期刊
JOURNAL OF CELL SCIENCE
卷 118, 期 8, 页码 1673-1685出版社
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jcs.02297
关键词
sperm; Ca2+-store; SERCA; SPCA; thapsigargin; bisphenol
类别
The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 mu M) and cyclopiazonic acid (10 mu M), failed to affect resting [Ca2+](i) in human spermatozoa. Slow progesterone-induced [Ca2+](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 mu M, 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 mu M 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 nonSERCA Ca2+ pump activity.
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