Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.

Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro.

Motile human sperm populations were prepared from liquefied semen (5 donors x 3 replicates) using Percoll gradients at 30-60 min post-ejaculation and preincubated in a complex 'synthetic tubal fluid' culture medium (STF) at 37 degrees C under 5% CO2 in air for 6 h. Aliquots of these suspensions were then incubated for a further 2 h in STF containing 0, 5, 10, 25, 50, 75 and 100% (v/v) pooled human follicular fluid (FF). Another aliquot was treated with 10 micron A23187 in STF for 20 min and then incubated in fresh STF medium for a further 2 h to induce maximal acrosome loss. Acrosome reactions were assessed using both the triple-stain technique and fluorescent peanut agglutinin lectin-labelling. Sperm motility and movement characteristics were assessed from videorecordings using digital image analysis (CellSoft). Exposure to FF caused only relatively small proportions of the preincubated spermatozoa to undergo acrosome reactions. The size of these responsive sub-populations was smaller than that capable of responding to a Ca2+ influx generated by A23187. Increased FF concentrations induced a progressive loss of motility and trends for changes in movement characteristics that may have been related to reduced intracellular Ca2+. This interpretation of these observations is that while FF may act to stimulate or promote the human sperm acrosome reaction it does not appear to be a specific inducer of it. Consequently, a precise role for FF at the relatively low concentrations that would be expected to be present in the tubal ampulla in the physiological regulation of human fertilization remains unproven.

The spontaneous acrosome reaction of human spermatozoa incubated in vitro.

Motile human sperm populations were prepared from liquefied semen (10 donors x 3 replicates) using Percoll density gradients at 30-60 min post-ejaculation. Sperm suspensions were incubated in a complex 'synthetic tubal fluid' culture medium (STF) at 37 degrees C under 5% CO2 in air for up to 36 h. Parallel aliquots were incubated with 50 microM A23187 to induce maximum acrosome loss (ARMAX). Acrosome reactions were assessed using both the triple-stain (TS) technique and fluorescent peanut agglutinin (PNA) lectin-labelling. During incubation, the proportion of TS acrosome reacted spermatozoa increased from 9.1 to 54.3% with ARMAX being 68.3%. Spermatozoa showing intact acrosomes by PNA labelling decreased from 68.4 to 26.1% over 36 h of incubation (ARMAX = 13.8%). Simultaneously, spermatozoa showing complete acrosomal loss (no PNA labelling) increased from 8.1 to 27.0% (ARMAX = 46.3%). Therefore, while only 23.5% of cells were actually undergoing acrosomal changes at the start of incubation, this had increased to 46.9% after 36 h (ARMAX = 40.7%). These experiments clearly show that even in selected populations, not all human spermatozoa are capable of undergoing an acrosome reaction. However, the incidence of acrosomal changes after 36 h of incubation did approach the ARMAX. These levels of spontaneous occurrence of the human sperm acrosome reaction were higher than those reported in many other in-vitro incubation studies: an improvement that may be attributable to the more physiological nature of the STF culture medium.