Evaluation of the acrosome reaction in human spermatozoa: comparison of cytochemical and fluorescence techniques

Abstract

The acrosome reaction, which is essential for fertilization, includes fusion and vesiculation of the plasma membrane with the outer acrosomal membrane of spermatozoa, thereby releasing the acrosomal content. Determination of the ability of spermatozoa to undergo the acrosome reaction has proved to be a useful parameter in evaluation of infertile patients. The objective of this study was to compare cytochemical techniques, such as double stain (Giemsa/trypan blue) and triple stain (Bismarck brown/rose bengal/trypan blue), with a fluorescence method using Pisum sativum agglutinin fluorescein conjugate and Hoechst dye N degrees 33258 (double fluorescence). Whereas the cytochemical methods are easy to perform in general laboratories, the fluorescence technique requires special and costly instrumentation. In semen obtained from fertile donors, spermatozoa were selected by the swim-up technique and the acrosome reaction was induced by incubation at low temperature. The percentages of vital and acrosome-reacted spermatozoa were determined after incubation at 4 degrees C and at room temperature. No statistically significant difference was found between double fluorescence (viability 86.3%, acrosome reaction 14.7%) and triple stain (viability 85.3%, acrosome reaction 17%) (P > 0.05). On the other hand, the double stain technique showed different values for viability (70.3%) and acrosome reaction (42.5%) (P < 0.05). In conclusion, triple stain yielded results similar to those obtained by the fluorescence technique in evaluating the acrosome reaction and can therefore easily be used in general or research laboratories.

Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods

“…In agreement with our results, Way et al (1995) observed lower percentages of sperm with detached acrosome as well as viable sperm in ejaculated bovine sperm stained with Trypan blue and Giemsa stain, as opposed to samples subjected to FITC-PSA labelling. The results of the present experiments are consistent with the data obtained by Risopatrón et al (2001) on human spermatozoa. In our experiments (Table 1) no significant differences (P > 0.05) were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%).…”

“…Two methods [double staining and Hoechst/FITC-Pisum sativum agglutinin (PSA) labelling] are commonly used for evaluating the acrosome reaction, but contradictory data have been obtained by these techniques. Risopatrón et al (2001) reported a decrease in the number of live and acrosome-reacted human spermatozoa after double staining in comparison with the FITC-PSA method in humans. In contrast, Kőhn et al (1997) found comparable results of viability as well as acrosome reaction rate with human sperm.…”

The Consequence of Short Insemination Strategy on Sperm Biological Characteristics, Embryo Morphokinetics, and Clinical Outcomes in the IVF Program

“…The spermatozoa were stained by 0.8% of Bismarck brown Y (B-2759, Sigma-Aldrich) for 10 min at 40°C, followed by washing with distilled water. Finally, they were incubated in 0.8% rose bengal 0.1 M Tris (R-3877, Sigma-Aldrich) for 40 min at RT, washed in distilled water, dehydrated in alcohol gradients (50%, 70%, 96%), and mounted with a coverslip [18]. Following staining, the intact acrosome showed a pink acrosomal region, while the white acrosomal region was a sign of degeneration.…”

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction

“…It should be noted that induction of the acrosome reaction does not result in the complete simultaneous removal of the acrosome from all such treated sperm cells, and that some cells display intermediate PSA-FITC levels. Sperm cells showing more than two-thirds of the acrosome without fluorescence or with a fluorescing band at the equatorial segment were considered to be AR (Risopatro ´n et al, 2001). A bandlike pattern was detected at the equatorial zone for the PATE and PATE-M proteins in both the AR and the AI sperm cells, whereas this pattern was seen at the post-acrosomal region for the PATE-B protein.…”