First published online 8 October 2008
doi: 10.1242/dev.024521
Development 135, 3677-3686 (2008)
Published by The Company of Biologists 2008
Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract
Gisela Machado-Oliveira1,2,
Linda Lefièvre2,3,
Christopher Ford2,3,
M. Belen Herrero4,
Christopher Barratt5,
Thomas J. Connolly2,3,
Katherine Nash1,2,
Aduen Morales-Garcia1,2,
Jackson Kirkman-Brown2,3 and
Steve Publicover1,2,*
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.
View larger version (75K):
[in this window]
[in a new window]
|
Fig. 1. Expression of eNOS in human oviductal and cumulus cells.
(A,C,E) Staining of human oviductal (ampullary) primary
culture (A), human cumulus (C) and human granulosa cell line (COV 434; E) for
eNOS. (B,D,F) Corresponding phase-contrast images of
these samples.
|
|
View larger version (63K):
[in this window]
[in a new window]
|
Fig. 2. NO mobilises stored Ca2+ in sperm. (A) Spermine
NONOate causes a slowly developing rise in [Ca2+]i in
human sperm. Responses of four separate cells are shown. Red trace shows
example of a cell generating [Ca2+]i oscillations.
(B) In low-Ca2+ medium ([Ca2+] 5 µM), the
response to NONOate was similar, but oscillations were rarely seen. Responses
of seven cells are shown. (C) Pseudocolour image series showing
NONOate-induced rise in [Ca2+]i in the sperm
neck/midpiece. Numbers show minutes since application of 100 µM spermine
NONOate. (D) Mean normalised increase in fluorescence 10 minutes after
application of 100 µM spermine NONOate to cells bathed in sEBSS (271 cells;
three experiments) and low-Ca2+ sEBSS (214 cells; three
experiments). (E) A rapid decrease in [Ca2+]i
followed washout of NONOate, followed by slow recovery. Upon re-introduction
of NONOate, many cells generated oscillations in the neck/midpiece region.
Responses of five individual cells shown. Lower panel shows pseudocolour
images series of a single [Ca2+]i oscillation (numbers
show time in seconds).
|
|
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3. Mobilisation of stored Ca2+ by NO does not involve cGMP.
(A) 100 µM 8-bromo cGMP causes rapid elevation of
[Ca2+]i in human sperm. Responses of six cells are
shown. (B) Response to 8-bromo cGMP is greatly reduced and slowed in
cells exposed to cGMP in low-Ca2+ saline. Responses of five cells
are shown. (C)Ca2+ dependence of the response to 100 µM
8-bromo cGMP. Light-grey bars show responses of cells bathed in sEBSS (72
cells; two experiments); dark-grey bars show cells bathed in
low-Ca2+ sEBSS (122 cells; three experiments). (D)
Pre-treatment with the sGC inhibitor ODQ (10 µM; white bar) does not
inhibit the increase in [Ca2+]i induced by exposure to
100 µM spermine NONOate (arrow). Responses of seven cells are shown.
(E) Mean normalised increase in fluorescence 10 minutes after
application of 100 µM spermine NONOate under control conditions (208 cells;
three experiments) and after pre-treatment with 10 µM ODQ (267 cells; three
experiments). Pre-treatment did not modify the amplitude of the response.
|
|
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4. NO and protein S-nitrosylation in sperm. (A) 100 µM GSNO,
an S-nitrosylating agent, causes a rise in [Ca2+]i
similar to that seen with NONOate but the onset of the effect is more rapid.
Responses of six cells are shown. (B) 100 µM GSH rapidly reverses
the action of 100 µM GSNO on sperm [Ca2+]i. Responses
of five cells are shown. (C) GSNO causes rapid S-nitrosylation of sperm
proteins: lane 1 shows background levels in cells processed immediately for
assay (indicated by *); lane 2 shows that, after 60 minutes of
incubation of the cells in sEBSS, this level does not change; lanes 3, 4, 5, 6
and 7 show increased S-nitrosylation in cells processed for assay immediately
upon exposure to 50 µM GSNO (*), and those incubated with GSNO
for 5, 10, 30 and 60 minutes, respectively. S-nitrosylation reaches near
steady-state levels in the sample processed immediately ( 5 minutes for
preliminary centrifugation; see Materials and methods). (D)
S-nitrosylation of sperm proteins is rapidly reversible. Left panel shows
S-nitrosylated proteins in untreated cells incubated for 10 minutes (lane 1),
cells exposed to GSNO and cys-SNO (lanes 2 and 4), and cells exposed to GSH
and exhausted cys-NO (lanes 3 and 5; controls). Right panel shows same
treatments but cells were washed in PBS immediately before processing for the
assay. S-nitrosylation caused by GSNO and CSNO is rapidly reversed upon
removal of the agent.
|
|
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5. Thiol reducing agents reverse NO effects. (A) DTT rapidly
reverses nitrosylation of sperm proteins. Lane 1 shows endogenous
S-nitrosylation in cells incubated in sEBSS for 60 minutes. Lanes 2 and 3 show
cells incubated in the presence of 1 mM GSH (control) and 100 µM GSNO. Lane
4 shows cells incubated as for lane 3 but 1 mM DTT was added to the incubation
5 minutes before processing for the assay. (B) DTT reverses the action
of 100 µM spermine NONOate. Upon application of 1 mM DTT, the increase in
fluorescence induced by spermine NONOate is rapidly reduced or completely
reversed. Responses of five separate cells are shown. (C) The
DTT-induced decrease in fluorescence is correlated with the preceding
NONOate-stimulated increase in fluorescence. Scattergram shows data from a
single experiment, representative of five repeats.
R2=0.33. (D) The action of DTT is not due to
e--dependent mitochondrial Ca2+ accumulation. After
application of 100 µM spermine NONOate to mobilise Ca2+, the
cells were exposed to 10 µM CCCP to collapse the mitochondrial inner
membrane potential. The effect of subsequent application of 1 mM DTT resembled
that seen in cells with functioning mitochondria. Responses of five cells are
shown.
|
|
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6. NO production by female reproductive tract cells induces S-nitrosylation
in human sperm. S-nitrosylated proteins were identified using
fluorescently tagged methanethiosulfonate, as described in the text.
Negligible levels of labelling were present in controls but treatment with 100
µM spermine NONOate or GSNO caused clear labelling, particularly at the
back of the sperm head. Incubation of sperm with primary cultures derived from
endometrial or tubal explants (ampulla and isthmus) induced levels of
S-nitrosyaltion at least as much as those seen with NONOate.
|
|
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7. Pre-treatment with 100 µM spermine NONOate potentiates
responses of sperm to 3 µM progesterone. (A) When sperm
were exposed to 3 µM progesterone after pre-treatment with spermine NONOate
(100 µM for 10 minutes), the initial [Ca2+]i
transient was enlarged (in some cells) and significantly prolonged compared
with that seen in control cells (inset shows three single cell responses,
scales as for main plot). Responses of eight cells are shown. (B)
Co-stimulation with spermine NONOate increases the proportion of cells in
which a prolonged [Ca2+]i transient occurs in response
to stimulation with 3 µM progesterone. Data are plotted as a percentage of
cells in each class (defined by [Ca2+]i transient
duration). Control cells (black bars; n=27) were from the same sample
as cells exposed to NO before and during progesterone stimulation (grey bars;
n=69) and cells in which NO was washed off as progesterone was
applied (white bars; 44 cells). (C) Progesterone (3 µM) causes a
brief increase in flagellar displacement. Red line and shading show the
mean±2s.d. of frame-to-frame midpiece displacement during the control
period. Graph shows the response of one cell (representative of over 150 cells
in two experiments). (D) Pre-treatment with spermine NONOate (100
µM) prolonged and intensified the effect of progesterone on flagellar
activity. Red line and shading show the mean±2s.d. of frame-to-frame
midpiece displacement during the control period. The graph shows response of
one sperm cell (representative of over 100 cells in two experiments).
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
