Effects of Phorbol Esters and a Diacyiglycerol on the Mouse Sperm. Acrosome. Reaction. Induced by the Zona Pellucida1. MICHAEL. A. LEE, GREGORY.
BIOLOGY
OF
36, 617-627
REPRODUCTION
Effects
of Phorbol Acrosome
MICHAEL
(1987)
Esters and a Diacyiglycerol
Reaction
A. LEE,
Induced
GREGORY
Department
by the Zona
S. KOPF,
of
University
and
Obstetrics
and
of Pennsylvania Philadelphia,
on the Mouse
Sperm
Pellucida1
BAYARD
T. STOREY2
Gynecology
School
Pennsylvania
of Medicine 19104
ABSTRACT Capacitated mouse sperm undergo the acrosome reaction when bound to isolated, mouse resembles, in part, ligand-receptor-mediated such processes have been shown the effects of phorbol esters, zona-induced acrosome reaction 13-acetate (TPA) chlortetracycline assay
with
cence on accelerated appearance
on
the
in part by protein kinase C-catalyzed activators of this kinase, on both At concentrations up to 10 pM,
examined.
the time course assay. Capacitated,
anterior
head;
fully
of
the spontaneous acrosome-intact
acrosome-reacted
nM
TPA
the fluorescence zona-induced are
mimicked
patterns, designated acrosome reaction, which by
60
nM
protein phosphorylation, the spontaneous and the 12-tetradecanoyl phorbol-
acrosome reaction as scored by the sperm display Pattern B in the CTC sperm
display
Pattern
AR
with
no fluores-
acrosome reaction was markedly of Pattern AR was retarded. The on the head and which marks an
between Pattern B and Pattern AR in the controls, was loss of Pattern B at early times post-binding to zonae. The to zonae was retarded by 65 nM TPA to the same extent
The transitions between define two stages of the of 65
were
in suspension and the zona-induced zona-induced acrosome reaction in the that occur in some somatic cells. Since
head. The time course of the loss of Pattern B in the zona-induced by 65 nM TPA as compared to controls, whereas the appearance of Pattern S, which is characterized by punctate fluorescence
intermediate state same extent as the times post-binding
effects
to be mediated which are potent
had no effect on (CTC) fluorescence
fluorescence the
spontaneous acrosome reaction intact zonae pellucidae. The exocytotic processes
accelerated by 65 nM TPA to the disappearance of Pattern S at later as the appearance of Pattern AR.
the B-to-S and the S-to-AR are affected in opposite
4-I’3-phorbol-12,13-didecanoate
(4-13-PDD)
transitions, directions by while
the
therefore TPA. The
4-a
isomer
is
without effect. Such stereospecificity is similar to that reported for the activation of protein kinase C. The diacyiglycerol, 1-oleyl-2-acetylglycerol, which is also known to activate protein kinase C, mimicked the effects of TPA and 4-13-PDD on the time courses of the B-to-S and S-to-AR transitions. These results suggest that protein kinase C may play an intermediary role in the zona-induced mouse sperm acrosome reaction.
INTRODUCTION
Capacitated reaction at and zona
at
mouse sperm undergo a slow rate spontaneously
a much pellucida
more rapid rate when of the egg (F!orman
1982a). The acrosome reaction the zona is induced by the zona by the compound 3-quinuclidiny! with unique potency (Florman 1982b). The acrosome reaction
the acrosome in suspension bound and
to the Storey,
of sperm bound to itself and is inhibited benzi!ate (QNB) and Storey, 1981, inducing activity
of the mouse zona pellucida
zona has been shown to reside with the g!ycoprotein ZP3 (Bleil and Wassarman,
1983), the sperm-binding 1983, 1986;
same glycoprotein that also possesses activity (Bleil and Wassarman, 1980, Florman et a!., 1984, Florman and
Wassarman, biological
1985;Wassarman activities have
eta!., been
1985). These differentiated
two ex-
perimentally. Binding to zonae was shown not to be affected by QNB, whereas induction of the acrosome reaction was inhibited (Florman and Storey, 1982a). The sperm-binding a discrete size class 3400-4500, molecule
Accepted September 29, 1986. Received July 31, 1986. ‘This research was supported by NIH grants HD-06274 and HD19096. 2 Reprint requests: Dr. Bayard T. Storey, Department of Obstetrics and Gynecology, Medical Labs 339, University of Pennsylvania, Philadelphia, PA 19104-6080.
Wassarman, ZP3, on
617
which with
the
of acrosome a!., 1984),
activity of 0-linked
retention 1985). other
can
has been localized o!igosaccharides
be
cleaved
of
activity
from
Minimal proteolytic hand, resulted in the
reaction-inducing implying that this
the
to of Mr parent
(Florman cleavage complete
activity (Florman activity is conferred
and of loss et by
LEE
618 functional groups on the and/or a very strict steric charide
groups
chain. The binding have requirements. receptor trypsin
protein backbone of ZP3 configuration of oligosac-
imposed
by
the
sperm-associated also been shown There appear to
sites: (Saling,
intact
polypeptide
receptors for zona to have distinct steric be two classes of such
one class is sensitive to inhibitors 1981) and the other is sensitive
using
125
I-labeled
sarman, 1986), in agreement of evidence that mouse mouse
zona
(Sa!ing
and
with
with sperm
a fully
Storey,
ZP3 the bind
intact
1979;
Saling
(Bleil
plasma et al.,
and
Was-
extensive to the
body intact
membrane
1979;
Florman
is an important
cells Ca2
is -
class.
apparently exocytotic
plays processes
protein phosphorylation and phospholipid-dependent
designated zuka et al.,
that
a!.,
1986).
diacy!g!ycerol
protein 1984; This
a key role in in somatic
catalyzed protein
by the kinase,
kinase C (Nishizuka, 1984; NishiBlumberg et a!., 1985; Kimura et enzyme
generated
is activated by
either
ligand-induced,
by
1,2-
receptor-
mediated polyphosphoinositide hydrolysis (Lapetina and Miche!!, 1973; Berridge and Irvine, 1984; Cockcroft, 1984; Sekar and Hokin, 1986; Smith et a!., 1986), latter, potent
or by a variety of the phorbol esters and useful (Nishizuka,
of
exogenous agents. Of the have proved particularly 1984; Blumberg et al.,
sequential
stages
by
the
as a fluorescent
probe
time first
courses of these stage and retard
two the
stages: second
AND
METHODS
Reagents
Glutaraldehyde solution from
was obtained Po!ysciences, Inc.
12-Tetradecanoy!phorbo!-1 racyc!ine (CTC), bovine
as a 25% (Warminster,
Phorbol-12,
13-didecanoate
1 3-didecanoate (OAG)
Jolla,
CA).
(4-a-PDD),
P.
F.
Nutley,
NJ.
4-13-phorbo!1-oleyl-2-acety!-
purchased
from
Calbiochern
for
Sorter,
from 4-a-
and
culture
from Flow Laboratories nuclidiny! benzilate (QNB) Dr.
were obtained Louis, MO).
(4-j3-PDD), were
Water
aqueous PA).
3-acetate (TPA), chlortetserum albumin (BSA), and
hyaluronidase (bovine, testicular) Sigma Chemical Company (St.
media
(McLean, was the
was
(La
obtained
VA). generous
Hoffmann-LaRoche
3-Quigift of
Laboratories,
9-Amino-3-chloro-7-methoxyacridine
(ACMA) was generously provided in purified Prof. C. P. Lee, Department of Biochemistry, State University School other reagents, obtained of analytical grade.
of Medicine, from various
form by Wayne
Detroit, suppliers,
MI.
All were
Media The culture medium (CM) used in all experiments was a modified Krebs-Ringer bicarbonate solution described by Toyoda et al. (1971), containing 25 mM sodium glucose, prepared
lactate, and 20 without
1 mM mg/rn! BSA,
a 0.20-pm in 20-rn!
filter aliquots
(Nalgene) for single
as needed, through a CO2 /95%
(1985).
two
the the
MATERIALS
has
into
(CTC)
1985). In this paper we report that, to zonae, phorbol esters are potent
stage.
were and
resolved
Storey, bound
perturbants of they accelerate
1985; Brock et al., 1985; Sharkey and Blumberg, 1986). If the zona-induced acrosome reaction were mediated by this mode of intracellular signaling, then one would expect perturbation of this process by phorbol esters. The zona-induced acrosome reaction been
ch!ortetracycline
(Lee and in sperm
glycerol
action of hormones with plasma membrane surface receptors on somatic cells. Sperm-zona pellucida interaction may, therefore, represent a process analogous to ligand-receptor interactions characterized in many somatic cells, of which ligand-induced One reaction receptor-mediated
use
12,
and Storey, 1982a; Storey et a!., 1984; Saling and Lakoski, 1985; Lee and Storey, 1985). The apparent specificity of sperm-zona interaction parallels the strict specificity known to be required for the inter-
exocytosis
AL.
of to
inhibitors of galactosy! transferase (Shur and Ha!!, 1982; Lopez et al., 1985). QNB does not affect sperm binding, as pointed out above, nor does it perturb the site sensitive to trypsin inhibitors (Benau and Storey, 1986). The sites for zona binding have been shown to be on the sperm plasma membrane by direct autoradiography
ET
sodium pyruvate, 5.57 mM BSA. Medium CM was first sterilized by passage through and use.
supplemented 0.20-pm filter,
then frozen at -80#{176} C Aliquots were thawed
with and
BSA, gassed
resterilized with 5%
air to pH 7.4.
Gametes Procedures based were All
for on those described handling
the
preparation of Inoue previously of
gametes
of and by
mouse
gametes
Wolf (1974, Lee and.
was
done
1975) Storey at
37#{176}C
PHORBOL
under
silicone
oil
Corp.,
Midland,
MI)
CO2 /95% were obtained (Harlan
Eggs
for
mature (Harlan
Sprague
zonae
had
with
Inc.,
were
Sperm
of
10.0
IU
and Zonae
(hCG;
mice super-
containing the 10-fold dilution
IN).
at
Binding
were prepared a narrow-bore
diameter
of
were washed adherent egg
the
to
Pellucidae
by forcing micropipette
zona-intact
3 times cytoplasm
egg).
comulus-free (i.d. The
isolated
eggs 0.75
=
zonae
in medium CM to remove and resuspended in 100-200
for
at 60
a concentration mm in the above
the appropriate 1 mM OAG;
60
compound nM 4-a
were added formamide
from (DMF)
0.1%
final
in the
of 10-25 X 10 medium containing
DMF
in dimethylwas present
alone
the control determinations. Comparative experiments showed that this concentration had no effect on the ability of the sperm
was
added
at in
control of DMF to bind to
was adjusted to 2 X 10 of the sperm suspension
cel!s/m! to a
100-pl drop containing 100-150 mechanically isolated zonae pel!ucidae plus the same compound at the concentration present during capacitation so as to maintain a constant concentration. The point of addition be the
of sperm zero time
compound so that the
sperm with the compound was at of the sperm-zona binding step. the test compound was present but was omitted from the drop
zonae, of the
allowed time
to the isolated zonae point in the experiments
was
sociated pM QNB mm
resulting compound
to
the
bind
zonae
taken to described
sperm. In was added
post-binding
in
an approximately as the sperm were
to the with
zonae
for
bound
a second set of to the sperm-zona
of sperm
Fluorescence
15
sperm
mm, were
experiments, suspension
50 60
to zonae.
Assays
The binding lucidae was (400 X) using
of sperm to the isolated scored by phase-contrast a Nikon Optiphot microscope
interchangeable optics. The
phase contrast bound sperm were
zonae pe!microscopy equipped
and then
epifluoresexamined
by epifluorescence microscopy (400 x) for patterns of CTC fluorescence that reflect the state of capacitation and the stages of the acrosome reaction, according to the method of Ward and Storey (1984). At the designated time point, 5 p! of a 500-pM CTC solution aded
washed
structurally intact zonae and undergo the acrosome reaction. Incubation of sperm with these test compounds during capacitation was used in the majority of the experiments. After capacitation, the final sperm concentration by adding an aliquot
which
was
to be tested (65 nM TPA; or 4-13-PDD). The compounds
stock solutions such that DMF
suspension;
were
with cence
pl CM (Lee and Storey, 1985). Sperm suspensions were prepared from excised caudae epididymides as described by Lee and Storey (1985). Sperm were capacitated, cells/rn!,
test step,
washed 3 times in CM containing 50 pM QNB using a wide bore micropipette to remove any loosely as-
All incubations were carried out in medium CM containing 20 mg/m! or 4 mg/mI BSA under silicone oil at 37#{176}Cin a humidified atmosphere of 5% CO2 in air. Mechanically isolated, structurally intact zonae pel!ucidae through
the
added to the zonae. In one set of experiments, QNB was added to a final concentration of 50 pM in the zonae suspension prior to the addition of sperm. Sperm
Chemical
619
In some experiments, from the capacitation
virgin,
pregnant
Sigma
REACTION
first contact of the the zero time point In other experiments, during capacitation
(PMSG) (Gesty!; Disoynth, 36-56 h later by 10.0 IU
gonadotropin MO).
Intact
with
Indianapolic, from
ACROSOME
in the figures. was omitted
Corning
Swiss Webster Mice were
Inc.).
THE
Spermatozoa Swiss Webster
obtained
injections
Capacitation
Structurally
Dow
AND
equilibrated
Dawley
mare’s serum gonadotropin Chicago, IL), followed human chorionic Co., St. Louis,
been humidity. breeder
12-week-old) Dawley, i.p.
200,
100% retired
isolation
(10to Sprague
ovulated
Corning
that
air at from
5%
mice
(Dow
ESTERS
to
an equal
zonae
with
volume
(37#{176} C) slide. Glutaraldehyde pH 7.8) was immediately tration placed zonae.
of over The
100% patterns.
humidity This
of CM containing
bound
sperm
on
5-10
a pre-warmed
(12.5% in 1 M Tris HC1, added to a final concen-
0.1%. A covers!ip was then carefully the 10-p1 drop containing sperm bound to slides were kept in light-tight chambers at until mode
assayed of fixation
for CTC maintains
fluorescent the patterns
without change for up to 12 h. In parallel experiments, the uptake of the dye ACMA was examined in sperm bound to zonae for different time intervals. A 500pM stock solution of ACMA in DMF was prepared and kept in a light-shielded container. At the time of assay, 9.85 pl of CM containing 5-10 washed isolated zonae with bound sperm was added to a warmed slide. A 0.1 pl aliquot of the ACMA stock solution was added with stirring to give a final concentration of 5 pM ACMA. Glutaraldehyde (12.5% in 1 M Tris-HC1, pH 7.8) was then added to a final con-
LEE
620 centration the drop container
of 0.05%. A covers!ip was then placed over and the slides were held in a light-tight at 100% humidity, as described for the CTC
procedure. from 15
mm
Determinations of to 5 h post-fixation
ACMA fluorescence showed that the
fluorescence patterns were stable to at least 5 h. The slides were examined under phase contrast (400 X) and then under epifluorescence (400 X) for fluorescence patterns of CTC and ACMA on the zonae-bound sperm.
Details
of
emission
system
Lee
Storey
and
time with
epifluorescence
both
(1985).
course 2-3
the
for
and
have
been
described
For
each
time
point
determinations, sperm/zona
excitation
dyes
a
were
total
of
by in the
30
zonae
scored.
of
In
TPA
a previous
acrosome
Sperm
CTC
study
of
reaction
sperm
bound
zonae pellucidae, in two stages, and
on
calibrated
Storey, 1985).
to
induced isolated,
in
ultrastructural
course
of
capacitated
the
mouse
intact
studies
mouse occurred probes
(Saling
1979; Lee and acrosome-intact
a pattern of fluorescence anterior head and midpiece
and
Storey, sperm
characterized by separated by a dark
band over the postequatorial segment. This banded pattern was designated Pattern “B.” It disappeared with time of incubation of sperm bound to zonae and was replaced by a punctate, or spotty, pattern of fluorescence
over
With
time
further
the
head,
designated
of incubation,
Pattern
Pattern
“S.”
and for convenience have been designated of CTC fluorescence patterns. The first stage B-to-S transition. The second stage is the
S-to-AR transition. stages was examined.
The
effect
When sperm were incubated (Nishizuka, 1984) during both zona-binding sequences, both S-to-AR course bound started
for
the
of TPA
cell
population
1). This characteristic lag period, with structurally intact zonae 1982a; Lee and Storey, 1985, TPA, population
and
by
the was
loss of complete
more rapid loss of Pattern particularly at the earlier mirrored by a more rapid Pattern S, when compared
Pattern in 150
mm
(Fig.
consistently and Storey, was abolished
B from the sperm mm (Fig. 1). The
B in the presence of TPA, times of incubation, was rise in the percentage of to the control (Fig. 2).
The presence of TPA therefore transition. No effect of TPA zonae was observed during these
the
in 180 observed (Florman 1986),
accelerates on sperm experiments.
the B-to-S binding to
effect was seen with TPA on the which marks the second stage of acrosome reaction. The percentage
displaying Pattern S remained elevated later incubation times when compared to
control,
and
did
not
decrease
below
30%
(Fig.
2).
The percentage of sperm displaying Pattern S in the presence of TPA remained constant for up to 260 mm, as determined in a separate set of incubatmons for extended times. Pattern AR, which approached 90% zero
as the in the
only 70% mained at
percentage control
of Pattern experiments
in the presence this percentage
S dropped towards at 210 mm, reached
of TPA (Fig. for up to 260
3) and reWhen
mm.
100
80
S disappeared
and was replaced by Pattern AR, corresponding to the completed acrosome reaction and characterized by dark heads and bright midpieces. The two stages of the zona-induced acrosome reaction occurred in sequence, in terms is the
completion
of sperm during the
Patterns
that the reaction with fluorescent
1979; Saling et a!., With CTC, capacitated
showed bright
time
structurally
we found as determined by
Fluorescence
the
AL.
The opposite S-to-AR transition, the zona-induced
RESULTS Effects
ET
on
both
60
40
20
these 0
with 65 nM TPA the capacitation and the B-to-S and the
transitions were affected with regard to time (Figs. 1-3). The loss of Pattern B from the sperm in the unperturbed control system only after a lag period of 30 mm and reached
0
30
60
90120150180210240270
Minutes FIG. 1. The effect of 65 nM TPA on the time course of the loss of CTC fluorescence Pattern B in mouse sperm bound to isolated, intact zonae pellucidae. For each time point, a total of 30 zonae with 2-3 sperm/zonae were scored. Error bars Control points in the absence of TPA: presence of 65 nM TPA: (0).
represent standard deviations. Experimental points in the
(.).
PI-IORBOL
ESTERS
AND
100
THE
ACROSOME
REACTION
strated that mechanically isolated zonae, incubated with phorbol esters for at least 1 h, still retain their ability to induce the acrosome reaction of bound sperm with a time course similar to that seen with control, untreated zonae (Endo et al., 1987b). In experiments where 65 nM TPA was absent
80
60
%s
during
capacitation
added TPA
40
0
I#{149}
0
30
I
11
60
1#{149}
90
I’I#{149}
I#{149}
sperm
I
120150180210240270
Minutes
reaction
in separate
was
experiments,
increase
there
in the
extended at the
monitored was
percentage
times, TPA
after no
further
of AR
there was concentration
300
mm
signifi-
patterns.
no effect used
Even on sperm in these
experiments. Pattern AR was observed in the sperm at earlier times in the presence of TPA when compared to the control sperm, which is the result expected from
an
states
characterized
elevation
(Fig.
2).
The
in the by increase
time was slower absence (Fig. 3).
percentage
Pattern in
in the
percent
presence
of
which (Wolf,
is optima! for capacitation 1979). Since BSA has been and
earlier
pattern
experiments out in medium
(Dicker
not
due
TPA
than
on
the
zonae.
same
as those
effects
were
of
CTC
the controls were
no
longer
fluorescent
in the carried
absence out to
incubation with TPA did its effect could be reversed
its effect
occurred
which
occurs
only
in the
was further exon the spontanein mouse
in medium CM in the absence at a rate far slower than that induced (Florman and Storey, 1982a). The
sperm
of zona by zona spontane-
reaction was unaffected by TPA at as high as 10 pM. No changes in the of the CTC fluorescent patterns
It has
experiments
been
80
were BSA,
and fertilization reported to bind
1980),
100
in its
to those observed with 20 mg/ml BSA (Figs. Both mediation and perturbation of the TPA by BSA can therefore be ruled out. effects of TPA on these transitions were due effects of this phorbol ester on the sperm, and to effects
TPA
This last point the effect of TPA
reaction,
ous acrosome concentrations rate or quality
with
were carried out to assess whether the differential effects of TPA on the B-to-S transitions could be due to the time-dependent binding of the phorbo! ester by BSA. When the BSA concentration was reduced to 4 mg/rn!, the effects of 65 nM TPA were essentially identical 1-3). effects The to the
the
effects of of CTC
times
AR
shown in Figures 1-3 CM containing 20 mg/rn!
Rozengurt,
by
acrosome
suspended protein protein
were
courses
that
zonae. testing
similar courses
were
of intermediate
S at these
The carried
TPA
and of
sperm
sperm in Figures 1-3. If during capacitation but was 10-fold upon addition of
that prolonged the sperm, that
dilution,
ous
the time
when
time point), The time
were the same as for These experiments
demonstrate not damage presence amined
added
changes
zonae, the
patterns of TPA.
by
acrosome
to
observed:
FIG. 2. The effect of 65 nM TPA on the time course of the appearance of CTC fluorescence Pattern S in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. Absence of TPA (U); 65 nM TPA (0).
at these motility
pattern
was
shown for the TPA-treated 65 nM TPA was present diluted approximately
20
cant
but
to zonae (zero were expressed.
fluorescent
the
621
demon-
60
%R 40
20
0 0
30
60
90
120150180210240270
Minutes FIG. 3. The effect of 65 nM TPA on the time course pearance of CTC fluorescence pattern AR in mouse sperm isolated, intact zonae pellucidae. The conditions were those 1. Absence of TPA (a); 65 nM TPA (0).
of the apbound to for Figure
LEE
622 were
observed
at
more than two needed to affect bound
to
this
zonae.
When
motility pM TPA
and morphology were unaffected
mm. These
results
interaction the
that
proteins
occur
and
the
B-to-S
the This
is a potent acrosome
transition,
is
sperm with 10 to 300
up
post-binding and
in order
S-to-AR
leading to were blocked.
with QNB, which the zona-induced
controls,
a specific,
zona
must
B-to-S
to
the for
sperm TPA
transitions.
implication were valid, it would effects of TPA would be blocked interactions reaction
which than that of sperm
in groups treated for time periods
the
membrane
affect
concentration,
compared
imply
between
plasma
TPA
orders of magnitude greater the CTC pattern transitions
If
to this
be expected that the when the sperm-zona zona-induced supposition
acrosome was tested
and specific inhibitor of reaction at its first stage,
but
does
not
affect
sufficient all of the
in the sperm
acrosome reaction by (Figs. 1-3). It has been must be present between
absence population
of
QNB to complete
have the
Pattern that 20 min
AR QNB after
progressing to shown previously zero time and
acrosome ionophore
reaction concerns Ca2 A23 187 at 5 pM
accelerate reaction
The diva!ent cation has been shown to
markedly the zona-induced in sperm bound to isolated intact
acrosome zonae (Lee
and Storey, 1985). The two stages, B-to-S and Sto-AR, can no longer be resolved: the entire population of bound sperm undergo a B-to-AR transition within the time resolution of the CTC assay. This suggested that A2 3187 might overcome the retardation of the S-to-AR transition induced by TPA. When this the
was tested, same result
it was found in the presence
absence: only a B-to-AR The final percentage playing the AR pattern A23
187
of TPA
of
that 5 pM A23 187 gave of 65 nM TPA as in its
transition could be observed. the sperm population diswas 100% in the presence of
plus
TPA,
compared
alone
(Fig.
3).
to
70%
in the
presence
sperm
binding to the zonae (Florman and Storey, 1982ab; Storey et al., 1984; Lee and Storey, 1985). As shown in Figure 4, QNB is as effective an inhibitor of the B-to-S transition in the presence of 65 nM TPA as in its absence. The sperm bound to zonae retain Pattern B over the entire 210-mm observation period, a time nearly
ET AL.
Stereospecificity
The was
of
isomeric
examined
the
Phorbol
specificity using
Ester
of the
available
Effects
phorbo!
phorbol-12-1
ester
effect
3-didecano-
ate stereoisomers. TPA has the (3 configuration at the 4-hydroxyl group with trans fusion of the A and B rings. The corresponding stereoisomer, 4j3-phorbol-12,13-didecanoate (4-j3-PDD; 60 nM) had very similar AR patterns didecanoate other hand,
effects on the time courses of B, 5, and as 65 nM TPA. The 4-a-phorbo!-12,13stereoisomer (4-a-PDD; 60 nM), on the had no effect on the time-course curves
sperm binding to zonae for effective inhibition of the zona-induced acrosome reaction to occur. Addition of QNB 60 mm after sperm binding has no inhibitory effect, and the sperm to have been occluded
binding at this
site for QNB time by zona
(Florman et al., 1982). The implication sperm-zona interaction is complete at 60 binding. This would lead to the expectation added at this of TPA. This ment. resulted
Addition of in time-course
patterns those served
in
the
QNB
presence
is
closely
effect effect coupled
glycoprotein acrosome
of
not affect borne out
at curves
60 mm post-binding for the three CTC
65
nM
TPA
the action by experi-
identical
needed reaction.
additional pellucida
on TPA. TPA on
These results indicate the B-to-S transition is
interaction for
successful
aspect of and sperm
to also direct
of sperm induction
the interaction leading to the
and
zona of
-
80
%B
would was
of
to the
that postQNB
mm that
shown in Figures 1-3. This experiment as a control to show that QNB had no
antagonistic that the
An zona
time point supposition
100
appears protein
I
I
-
60 40
-
20
00
30
60
90
120150180210240270
Minutes
the
between induced
FIG. 4. Effect of 50 MM QNB on the loss of CTC fluorescence Pattern B in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. Absence of TPA (#{149}); 65 nM TPA (0).
PHORBOL
of of
the CTC time-course
fluorescence curves
disappearance essentially
of the superimposable
1-3,
4-a-PDD
with
1984;
the
control
the TPA curves. The of the zona-induced sperm has the same
Blumberg
a!.,
activation stereospecificity
likely
this
THE
ACROSOME
1985).
Since
100
were Figures
80
curves
and
of protein (Niedel
enzyme
is the
target
kinase et a!.,
40
20
phorbol
C also 1983),
of these
disit is
agents
0 0
in
30
60
Effects
of
The kinase
physiological C is considered
intracellular to be the
from
hydrolysis phorbo! protein
100
1, 2-Diacyiglycerol
generated
membrane
regulator diacylglycero!
of protein moiety
65
nM
1985; courses
acrosome disappearance than with
If the sperm that
et a!., 1984; Brock et al., 1985). OAG, the concentration expected
Effect Associated
of
180
150
180
210
240
TPA
(Blumberg
Sharkey of the
and two
%S 40
et a!.,
Blumberg, stages of
1985;
20
Brock
et
1986), on the zona-induced
the
0
TPA with
on
Sperm the
B-to-S
was observed on of zonae pellucidae. ACMA
Fluorescent
sperm
100
a pKa within
120
1
210
240
-
0/
40
20-
0
.
0
is a weak base with 1983), accumulates
90
60-
Transition
1985). of 8.8
I’E’
60
-
80
Patterns
ACMA has been shown to for the first stage of the reaction (Lee and Storey,
30
Minutes
in
The fluorescent probe be an additional marker zona-induced acrosome This probe, which (Huang et al.,
11II1
0
reaction is shown in Figure 5. The rate of of Pattern B is somewhat more rapid TPA, but otherwise the effects of OAG,
1 mM OAG in the absence
-
60
The to
TPA, and 4-13-PDD are very similar. The B-to-S transition is accelerated while the S-to-AR transition is retarded. Incubations extended to 260 mm in the presence of OAG also displayed a plateau of both Pattern AR (7 0%) and Pattern 5 (3 0%) of the sperm population. As observed with TPA and 4-(3-PDD, no effect of suspension
150
polyphosphoinositide
(Nishizuka, 1984; Berridge, 1984). esters were acting by stimulating kinase C activity, it would be expected
(Nishizuka of 1 mM
mimic
120
-
80
an exogenous 1,2,-diacy!g!ycerol would have effects similar to TPA and 4-13-PDD. A diacy!g!ycero! frequently used as a test compound for comparison is OAG effect
90
Minutes
sperm.
mouse
a!., time
623
60
perturbation acrosome stereoisomer
the
REACTION
sets and/or
esters as do the various effects in other cell types (Nishizuka,
et
ester-induced plays such
AND
two
and AR patterns the curves of
on
for phorbol by these agents
that
5,
mimicking
4-#{237}3-PDDmimicking ot the two stages reaction in mouse specificity induced
patterns. The the appearance
for B,
ESTERS
IIIIIII
30
I
60
90
120
150
180
210
240
Minutes FIG. 5. Effect of 1 mM OAG on the time course of the loss of fluorescence B (top panel), the appearance of Pattern S (middle panel), and the appearance of Pattern AR (bottom panel) in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. CTC
LEE
624 100
ET AL.
Pattern
5, was
reaction, through
80
60
obligatory.
defined by intermediate
B-to-S-to-AR.
This
a sequential
first-order
Completion Pattern state
AR, 5,
formulation
of the acrosome could with
is the
chemical
only proceed the sequence same
as that
reaction
written
B
As
0
30
60
90
120
150
180
210
Minutes FIG.
6.
Effect
of
65
nM
TPA
ACMA fluorescence Pattern intact zonae. The conditions (#{149}); 65 nM TPA (a).
on
the
time
A in mouse are those for
course
sperm Figure
of
the
bound to 1. Absence
loss
of
isolated, of TPA
the
reaction
mouse
fluorescence some. ACMA
because
This has fluorescence
time
sperm
course
of
of
been the
with
pH
within
a lower
designated was shown loss
heads
of
Pattern to follow
CTC
Pattern
enhanced the
acro-
distribution different with this
B both of 50
tion
that
both
(Lee
and
Storey,
support
probes
monitor
for the
the
earlier B-to-S
contentransition
1985). DISCUSSION
The acrosome the time patterns population and that
division reaction
of
the zona-induced mouse sperm into stages was originally based on
course of the changes in CTC fluorescence observed as the reaction proceeded in a of sperm bound to isolated zonae (Lee
Storey, 1985). an intermediate
The essence of such state, experimentally
a model defined
was by
B
of zona-bound sperm displaying the fluorescent patterns was fully consistent formulation, if the rate constants k1 and k2
model consisting regulated
for
But in the original study, means of distinguishing
there was between
and
consisting was seen
inhibitory
the first formulated decrease
k2.
of a single experimentally
effects
view.
cence patterns Pattern S at rapid decrease in Pattern intermediate that Pattern
no a
the zona-induced acrosome reaction of two sequential stages, with each stage by different regulatory processes, and a
as two sequential membrane changes. The data Figures 1-3 support the idea that the biologically active phorbo! esters and OAG have both stimulatory
1 for loss of CTC Pattern presence of TPA. Addition
further
of
B in sperm
for Pattern B. The coincidence of the time-dependent loss of Patterns A and B in both the absence and presence of TPA, and the inhibition of both losses by provide
concentration
model progress
pM QNB at zero time of binding completely inhibited the loss of ACMA fluorescence; the results with Pattern A were the same as those shown in Figure 4
QNB,
the
A. Loss of closely the
bound to zonae and to be associated with loss of the H+ permeability barrier across the plasma membrane (Lee and Storey, 1985). The effect of 65 nM TPA on the time course of ACMA fluorescence loss is shown in Figure 6. The time course is essentially identical to that shown in Figure in the absence and
proceeds,
falls monotonical!y with time; the concentration of S rises as it is formed from B, then falls as it is converted to AR; the concentration of AR rises after a lag time, then levels off as the reaction is completed. The
were similar. experimental acrosome-intact
AR
k2
k1
0
as:
F-
40
20
for
In above, The
regulated reaction whose by CTC fluorescence
on these terms these
effect
of
transitions
the agents
on the
sequential increase
distribution
and
in
support reaction k1 and
of fluores-
is observed as a more rapid increase in earlier times, with a concomitant more in Pattern B. This more rapid increase S at earlier times provides more of this state leading to Pattern AR. The result is AR is observed with TPA at earlier times
than in the control, although its rate of formation from the intermediate, as shown by the slope of the curves tracing the increase of AR with time in Figure 3, is less. The B-to-S transition is closely correlated in time course with the loss of ACMA fluorescence both in the presence and absence of TPA. Since the loss of ACMA fluorescence represents a loss of the permeability barrier of the plasma membrane, such that a pH gradient can no longer be maintained, the B-to-S transition can be defined operationally in terms of an “intact-to-leaky” transition for the plasma membrane over the sperm head. The next stage, S-to-AR, can
PHORBOL
then Since
be defined each of
operationally these transitions
ESTERS
AND
as “leaky-to-reacted.” comprises a group
THE
of
closely linked reactions, at least two plausible sites of regulation of the zona-induced acrosome reaction by phorbol
esters
may
kinase C activity in mouse sperm, these compounds
the
acrosome
reaction
processes reported,
do involve moreover,
boar
sperm
directed protein
be
postulated.
protein
has not yet been demonstrated potency and stereospecificity in affecting the zona-induced suggest
that
this enzyme. that this
(Adeyemo
at the kinase
Although
and
these
It has enzyme Koide,
identification C in mouse
regulatory
Studies
and characterization sperm are currently
of in
625
REACTION
compelling pe!!ucida Lakoski,
that mouse sperm bind to the with an intact plasma membrane (Saling 1985; Lee and Storey, 1985; Bleil
Wassarman, microscopy, zona
of
been recently is present in 1986).
ACROSOME
1986). When all sperm bound
of
whereas vitelline 1979).
zona-intact
examined to the
eggs
had
by outside
intact
those sperm that had penetrated the penspace were acrosome reacted (Saling et a!., The two CTC patterns corresponding to these
states of the mouse sperm were the B and AR patterns, respectively. At present, there is no firm evidence that, in the absence of perturbants, sperm displaying CTC Pattern the zona. It
S still remain bound to the outside of has been shown, however, that mouse
sperm
displaying
The effects interaction of
outside treated to the
of the zonae of mouse eggs that with TPA, a condition in which the full acrosome reaction does not occur
membrane,
since
the
on mouse pellucida effects
sperm require the and sperm plasma
are
manifest
at
60
nM
Pattern
TPA in the presence of zonae but are not observed at concentrations as high as 10pM in sperm in suspension in the absence of zonae. The presence of zonae
a!., 1987b). This implies ligand groups may mediate
pel!ucidae
division described
of labor above,
cellular for the
messenger zona with
is
also
necessary
for
the
OAG
effects
shown in Figure 5. These data support the concept that the sites regulated by phorbo! esters and diacylglycerol are affected only when the sperm interact with
a
physiologically
relevant
pellucida. Since ZP3 both sperm-receptor activities
(Bleil
be anticipated protein and required phorbo! remains
and
Wassarman,
that the the mouse
for the esters to be
specificity of that coupling
1980,
interaction sperm
this of
interaction the zona
receptor Both senger
which in
two types an intracellular
occupancy interact regulates
zona
1983),
with
to
of
The
zona is
concept receptor
ester-sensitive sites of a series of defined model based experimental
on this data, is
messenger molecules signal chain linking
protein
protein enzyme
the
kinase activity
kinase
C activation.
C, but involved
one in
mesthe
B-to-S transition, while the other messenger regulates enzyme activity involved in the S-to-AR transition. Definition of each of these transitions, or stages, of the still
zona-induced at very coarse
acrosome resolution.
reaction is, of necessity, The evidence is by now
another
reaction cellular
it would
between this plasma membrane
supports ligand/sperm
messengers. The simplest and consistent with the
one in participate
the
expression of the observed effects of and diacylglycerol. However, this demonstrated experimentally. The
interaction with the phorbol occurs through the generation cellular concept,
ligand,
is the zona protein that possesses and acrosome-reaction-inducing
electron of the
acrosomes,
progress. of TPA the zona
zona and and
may
mediate
S remained
the
S-to-AR
be consistent postulates
systems linking the activation that
to
the
operates
protein while
transition.
This
with the model two sets of intra-
the sperm of protein
zona-induced
in mouse sperm signal transduction
the
had been transition (Endo et
that one set of zona the B-to-S transition
would which
concept
bound
receptor kinase C. acrosome
through mechanism
an intrainvolving
second messengers, having some features in common with those intracellular regulatory systems characterized for hormone-induced actions in somatic cells (Rodbe!l, 1980; Ross and Gilman, 1980; Bernidge, 1984), receives further support from the observation that mouse sperm contain a protein with properties similar to the inhibitory guanine nucleotide-binding regulatory protein, Gm (Kopf et a!., 1986). This protein appears to mediate signal transduction distal to the binding of cell surface receptors by their respective ligands in somatic cells (Gi!man, 1984), and has been implicated in such cellular functions as receptor-mediated (Gomperts, Davidson, cyc!ase 1985).
changes
Ca2
+
mobilization
1983; Moiski et al., 1984; Haslam and 1984; Turner et al., 1986) and adenylate regulation Functional
(Gilman, inactivation
in mouse sperm by inhibits the zona-induced B-to-S hibitory
in
transition effect
5’0(2-thiotriphosphate)
of
stage; lAP
1984; of the
islet-activating acrosome is
protection conferred
(Endo
et
Smigel Gi-like
et al., protein
protein reaction
(lAP) at the
against the inby guanosineal.,
1987a).
The
LEE
626 means provide
by the
which
these
regulatory
elements signals
link
that
ultimately
together
to
mediate
the stages of zona-induced acrosome reaction have not yet been elucidated. Enough evidence is at hand, however, to include the zona-induced acrosome reaction responses signal
in mouse utilizing transduction
sperm a
in the
large
ligand-activated
group
of cellular intracellular
chain. ACKNOWLEDGMENTS
We thank Ms. Cynthia Ward for consultation and organization of ovulation schedules and Dr. Danny Benau for literate and graphic discussions. Excellent secretarial help was provided by Ms. Elvira Walker.
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