoa with normal intact acrosomes determined with Pisum sativum agglutinin labelled with fluorescein. However, acrosin levels and the proportion of spermatozoa with normal intact acrosomes in semen were not significantly related to the fertilization rate in vitro. Only the percentage normal morphology and sperm concentration in the insemination medium were independently significantly related to the fertilization rate by logistic regression analysis. In patients with fewer than 30% of spermatozoa with normal morphology, although acrosin levels were not correlated with the fertilization rate, the proportion of spermatozoa with normal intact acrosomes in the insemination medium was the only significant factor in the logistic regression analysis. In conclusion, acrosin levels have no prognostic value for fertilization in vitro but the proportion of spermatozoa with normal intact acrosomes may be a useful clinical marker of fertilizing ability in men with poor sperm morphology.

The human acrosome reaction was induced with the calcium ionophore A23187 and the proportion of reacted spermatozoa was determined with fluorescein-labelled Pisum sativum agglutinin. Human oocytes that failed to fertilize in vitro were used to test binding of spermatozoa to the zona pellucida (ZP) and oolemma. Differential labelling of spermatozoa with fluorescein and rhodamine was used to control for variability in the oocytes. Spermatozoa labelled with one fluorochrome were treated with A23187 and mixed with equal numbers of motile untreated control spermatozoa labelled with the other fluorochrome. The mixture was incubated with zona-intact and zona-free oocytes for 4 h. The sperm-ZP binding ratio of test to control spermatozoa was significantly decreased with increasing proportions of acrosome reacted spermatozoa. In contrast, the sperm-oolemma binding ratio was significantly increased with A23187 treatment. This suggests that acrosome-reacted spermatozoa do not bind to the human ZP.

To investigate the involvement of protein kinases in signal transduction in the human zona pellucida (ZP)-induced acrosome reaction (AR), the effects of protein kinase (PK) activators, dibutyryl cAMP (PKA) and cGMP (PKG), phorbol 12-myristate 13-acetate (PMA, PKC), and the PKC inhibitor, staurosporine were studied. Sperm samples were obtained from normozoospermic men with normal sperm-ZP binding. Oocytes were obtained from other patients with failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were pre-incubated with 2.5-20 microM PMA, 1 mM dibutyryl cAMP or cGMP, 3 mM pentoxifylline or 0.125-2.0 microM staurosporine for 30 min and then incubated with four oocytes for 2 h in human tubal fluid supplemented with bovine serum albumin. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small bore pipette and the state of the AR was determined by fluorescein-labelled Pisum sativum agglutinin. Motility and movement characteristics were assessed by computer-assisted sperm analysis (CASA) after incubation of spermatozoa with PMA for 30 min and 2 h. The dibutyryl cAMP and cGMP analogues had a small positive effect (P < 0.05) but pentoxifylline had no effect on stimulating the ZP-induced AR (P > 0.05). In contrast, PMA stimulated ZP-induced AR in a marked dose-dependent manner. Only the highest concentrations (15-20 microM) of PMA significantly decreased percentage motility (P < 0.001). Doses of 2.5-15 microM of PMA significantly stimulated ZP-induced AR without decreasing motility (P < 0.001). The PKC inhibitor, staurosporine (0.125-0.25 microM) significantly inhibited ZP-induced AR without affecting motility (P < 0.001). Sperm samples from 33 normozoospermic men were used for studies of the ZP-induced AR augmented with 15 microM PMA. One sample did not show a response to PMA stimulation. Among the 14 men with low ZP-induced AR, half had normal responses to PMA and other half had low responses to PMA. In conclusion, activation or inhibition of PKC significantly increases or decreases human ZP-induced AR suggesting that PKC plays a important role in the signal transduction pathway for the physiological AR.