Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

The study of the acrosome reaction (AR) of mammalian species is seriously limited by the fact that putative important macromolecules are completely unable of entering the cell. In order to avoid such a technical limitation we developed a method of permeabilization of human spermatozoa with streptolysin-O (SLO). After swim-up, human spermatozoa from normal donors were incubated under capacitating conditions for 3 h, and the concentration adjusted to 5-10 millions/ml. After permeabilization with SLO at 4ºC, 100 microliters aliquots were used in order to test the following reagents, alone or in different combinations: 1) calcium; 2) calcium ionophore A23187; 3) lysophosphatidylcholine (LPC); 4) GTPgS; 5) GDPbS; 6)[AlF4]-; and 7) BAPTA, a calcium chelator. At least 200 spermatozoa were evaluated by fluorescence microscopy (PSA-FITC). Almost all spermatozoa resulted permeabilized since most of them were dead. Calcium increased AR to levels comparable to those reached by intact spermatozoa treated with well known stimulants like LPC and A23187. Also, G-protein stimulation with GTPgS or AlF increased AR. On the other hand, GDPbS, a G-protein inhibitor, was able of blocking the increase of AR induced by calcium. Our results suggest that permeabilization of human spermatozoa with SLO does not affect the intrinsic capability of the acrosome to respond to usual stimuli. Also, from our data, we reinforce the idea about a G-protein and calcium participating during the AR. It is concluded that the model could be eventually useful to study macromolecules involved on the acrosome reaction of human spermatozoa.

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Poster Number PAyunes0152
Keywords: human spermatozoa, streptolysin-O, G proteins, calcium, permeabilization