Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC137

Poster Communications

4-Aminopyridine mobilises Ca2+ in human sperm

S. Costello1, S. J. Publicover1, C. L. Barratt2, J. C. Kirkman-Brown3

1. Department of Biosciences, University of Birmingham, Birmingham, United Kingdom. 2. Division of Maternal and Child Health Sciences, Medical School, University of Dundee, Dundee, United Kingdom. 3. Centre for Human Reproductive Science (ChRS) and Assisted Conception Unit, Birmingham Women's NHS Foundation Trust, Birmingham, United Kingdom.

4-Aminopyridine (4-AP; 2mM), a blocker of voltage-activated K+ channels, is one of the most potent inducers of human sperm hyperactivation yet described. The proportion of hyperactivated cells (Sort 7 assessed by CASA) in a population incubated in capacitating medium increases rapidly from 3-5% to 40-50% (Gu et al, 2004). Since data from our own studies and those of other laboratories have shown that [Ca2+]i plays a central role in regulating activity of the flagellum, we have investigated the effect of 4-AP on [Ca2+]i in human sperm using Oregon Green BAPTA. The application of 2mM 4-AP to human sperm caused a dose dependent, tonic increase in [Ca2+]i fluorescence of 34.5% 1.1 s.e.m. in more than 90% of cells. Application of 4-AP to cells bathed in medium with no added Ca2+(≈5*10-6 M) caused, in most cells , a similar response to that of standard medium, though a proportion of cells (10-30%) showed no change in [Ca2+]i. Application of 4-AP in EGTA-buffered saline (<10-8M) induced a clear response in ~25% of cells but [Ca2+]i elevation was transient and was of diminished amplitude. This response was abolished after 10-15 min incubation under these conditions. We used CASA to examine whether mobilisation of stored Ca2+ might contribute to the strong hyperactivating action of 4-AP on human sperm. The ability of 4-AP to increase the proportion of hyperactivated cells was not inhibited by simple omission of Ca2+ from the medium (standard medium 43.3% ± 2.9 s.e.m., 'nominally Ca2+ free' medium 48.0% ± 8.5 s.e.m.). Furthermore, following brief re-suspension in EGTA buffered saline, application of 2mM 4-AP induced hyperactivation in a proportion of cells only slightly lower than controls (39.7% ± 8.25 s.e.m.). However, after incubation in EGTA-buffered medium for 20 mins the proportion of motile cells was decreased and the hyperactivation effect of 4-AP was significantly reduced (19.8% ± 5.3 s.e.m.). Treatment of human sperm with 4-AP did not increase the occurrence of acrosome reaction in cells incubated under capacitating conditions. 4-AP is a weak base and our findings thus might reflect activation of the pH-sensitive Ca2+ current ICatSper. We super-fused sperm with saline adjusted to pH8.5 (replacing standard medium at pH 7.4). This induced a sustained increase in [Ca2+]i, consistent with tonic activation of CatSper channels. This pre-treatment did not occlude the response to subsequent application of 4-aminopyridine. We propose that 4-AP induced elevation of [Ca2+]i and hyperactivation of human sperm reflects the mobilisation of labile, EGTA-sensitive store and also a sustained influx of Ca2+. Our results suggest that the 4-aminopyridine induced Ca2+-influx may not be solely pH/CatSper dependent and that modulation of capacitative Ca2+ influx may play a role in this effect.

Where applicable, experiments conform with Society ethical requirements