Capacitation is a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus-oocyte complex. The attainment of a capacitated state is dependent upon an increase in tyrosine phosphorylation and results in the acquisition of responsiveness to physiological agonists such as progesterone and ZP3. In this study we have shown that this capacitation-dependent increase in tyrosine phosphorylation is controlled by a unique redox-regulated, cAMP-mediated, signal transduction cascade. Either stimulation of reactive oxygen species generation or elevation of intracellular cAMP induced increases in phosphotyrosine expression by human spermatozoa and enhanced their responsiveness to progesterone. Ultimate convergence of the redox- and cAMP-regulated pathways was indicated by the ability of the protein kinase A inhibitor, H89, to block both modes of signal transduction. Furthermore, the fact that the redox-regulated pathway could be silenced by catalase, while this enzyme had no effect on the cAMP-mediated response, indicated that oxidant generation must lie upstream from cAMP in the reaction sequence. In keeping with this conclusion, a functional association was demonstrated between the redox status of human spermatozoa and their cAMP content. The continuous production of reactive oxygen species was also shown to be necessary for the protein kinase A-tyrosine phosphorylation axis to remain functional. If the generation of oxidising conditions during capacitation was prevented with 2-mercaptoethanol, 2-deoxyglucose or the flavoprotein inhibitor, diphenylene iodonium, then cAMP could no longer trigger tyrosine phosphorylation. These data support a model for human sperm capacitation as a redox-regulated process, involving a unique sequence of interactive events including reactive oxygen species production, elevation of intracellular cAMP, stimulation of protein kinase A and the induction of tyrosine phosphorylation. This is the first report of such a signal transduction cascade and may have implications for the functional significance of reactive oxygen metabolites in other cell types.

Adeoya-Osiguwa
S. A.
,
Fraser
L. R.
(
1996
).
Evidence for Ca2+-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm.
Mol. Reprod. Dev
44
,
111
120
Ahuja
K. K.
(
1985
).
Inhibitors of glycoprotein biosynthesis block fertilization in the hamster.
Gamete Res
11
,
179
189
Aitken
R. J.
,
Clarkson
J. S.
(
1987
).
Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
J. Reprod. Fertil
81
,
459
469
Aitken
R. J.
,
Buckingham
D.
,
Brindle
J.
,
Gomez
E.
,
Baker
G.
,
Irvine
D. S.
(
1995
).
Analysis of sperm movement in relation to the oxidative stress created by leucocytes in washed sperm preparations and seminal plasma.
Human Reprod
10
,
2061
2071
Aitken
R. J.
,
Buckingham
D. W.
,
Harkiss
D.
,
Fisher
H.
,
Paterson
M.
,
Irvine
D. S.
(
1996
).
The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation.
Mol. Cell. Endocrinol
117
,
83
93
Aitken
R. J.
,
Buckingham
D. W.
,
Huang
G.-F.
(
1993
).
Analysis of the responses of human spermatozoa to A23187 employing a novel technique to monitor the acrosome reaction.
J. Androl
14
,
132
141
Aitken
R. J.
,
Buckingham
D. W.
,
Irvine
D. S.
(
1996
).
The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated.
Endocrinology
137
,
3999
4009
Aitken
R. J.
,
Buckingham
D. W.
,
West
K. M.
(
1992
).
Reactive oxygen species and human spermatozoa; analysis of the cellular mechanisms involved in luminol-and lucigenin-dependent chemiluminescence.
J. Cell. Physiol
151
,
466
477
Aitken
R. J.
,
Fisher
H.
,
Fulton
N.
,
Knox
W.
,
Lewis
B.
(
1997
).
Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine.
Mol. Reprod. Dev
47
,
468
482
Aitken
R. J.
,
Paterson
M.
,
Fisher
H.
,
Buckingham
D. W.
,
van Duin
M.
(
1995
).
Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function.
J. Cell Sci
108
,
2017
2025
Blackmore
P. F.
,
Beebe
S. J.
,
Danforth
D. R.
,
Alexander
N.
(
1990
).
Progesterone and 17-hydroxyprogesterone: novel stimulators of calcium influx in human sperm.
J Biol. Chem
265
,
1376
1380
Carrera
A.
,
Moos
J.
,
Ning
X. P.
,
Gerton
G. L.
,
Tesarik
J.
,
Kopf
G. S.
,
Moss
S. B.
(
1996
).
Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation.
Dev. Biol
180
,
284
296
Demaurex
N.
,
Downey
G. P.
,
Waddell
T. K.
,
Grinstein
S.
(
1996
).
Intracellular pH regulation during spreading of human neutrophils.
J. Cell Biol
133
,
1391
1402
Florman
H. M.
(
1994
).
Sequential focal and global elevations of sperm intracellular Ca2+are initiated by the zona pellucida during acrosomal exocytosis.
Dev. Biol
165
,
152
164
Fraser
L. R.
,
Herod
J. E.
(
1990
).
Expression of capacitation-dependent changes in chlorotetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycolysable substrate.
J. Reprod. Fertil
88
,
611
621
Galantino-Homer
H.L.
,
Visconti
P.E.
,
Kopf
G. S.
(
1997
).
Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3,5 -monophosphate-dependent pathway.
Biol. Reprod
56
,
707
719
Gomez
E.
,
Buckingham
D. W.
,
Brindle
J.
,
Lanzafame
F.
,
Irvine
D. S.
,
Aitken
R. J.
(
1996
).
Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress and sperm function.
J. Androl
17
,
276
287
Griveau
J. F.
,
Renard
P.
,
Le Lannou
D.
(
1995
).
Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process.
Int. J. Androl
18
,
67
74
Hecht
D.
,
Zick
Y.
(
1992
).
Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro.
Biochem. Biophys. Res. Commun
188
,
773
779
Heffetz
D.
,
Zick
Y.
(
1989
).
H2O2potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells.
J. Biol. Chem
264
,
10126
10132
Henderson
L. M.
,
Banting
G.
,
Chappell
J. B.
(
1995
).
The arachidonate-activable, NADPH oxidase-associated H+channel-evidence that gp91-phox functions as an essential part of the channel.
J. Biol. Chem
270
,
5909
5916
Hirono
A.
,
Beutler
E.
(
1989
).
Alternative splicing of human glucose-6-phosphate dehydrogenase messenger RNA in different tissues.
J. Clin. Invest
83
,
343
346
Hyne
R. E.
,
Garbers
D. L.
(
1979
).
Regulation of guinea pig sperm adenylate cyclase by calcium.
Biol. Reprod
21
,
1135
1142
Kleinveld
H. A.
,
Sluiter
W.
,
Boonman
A. M. C.
,
Swaak
A. J. G.
,
Hack
C. E.
,
Koster
J. F.
(
1991
).
Differential stimulation of oxygen-free-radical-altered immunoglobulin G of the production of superoxide and hydrogen peroxide by human polymorphonuclear leucocytes.
Clin. Sci
80
,
385
391
Langlais
J.
,
Roberts
K. D.
(
1985
).
A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa.
Gamete Res
12
,
183
224
Leclerc
P.
,
De Lamirande
E.
,
Gagnon
C.
(
1996
).
Cyclic adenosine 3,5 monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility.
Biol. Reprod
55
,
684
692
Leclerc
P.
,
De Lamirande
E.
,
Gagnon
C.
(
1997
).
Regulation of protein-tyrosine phophorylation and human sperm capacitation by reactive oxygen derivatives.
Free Rad. Biol. Med
22
,
643
656
Leyton
L.
,
Saling
P. M.
(
1989
).
95 kD sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.
Cell
57
,
1123
1130
MacLeod
J.
(
1943
).
The role of oxygen in the metabolism and motility of human spermatozoa.
Am. J. Physiol
138
,
512
518
Meizel
S.
,
Turner
K. O.
(
1996
).
Chloride efflux during the progesterone-initiated human sperm acrosome reaction is inhibited by lavendustin A, a tyrosine kinase inhibitor.
J. Androl
17
,
327
330
Monteiro
H. P.
,
Stern
A.
(
1996
).
Redox regulation of tyrosine phosphorylation-dependent signal transduction pathways.
Free Rad. Biol. Med
21
,
323
333
Naftalin
R. J.
,
Rist
R. J.
(
1993
).
The relationship between sugar metabolism transport and superoxide radical production in rat peritoneal macrophages.
Biochim. Biophys. Acta
1148
,
39
50
Nanda
A.
,
Romanek
R.
,
Curnutte
J. T.
,
Grinstein
S.
(
1994
).
Assessment of the contribution of the cytochrome b moiety of the NADPH oxidase to the transmembrane H+conductance of leukocytes.
J. Biol. Chem
269
,
27280
27285
Osman
R. A.
,
Andria
M. L.
,
Jones
A. D.
,
Meizel
M.
(
1989
).
Steroid induced exocytosis: the human sperm acrosome reaction.
Biochem. Biophys. Res. Commun
160
,
828
34
Parrish
J. J.
,
Susko-Parrish
J. L.
,
Uguz
C.
,
First
N. L.
(
1994
).
Differences in the role of cyclic adenosine 3,5 -monophosphate during capacitation of bovine sperm by heparin or oviduct fluid.
Biol. Reprod
51
,
1099
1108
Segal
A. W.
(
1995
).
The NADPH oxidase of phagocytic cells is an electron pump that alkalinizes the phagocytic vacuole.
Protoplasma
184
,
86
103
Shibanuma
M.
,
Kuroki
T.
,
Nose
K.
(
1988
).
Superoxide as a signal for increase in intracellular pH.
J. Cell. Physiol
136
,
379
383
Spungin
B.
,
Breitbart
H.
(
1996
).
Calcium mobilization and influx during sperm exocytosis.
J. Cell Sci
109
,
1947
1955
Suter
D.
,
Chow
P.Y.W.
,
Martin
I. C. A.
(
1979
).
Maintenance of motility in human spermatozoa by energy derived through oxidative phosphorylation and addition of albumin.
Biol. Reprod
20
,
505
510
Tan
C. M.
,
Xenoyannis
S.
,
Feldman
R. D.
(
1995
).
Oxidant stress enhances adenylyl cyclase activation.
Circ. Res
77
,
710
717
Traxinger
R. R.
,
Marshall
S.
(
1990
).
Suitability of 2-deoxyglucose for measuring initial rates of glucose uptake in isolated adipocytes.
Biochem. Int
22
,
607
615
Visconti
P. E.
,
Moore
G. D.
,
Bailey
J. L.
,
Leclerc
P.
,
Connors
S. A.
,
Pan
D.
,
Olds-Clarke
P.
,
Kopf
G. S.
(
1995
).
Capacitation in mouse spermatozoa, II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.
Development
121
,
1139
1150
Yanagimachi
R.
,
Yanagimachi
H.
,
Rogers
B. J.
(
1976
).
The use of zona free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa.
Biol. Reprod
15
,
471
476
Wassarman
P.
(
1988
).
Zona pellucida glycoproteins.
Ann. Rev. Biochem
57
,
415
442
White
D. R.
,
Aitken
R. J.
(
1989
).
Relationship between calcium, cAMP, ATP and intracellular pH and the capacity to express hyperactivated motility by hamster spermatozoa.
Gamete Res
22
,
163
178
Zeng
Y.
,
Oberdorf
J. A.
,
Florman
H. M.
(
1996
).
pH regulation in mouse spermidentification of Na+-dependent, Cl -dependent, and HCO3-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation.
Dev. Biol
173
,
510
520
This content is only available via PDF.