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First published online April 5, 2005
doi: 10.1242/10.1242/jcs.02297

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Secretory pathway Ca²⁺-ATPase (SPCA1) Ca²⁺ pumps, not SERCAs, regulate complex [Ca²⁺]_i signals in human spermatozoa

Claire Harper^1,2,*, Laura Wootton¹, Francesco Michelangeli¹, Linda Lefièvre², Christopher Barratt^2,3 and Stephen Publicover^1,

¹ School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
² Reproductive Biology and Genetics Research Group, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
³ Assisted Conception Unit, Birmingham Women's Hospital, Edgbaston, Birmingham, B15 2TG, UK

Author for correspondence (e-mail: s.j.publicover{at}bham.ac.uk)

Accepted 31 January 2005

The sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitors thapsigargin (0.1-1 µM) and cyclopiazonic acid (10 µM), failed to affect resting [Ca²⁺] in human spermatozoa. Slow progesterone-induced [Ca^{2+ i}]_i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca²⁺ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 µM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca²⁺]_i and partial, dose-dependent disruption of oscillations. A 10-40 µM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca²⁺ ATPases, caused elevation of resting [Ca²⁺]_i and inhibition of [Ca²⁺]_i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca²⁺. Low doses of bis-phenol had marked effects on [Ca²⁺]_i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca²⁺ influx induced by progesterone is not mediated via mobilisation of Ca²⁺ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca²⁺ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca²⁺ store(s) and store-dependent [Ca²⁺]_i oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca²⁺ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca²⁺ ATPases contribute at least part of this non-SERCA Ca²⁺ pump activity.

Key words: Sperm, Ca²⁺-store, SERCA, SPCA, Thapsigargin, bis-phenol

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