Summary. The Ba2+, Mg2+ and Sr2+ (each at 1\m=.\80mm) to substitute for 1\m=.\80 mm-Ca2+ in promoting the AR was also assessed. Of these, only Sr2+ provided a response greater than that observed in unsupplemented Ca2+-deficient medium. In Sr2+ the proportion of responding cells after 24h (\m=~\13%) was similar to that obtained in Ca2+( \ m=\ 1 5 %) , although a majority of those in Sr2+ were at intermediate stages. In 3\m=.\60mm-Sr2+ the response was significantly higher than that observed in both 1\m=.\80mm-Ca2+ and 1 \m=.\80mm-Sr2+, but significantly lower than that in 3\m=.\60mm-Ca2+. Under all conditions motility was maintained at >90% for 24 h. The introduction of the Ca2+ ionophore ionomycin, in the presence of 1\m=.\80mm-Ca2+ , induced the AR in a concentration\x=req-\ dependent but preincubation time-independent manner, with the maximum response of \m=~\60% being obtained with 30 \g=m\m-ionomycin.Finally, incubation in the presence of 1 \m=.\80mm-Ca2+ and verapamil, generally considered to be a calcium channel antagonist, resulted in a concentration-and incubation time-dependent increase in the AR, the maximum response in all groups being observed only after 24 h incubation. Recent evidence from other species suggests that this may represent an agonistic interaction with calcium channels. We conclude that optimal conditions for capacitation and the AR in human spermatozoa require extracellular Ca2+ at > 1 \m=.\80mm. AR. This may have relevance to other reports that zona binding and gamete fusion in the human are lower in the presence of Sr2 + than Ca2 +.

Suspensions of motile sperm were prepared from semen samples donated by 40 men of recent, proven fertility and incubated under capacitating conditions for 24 h. At selected time-points aliquots were removed, assessed for motility, fixed and examined with the electron microscope to determine the rate of acrosome loss. Data indicate that acrosome loss increases significantly with time, but absolute values are relatively low. After 24 h a mean of 15.4% sperm had initiated the acrosome reaction; this figure included 9.7% which had completed it. The proportion of cells at intermediate stages was similar throughout incubation (approximately 5%), indicating that initiation of the acrosome reaction occurs at a fairly constant rate. In four samples motility declined over 24 h and in six, contaminating cells were observed. In the majority of these 10, acrosome loss was higher than that observed in the remaining 30 samples. Additionally, the assessment of greater than 25,000 cells during this study made it possible to evaluate specific ultrastructural features of the normal acrosome reaction in human sperm. Six stages were identified, with the intermediate ones involving loss of acrosomal matrix material while outer membranes appear to retain their integrity; this contrasts sharply with the current view of the generalized mammalian sperm acrosome reaction.

Summary. The effect of human follicular fluid (FF) on the incidence of spontaneous acrosome reactions (AR) in human spermatozoa was examined over a 24\p=n-\25-h period using electron microscopy. Suspensions of motile spermatozoa were prepared by a swim-up method in Earle's medium, known to support in-vitro fertilization. After adjusting the concentration to 10 \m=x\106 cells/ml, suspensions were diluted 1:1 with medium (control) or FF, the latter giving a final concentration of 50% FF. In addition, at 5 h and 24 h an aliquant of the control suspension was removed, diluted 1:1 with FF and incubated for 1 h; the three suspensions were examined at 6 h and 25 h. Continuous exposure to 50% FF stimulated the AR, the effect being significant (P < 0\m=.\001) at 25 h. However, the 1-h short exposure of spermatozoa to FF did not produce an increase in AR, even after 24 h preincubation. In a separate series of experiments, the effect of continuous incubation for 24 h in increasing concentrations of FF was investigated. A significant linear dose-dependent effect on the AR was observed with all concentrations assessed (P < 0\m=.\01for 12\m=.\5%FF and P < 0\m=.\001for 25, 50, 75 and 100% FF, compared with FF-free control). Therefore, human FF can stimulate the AR, but only after a continuous exposure to FF. A short exposure to FF, even after 24 h preincubation, does not trigger an increased AR response.

Oocyte-cumulus complexes were obtained, after induced ovulation, from infertile patients participating in an in-vitro fertilization programme. About 6 h after retrieval and depending on the expansion of the cumulus, 100,000 motile spermatozoa, prepared by a migration-centrifugation method, were added. After 14-18 h incubation at 37 degrees C, oocytes were examined for signs of fertilization (pronuclei and polar body formation) and then removed; spermatozoa remaining in the incubation medium were fixed for transmission electron microscopy. To provide an adequate number of cells for observation, spermatozoa from a minimum of 3-5 oocytes from the same patient were pooled. When sufficient spermatozoa were available after insemination, the remainder of the suspension was incubated at 37 degrees C and fixed along with the corresponding oocyte-incubated sample. In all, 32 sperm samples were assessed and fertilized oocytes were obtained with 29 of these. In the 24 samples in which greater than 100 spermatozoa (mean of 192) could be assessed, 32% of spermatozoa had initiated or completed the acrosome reaction. In the 15 of these 24 samples for which oocyte-free controls were available, 31% of cells were reacting or reacted, compared with 15% of cells (P less than 0.001) in the controls. In the remaining 8 samples, incubated with oocyte-cumulus complexes, less than 100 but greater than or equal to 20 spermatozoa (mean of 42) were assessed and again 32% of spermatozoa were reacted.(ABSTRACT TRUNCATED AT 250 WORDS)

Human sperm were incubated under capacitating conditions and, at selected points up to 24 h of incubation, motile cells were fixed and assessed with the electron microscope for the presence or absence of the acrosome. Two methods of sample preparation were compared. In the first, semen samples were washed, incubated and filtered through glass beads to select motile cells before fixation. In the second, motile sperm were allowed to swim up into medium; this produced greater than 95% motile cells which were then incubated and fixed as required. In both series of experiments a significant increase in acrosome loss with time was observed (P = 0.00013), although only 10.5% of cells had lost the acrosome after 24 h. It is concluded that overt acrosome loss occurs less frequently in human sperm than in those of commonly used laboratory animals.