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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

View larger version (21K): [in a new window]   Fig. 1. Effects of store Ca2+ ATPase inhibitors on resting [Ca2+]i in human sperm populations loaded with Fura-2. Horizontal axes in all traces show time in minutes. (A) Main trace shows effect of sequential additions of 1 µM and 10 µM (total final concentration) thapsigargin (arrows). 10 µM thapsigargin regularly caused a sustained increase in [Ca2+]i of 100-300 nM. In most experiments 1 µM thapsigargin had no discernible effect or apparently caused a slight fall in [Ca2+]i (inset). Occasionally we saw a small sustained increase of 20-30 nM and/or (in two experiments) a small (10-20 nM) transient increase in [Ca2+]i as shown here. (B) treatment of cell populations with 10 µM and 40 µM (total final concentration) bis-phenol (arrows) caused a dose-dependent, sustained increase in [Ca2+]i; the first application usually inducing an initial transient elevation of [Ca2+]i lasting 1-2 minutes. (C) A normal response to 10 µM and 40 µM bis-phenol was seen in cell suspensions that generated no elevation of [Ca2+]i (or a transient decrease) in response to 1 µM thapsigargin. Arrows indicate times of drug additions.

View larger version (41K): [in a new window]   Fig. 2. Effects of store Ca2+-ATPase inhibitors on resting [Ca2+]i in individual human sperm. (A) Responses of eight cells to 1 µM thapsigargin (thaps, bar above traces). Most cells failed to respond at this dose (lines without symbols) but a small proportion (approx 5%) generated a brief increase in fluorescence lasting approximately 1 minute (). Inset shows mean response of all cells in this experiment (thapsigargin added at arrow). (B) Responses of seven cells to 10 µM thapsigargin At this concentration thapsigargin (bar above traces) caused a sustained elevation of [Ca2+]i in 35% of cells, a small proportion generating an initial transient (). An example of a cell that failed to respond to 10 µM thapsigargin is shown (). Inset, mean response of all cells in this experiment (thapsigargin added at arrow). (C) Responses of six cells to 10 µM bis-phenol (bar above traces). Bis-phenol caused a clear rise in [Ca2+]i in 70% of cells. Most cells generated a sustained response that was preceded by a transient elevation (,). Inset, mean response of all cells in this experiment (bis-phenol added at arrow). (D) Responses of four cells to 40 µM bis-phenol (bar above traces) in `Ca2+-free' sEBSS (no added Ca2+). In this saline bis-phenol caused a clear rise in [Ca2+]i in 74% of cells. Responses resembled those in standard EBSS. Upon reintroduction of standard EBSS containing Ca2+ (upward arrow) all cells showed a marked rise in [Ca2+]i. Inset, mean response of all cells in this experiment (bis-phenol added at arrow).

View larger version (44K): [in a new window]   Fig. 3. Effects of store Ca2+-ATPase inhibitors on [Ca2+]i in individual cells after establishment of the sustained phase of the progesterone-induced response. (A) Application of 1 µM thapsigargin (bar above traces) had no effect on [Ca2+]i in most cells (lines without symbols) but in approximately 2% of cells there was a sustained increase in [Ca2+]i (). Responses of 15 cells are shown. (B) 10 µM thapsigargin (bar above traces) was similarly ineffective in most cells (lines without symbols) but induced a sustained elevation of [Ca2+]i in approximately one third of cells (,,). Responses of six cells are shown. (C) 40 µM bis-phenol added 10 minutes after progesterone (arrow) caused a sustained elevation of [Ca2+]i in 74% of cells (lines without symbols), occasionally preceded by a [Ca2+]i transient (), a few cells showing no clear response (). Responses of six cells are shown. (D) After application of 1 µM thapsigargin (bar above traces), subsequent application of 40 µM bis-phenol (bar above traces) caused a sustained rise in [Ca2+]i in 40% of thapsigargin-insensitive cells. Responses of five cells are shown. Trace without symbols is a non-responsive cell.

View larger version (48K): [in a new window]   Fig. 4 . Thapsigargin inhibits [Ca2+]i oscillations at non-SERCA-specific doses. (A) Application of 5 µM thapsigargin to cells generating oscillations after progesterone stimulation had no effect in most instances (). In just 2.5% of these cells there was a failure to clear Ca2+ from the cytoplasm between cycles, such that oscillation amplitude was reduced (). Upon washout of thapsigargin the amplitude of oscillations recovered. (B) Two single cell records from cells stimulated with 3 µM progesterone followed by application of 10 µM thapsigargin. In these two cells oscillations arrested at peak. (C) Records from two cells generating progesterone-induced oscillations which responded to 10 µM thapsigargin as in B. Subsequent application of 10 µM A23187 caused a further significant increase in fluorescence, showing that the `stable' elevation of [Ca2+]i was not an artefact due to saturation of the fluorescent probe. (D) Records from three cells in an experiment in which cells were exposed to a series of increasing doses of thapsigargin. In these cells 10 µM thapsigargin failed to inhibit [Ca2+]i oscillations but raising the concentration to 30 µM caused [Ca2+]i to stabilise at an elevated level.

View larger version (62K): [in a new window]   Fig. 5. Bis-phenol inhibits [Ca2+]i oscillations. Application of 10 µM bis-phenol (bar above traces) to cells in which [Ca2+]i oscillations were already established in response to progesterone caused some cells to arrest immediately (A, records from two cells) or gradually (B, records from two cells). Activity in some cells recovered upon washout of bis-phenol (shown by bar above A). (C) In half of the cells in which oscillations persisted in the presence of 10 µM bis-phenol there was a significant prolongation of [Ca2+]i peaks (records from two cells from same experiment as A and B). (D) Mean oscillation transients (mathematical averages of three oscillation cycles synchronised to peak [Ca2+]i) from two cells. Transients generated before () and during () superfusion with 10 µM bis-phenol (b-p) have been overlaid to show the change in kinetics. Application of the drug extends the [Ca2+]i, peak, apparently revealing two phases of Ca2+ clearance. (E) Application of 30 µM bis-phenol to cells in which oscillations persist in the presence of 10 µM bis-phenol results in immediate arrest at or near peak [Ca2+]i. Records from two cells are shown. (F) Application of bis-phenol (40 µM) caused immediate arrest of [Ca2+]i oscillations in cells superfused with `Ca2+-free' sEBSS (no added Ca2+). Records from two cells are shown. Under these conditions, upon cessation of oscillation [Ca2+]i settled at a level at or below that occurring during oscillation troughs (compare to panels A, B and E).

View larger version (32K): [in a new window]   Fig. 6. Comparison of the potencies of thapsigargin and bis-phenol in their effects on [Ca2+]i oscillations in human spermatozoa, Ca2+ store ATPase activity and mobilisation of stored Ca2+. (A) Dose-dependence of the percentage inhibition by thapsigargin of ATP-dependent Ca2+ uptake by rat cerebellar microsomes () and arrest of progesterone-induced [Ca2+]i oscillations in human spermatozoa (). Results are the mean±s.e.m. percentage of cells continuing to oscillate after application of thapsigargin; three to six experiments were performed. (B) Dose-dependence (expressed as a percentage of the maximum) of thapsigargin-induced elevation of [Ca2+]i in intact HL-60 cells () (Demaurex et al., 1992) and adrenal glomerulosa cells () (Ely et al., 1991) and dose-dependence of the percentage of oscillating cells in which thapsigargin caused arrest (). (C) Dose-dependence of effects of inhibition by bis-phenol of ATP-dependent Ca2+ uptake by rat cerebellar microsomes (), Ca2+ ATPase activity in pig cerebellar microsomes (whole, ; or in the presence of 1 µM thapsigargin, ) and arrest by bis-phenol of progesterone-induced [Ca2+]i oscillations in human spermatozoa (; mean±s.e.m. percentage of cells continuing to oscillate after application of bis-phenol). (D) Dose-dependence of the mobilisation of Ca2+ by bis-phenol in HL-60 cells (in EGTA-buffered Ca2+-free saline; ) (Brown et al., 1994) and arrest of progesterone-induced [Ca2+]i oscillations in human spermatozoa by bis-phenol ().

View larger version (116K): [in a new window]   Fig. 7. Expression and localisation of Ca2+ store ATPases in human spermatozoa. Western blotting was used to detect SERCAs (A) and SPCA1 (B) in rat brain (br) and sperm (sp) lysates. A robust signal, at the appropriate molecular weight, was obtained with the SERCA antibody (Y1F4) in brain lysates but no staining was detected with sperm (three experiments). In contrast, using the same lysates, we detected SPCA1 both in rat brain and in sperm. Though the intensity of the band was considerably lighter in sperm than in brain, the band was found consistently. The blot has two sperm protein lanes to establish that the band was not due to `bleed' from the brain lysate lane. (C-F) In-situ localisation of SERCA and SPCA1 in human spermatozoa. Pictures show overlays of fluorescence and phase-contrast images. All fluorescent images were obtained and processed using identical procedures. Immunolocalisation using antibody Y1F4 showed no significant staining (C) whereas the antibody directed against SPCA1 (D) localised clearly to the rear head and midpiece. Incubation with the secondary antibody alone gave no significant labelling in either case (E and F). Scale bar in C applies to C-F.

View larger version (13K): [in a new window]   Fig. 8. Intracellular manganese clearance in human spermatozoa. One of three repeat experiments in which Fura-2-loaded human spermatozoa were suspended in sEBBSS containing 1 mM MnCl2 and stimulated with progesterone. Measurement of fluorescence at 360 nm (isobestic point for Fura-2) shows rapid quenching of fluorescence due to the initial rapid progesterone-induced Mn2+ influx followed by a slower quench due to the subsequent slower influx. Subsequent addition of 1 mM La3+ (to block Mn2+ influx) not only prevented further quench but resulted in partial recovery of fluorescence. The initial fast phase (see inset showing detail of response upon La3+ application) is probably, at least in part, artefactual (see text). A subsequent slower phase appears to reflect removal of Mn2+ from the cytoplasm, consistent with activity of a Mn2+ transporting pump (such as SPCA1) in spermatozoa. Subsequent application of 40 µM bis-phenol caused a rapid, partial reversal of the fluorescence recovery with an amplitude corresponding to the slow phase.

View larger version (23K): [in a new window]   Fig. 9. Tentative model for intracellular Ca2+ storage and generation of [Ca2+]i signals and cellular responses in progesterone-stimulated human spermatozoa. The initial [Ca2+]i transient that occurs immediately upon application of progesterone is mediated primarily by Ca2+-influx and induces acrosome reaction in a proportion of cells (grey arrow). Ca2+ is removed from the cytoplasm primarily by pumping at the plasma membrane (PMCA and Na+-Ca2+ exchanger, blue). Acrosome reaction may involve mobilisation of Ca2+ stored in the acrosome itself (De Blas et al., 2002; Herrick et al., 2005). Sustained elevation of [Ca2+]i, which follows the transient (or can be induced by a progesterone concentration gradient to simulate approach to the oocyte, a procedure that induces [Ca2+]i oscillations without a preceding [Ca2+]i transient) (Harper et al., 2004), acts on RyRs (red) on a Ca2+ store in the caudal part of the head or midpiece, causing Ca2+-induced Ca2+ release. Cyclic release and re-uptake occur as a result of refilling of this store by bis-phenol-sensitive SPCA1 (green), generating slow [Ca2+]i oscillations at the base of the flagellum and causing alternation of flagellar beat pattern (grey arrow).