1 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK 2 Present address: School of Biological Sciences, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK
3 Corresponding author. E-mail: S.J.PUBLICOVER{at}bham.ac.uk
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Abstract |
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Key words: calcium/motility/oscillation/progesterone/sperm
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Sperm responses to oocyte-derived factors |
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At least two components of the egg vestments, zona pellucida and progesterone (secreted at high concentrations by the cumulus cells), activate Ca2+ signalling in mammalian spermatozoa (Florman et al., 1989; Blackmore et al., 1990)
. The induction of the AR by solubilized zona pellucida has been studied in detail in the mouse model and involves activation of a T-type voltage-operated calcium chanel (VOCC) followed by store mobilization and prolonged (probably) capacitative Ca2+ influx (Florman et al., 1992; Evans and Florman, 2002
). In contrast, the significance of progesterone is much less clear. The responsiveness of human spermatozoa to progesterone correlates with the fertilization rate at IVF (Krausz et al., 1996
; Forti et al., 1999
; Giojalas et al., 2004
), and removal of cumulus cells from oocytes significantly reduces the success rate of IVF in most mammals (Tanghe et al., 2002
; van Soom et al., 2002). However, the most commonly observed effect of progesterone-induced Ca2+ influx, stimulation of the AR, is arguably not an adaptive response. Human spermatozoa respond with elevation of [Ca2+]i and the AR to doses well below the 110 µmol/l that is believed to occur adjacent to the oocyte (Osman et al., 1989
; Baldi et al., 1991
; Harper et al., 2003)
. The AR should therefore occur in many cells before they enter the cumulus, potentially compromising their ability subsequently to penetrate the zona.
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Effect of progesterone on sperm [Ca2+]i |
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The receptor that mediates the response of spermatozoa to progesterone is yet to be characterized, but the rapidity of the response, and the efficacy of progesterone conjugated to bovine serum albumin (BSA) (Meizel and Turner, 1991), lead to the conclusion that it is a cell surface, non-genomic binding site. Rapid, signalling events activated by progesterone (and estrogen) are known to occur in a range of cells, through either activation of a subpopulation of conventional steroid receptors, activation of modified/truncated conventional receptors, activation of novel transmembrane receptors or possibly by allosteric modulation of receptors for other agonists (Edwards, 2005)
. Using an antibody to the C-terminus of the conventional progesterone receptor (C262), two proteins resembling N-terminal truncated receptors have been detected in human spermatozoa (Luconi et al., 1998
, 2002
). This antibody is able to inhibit the progesterone-induced AR (Sabeur et al., 1996
; Luconi et al., 1998
). A progesterone-binding protein unrelated to the nuclear progesterone receptor (mPR), cloned from porcine liver, has also been proposed as the receptor in sperm. Antibodies against this receptor have effects on both the progesterone-induced [Ca2+]i signal and the AR (Buddkikot et al., 1999; Falkenstein et al., 1999
). These authors recently have proposed that porcine spermatozoa possess both mPR and another receptor that is structurally different (Losel et al., 2004
). Attempts to localize the sperm progesterone receptor have employed primarily antibodies against the conventional progesterone receptor or fluorescently tagged progesterone. These studies have identified binding on the head, mostly the acrosomal region (Gadkar et al., 2002
; Huo et al., 2002
; Shah et al., 2005
), though Sabeur et al. (1996)
observed equatorial binding of the C262 antibody. Antisera against the porcine liver progesterone-binding protein (see above) initially localized to a site on the posterior head of human spermatozoa which moved equatorially during capacitation (Buddkikot et al., 1999). The finding that stimulation of human spermatozoa with 3 µmol/l progesterone induces an initial transient that starts in the equatorial region (Meizel et al., 1997
) is consistent with these observations.
Doseeffect and binding assays identify two receptors for progesterone on human spermatozoa. One is poorly specific and has a Kd of 40 µmol/l, but the other is specific for progesterone and has a much higher affinity (Kd in the nmol/l range; Luconi et al., 1998). The progesterone-induced [Ca2+]i response, measured fluorimetrically in populations of human spermatozoa, is dose dependent. An EC50 for the amplitude of the progesterone-induced transient of 50100 nmol/l has been reported (Baldi et al., 1991
, Luconi et al., 1998
; Kumar et al., 2000
; Schaefer et al., 2000
; Harper et al., 2003)
, possibly corresponding to activation of the specific progesterone receptor. We find that both the [Ca2+]i response and induction of the AR saturate at
300 nmol/l progesterone (Harper et al., 2003)
. Doses of 1100 µmol/l progesterone can exert further effects on [Ca2+]i, maybe through the low-affinity receptor (Luconi et al., 1998
). Although such doses might be encountered within the cumulus, the extreme sensitivity of human spermatozoa to progesterone suggests that this hormone will exert major effects at much lower doses, encountered before the sperm contacts the oocytecumulus complex. The stimulus will therefore occur not as a sudden rise in the concentration of progesterone (as is used in pharmacological investigations) but as a gradient generated by diffusion and/or ciliary currents which the cell ascends as it approaches the oocyte (Harper et al., 2004
).
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Response of human spermatozoa to a progesterone gradient |
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To attempt to mimic the stimulus encountered by sperm approaching the oocyte and to understand better what effect remote detection of progesterone by spermatozoa in vivo may have, we investigated the effect of applying progesterone to human spermatozoa as a logarithmic concentration gradient (Harper et al., 2004). As the progesterone concentration increased (gradient raised from 0 to 3 µmol/l over a period of 1525 min), there was a concomitant increase in [Ca2+]i in almost all cells, initiated when progesterone was 110 nmol/l and saturating at micromolar levels, possibly reflecting desensitization of the high-affinity progesterone receptor (Aitken et al., 1996
; Luconi et al., 1998
; Harper et al., 2003
). Interestingly, early transient calcium increases were never seen, suggesting that previous studies, conducted by others and ourselves, using stepped progesterone additions were unrepresentative of responses occurring in vivo. The most striking characteristic of progesterone gradient stimulation was the generation of slow [Ca2+]i oscillations in over one-third of cells, typically starting when the progesterone concentration was between 5 and 50 nmol/l. Signals of this complexity in spermatozoa have been described only recently and were thought to occur only rarely (Fukami et al., 2003
; Wood et al., 2003
; Kirkman-Brown et al., 2004
).
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Generation of [Ca2+]i oscillations |
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Role of progesterone-induced [Ca2+]i oscillations |
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Compartmentalized [Ca2+]I signalling in human spermatozoa |
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Submitted on March 24, 2005; resubmitted on April 29, 2005; accepted on May 24, 2005.
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