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First published online 8 October 2008
doi: 10.1242/dev.024521
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1 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
2 Centre for Human Reproductive Science, Birmingham Women's Hospital, Birmingham
B15 2TG, UK.
3 Reproductive Biology and Genetics Research Group, The Medical School,
University of Birmingham, Birmingham B15 2TT, UK.
4 Center for Research in Contraceptive and Reproductive Health, Department of
Cell Biology, PO Box 800732, University of Virginia, Charlottesville, VA
22908, USA.
5 Division of Maternal and Child Health Sciences, Medical School, University of
Dundee, Ninewells Hospital, Dundee DD1 9SY, UK.
* Author for correspondence (e-mail: s.j.publicover{at}bham.ac.uk)
Accepted 22 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Calcium, Cumulus, Motility, Nitric oxide, Oviduct, Sperm, Human
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Miki et al., 1977;
Braughler et al., 1979;
Ahern et al., 2002). It is a
small, uncharged molecule and therefore highly diffusible. However, NO is a
free radical and its reactivity is such that the lifetime of the molecule is
brief, with biological effects probably being confined to a volume with a
radius of 100-200 µm from the point of synthesis
(Lancaster, 1997). Though the
action of NO is of particular importance in the cardiovascular tissue [where
it was first called endothelium-derived relaxing factor (EDRF)] and nervous
system (Calebrese et al., 2007; Li and
Moore, 2007; Rastaldo et al.,
2007), it is now clear that NO plays a role in most tissue and
cell types. The expression of NOS has been demonstrated both in cells of the
male and female mammalian reproductive tracts and also in gametes of
vertebrates and invertebrates, leading to the suggestion that NO may play
important physiological roles in fertilisation
(Creech et al., 1998;
Rosselli et al., 1998;
Thaler and Epel, 2003;
Kim et al., 2004).
In mammalian sperm, production of NO endogenously and/or by cells of the
female reproductive tract may contribute to capacitation, inducing tyrosine
phosphorylation by mechanisms involving and/or independent of the cAMP-protein
kinase A pathway (Funahashi,
2002; Thundathil et al.,
2003; O'Flaherty et al.,
2004; O'Flaherty et al.,
2005; O'Flaherty et al.,
2006; Roy and Atreja,
2008). NO also induces or contributes to induction of acrosome
reaction (Revelli et al.,
2001; Funahashi,
2002; Herrero et al.,
2003; O'Flaherty et al.,
2004; Yang et al.,
2005). With regard to effects of NO on motility of mammalian
sperm, a number of studies have shown that application of NO in vitro has
functional effects, but the data here are complex. Treatment with NO donors at
high doses, or prolonged exposure to NO, suppresses motility, probably
simulating cytotoxic effects that may occur in the testis or in sperm held in
semen (Herrero et al., 1994;
Weinberg et al., 1995;
Zhang and Zheng, 1996;
Joo et al., 1999;
Calabrese, 2001;
Wu et al., 2004). However, low
concentrations of NO may stimulate motility
(Herrero et al., 1994;
Zhang and Zheng, 1996;
Calabrese, 2001). In this
context, the study of Creech et al.
(Creech et al., 1998) on sperm
of the Fathead Minnow (Pimephelus promelus) is particularly interesting. In
ova of this fish, NOS is localised to the micropyle, the route of entry into
the oocyte for the sperm. NO is produced during a crucial 5 minute period
after laying of the eggs and enhances sperm motility. The spatial and temporal
`precision' of the NO signal thus potentially plays a key role in
fertilisation in this species (Creech et
al., 1998). Reports that NOS is present in the mammalian oviduct
(Rosselli et al., 1996;
Ekerhovd et al., 1999;
Lapointe et al, 2006), and
also in the oocyte and the cumulus and corona cells that surround it
(Hattori et al., 2001;
Reyes et al., 2004;
Tao et al., 2004), raise the
intriguing possibility that NO plays a similar role in mammalian
fertilisation, regulating sperm motility or even inducing chemotaxis
(Miraglia et al., 2007).
Participation of Ca2+, a key regulator of sperm motility and
hyperactivation (Darszon et al.,
2007; Publicover et al.,
2007), in modulation by NO has not yet been investigated. Here, we
report that NO is a potent regulator of Ca2+ signalling in human
sperm, acting synergistically with progesterone, and we propose a role for NO
in regulating the interaction of human gametes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surplus cumulus cells were obtained during intracytoplasmic sperm injection (ICSI) cycles performed at The Assisted Conception Unit, Birmingham Women's Hospital [Human Fertilization and Embryology Authority (HFEA) Centre 0199].
COV434 cells (immortalised human granulosa cell line; a gift from the
Clinical Oncology Unit, LUMC, The Netherlands)
(Zhang et al., 2000) were
grown in DMEM-F12 (10% foetal bovine serum; 1% penicillin/streptomycin; 1% non
essential amino acids) at 37°C, 5% CO2.
Detection of NOS in human female reproductive tract and cumulus
Loose human cumulus from oocyte retrieval were stored in phosphate-buffered
saline (PBS) at 4°C. Cells were then smeared onto standard microscope
slides, air-dried and fixed with 100% methanol (-20°C for 6 minutes). The
slides were treated with 50% (v/v) methanol in PBS (20°C for 5 minutes)
and washed three times in 0.1% (v/v) Triton X-100 in PBS and subsequently
re-hydrated with PBS for 15 minutes (20°C).
COV434 cells were released by scraping and centrifuged at 300 g for 5 minutes at room temperature. Cells were then resuspended in PBS, smeared onto standard microscope slides, air-dried and fixed with 4% formaldehyde for 6 minutes at room temperature then permeabilised using 0.2% Triton X-100 for 15 minutes and washed with 0.1% (v/v) Triton X-100 in PBS.
Ampullary explants were washed in Hanks balanced salt solution (HBSS, Gibco) before being incubated with 0.25% collagenase type I (Gibco: 17100-017) in Dulbecco's phosphate-buffered saline (DPBS) w/o CaCl2 or MgCl2 (Gibco 14190) for 1 hour at 37°C with gentle agitation. The supernatant was collected and pelleted by centrifugation at 500 g for 5 minutes. This was then plated in DMEM F12 supplemented with 150 pg/ml 17β-oestradiol and left to adhere and grow at 37°C in 6% CO2 for 2 days. These cells formed a monolayer and retained functioning cilia. Fixation/permeabilisation was as for COV434 cells.
Slides were blocked in 1% (w/v) bovine serum albumin (BSA), 5% (v/v) goat serum in PBS (30 minutes, 37°C in 5% CO2 in air) then incubated with rabbit polyclonal anti-eNOS, -nNOS or -iNOS (1:50 dilution in 1% (w/v) BSA in PBS, 37°C in 5% CO2 in air, 60 minutes). Slides were washed with PBS then secondary antibody [donkey anti-rabbit Texas Red or FITC, 1:200 dilution in 1% (w/v) BSA in PBS] was applied (37°C in 5% CO2 in air for 60 minutes). Finally, slides were washed and coverslips mounted using DakoCytomation fluorescence mounting medium.
Detection of NO production in cumulus and oviductal epithelium
Human cumulus masses and ampullary explants were washed in supplemented
Earle's Balanced Salt Solution (sEBSS) and incubated in the dark at 37°C
and 6% CO2 with 5 µM 4,5-diaminofluorescein (DAF)-FM diacetate
for 30 minutes. Excess DAF-FM was removed by three washes in sEBSS and the
cumulus was transferred to microscope slides under a cover slip supported on
spots of vacuum grease so as to compress it gently. The slides were examined
under a Nikon inverted fluorescence microscope (488 nm excitation/540 nm
emission).
Sperm preparation and capacitation
Donors were recruited at the Birmingham Women's Hospital (HFEA Centre
0119), in accordance with the Human and Embryology Authority Code of Practice.
All donors gave informed consent (LREC 2003/239) and sperm were obtained by
direct swim-up into sEBSS (pH 7.3-7.4) with 0.3% BSA and adjusted to 6 million
cells/ml (Kirkman-Brown et al.,
2000). Sperm were allowed to capacitate at 37°C and 5%
CO2 for 5-6 hours. For the biotin switch assay, semen was layered
over 1 ml fractions of 45 and 90% Percoll [made isotonic with M medium
1x: 137 mM NaCl, 2.5 mM KCl, 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM glucose].
Samples were centrifuged (2000 g for 20 minutes), further
washed with PBS, then diluted and incubated in PBS.
Single cell imaging
Cell density was reduced to 4 million cells/ml and 200 µl aliquots were
then loaded with 12 µM Oregon Green BAPTA (OGB) 1-AM (0.6% dimethyl
sulfoxide (DMSO), 0.12% Pluronic F-127) for 40 minutes and transferred to an
imaging chamber (180 µl), incorporating a coverslip coated with 1%
poly-D-lysine (PDL), for further 20 minutes (all at 37°C and 5%
CO2). The imaging chamber was then perfused with fresh medium
(25°C) to remove unattached cells and excess dye. All experiments were
performed at 25±1°C, with a perfusion rate of 
0.4 ml/minute.
Cells were imaged with a Nikon TE200 inverted fluorescence microscope. Images
were obtained every 10 seconds using a x40 objective and a Hamamatsu
Orca 1 cooled CCD camera controlled by iQ software (Andor Technology, Belfast,
UK).
Data were processed offline using iQ as described previously
(Kirkman-Brown et al., 2000).
For each cell, Microsoft Excel was used to calculate the mean and 95%
confidence interval of fluorescence intensity for (1) 14 images during the
control period (Con ± con), (2) 14 images from minute 3 after
treatment (A ± a) and, (3) 14 images from minute 6 after
treatment (B ± b). At each sampling point the response was
considered significant if:
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Assay of sperm protein S-nitrosylation and visualisation of S-nitrosoproteins
S-nitrosylation of proteins in human spermatozoa was assessed using the
biotin switch assay as described previously
(Lefièvre et al.,
2007).
To visualise S-nitrosoproteins in sperm exposed to female reproductive
tract-synthesised NO, sperm (50 million cells/ml) were incubated with fresh
human tubal and endometrial explants (fragments 3 mm3) in 50
µl DMEM F12 medium (Gibco # 11320), supplemented with 150 pg/ml
17β-oestradiol (Sigma, E8875) at 37°C in 5% O2/6%
CO2 balance N2 for 2 hours. Sperm were then retrieved
and fixed on slides using 4% formaldehyde and S-nitrosoproteins were detected
using a method adapted from Yang and Loscalzo
(Yang and Loscalzo, 2005), as
described previously (Lefièvre et
al., 2007). This method depends on blocking thiols with a
thiol-reactive agent (MMTS) followed by reduction of S-nitrosothiols with
ascorbate, and labelling with fluorescently tagged methanethiosulfonate
(MTSEA).
Flagellar activity assessment
Samples were prepared and capacitated as described above and spermatozoa
were introduced into the chamber and observed under phase-contrast microscopy.
Loosely attached cells with a freely motile flagellum were then selected to
assess flagellar activity (images acquired at 1 Hz). Using the mid-point of
the midpiece as a reference point, frame-to-frame displacement was measured
throughout the experiments using the ImageJ MTrackJ plugin and plotted against
time.
Materials
sEBSS contained (in mM): 1 NaH2PO4, 5.4 KCl, 0.81
MgSO4.7H2O, 5.5
C6H12O6, 2.5
C3H3NaO3, 19 CH3CH(OH)COONa, 1.8
CaCl2.2H2O, 25 NaHCO3 and 116.4 NaCl (pH
7.3-7.4, 285-295 mOsm). In Ca2+-free sEBSS, CaCl2 was
replaced with NaCl (118.4 mM). Fatty acid-free BSA was from SAFC Biosciences
(Lenexa, KS, USA; catalogue number 85041C-50G).
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OGB 1-AM was from Invitrogen Molecular Probes (Paisley, UK). PDL was from BD Biosciences (Oxford, UK). Protease inhibitor cocktail tablets were from Roche Diagnostics (Lewes, East Sussex, UK) and EZ-Link Biotin-N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (EZ-Link Biotin-HPDP) was from Perbio Science UK (Cramlington, Northumberland, UK). Nitrocellulose membrane was supplied by GE Healthcare UK (St Giles, Bucks, UK), IgG Fraction Monoclonal Mouse Anti-Biotin was supplied by Jackson ImmunoResearch Laboratories (Stratech Scientific, Soham, Cambridgeshire, UK) and Lumi-GLO, an enhanced chemiluminescence kit, was from Insight Biotechnology (Wembley, Middlesex, UK).
All other chemicals referred in the text were from Merck Biosciences (Beeston, Nottingham, UK), except progesterone, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), DMSO, Pluronic F-127 and 8-bromoguanosine-3',5'-cyclophosphate sodium salt (8-bromo cGMP), which were from Sigma-Aldrich (Poole, Dorset, UK).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10%) became
stained by DAF (see Fig. S3 in the supplementary material).
NO donors cause elevation of human sperm [Ca2+]i
Expression of active NOS in oviduct and cumulus indicates that sperm will
encounter NO as they approach the oocyte. To investigate the possible effect
of this stimulus on sperm [Ca2+]i (which regulates
motility), we applied NO donors to sperm loaded with OGB. Spermine NONOate
(100 µM) caused a gradual but significant rise in
[Ca2+]i (73±5% of cells; n=8). The
latency of the effect was 0-2 minutes and fluorescence typically stabilised at
20% above control levels after 10 minutes
(Fig. 2A). In 19% of cells,
oscillations were superimposed on the NO-induced elevation of
[Ca2+]i (Fig.
2A, red trace). This action of NONOate was dose dependent:
application of 1 µM induced a discernible increase in fluorescence in only
25% of cells. When NONOate concentration was subsequently raised to 10 µM,
the majority of cells (>70%) showed a gradual increase in fluorescence of
1-30% over 5-10 minutes. Subsequent application of 100 µM NONOate caused
little further increase in fluorescence but clearly enhanced the occurrence of
[Ca2+]i oscillations (see Fig. S4 in the supplementary
material). Similar results were obtained in three experiments. To determine
whether this rise in [Ca2+]i was dependent primarily
upon influx of Ca2+ at the plasmalemma, we incubated cells in
saline with no added Ca2+ (Fig.
2B), where [Ca2+]
5 µM
(Harper et al., 2004). Under
these conditions, a slow elevation in [Ca2+]i occurred
in 64±12% of cells (n=4; NS compared with sEBSS) but
oscillations were rarely seen. The localisation (primarily neck/midpiece,
spreading into the posterior head) (Fig.
2C) and mean amplitude (Fig.
2D) of the response to NO were similar under the two conditions.
Upon washout of spermine NONOate, [Ca2+]i fell rapidly
but then showed a partial recovery in many cells. When the NONOate was
reintroduced, most cells again responded, the increase in
[Ca2+]i being more pronounced and usually occurring as a
series of oscillations in the neck/midpiece
(Fig. 2E).
Mobilisation of Ca2+ by NO is not dependent on stimulation of guanylate cyclase
The `classic' target for NO in its role as a messenger is soluble guanylate
cyclase (sGC). Though cGMP (and therefore sGC activity) is low in mammalian
sperm, there is evidence that effects of NO on acrosome reaction and possibly
other sperm functions are exerted through this pathway
(Herrero et al., 1998;
Revelli et al., 2001). To
investigate whether sGC might mediate NO-induced elevation of
[Ca2+]i, we first examined the effects of the
membrane-permeant analogue 8-bromo cGMP (100 µM). Upon application of
8-bromo cGMP, OGB fluorescence increased (77±9% of cells; n=4)
to a plateau, stabilising after 2-3 minutes
(Fig. 3A). However, unlike the
action of NONOate, when the experiments were repeated in low-Ca2+
saline the effect of cGMP, though detectable (63±9% of cells;
n=3), was reduced in amplitude by over 70%
(Fig. 3B,C). Thus, cGMP does
elevate [Ca2+]i in human sperm but appears to do so by
increased Ca2+ influx rather than by mobilisation of intracellular
stores. To confirm that the response to NONOate was not due to activation of
sGC, we used the sGC inhibitor ODQ. Approximately 10 minutes pre-treatment
with 10 µM ODQ, a dose in excess of that required to inactivate the enzyme
(Schrammel et al., 1996;
Garthwaite et al., 1995),
exerted no inhibitory effect on the response to NO
(Fig. 3D,E). We repeated these
experiments using 100 µM ODQ and again a clear response to NO was apparent
(data not shown).
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|
). S-nitrosylglutathione (GSNO), a membrane-impermeant
protein S-nitrosylating agent (Ji et al.,
1999; Zaman et al.,
2006) can act at intracellular targets, probably by generation of
the membrane permeant product cys-NO
(Zhang and Hogg, 2004). Upon
application of 100 µM GSNO, a significant elevation of
[Ca2+]i occurred in 70±5% of sperm
(n=7). This effect was rapid in comparison to the action of NO,
reaching a plateau in 3 minutes in most cells
(Fig. 4A).
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), but
formation of membrane permeant product cys-NO, leading to direct
S-nitrosylation of intracellular proteins is suppressed
(Zhang and Hogg, 2004). When
GSH was applied in the presence of GSNO there was a rapid fall in
[Ca2+]i (Fig.
4B). We conclude that mobilisation of Ca2+ by GSNO is
by a `direct' action to nitrosylate target proteins in the sperm.
Kinetics of sperm protein S-nitrosylation parallel those of Ca2+ mobilisation
[Ca2+]i responses to GSNO were rapid (3 minutes
to peak) (Fig. 4A). Reversal of
[Ca2+]i elevation in the presence of GSH was similarly
rapid (Fig. 4B), as was
reduction in [Ca2+]i upon washout of spermine NONOate
(Fig. 2E). We therefore
investigated the kinetics and reversibility of protein S-nitrosylation in
sperm exposed to GSNO. When sperm were processed for the biotin switch assay
immediately after exposure to 50 µM GSNO (5 minutes for preliminary
centrifugation; see Materials and methods), S-nitrosylation was already at
steady-state, with further incubation (up to 60 minutes) having very little
effect (Fig. 4C). Conversely,
when cells incubated under S-nitrosylating conditions were washed in PBS,
S-nitrosylation was immediately reversed
(Fig. 4D).
Mobilisation of Ca2+ by NO and GSNO are reversed by DTT
Dithiothreitol (DTT) is a cell-permeant thiol-reducing agent that, even at
low doses (1 mM), effectively reverses biological effects induced by protein
S-nitrosylation (Stoyanovsky et al.,
1997). After a 1 hour exposure of intact sperm to 100 µM GSNO,
application of 1 mM DTT caused complete reversal of S-nitrosylation within 5
minutes (Fig. 5A). Similarly,
when 1 mM DTT was applied to sperm 10-15 minutes after exposure to spermine
NONOate (when mobilisation of Ca2+ by NO was well established), we
observed a rapid fall in [Ca2+]i. Mean fluorescence
(Rtot) fell to 5% above control levels and some cells returned
to levels recorded before application of NO
(Fig. 5B). Similar effects were
seen in four other experiments. The amplitude of the fall in fluorescence
induced by DTT was correlated with that of the preceding NONOate-induced rise
(Fig. 5C), consistent with an
action of DTT to reverse the effect of exposure to NO. In most cells, there
was then a small increase in fluorescence of 5-10% over the following 10
minutes. A rapid reversal of the action of GSNO on
[Ca2+]i also occurred upon application of 1 mM DTT (not
shown).
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;
Pariente et al., 2001;
McStay et al., 2002) and might
thus contribute to the observed [Ca2+]i responses. We
therefore exposed NO-treated cells to 10 µM carbonyl cyanide
m-chlorophenylhydrazone (CCCP) prior to DTT exposure to collapse the
mitochondrial inner membrane potential and mobilise mitochondrial
Ca2+ (Konji et al.,
1985). In most cells, there was a visible increase in
[Ca2+]i upon CCCP application, consistent with
participation of mitochondria in [Ca2+]i buffering
(Wennemuth et al., 2003) and
many of the cells then showed [Ca2+]i oscillations
(Fig. 5D, red and blue traces).
When 1 mM DTT was then applied to these cells, recovery of
[Ca2+]i occurred as before, despite the inability of
uncoupled mitochondria to accumulate Ca2+
(Fig. 5D).
The efficacy of DTT to reduce [Ca2+]i was such that
we investigated whether an effect could be seen in cells not previously
exposed to NO. In most cells (63±7%, n=3) this was the case,
but the amplitude of this effect was small (5%). In most of these cells,
there was then a slight recovery (2%) over the following 5 minutes (not
shown).
Incubation of sperm with human oviduct explants causes protein S-nitrosylation
To determine whether NO production by tissues encountered by the sperm is
sufficient to induce protein S-nitrosylation, we incubated sperm with human
oviduct explants. Sperm retrieved from these incubations and processed for
labelling of S-nitrosothiols (Lefievre et
al., 2007) showed levels of labelling equivalent in intensity and
distribution to that induced by parallel incubation with 100 µM GSNO and
slightly greater than that seen with 100 µM NONOate
(Fig. 6). Sperm incubated with
oviduct showed higher levels of sperm S-nitrosylation (labelling with MTSEA)
than did those incubated with endometrium.
Interaction of the Ca2+-mobilising effects of NO and progesterone
We have shown previously that progesterone cyclically mobilises
Ca2+ stored in a membranous compartment in the sperm neck/midpiece
region (Harper et al., 2004;
Harper and Publicover, 2005;
Bedu-Addo et al., 2007), an
effect that involves activation of ryanodine receptors (RyRs)
(Harper et al., 2004)
(reviewed by Harper and Publicover,
2005). RyRs are known to be positively regulated by
S-nitrosylation (Stoyanovsky et al.,
1997; Meissner,
2004) and RyR2 was identified in the nitrosoproteome of human
sperm (Lefièvre et al.,
2007). As the action of NO on sperm [Ca2+]i
is by S-nitrosylation (leading to mobilisation of stored Ca2+),
interaction or synergism between the effects of these two agents, both of
which will be encountered by sperm approaching the oocyte, might be
anticipated.
|
)
(Fig. 7A, insert). When cells
were pre-treated with 100 µM spermine NONOate (10 minutes) then exposed to
progesterone in the continued presence of the NO donor, this response were
clearly altered. Although 2- to 3-minute transients were still observed
(Fig. 7A, brown and yellow
traces), the majority of cells showed a [Ca2+]i plateau
(Fig. 7A, red and green traces)
or a normal peak with a pronounced `shoulder'
(Fig. 7A, orange, pink and blue
traces). Analysis of transient duration (from start of rise to inflexion at
end of falling phase) showed that after exposure to NONOate prolonged
responses became much more common (Fig.
7B). In three pairs of experiments (control and NO treated cells
from the same sample) (see Fig.
7B), pre-treatment with NONOate increased the proportion of
responses of at least 2.5 minutes from 42±8% to 92±3%
(P<0.025, paired t-test). To investigate whether the
effect of pre-treatment with NONOate would persist in the absence of the NO
donor, we carried out parallel experiments in which NONOate was washed off
simultaneously with the introduction of 3 µM progesterone. In these
experiments, responses to progesterone resembled those of non-pretreated cells
(Fig. 7B): the proportion of
transients of at least 2.5 minutes in two experiments were 40% and 45% (not
significant compared with parallel experiments without pretreatment; paired
t-test).
Synergism of NO and progesterone in regulating flagellar beat mode
Mobilisation of Ca2+ stored in the neck/midpiece region of human
sperm by progesterone or by 4-aminopyridine causes an increase in midpiece
bending and flagellar displacement, which is clearly visible in loosely
tethered cells (Harper et al.,
2004; Bedu-Addo et al.,
2007; Bedu-Addo et al.,
2008). We therefore imaged cells under phase contrast (1 Hz
acquisition rate) to assess the midpiece (and thus flagellar) displacement. In
70% of cells exposed to 3 µM progesterone, there was a brief (30-50
seconds) increase in frame-to-frame flagellar displacement
(Fig. 7C) (representative cell
from over 150 cells in two experiments), consistent with increased flagellar
activity during the [Ca2+]i transient
(Fig. 7A, insert). When cells
were treated with 100 µM spermine NONOate for 10 minutes, there was no
significant effect on flagellar beat mode. Subsequent application of 3 µM
progesterone (in the continued presence of the NO donor) did not alter the
proportion of cells showing a response (80%), but the enhancement of
flagellar activity (measured as an increase in frame-to-frame midpiece
displacement) was maintained for the duration of recording (4 minutes),
including a series of peaks (Fig.
7D) (representative cell from over 100 cells in two experiments).
The kinetics of this increase in midpiece displacement were consistent with
those of the enhanced [Ca2+]i response to progesterone
seen in sperm pre-treated with NONOate
(Fig. 7A,B). Supplementary
movies show examples of cells responding to progesterone in the presence and
absence of 100 µM spermine NONOate.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1000-fold reduction in
[Ca2+]o) (Fig.
2B,D), so this effect reflects mobilisation of stored
Ca2+ by NO.
The primary actions of NO in target tissues are: (1) activation of soluble
guanylate cyclase (sGC), leading to a rise in [cGMP] and actions mediated
through PKG or through direct action on cyclic nucleotide-gated channels; and
(2) direct modulation of protein function by S-nitrosylation of exposed
cysteine residues (Davis et al.,
2001; Ahern et al.,
2002). It has been suggested that NO acts as a chemoattractant for
human sperm, acting through stimulation of sGC
(Miraglia et al., 2007) and
when we exposed human sperm to cGMP, we observed a sustained rise in
[Ca2+]i not dissimilar to that seen with NO. However,
this effect was clearly dependent on [Ca2+]o, consistent
with generation by Ca2+ influx and not store mobilisation
(Fig. 3A-C). Furthermore,
saturating doses of ODQ, an effective inhibitor of sGC, did not modify the
response to NO (Fig. 3D,E). By
contrast, induction of AR by NO was blocked by the same drug (W. C. Ford,
unpublished). We have shown recently that NO, at the concentrations used here,
causes S-nitrosylation of a number of sperm proteins
(Lefièvre et al.,
2007), suggesting that this alternative effect of NO could
underlie our observations. Consistent with this interpretation we found
that:
) acted
similarly to NO but more rapidly (Fig.
4A). ;
Lefievre et al., 2007) showed
that, like the effect of GSNO on [Ca2+]i, GSNO-induced
S-nitrosylation was rapid, occurring in 5 minutes or less (the minimum period
that could be assessed using the assay)
(Fig. 4C) and was immediately
reversed by washout of GSNO (Fig.
4D) or co-application of 1 mM DTT
(Fig. 5A). ) but will
inhibit generation of membrane permeant cys-NO, which is required for protein
S-nitrosylation by GSNO in intact cells
(Zhang and Hogg, 2004). We conclude that DTT reverses the action of NO and that NO-induced mobilisation of stored Ca2+ reflects direct modulation of protein function by S-nitrosylation. It is of interest that, when NONOate was washed off and reintroduced after 5-10 minutes, the effect of NO was apparently enhanced, particularly the generation of [Ca2+]i oscillations (Fig. 2E). Protein S-nitrosylation reverses rapidly upon washout of NONOate (Fig. 4D) so this persistence of effect may reflect increased Ca2+ leak at the plasmalemma (and consequent filling of the store), perhaps owing to increased [cGMP].
Progesterone mobilises Ca2+ stored in the neck/midpiece of human
sperm, by a mechanism involving activation of RyRs, leading to
[Ca2+]i oscillations strikingly similar to those
described here (Harper et al.,
2004) (Fig. 2A).
RyRs are localised to the neck/midpiece
(Harper et al, 2004;
Lefièvre et al., 2007)
and we have shown recently that RyR2 is a target for S-nitrosylation in human
sperm (Lefièvre et al.,
2007). As S-nitrosylation (or S-oxidation by HNO) of RyRs
increases open probability of these channels and mobilises microsomal
Ca2+ (Stoyanovsky et al.,
1997; Cheong et al.,
2005), we suggest that an action on these receptors is the most
likely cause of the Ca2+-mobilising abilities of NO and GSNO in
human sperm. Consistent with convergence of the actions of progesterone and
NO, pre-treatment of cells with spermine NONOate prolonged significantly the
[Ca2+]i transient induced by 3 µM progesterone
(Fig. 7A,B). This effect was
dependent upon the continued presence of NO, with no synergism being observed
when the NO donor was washed off simultaneously with introduction of
progesterone. In effect, this means that the actions of NO are reversed within
2.5 minutes (duration of the `control' action of progesterone), consistent
with the rapid reversibility of protein S-nitrosylation in sperm.
Though potential sources of NO are present throughout the female
reproductive tract, it is probable that NO encountered by sperm in the
fallopian tube and upon approaching and entering the cumulus oophorus provides
a particularly potent stimulus (Rosselli
et al., 1996; Ekerhovd et al.,
1999; Hattori et al.,
2001; Reyes et al.,
2004; Tao et al.,
2004; Lapointe et al,
2006). Human cumulus samples expressed constitutive forms of NOS
(as did COV434 human granulosa cells) and all three NOS isoforms were present
in the oviduct (Fig. 1; see
Figs S1 and S2 in the supplementary material). Co-incubation of human sperm
with human oviductal explants was at least as effective in inducing
S-nitrosylation as was the exposure of sperm to spermine NONOate or GSNO
(Fig. 6). Thus, the reversible
NO-induced mobilisation of Ca2+ stored in the neck region of the
sperm, which we describe here, can occur in vivo. The recent observation that
NO induces chemotaxis (Miraglia et al.,
2007), though of great interest, is most unlikely to relate to our
findings. The effect was at a dose of GSNO 500-1000x lower than that
used here. Chemotactic effects are highly concentration specific, being lost
when the concentration of the attractant is increased above the effective
dose. Furthermore, chemotaxis was exerted through activation of sGC (mimicked
by cGMP and sensitive to ODQ). The effect described here is seen with 50-100
µM GSNO (and 100 µM NONOate) and is exerted through protein
S-nitrosylation (not mimicked by cGMP, insensitive to ODQ).
Our observation that progesterone, which is also present in the female
reproductive tract and is synthesised by cells of the cumulus
(Chian et al., 1999;
Mingoti et al., 2002;
Yamashita et al., 2003), can
act synergistically with NO to mobilise Ca2+ is intriguing.
Progesterone has been reported to have a weak hyperactivating effect on human
sperm (Uhler et al., 1992;
Yang et al., 1994;
Jaiswal et al., 1999). In the
presence of NO, this effect might be expected to be enhanced, reflecting the
increased duration of [Ca2+]i elevation that occurs
under these circumstances. Examination of cells exposed to progesterone in the
presence of NO confirmed that, though 100 µM spermine NONOate alone had
little effect on activity of the flagellum, the transient action of
progesterone on flagellar beating was transformed into a prolonged enhancement
characterised by increased excursion of the midpiece
(Fig. 7D). We propose that,
within the oviduct, the synergistic actions of NO (by S-nitrosylation) and
progesterone to mobilise stored Ca2+ in the sperm neck/midpiece
(probably by activation of RyRs) will modulate flagellar activity,
particularly bending in the midpiece
(Bedu-Addo et al., 2008),
contributing to the hyperactivation that is vital for penetration of the egg
vestments.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3677/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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