Vol. 91, No. 1, 1979 November
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 7-Q
14, 1979
EFFECTS OF CHOLERA TOXIN AND 5'-GUANYLYLIMIDODIPHOSPHATE ADENYLATE CYCLASE ACTIVITY Cheng,
C. Y. and Boettcher,
ON HUMAN SPERMATOZOAL
B.
Department of Biological Sciences, The University of Newcastle, New South Wales, 2308, AUSTRALIA
Received
June
29,1979 SUMMARY
The effects of cholera toxin and 5'-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoa1 adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 ug/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase where the adenylate cyclase would be assay, or was added to lysed spermatozoa, accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 uM was effective in activating human spermatozoa1 adenylate cyclase activity. INTRODUCTION The presence zing)
EC 4.6.1.1.) for
specific
out with
spermatozoa
can be judged
6-8).
hormonal from
cyclases monkey
(3,4),
(3,4),
but
has been demonstrated
human spermatozoa
is
inhibited
number the
acids of other
effects
Abbreviatiggs: buffer (Ca
(9),
but
species
of cholera
this (10,ll).
toxin
Gpp(NH)p,
by sodium could This
(6).
cyclase
(for
review,
cyclase
a number
presents
in results
see
by thyroxine
be demonstrated
and 5' -guanylylimidodiphosphate 5'-guanylylimidodiphosphate;
spermatozo-
can be activated
not be demonstrated report
The attempts
Mammalian
adenylate
fluoride,
(1,Z).
has been carried
hormones
cannot
that
1971
and man (1).
to steroid
such activation
It
(5)
(cycliin
cyclase
successful
adenylate
(5).
carboxylic
bull
marginally
spermatozoa1
demonstrated
of adenylate
seem to be insensitive
and triiodothyronine spermatozoa
only
(ATP pyrophosphate-lyase
was first
activators
the monkey
to have been
However,
cyclase
in human spermatozoa
A search
al adenylate
of adenylate
in bull
from ejaculated
of metallic
ions
spermatozoa
of a
of studies (Gpp(NH)p)
and
on *
on
RF@, Krebs-Ringer-Phosphate
-free). 0006-291 1
Copyright All rights
X/79/21
OOOl -09$01.00/O
@ 1979 by Academic Press. Inc. of reproduction in anyform reserved.
BIOCHEMICAL
Vol. 91, No. 1, 1979
associated ing
constituents
a secretagogue
cellular cologic
to the granule
by numerous
stimuli.
Nevertheless,
such as PMA, whose mode of action we are able
calcium, tool
induced
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
which
will
enzyme
aid
to employ
an agent
in the elucidation
secretory
process
which
is
by utiliz-
independent
of extra-
such as TMB-8 as a pharmaof the true
heretofore
relevance has not
of calcium
been possible.
REFERENCES
:: i: 5. 6. 7. ;: 10.
18. ii: 21. 22. 23. 24. 25. 26. % 29. 30. 31. i5: 34. 35.
Henson, P. M. (1971a) J. Imnunol. 107, 1535-1546. Henson, P. M. (1971b) J. Imnunol. 107, 1547-1557. Hawkins, D. (1972) J. Imnunol. 108, 310-317. Zurier, R. B., Hoffstein, S. and Weissmann, G. (1973) J. Cell Biol. 58, 27-41. Henson, P. M. (1972) Amer. J. Pathol. 68, 593-606. Showell, H. J., Freer,R. J., Zigmond, S. H., Schiffmann, E. Aswanikumar, S Corcoran, B. and Becker, E. L. (1976) J. Exp. Med. 143, 1154-1169. Bajnton, Il. F., Ullyot, J. L. and Farquhar, M. G. (1971) J. Exp. Med. 134, 907-934. Avila, J. L. and Convit, J. (1973a) Biochim, Biophys, Acta 293, 397-408. Spitznagel, J. K., Daldorf, F. G., Leffell, M. S., Folds, J. D., Welsh, I. R. H., Cooney, M. H., and Martin, L. E. (1974) Lab. Invest. 30, 774-785. West, B. C., Rosenthal, A. S., Gelb, N. A., and Kimball, H. R. (1974) Amer. J. Pathol. 77, 41-62. Woodin, A. M. and Wieneke, A. A. (1963) Biochem. J. 87, 487-495. Smith, R. J. and Ignarro, L. J. (1975) Proc. Natn. Acac, Sci. 72, 108-112. Northover, B. J. (1977) Br. J. Pharmacol. 59, 253-259. Smith, R. J. (1979) Biochem, Pharmacol. (in press). Hecker, E. (1968) Cancer Res. 28, 2338-2348. Smith, R. J. (1978) J. Pharmacol. Exp. Ther. 207, 618-629. Worthington Enzyme Manual (1972) pp. 100-101, Worthington Biochemical Corp., Freehold, N. J. Sastry, B. V. R. and Lasslo, A. (1958) J. Org. Chem. 23, 1577-1578. Malagodi, M. H. and Chiou, C. Y. (1947b) Europ. J. Pharmacol. 27, 25-33. Estensen, R. D., White, J. G., and Holmes, B. (1974) Nature (London) 248, 347-348. Goldstein, I., Hoffstein, S. T., and Weissmann, G.(l975) J. Cell Biol. 66, 647-652. Bainton, 0. F. (1973) J. Cell Biol. 58, 249-264. Bentwood, B. J. (1979) Fed. Proc. 38 (Part 2), 1362. Nachman, R., Hirsch, J. G., and Baggiolini, M. (1972) J. Cell Biol. 54, 133-140. Avila, J. L. and Convit, J. (1937b) Biochim, Biophys. Acta 293, 409-423. Malagodi, M. H. and Chiou, C. Y. (1974a) Pharmacology 12, 20-31. Chiou, C. Y. and Malagodi, M. H. (1975) Brit. J. Pharmacol. 53, 279-285. Charo, I. F., Feinman, R. D. and Detwiler, T. C. (1976) Biochem, Biophys. Res. Comnun. 72, 1462-1467. Gorman, R. R., Wierenga, W. and Miller, 0. V. (1979) Biochim, Biophys. Acta 572, 95-104. Goldstein, I. M., Horn, J. K., Kaplan, H. B. and Weissmann, G.(1974) Biochem, Biophys. Res. Comnun. 60, 807-812. Manery, J. F. (1966) Fed. Proc. 25, 1804-1810. Poste, G. and Allison, A. C. (1973) Biochim. Biophys. Acta 300, 421-465. Poste, G. and Allison, A. C. (1971) J. Theor, Biol. 32, 165-184. Satir, B. (1974) J. Supramol. Struct. 2, 529-537. Vollet, J. J., Roth, L. E. and Davidson, M. (1972) J. Cell Biol 55, 269a.
271
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The activity of adenylate cyclase was measured by Method C (in "Note Added in Proof") of Salomon ef2al. (19). Tg3.s is a labelled substrate assay method, based on the corjyersion of [a PI-ATP to [a PI-CAMP by adenylate cyclase, with isolation of [a PI-CAMP by passing the reaction product through Dowex cationexchange and alumina columns. Each assay tube contained 50 ul of either erythrocyte or sperm suspension which had (test) or had not (control) been treated with cholera toxig for va,$ous incubation periods; 50 mM Tris-HCl, pH 7.4; 10 mM KCl; 3 x 10 cpm [a P]-ATP (20-26.3 Ci/mmole), (The Radiochemical Centre, Amersham, England); 1 mM ATP; 1 mM CAMP (A grade, Calbiochem); 6 mM MgC12; 5 mM 2-phosphoenolpyruvate (A grade, Calbiochem), 2 units pyruvate kinase (600 I.U./ml, A grade, Calbiochem) and 10 mM caffeine (Sigma), in a total volume of 100 Reaction mixtures were incubated at 37OC for 20-30 min in an oscillating !Jl. water bath (100 rpm, Paton Industries Pty. Ltd., South Australia). The reaction was stopped by the addition of 100 ul of stopping soluti 9 n , which contained 40 mM ATP, 1.4 mM CAMP, 2% sodium dodecyl sulfate at pH 7.5. H-CAMP (25,000 cpm) (30 Ci/mmole from Radiochemical Centre, Amersham) in 50 ul was added, to determine the efficiency of recovery of CAMP. The total 250 ul volume was placed in a boiling water bath for 1.5 min. After cooling, 0.8 ml of distilled water was agged to each tube and the sample was then applied to the columns for isolation of [a PI-CAMP. It was found that if the lysed or homogenized cells were sedimented (centrifyaed at 20,000 g for 10 min), and only the supernatant, where more than 95% of [a PICAMP was present, was allowed to pass through the columns, the flow rate was better. Eluates from the Dowex cation-exchange resin and alumina columns were then collected into scintillation vials, each containing 10 ml Insta-gel, and were counted in a Packard Tri-Carb (Model 3255) Scintillation Counter. Experiment B. 1 ml of washed human spermatozoa were sonicated, as stated abovg, and 50 ul aliquots were added to separate glass test-tubes and incubated at 37 C. The standard assay mixture, as used above , and lo-100 uM Gpp(NH)p (test) or the same amount of distilled water (control) was added to each tube in a final reaction volume of 100 ul. The tubes were then incubated at 37OC in an oscillating water bath (100 rpm),2for between 1 and 30 m,Q. The subsequent procedures for the measurement of [a PI-CAMP formed from [a P]-ATP by the adenylate cyclase were the same as described above.
In the found
for
absence
adenylate
cyclase
or freezed-thawed, determinations erase
ranged from
inhibitor)
was between cyclase
activity
The recovery
between
spermatozoa effect
between
8 different
and 37.05 decayed
human spermatozoa
experiments. from
rapidly
the
was incubated
on adenylate
and the rate cyclase
activity
with
the toxin
3
had been sonicated,
sperm/20
min in 20 (a phosphodiest-
the
of CAMP production
It were
columns
rate
was found incubated
adenylate
at 37OC (Fig.
removed
1).
48% and 58.8%.
human spermatozoa
was then
(Fig.
that
was between
intact
of CAMP formation was observed
activities
10 mM caffeine
min.
when spermatozoa
specific
which
assay mixture, sperm/20
5 min and 5 h at 20 ug/ml, sonicated,
If
the Dowex and alumina toxin
the
102 and 114 pmoles/108
pmoles/108
of CAMP from Where cholera
for
in washed
was excluded 24.8
RESULTS toxin and Gpp(NH)p,
of cholera
and the
assayed,
no demonstrable
1).
In some experiments,
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
cant test
-r~-----
-
lh
Time toxin
Fig. 1. Adenylate cyclase cubated with cholera toxin either with cholera toxin,
~
%
of incubation of at 37% before
sperm with the assay
rol
-
cholera
activity of human spermatozoa (1 x 10' cells/ml) in(20 us/ml). Washed human spermatozoa were incubated 5 mW ATP, 1 mM DTT and 1 mM NAD 60-0) or buffer
without toxin, 5 rM ATP, 1 mM DTT and 1 mM NAD (0-O) at 37 C for between and 5 h. After each incubation period, 1.5 ml ice-cold KRP was added to assay tube, the cells were sedimented and were subjected to sonication.
cyclase
activity
was then assayed by the addition
20 min at 37'C. Note the points are Mean+SEM of at where pooled semen samples
there
was no attempt
been
incubated
cyclase But,
of the
the
(Table
susceptible
increased
activity 1.56 pmoles
the
cholera
activity
to cholera
toxin
(Table
lb).
cholera
toxin,
the
400%, but
similar
in
cyclase
cyclase
cyclase
in pigeon
of adenylate
of CAMP formed/
of CAMP formed/u1
both
(Table
by Gill
had
the adenylate
CAMP formation
assay.
to cholera erythrocytes
of adenylate
in human spermatozoa
comparable
it
toxin was
5 min and 1 h preincubation
activity
erythrocytes
4
min
after
with
no response
activity
reported
experiments,
spermatozoa
of pigeon
With
1.11 of cells/h,
of cells/h
20
activity
basal
Adenylate assay mixture for at 37OC. All
be in contact
showed
treatment
adenylate
from
the
still
adenylate
any change
could
during
However,
with
cyclase activity from 3 different were used.
toxin
and so it
spermatozoa
cyclase
by about
pmoles
sperm,
disrupted
of erythrocytes
elicit
the
adenylate
la).
decrease of adenylate least 6 determinations from different donors
to remove
with
of standard
5 min each
la,b).
did
not
The basal
was found with
cyclase
the
and King
between
1.24
results
of l-3
(20).
and
Vol. 91, No. 1, 1979
BIOCHEMICAL
Table 1. The activity erythrocytes (b) after
a.
AND BIOPHYSICAL
of adenylate cyc&ase in human spermatozoa (a) and pigeon incubation at 37 C with cholera toxin (5 rig/ml).*
Time of incubation before cell and adenylate cyclase assay
lysis
Adenylate
activity
of CAMP formed/lo' cells/20 min Without Toxin With Cholera Toxin -(Control) (Test) 109.90+6.22 101.44+7.11
lh
43.45f3.11
b.
42.2Sk2.41
pmoles of CAMP formed/SO ~1 of cells/20 Without Toxin With Cholera Toxin (Control) (Test) 26.00f2.11 85.16k2.17
5 min lh
20.72f2.62
min
86.95k3.41
at -7OOC for 3 min and thawing Afteroincubation, the cel122were lysed by incubating at 37 C and the rate of [a PI-CAMP synthesized was assayed. During the 20 min assay period, cholera toxin, presumably, could contact the adenylate cyclase. All data are Mean+SEM of 8 determinations. Gpp(NH)p
cyclase
activity
dependent. for
at a concentration
The adenylate
incubation
cyclase
activity
min at 37Oc in cyclase in basal
cyclase
This
activity
not be any greater
for
(Table
2).
the standard
After
by 25%, but
of sperm after
incubation
that
was about
150% (Fig.
Table 2. Adenylate cyclasg activity Gpp(NH)p for 20 min at 37 C.* Additions
incubated
the a period
2).
where
to produce
sperm were
after
of adenylate
was found
than
assay mixture,
stimulation
stimulation
5 min or more was found
was increased activity
of 10 uM caused
in human spermatozoa.
1 min was found
but,
basal
no Gpp(NH)p increasing with
to be timewith
was added
Gpp(NH)p
activity
for
of human spermatozoa
after
Gpp(NH)p,
10 0
133.12k5.44
Gpp(NH)p,
100 IIM
152.43t6.11
20
of adenylate
of 30 min at 37OC, the
Where Gpp(NH)p
Gpp(NH)p
adenylate
increase
was used at a concent-
incubation
Adenylate cyclase activity (pmoles of CAMP formed/20 min/lO 106.66+8.71
None
*
cyclase
@moles
5 min
*
RESEARCH COMMUNICATIONS
with
sperm)
Gpp(NH)p was included in the standard assay mixture and incubated with human spermatozoa for 20 min at 37OC. All data are MeankSEM of 6 determinations.
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
400
300
E Q “0 5
200
2 E” a i
0 100
minutes
Fig. 2. Adenylate cyclase activity of human spermatozoa (1 x 1OS cells/ml) incubated with Gpp(Wii)p (10 PM). Washed human spermatozoa were sonicated, 50 ~1 was then incubated with standard assay mixture either including 10 yy Gpp(WH)p ( 0-o ) or not (-1, for between 1 and 30 min, during which [a P]CAMP was synthesized from [a PI-ATP. All data reported here are results of 3 different experiments where pooled semen samples from different donors were used. Each experiment had at least 2 replicates.
ration
of
adenylate
100
uM,
it
cyclase
was
than
more it
effective
was
at
in
10
activating
uM during
the
the
20 min
basal
activity
assay
period
of (Table
2).
DISCUSSION It spermatozoa
is
epinephrine,
spermatozoa
been
demonstrated
insensitive
to
isoproterenol,
trophin, search
has
luteinizing for
can
be
a number
the of
adenylate
including
judged
hormone,
prostaglandin
hormonal to
E
activator be
cyclase
hormones
follicle-stimulating hormone,
a specific
that
of
marginally
1'
and
adenylate successful
6
activity
of
epinephrine, human
insulin
chorionic
(1).
human norgonado-
The
cyclase
in
other
(6-8).
Therefore,
mammalian
Vol. 91, No. 1, 1979
cholera
toxin
cyclase
activity
and Gpp(NH)p
lack
cholera the
toxin
of response toxin,
toxin
adenylate that
that
there
by its
cyclase
system
the ability
in some experiments
toxin
likely
adenylate
relatively
by the
cholera
also
observed
with
cholera
assayed,
amount ation
toxin that
that
for
lb).
with
cholera
the
the cholera cyclase
toxin
during
response
were
of pigeon
the toxin
lysed
was not
the
in other
in erythrocytes (13)
but,
the
period.
erythrocytes
to cholera the adenylate
rate
and cho-
cyclase,
it
still
though action
of response
unknown,
(21).
of of human
though
it
seems
in human spermatozoa
spermatozoa. erythrocytes
was found (Table
la,b).
were
lysed
studies increases
has been with
studies
cholera
In the
toxin cyclase.
is
incubated
time
of 1 h
that
the
time
of incub-
toxin
studies
have been able has been
found
increasing
the
was
and CAMP formation
an incubation
it
to be activated It
of the erythrocytes
from where
It
lack
a con-
lysed
the
factor(s)
activity
and would
cause
were
for
remains
and CAMP was assayed. removed,
cells
treatments
CAMP production
spermatozoa
in those
20 min assay
can contact
in
the cells
different
However,
toxin
cells
cyclase
that
of CAMP production,
cytosol
with
5 min before
significantly
(Table
toxin
of pigeon
was inactive
and to which
to the adenylate
for
for
the underlying
spermatozoa
rate
to
receptors
erythrocyte,
to be essential
is present cyclase
surface
been reported
access on the
was unable
can activate
the
The reason
the adenylate
toxin
where
of a specific
cytosol
of CAMP produced
before
where
lack
lack
to mouse thymus
to cholera
adenylate
was not
was used
on adenylate
in human spermatozoa.
to activate
la)
effect
present.
little
However,
to bind
have direct
cyclase
it
has also
are considered
were
cyclase
before
ability
any observable
to be due to the
since
effects
tested
30 per pigeon
It
(Table
presumably
(131,
spermatozoa1
20).
toxin's
ATP and cytosol,which
cholera
about
of toxin
in the
not produce
are
(13,
However,
NAD,
their
spermatozoa
B components
reduction
did
RESEARCH COMMUNICATIONS
of concentrations
may indicate
attaches
could
range
of adenylate
comitant
toxin
to study
response
of which
decrease
lera
used here
at the
any significant
This
were
AND BIOPHYSICAL
in human spermatozoa.
Cholera elicit
BIOCHEMICAL
undertaken
to contact
reported more rapid
was removed
(13)
that
the the
in conditions
here adenylate
Vol. 91, No. 1, 1979
BIOCHEMICAL
The results human spermatozoa It
has also
bovine
that
receptor
mechanism stimulates
indicated
(22)
that
the
this
activation
is
adenylate
cyclase
activity
and that
the
response
range
epididymis unknown,
and is
activity
of
of concentrations cyclase
but
it
of
by Gpp(NH)p.
has been
by a mechanism cyclase
tested.
activity
was activated
of adenylate
process
cyclase
adenylate
the caput
for
RESEARCH COMMUNICATIONS
adenylate
in the
from
by means of a time-dependent found
that
by Gpp(NH)p
recently
recovered
occupation,
occurs been
reported
spermatozoa
Gpp(NH)p
here
can be increased
been
The underlying
reported
AND BIOPHYSICAL
reported
independent
of
to the nucleotide
dose-dependent
(15),
as has
here.
ACKNOWLEDGMENTS The authors D.J.
Kay
and H. -R.
study
was supported
World
Health
wish
to express
Tinneberg
for
by grants
Organization
their
from
(Grant
the
no.
their helpful Ford
appreciation suggestions Foundation
to Drs. and (Grant
R.J.
Rose,
criticism.
no.
760-0528)
This and
74250).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Gray, J.P. (1971) Ph.D. thesis. Univ. of Vanderbilt, Nashville, Tenn. Gray, J-P., Hardman, J.G., Hammer, J.L., Hoes, R.T. and Sutherland, E.W. (1971)'Fed. Proc. 30:1267 Abstr. Casillas, E.R. and%oskins, D.D. (1970) Biochem. Biophys. Res. Commun. -40: 255-262. Casillas, E.R. and Hoskins, D.D. (1971) Arch. Biochem. Biophys. 147:148-155. Herman, C.A., Zahler, W.L., Doak, G.A. and Csmpb-ell, B.J. (1976)Arch. Biochem. Biophys. 177:622-629. Hoskins, D.D. and allas, E.R. (1975) in Advances in Sex Hormone Research, Vol. 1 (Singhal, R.L. and Thosma, J-A., eds.) pp. 283-324, University Park Press, Baltimore. Hoskins, D.D. and Casillas, E.R. (1975) in Handbook of Physiology, Vol. 5 Sect. 7 (Greep, R-O., Astwood, E.B., Hamilton, D.W., and Geiger, S-R., eds.) pp. 453-460, American Physiological Society, Washington, D.C. Harrison, R.A.P. (1975) in The Biology of the Male Gamete (Duckett, J.G. and Racey, P.A., eds.) pp. 301-316, Academic Press, London. Haesungcharern, A. and Chulavatnatol, M. (1978) J. Reprod. Fertil. 53:59-61. Gray, J.P., Drunnnond, G-1. and Luk, D.W.T. (1976) Arch. Biochem. Biophys. 172:20-30. Gbers, D.L. (1977) Biol. Reprod. 16:377-384. Finkelstein,R.A. (1973) CRC CriticarPev. Microbial. 2:553-623. Gill, D-M. (1977) Adv. Cyclic Nucleotide Res. 8:85-118. Vaughan, M. and Moss. J. (1978) J. Suprsmol. Srruct. 8:473-488. Londos, C., Salomon, Y., Lin, M., Harwood, J.P., Schr&n, M., Wolff, J. and Rodbell, M. (1974) Proc. Natl. Acad. Sci. U.S.A. 71:3087-3090. Harrison, R.A.P. (1976) J. Reprod. Fertil. -48:347%53.
8
Vol. 91, No. 1, 1979
17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DeLuca, H.F. (1972) in Manometric and Biochemical Techniques, 5th eds. (Umbreit, W-W., Burris, R.H. and Stauffer, J.F. eds) pp. 146-147, Burgess Publishing Company, Minneapolis. Londesborough, J. (1976) Anal. Biochem. 71:623-628. Salomon, Y., Londos, C. and Rodbell, M. fl974) Anal. Biochem. 58:541-548. Gill, D.M. and King, C.A. (1975) J. Biol. Chem. 250:6424-6432. Holmgren, J. and LiSnnroth, I. (1976) J. Infect. z. 133:S64-S74. Casillas, E.R., Elder, C.M. and Hoskins, D.D. (1978) Fed. Proc. -37:1688 Abstr.
9
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 7-Q
14, 1979
EFFECTS OF CHOLERA TOXIN AND 5'-GUANYLYLIMIDODIPHOSPHATE ADENYLATE CYCLASE ACTIVITY Cheng,
C. Y. and Boettcher,
ON HUMAN SPERMATOZOAL
B.
Department of Biological Sciences, The University of Newcastle, New South Wales, 2308, AUSTRALIA
Received
June
29,1979 SUMMARY
The effects of cholera toxin and 5'-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoa1 adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 ug/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase where the adenylate cyclase would be assay, or was added to lysed spermatozoa, accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 uM was effective in activating human spermatozoa1 adenylate cyclase activity. INTRODUCTION The presence zing)
EC 4.6.1.1.) for
specific
out with
spermatozoa
can be judged
6-8).
hormonal from
cyclases monkey
(3,4),
(3,4),
but
has been demonstrated
human spermatozoa
is
inhibited
number the
acids of other
effects
Abbreviatiggs: buffer (Ca
(9),
but
species
of cholera
this (10,ll).
toxin
Gpp(NH)p,
by sodium could This
(6).
cyclase
(for
review,
cyclase
a number
presents
in results
see
by thyroxine
be demonstrated
and 5' -guanylylimidodiphosphate 5'-guanylylimidodiphosphate;
spermatozo-
can be activated
not be demonstrated report
The attempts
Mammalian
adenylate
fluoride,
(1,Z).
has been carried
hormones
cannot
that
1971
and man (1).
to steroid
such activation
It
(5)
(cycliin
cyclase
successful
adenylate
(5).
carboxylic
bull
marginally
spermatozoa1
demonstrated
of adenylate
seem to be insensitive
and triiodothyronine spermatozoa
only
(ATP pyrophosphate-lyase
was first
activators
the monkey
to have been
However,
cyclase
in human spermatozoa
A search
al adenylate
of adenylate
in bull
from ejaculated
of metallic
ions
spermatozoa
of a
of studies (Gpp(NH)p)
and
on *
on
RF@, Krebs-Ringer-Phosphate
-free). 0006-291 1
Copyright All rights
X/79/21
OOOl -09$01.00/O
@ 1979 by Academic Press. Inc. of reproduction in anyform reserved.
BIOCHEMICAL
Vol. 91, No. 1, 1979
associated ing
constituents
a secretagogue
cellular cologic
to the granule
by numerous
stimuli.
Nevertheless,
such as PMA, whose mode of action we are able
calcium, tool
induced
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
which
will
enzyme
aid
to employ
an agent
in the elucidation
secretory
process
which
is
by utiliz-
independent
of extra-
such as TMB-8 as a pharmaof the true
heretofore
relevance has not
of calcium
been possible.
REFERENCES
:: i: 5. 6. 7. ;: 10.
18. ii: 21. 22. 23. 24. 25. 26. % 29. 30. 31. i5: 34. 35.
Henson, P. M. (1971a) J. Imnunol. 107, 1535-1546. Henson, P. M. (1971b) J. Imnunol. 107, 1547-1557. Hawkins, D. (1972) J. Imnunol. 108, 310-317. Zurier, R. B., Hoffstein, S. and Weissmann, G. (1973) J. Cell Biol. 58, 27-41. Henson, P. M. (1972) Amer. J. Pathol. 68, 593-606. Showell, H. J., Freer,R. J., Zigmond, S. H., Schiffmann, E. Aswanikumar, S Corcoran, B. and Becker, E. L. (1976) J. Exp. Med. 143, 1154-1169. Bajnton, Il. F., Ullyot, J. L. and Farquhar, M. G. (1971) J. Exp. Med. 134, 907-934. Avila, J. L. and Convit, J. (1973a) Biochim, Biophys, Acta 293, 397-408. Spitznagel, J. K., Daldorf, F. G., Leffell, M. S., Folds, J. D., Welsh, I. R. H., Cooney, M. H., and Martin, L. E. (1974) Lab. Invest. 30, 774-785. West, B. C., Rosenthal, A. S., Gelb, N. A., and Kimball, H. R. (1974) Amer. J. Pathol. 77, 41-62. Woodin, A. M. and Wieneke, A. A. (1963) Biochem. J. 87, 487-495. Smith, R. J. and Ignarro, L. J. (1975) Proc. Natn. Acac, Sci. 72, 108-112. Northover, B. J. (1977) Br. J. Pharmacol. 59, 253-259. Smith, R. J. (1979) Biochem, Pharmacol. (in press). Hecker, E. (1968) Cancer Res. 28, 2338-2348. Smith, R. J. (1978) J. Pharmacol. Exp. Ther. 207, 618-629. Worthington Enzyme Manual (1972) pp. 100-101, Worthington Biochemical Corp., Freehold, N. J. Sastry, B. V. R. and Lasslo, A. (1958) J. Org. Chem. 23, 1577-1578. Malagodi, M. H. and Chiou, C. Y. (1947b) Europ. J. Pharmacol. 27, 25-33. Estensen, R. D., White, J. G., and Holmes, B. (1974) Nature (London) 248, 347-348. Goldstein, I., Hoffstein, S. T., and Weissmann, G.(l975) J. Cell Biol. 66, 647-652. Bainton, 0. F. (1973) J. Cell Biol. 58, 249-264. Bentwood, B. J. (1979) Fed. Proc. 38 (Part 2), 1362. Nachman, R., Hirsch, J. G., and Baggiolini, M. (1972) J. Cell Biol. 54, 133-140. Avila, J. L. and Convit, J. (1937b) Biochim, Biophys. Acta 293, 409-423. Malagodi, M. H. and Chiou, C. Y. (1974a) Pharmacology 12, 20-31. Chiou, C. Y. and Malagodi, M. H. (1975) Brit. J. Pharmacol. 53, 279-285. Charo, I. F., Feinman, R. D. and Detwiler, T. C. (1976) Biochem, Biophys. Res. Comnun. 72, 1462-1467. Gorman, R. R., Wierenga, W. and Miller, 0. V. (1979) Biochim, Biophys. Acta 572, 95-104. Goldstein, I. M., Horn, J. K., Kaplan, H. B. and Weissmann, G.(1974) Biochem, Biophys. Res. Comnun. 60, 807-812. Manery, J. F. (1966) Fed. Proc. 25, 1804-1810. Poste, G. and Allison, A. C. (1973) Biochim. Biophys. Acta 300, 421-465. Poste, G. and Allison, A. C. (1971) J. Theor, Biol. 32, 165-184. Satir, B. (1974) J. Supramol. Struct. 2, 529-537. Vollet, J. J., Roth, L. E. and Davidson, M. (1972) J. Cell Biol 55, 269a.
271
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The activity of adenylate cyclase was measured by Method C (in "Note Added in Proof") of Salomon ef2al. (19). Tg3.s is a labelled substrate assay method, based on the corjyersion of [a PI-ATP to [a PI-CAMP by adenylate cyclase, with isolation of [a PI-CAMP by passing the reaction product through Dowex cationexchange and alumina columns. Each assay tube contained 50 ul of either erythrocyte or sperm suspension which had (test) or had not (control) been treated with cholera toxig for va,$ous incubation periods; 50 mM Tris-HCl, pH 7.4; 10 mM KCl; 3 x 10 cpm [a P]-ATP (20-26.3 Ci/mmole), (The Radiochemical Centre, Amersham, England); 1 mM ATP; 1 mM CAMP (A grade, Calbiochem); 6 mM MgC12; 5 mM 2-phosphoenolpyruvate (A grade, Calbiochem), 2 units pyruvate kinase (600 I.U./ml, A grade, Calbiochem) and 10 mM caffeine (Sigma), in a total volume of 100 Reaction mixtures were incubated at 37OC for 20-30 min in an oscillating !Jl. water bath (100 rpm, Paton Industries Pty. Ltd., South Australia). The reaction was stopped by the addition of 100 ul of stopping soluti 9 n , which contained 40 mM ATP, 1.4 mM CAMP, 2% sodium dodecyl sulfate at pH 7.5. H-CAMP (25,000 cpm) (30 Ci/mmole from Radiochemical Centre, Amersham) in 50 ul was added, to determine the efficiency of recovery of CAMP. The total 250 ul volume was placed in a boiling water bath for 1.5 min. After cooling, 0.8 ml of distilled water was agged to each tube and the sample was then applied to the columns for isolation of [a PI-CAMP. It was found that if the lysed or homogenized cells were sedimented (centrifyaed at 20,000 g for 10 min), and only the supernatant, where more than 95% of [a PICAMP was present, was allowed to pass through the columns, the flow rate was better. Eluates from the Dowex cation-exchange resin and alumina columns were then collected into scintillation vials, each containing 10 ml Insta-gel, and were counted in a Packard Tri-Carb (Model 3255) Scintillation Counter. Experiment B. 1 ml of washed human spermatozoa were sonicated, as stated abovg, and 50 ul aliquots were added to separate glass test-tubes and incubated at 37 C. The standard assay mixture, as used above , and lo-100 uM Gpp(NH)p (test) or the same amount of distilled water (control) was added to each tube in a final reaction volume of 100 ul. The tubes were then incubated at 37OC in an oscillating water bath (100 rpm),2for between 1 and 30 m,Q. The subsequent procedures for the measurement of [a PI-CAMP formed from [a P]-ATP by the adenylate cyclase were the same as described above.
In the found
for
absence
adenylate
cyclase
or freezed-thawed, determinations erase
ranged from
inhibitor)
was between cyclase
activity
The recovery
between
spermatozoa effect
between
8 different
and 37.05 decayed
human spermatozoa
experiments. from
rapidly
the
was incubated
on adenylate
and the rate cyclase
activity
with
the toxin
3
had been sonicated,
sperm/20
min in 20 (a phosphodiest-
the
of CAMP production
It were
columns
rate
was found incubated
adenylate
at 37OC (Fig.
removed
1).
48% and 58.8%.
human spermatozoa
was then
(Fig.
that
was between
intact
of CAMP formation was observed
activities
10 mM caffeine
min.
when spermatozoa
specific
which
assay mixture, sperm/20
5 min and 5 h at 20 ug/ml, sonicated,
If
the Dowex and alumina toxin
the
102 and 114 pmoles/108
pmoles/108
of CAMP from Where cholera
for
in washed
was excluded 24.8
RESULTS toxin and Gpp(NH)p,
of cholera
and the
assayed,
no demonstrable
1).
In some experiments,
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
cant test
-r~-----
-
lh
Time toxin
Fig. 1. Adenylate cyclase cubated with cholera toxin either with cholera toxin,
~
%
of incubation of at 37% before
sperm with the assay
rol
-
cholera
activity of human spermatozoa (1 x 10' cells/ml) in(20 us/ml). Washed human spermatozoa were incubated 5 mW ATP, 1 mM DTT and 1 mM NAD 60-0) or buffer
without toxin, 5 rM ATP, 1 mM DTT and 1 mM NAD (0-O) at 37 C for between and 5 h. After each incubation period, 1.5 ml ice-cold KRP was added to assay tube, the cells were sedimented and were subjected to sonication.
cyclase
activity
was then assayed by the addition
20 min at 37'C. Note the points are Mean+SEM of at where pooled semen samples
there
was no attempt
been
incubated
cyclase But,
of the
the
(Table
susceptible
increased
activity 1.56 pmoles
the
cholera
activity
to cholera
toxin
(Table
lb).
cholera
toxin,
the
400%, but
similar
in
cyclase
cyclase
cyclase
in pigeon
of adenylate
of CAMP formed/
of CAMP formed/u1
both
(Table
by Gill
had
the adenylate
CAMP formation
assay.
to cholera erythrocytes
of adenylate
in human spermatozoa
comparable
it
toxin was
5 min and 1 h preincubation
activity
erythrocytes
4
min
after
with
no response
activity
reported
experiments,
spermatozoa
of pigeon
With
1.11 of cells/h,
of cells/h
20
activity
basal
Adenylate assay mixture for at 37OC. All
be in contact
showed
treatment
adenylate
from
the
still
adenylate
any change
could
during
However,
with
cyclase activity from 3 different were used.
toxin
and so it
spermatozoa
cyclase
by about
pmoles
sperm,
disrupted
of erythrocytes
elicit
the
adenylate
la).
decrease of adenylate least 6 determinations from different donors
to remove
with
of standard
5 min each
la,b).
did
not
The basal
was found with
cyclase
the
and King
between
1.24
results
of l-3
(20).
and
Vol. 91, No. 1, 1979
BIOCHEMICAL
Table 1. The activity erythrocytes (b) after
a.
AND BIOPHYSICAL
of adenylate cyc&ase in human spermatozoa (a) and pigeon incubation at 37 C with cholera toxin (5 rig/ml).*
Time of incubation before cell and adenylate cyclase assay
lysis
Adenylate
activity
of CAMP formed/lo' cells/20 min Without Toxin With Cholera Toxin -(Control) (Test) 109.90+6.22 101.44+7.11
lh
43.45f3.11
b.
42.2Sk2.41
pmoles of CAMP formed/SO ~1 of cells/20 Without Toxin With Cholera Toxin (Control) (Test) 26.00f2.11 85.16k2.17
5 min lh
20.72f2.62
min
86.95k3.41
at -7OOC for 3 min and thawing Afteroincubation, the cel122were lysed by incubating at 37 C and the rate of [a PI-CAMP synthesized was assayed. During the 20 min assay period, cholera toxin, presumably, could contact the adenylate cyclase. All data are Mean+SEM of 8 determinations. Gpp(NH)p
cyclase
activity
dependent. for
at a concentration
The adenylate
incubation
cyclase
activity
min at 37Oc in cyclase in basal
cyclase
This
activity
not be any greater
for
(Table
2).
the standard
After
by 25%, but
of sperm after
incubation
that
was about
150% (Fig.
Table 2. Adenylate cyclasg activity Gpp(NH)p for 20 min at 37 C.* Additions
incubated
the a period
2).
where
to produce
sperm were
after
of adenylate
was found
than
assay mixture,
stimulation
stimulation
5 min or more was found
was increased activity
of 10 uM caused
in human spermatozoa.
1 min was found
but,
basal
no Gpp(NH)p increasing with
to be timewith
was added
Gpp(NH)p
activity
for
of human spermatozoa
after
Gpp(NH)p,
10 0
133.12k5.44
Gpp(NH)p,
100 IIM
152.43t6.11
20
of adenylate
of 30 min at 37OC, the
Where Gpp(NH)p
Gpp(NH)p
adenylate
increase
was used at a concent-
incubation
Adenylate cyclase activity (pmoles of CAMP formed/20 min/lO 106.66+8.71
None
*
cyclase
@moles
5 min
*
RESEARCH COMMUNICATIONS
with
sperm)
Gpp(NH)p was included in the standard assay mixture and incubated with human spermatozoa for 20 min at 37OC. All data are MeankSEM of 6 determinations.
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
400
300
E Q “0 5
200
2 E” a i
0 100
minutes
Fig. 2. Adenylate cyclase activity of human spermatozoa (1 x 1OS cells/ml) incubated with Gpp(Wii)p (10 PM). Washed human spermatozoa were sonicated, 50 ~1 was then incubated with standard assay mixture either including 10 yy Gpp(WH)p ( 0-o ) or not (-1, for between 1 and 30 min, during which [a P]CAMP was synthesized from [a PI-ATP. All data reported here are results of 3 different experiments where pooled semen samples from different donors were used. Each experiment had at least 2 replicates.
ration
of
adenylate
100
uM,
it
cyclase
was
than
more it
effective
was
at
in
10
activating
uM during
the
the
20 min
basal
activity
assay
period
of (Table
2).
DISCUSSION It spermatozoa
is
epinephrine,
spermatozoa
been
demonstrated
insensitive
to
isoproterenol,
trophin, search
has
luteinizing for
can
be
a number
the of
adenylate
including
judged
hormone,
prostaglandin
hormonal to
E
activator be
cyclase
hormones
follicle-stimulating hormone,
a specific
that
of
marginally
1'
and
adenylate successful
6
activity
of
epinephrine, human
insulin
chorionic
(1).
human norgonado-
The
cyclase
in
other
(6-8).
Therefore,
mammalian
Vol. 91, No. 1, 1979
cholera
toxin
cyclase
activity
and Gpp(NH)p
lack
cholera the
toxin
of response toxin,
toxin
adenylate that
that
there
by its
cyclase
system
the ability
in some experiments
toxin
likely
adenylate
relatively
by the
cholera
also
observed
with
cholera
assayed,
amount ation
toxin that
that
for
lb).
with
cholera
the
the cholera cyclase
toxin
during
response
were
of pigeon
the toxin
lysed
was not
the
in other
in erythrocytes (13)
but,
the
period.
erythrocytes
to cholera the adenylate
rate
and cho-
cyclase,
it
still
though action
of response
unknown,
(21).
of of human
though
it
seems
in human spermatozoa
spermatozoa. erythrocytes
was found (Table
la,b).
were
lysed
studies increases
has been with
studies
cholera
In the
toxin cyclase.
is
incubated
time
of 1 h
that
the
time
of incub-
toxin
studies
have been able has been
found
increasing
the
was
and CAMP formation
an incubation
it
to be activated It
of the erythrocytes
from where
It
lack
a con-
lysed
the
factor(s)
activity
and would
cause
were
for
remains
and CAMP was assayed. removed,
cells
treatments
CAMP production
spermatozoa
in those
20 min assay
can contact
in
the cells
different
However,
toxin
cells
cyclase
that
of CAMP production,
cytosol
with
5 min before
significantly
(Table
toxin
of pigeon
was inactive
and to which
to the adenylate
for
for
the underlying
spermatozoa
rate
to
receptors
erythrocyte,
to be essential
is present cyclase
surface
been reported
access on the
was unable
can activate
the
The reason
the adenylate
toxin
where
of a specific
cytosol
of CAMP produced
before
where
lack
lack
to mouse thymus
to cholera
adenylate
was not
was used
on adenylate
in human spermatozoa.
to activate
la)
effect
present.
little
However,
to bind
have direct
cyclase
it
has also
are considered
were
cyclase
before
ability
any observable
to be due to the
since
effects
tested
30 per pigeon
It
(Table
presumably
(131,
spermatozoa1
20).
toxin's
ATP and cytosol,which
cholera
about
of toxin
in the
not produce
are
(13,
However,
NAD,
their
spermatozoa
B components
reduction
did
RESEARCH COMMUNICATIONS
of concentrations
may indicate
attaches
could
range
of adenylate
comitant
toxin
to study
response
of which
decrease
lera
used here
at the
any significant
This
were
AND BIOPHYSICAL
in human spermatozoa.
Cholera elicit
BIOCHEMICAL
undertaken
to contact
reported more rapid
was removed
(13)
that
the the
in conditions
here adenylate
Vol. 91, No. 1, 1979
BIOCHEMICAL
The results human spermatozoa It
has also
bovine
that
receptor
mechanism stimulates
indicated
(22)
that
the
this
activation
is
adenylate
cyclase
activity
and that
the
response
range
epididymis unknown,
and is
activity
of
of concentrations cyclase
but
it
of
by Gpp(NH)p.
has been
by a mechanism cyclase
tested.
activity
was activated
of adenylate
process
cyclase
adenylate
the caput
for
RESEARCH COMMUNICATIONS
adenylate
in the
from
by means of a time-dependent found
that
by Gpp(NH)p
recently
recovered
occupation,
occurs been
reported
spermatozoa
Gpp(NH)p
here
can be increased
been
The underlying
reported
AND BIOPHYSICAL
reported
independent
of
to the nucleotide
dose-dependent
(15),
as has
here.
ACKNOWLEDGMENTS The authors D.J.
Kay
and H. -R.
study
was supported
World
Health
wish
to express
Tinneberg
for
by grants
Organization
their
from
(Grant
the
no.
their helpful Ford
appreciation suggestions Foundation
to Drs. and (Grant
R.J.
Rose,
criticism.
no.
760-0528)
This and
74250).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Gray, J.P. (1971) Ph.D. thesis. Univ. of Vanderbilt, Nashville, Tenn. Gray, J-P., Hardman, J.G., Hammer, J.L., Hoes, R.T. and Sutherland, E.W. (1971)'Fed. Proc. 30:1267 Abstr. Casillas, E.R. and%oskins, D.D. (1970) Biochem. Biophys. Res. Commun. -40: 255-262. Casillas, E.R. and Hoskins, D.D. (1971) Arch. Biochem. Biophys. 147:148-155. Herman, C.A., Zahler, W.L., Doak, G.A. and Csmpb-ell, B.J. (1976)Arch. Biochem. Biophys. 177:622-629. Hoskins, D.D. and allas, E.R. (1975) in Advances in Sex Hormone Research, Vol. 1 (Singhal, R.L. and Thosma, J-A., eds.) pp. 283-324, University Park Press, Baltimore. Hoskins, D.D. and Casillas, E.R. (1975) in Handbook of Physiology, Vol. 5 Sect. 7 (Greep, R-O., Astwood, E.B., Hamilton, D.W., and Geiger, S-R., eds.) pp. 453-460, American Physiological Society, Washington, D.C. Harrison, R.A.P. (1975) in The Biology of the Male Gamete (Duckett, J.G. and Racey, P.A., eds.) pp. 301-316, Academic Press, London. Haesungcharern, A. and Chulavatnatol, M. (1978) J. Reprod. Fertil. 53:59-61. Gray, J.P., Drunnnond, G-1. and Luk, D.W.T. (1976) Arch. Biochem. Biophys. 172:20-30. Gbers, D.L. (1977) Biol. Reprod. 16:377-384. Finkelstein,R.A. (1973) CRC CriticarPev. Microbial. 2:553-623. Gill, D-M. (1977) Adv. Cyclic Nucleotide Res. 8:85-118. Vaughan, M. and Moss. J. (1978) J. Suprsmol. Srruct. 8:473-488. Londos, C., Salomon, Y., Lin, M., Harwood, J.P., Schr&n, M., Wolff, J. and Rodbell, M. (1974) Proc. Natl. Acad. Sci. U.S.A. 71:3087-3090. Harrison, R.A.P. (1976) J. Reprod. Fertil. -48:347%53.
8
Vol. 91, No. 1, 1979
17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DeLuca, H.F. (1972) in Manometric and Biochemical Techniques, 5th eds. (Umbreit, W-W., Burris, R.H. and Stauffer, J.F. eds) pp. 146-147, Burgess Publishing Company, Minneapolis. Londesborough, J. (1976) Anal. Biochem. 71:623-628. Salomon, Y., Londos, C. and Rodbell, M. fl974) Anal. Biochem. 58:541-548. Gill, D.M. and King, C.A. (1975) J. Biol. Chem. 250:6424-6432. Holmgren, J. and LiSnnroth, I. (1976) J. Infect. z. 133:S64-S74. Casillas, E.R., Elder, C.M. and Hoskins, D.D. (1978) Fed. Proc. -37:1688 Abstr.
9
