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

Abstract

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 [Ca2+ 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.

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

“…intracellular Ca 2þ pump in these cells because both functional and immunocytochemical tests failed to show the presence of SERCAs (Harper et al 2005). A similar picture arises from sea-urchin sperm cells, which lack SERCAs.…”

Calcium Channels in the Development, Maturation, and Function of Spermatozoa

“…Western blotting detected secretory pathway Ca 2ϩ -ATPase (SPCA1) in human spermatozoa, but not SERCA, and immunostaining showed that SPCA1 is localized in the head-tail junction, where the Ca 2ϩ oscillations are observed (238). A fluorescently labeled ryanodine binds to the same sites (238). These results strongly suggest that SPCA1 is involved in refilling Ca 2ϩ during the Ca 2ϩ oscillations.…”

“…Low concentrations (50 -100 M) of ryanodine accelerated the oscillations, while high doses (500 M) reduced them. TMB-8 and tetracaine, inhibitors of Ca 2ϩ release from Ca 2ϩ stores, arrested the oscillations, suggesting the importance of store emptying/refilling cycles in this process (238). The progesterone-induced oscillations are relatively insensitive to thapsigargin or cyclopiazonic acid, inhibitors of SERCA, but sensitive to bis-phenol, a nonspecific Ca 2ϩ -ATPase inhibitor.…”

“…The progesterone-induced oscillations are relatively insensitive to thapsigargin or cyclopiazonic acid, inhibitors of SERCA, but sensitive to bis-phenol, a nonspecific Ca 2ϩ -ATPase inhibitor. Western blotting detected secretory pathway Ca 2ϩ -ATPase (SPCA1) in human spermatozoa, but not SERCA, and immunostaining showed that SPCA1 is localized in the head-tail junction, where the Ca 2ϩ oscillations are observed (238). A fluorescently labeled ryanodine binds to the same sites (238).…”

Biochemical Identification of Dynein-ATPase Activity in Human Sperm

“…Since vanadate inhibits the dynein-ATPase and also the sperm fl agellar motility (Gibbons et al, 1978;Kobayashi et al, 1978), the vanadate-sensitive Mg 2+ -ATPase activity of the axoneme-containing fractions shown in our results might represent the dynein-ATPase activity of the human sperm. The remaining vanadate-insensitive Mg 2+ -ATPase activity of the preparations is probably the result of the activity of different ATPases and phosphatases present in the human sperm (Harper et al, 2005;Tomes et al, 2004).…”