BIOLOGY
OF
REPRODUCTION
Stimulation
Sperm
40, 133-141
(1989)
of Phospholipid
Turnover in Isolated Sea Urchin
Heads by the Fucose-Sulfate
Glycoconjugate
That Induces an Acrosome Reactionl
STEVEN
and
E. DOMINO3
DAVID
L. GARBERS2’3’4’5
Departments
and Molecular
and Howard
Vanderbilt
of Pharmacology
Physiology
and Biophysics4
Hughes
Medical
Institute
University
Medical
Center5
Nashville,
Tennessee
37232
ABSTRACT
The fucose-sulfate
glycoconjugate
(FSG) component
of sea urchin egg jelly that induces
an acrosome
reaction
in spermatozoa-stimulated
multiple Cafl-dependent
phospholipid
changes in the sperm cell head and
flagellum.
When cells were radiolabeled
with myo-f3Hjinositol,
FSG treatment
decreased
radioactivity
in
phosphatidylinositol
4-phosphate
and phosphatidylinositol
4,5-bisphosphate
within 30 s. In addition,
FSG
treatment
elevated
concentrations
of phosphatidic
acid in spermatozoa.
The Cafl-channel
antagonist,
verapamil,
inhibited
the effects of FSG on (3Hjpolyphosphoinositides
and phosphatidic
acid. To investigate
the possible
compartmentalization
of phospholipid
turnover,
isolated
heads and flagella
were prepared.
Treatment
of sperm heads with FSG or the monovalent
cation ionophore,
gramicidin
S, caused increased
(3Hjinositol
phosphate
and phosphasidic
acid accwnulation
and induction of an acrosome reaction. Effects of
FSG and grainicidin
S on phosphatidic
acid elevations
in sperm heads and intact cells were inhibited
by
verapamil.
FSG failed to cause detectable
changes in (3Hjinositol
phosphate
or phosphatidic
acid concentrations in isolated flagellar
preparations.
However,
when cells were treated with FSG and the flagella
were
isolated
subsequently,
phosphatidic
acid concentrations
in the flagellar
preparations
were increased.
INTRODUcTION
histone Hi on a single site by the cyclic AMP-dependent protein kinase (Porter and Vacquier, 1986; Porter et
aL, 1988).
In addition
to changes
in cyclic nucleotide
metabolism, rapid ionic flux changes
occur during the acrosome reaction. Upon addition of egg jelly, sperm cells
release K and H and take up Na and Ca2 (Schackmann et a!., 1978; Tilney et al., 1978; Schackmann
et
al., 1981,
1984;
Gonzalez-Martinez
and
Darszon,
1987), transient
intracellular
alkalinization
(Christen
et
a!., 1983; Lee et a!., 1983; Garcia-Soto
et a!., 1987),
and increases
in intracellular
free calcium
concentrations (Trimmer
et a!., 1986). The acrosome
reaction can
be induced in the absence of egg jelly by treatments
that affect ion conductance,
including
the divalent ionophore, A23187 (Decker et al., 1976; Talbot et al., 1976;
Collins
and Epel, 1977); the monovalent
cation ionophores, mgericin and gramicidin
S (Schackmann
et a!.,
1978); elevated
external
pH (Gregg and Metz, 1976);
and monoclonal
antibodies
to sperm
cell membrane
proteins
(Trimmer
et a!., 1986, 1987).
The hydrated
extracellular
matrix
surrounding
sea
urchin eggs, called the egg jelly layer, contains
molecules that have dramatic
effects on sperm cell behavior.
A component
of egg jelly, the fucose-sulfate
glycoconjugate (FSG), induces an acrosome
reaction
with relative species
specificity
(SeGall
and Lennarz,
1979,
1981). FSG has been shown to elevate cyclic aiienosine
monophosphate
(cyclic
AMP) concentrations
in intact
cells (Kopf and Garbers,
1980; Garbers
et a!., 1983)
and isolated sperm heads
(Garbers,
1981), stimulate
cyclic AMP-dependent
protein kinase activity
(Garbers
et al., 1980), and increase
phosphorylation
of sperm
Accepted March 7, 1989.
Received November
7, 1988.
1Supponed
by National Institutes of Health Grants 10)10254,
HD05797,
and GM07347,
and grant 87-CRCR-1-2569
from the United States Department
of Agriculture.
A portion of this work will be submitted to Vanderbilt
University in partial fullilintent
for the requirements
for the degree of Ph.D. in Pharmacology
(S.ED.)
2Reprint requests.
133
134
DOMINO
AND
The morphology
of the sea urchin
spermatozoon
during the acrosome
reaction has been well described
(Eddy and Shapiro, 1979), but relatively
little is known
about the biochemical
changes in the membrane
during
this exocytotic
event. Studies with mammalian
spermatozoa have shown changes
in plasma membrane
phospho!ipids
when an acrosome
reaction
is induced
by
ionophore
or bovine serum albumin treatment
In human spermatozoa
radiolabeled
with [14C]arachidonic
acid, treatment with A23 187 increased
radioactivity
in
free arachidonic
acid and diacylg!ycerol
and decreased
radioactivity
in a total phospholipid
fraction (Bennet et
a!., 1987). In addition, plasma membranes
isolated from
boar spermatozoa
incubated with albumin had increased
concentrations
of diaclyglycerols
and free fatty acids
and decreased
concentrations
of phosphatidylinositol
and sphingomyelin
(Nikolopou!ou
et a!., 1986).
Sea
urchin egg jelly has been reported to increase
concentrations of free fatty acids associated
with spermatozoa
(SeGal! and Lennarz,
1981).
We have previously
reported that FSG caused Ca2dependent
increases
in [3H]inositol
phosphate
accumulation,
suggesting
that phosphatidy!inositol
turnover
(P1) is associated
with the acrosome
reaction (Domino
and
Garbers,
1988).
Whether
or not
[3H]phosphoinositide
accumulation
was altered and whether the
changes in phosphatidylinositol
turnover occurred
in the
head or tail region were not known. Here, we report
that FSG alters
[3H]phosphoinositides,
[3H1inositol
phosphates,
and phosphatidic
acid concentrations
in
iso!ated sea urchin sperm heads.
MATERIALS
AND
METHODS
Chemicals
Tritium-!abe!ed
myo-inositol
(15 Ci/mmol)
was purchased from American
Radiolabeled
Chemicals
Inc. (St.
Louis,
MO).
[3H]Inositol
1-phosphate
(I(1)P1),
[3H]inosito!
1,4-bisphosphate
(1(1 ,4)P2),
[3H]inosito!
1 ,4,5-trisphosphate
(1(1 ,4,5)P3),
and
[3H}phosphatidy!inositol
4,5-bisphosphate
(PIP2) were from Du
Pont-New
England Nuclear (Boston,
MA). Carrier-free
[32P]orthophosphate
(32P1) was purchased
from ICN
Radiochemica!s
(Irvine,
CA).
Gramicidin
S and
(±)verapamil
hydrochloride
were obtained
from Sigma
Chemical
Co. (St. Louis, MO). lonomycin
was from
Ca!biochem
(La Jolla, CA). Merck Silica Gel 60 and
Silica Ge! 60 F-254 thin-layer
plates were purchased
GARBERS
from Bodman
Chemicals
(Aston, PA) and were stored
in a desiccator
overnight
before use. Egg yolk phosphatidyicholine-derived
phosphatidic
acid
standards
were from Avanti
Polar Lipids,
Inc. (Peiham,
AL).
Osmium
tetraoxide
and po!y-L-!ysine
were from Po!ysciences, Inc. (Warrington,
PA). All common
chemicals
were the highest
grade available
and were purchased
from Sigma or Fisher Scientific
Co. (Pittsburg,
PA).
Animals
and
Collection
of Gametes
Sea urchins
(Strongylocentrotus
purpuratus)
were
obtained
from Marinus,
Inc. (Long Beach, CA). Gametes were collected
by injecting
urchins with 3-5 ml
of 0.5 M KC1 and washing
them in cold artificial
seawater
(ASW), pH 7.9, containing
454 mM NaC!, 9.7
mM KCI, 9.6 mM CaC12, 24.9 mM MgCl2, 27.1 mM
MgSO4, 4.4 mM NaHCO3,
and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfomc
acid (HEPES).
“Ca!cium-free”
seawater
was prepared by replacing
CaC12
with MgCl2.
Egg jelly was collected
from eggs by
treatment
with pH 5.0 ASW, and FSG was purified
by
ethanol precipitation,
dialysis
against water, treatment
with 6 M guanidine
hydrochloride
(pH 7.9), and 60%
CaC1 discontinuous
density centrifugation
(Domino
and
Garbers,
1988). FSG preparations
were analyzed
for
fucose content (Dische et a!., 1949) and stored at -70#{176}C
at a final concentration
of 300 .Lg fucose/mi
of water.
Radiolabeling
of Spermatozoa
and High-Performance
Liquid
(HPLC)
Phosphate
Analysis
with myo-[3H]inositol
Chromatographic
of [3H]inositol
Metabolites
Spermatozoa
were
radio!abeled
with
myo[3H]inosito!
as previously
described
(Domino
and Oarbers, 1988). After 6-8 h at 15#{176}C,
approximately
1.5 l.LCi
of [3H]inositol
were incorporated
in the chloroform
fraction of a 25-mg (wet weight) aliquot of spermatozoa. When labeled under pulse-chase
conditions,
spermatozoa
were incubated
with [3H]inosito!
for 3 h,
washed with seawater, and subsequently
incubated with
unlabeled
myo-inosito!
(13 pM) for 5 h. Reactions
of
spermatozoa
in seawater with various treatments
were
stopped by addition of 20% ice-cold trichloroacetic
acid
(10% final concentration).
Insoluble
materials were removed by centrifugation
at 2000 x g for 30 mm, and
the supernatant fractions containing
water-soluble
inosito! phosphates
were chromatographed
on HPLC anion-
COMPARTMENTALIZATION
OF MEMBRANE
exchange
columns
(Whatman
Partisphere
SAX, 4.6 mm
ID x 12.5 cm, 5 pm particle size; Whatman,
Clifton,
NJ) with ammonium
formate
gradients
exactly
as described previously
(Domino
and Garbers,
1988). Standards of [3H]I(1)P1,
[3H]I(1,4)P2,
and [3H]I(1,4,5)P3
were chromatographed
before and after each set of
experimental
samples.
Extraction
and
Chromatography
Thin-Layer
of Phospholipids
In preliminary
experiments,
reaction
mixtures
containing spermatozoa
were stopped in 10% trichioroacetic acid and cell membranes
were pelleted and extracted
in chloroform:methanol:12
M HC1 (4.0:80:1)
(Downes
and Wusteman,
1983). However,
recoveries
of phospholipids
from the irichloroacetic
acid pellets
by this
method varied greatly. Recovery
was increased
to about
85% for exogenous
[3HJPIP2
by terminating
1.0-mi
reaction
mixtures
in seawater
with 3.75 ml of chioroform:methano!:HCI
(100:200:1)
and extracting
the
phospholipids
in the chloroform
phase of a Bligh and
Dyer extraction
(Bligh and Dyer, 1959). After stopping
the reactions,
samples were kept on ice and sonicated
for 5 s, 3 times each, to break up clumps of cells. Bligh
and Dyer extractions
of the lipids were made by the
addition of 1.25 ml chloroform
and 1.25 ml water and
separating
the aqueous and chloroform
phases by centrifugation
at 1000 x g for 30 mm (Bligh and Dyer,
1959). The aqueous fraction (top) was aspirated
and the
chloroform
fraction
was stored overnight at -70C.
Chloroform
extracts were evaporated
to dryness under a
stream of nitrogen gas. The tubes were quickly transferred to an ice bath and 50 p1 of chloroform
were
added. To separate P1, phosphatidylinositol
4-phosphate
(PIP), and PIP2, aliquots were spotted on 20 x 20-cm
Merck
Silica Ge! 60 plates (pretreated
by dipping in
50% methanol
containing
1.0% potassium oxa!ate and
dried at 100#{176}C
for 1 h), and developed in chloroform:
acetone:methanol:acetic
acid:water
(120:45:39:36:22)
(Jo!les et a!., 1981). Lipids were identified
by comparing relative
mobiities
to standards
and by specific
sprays (Kates, 1986). To isolate phosphatidic acid, allquota of 10 p1 or 20 p1 were spotted on 20 x 10-cm
Merck Silica Gel 60 F-254 thin-layer
plates and developed in ethyl acetate:iso-octane:acetic
acid (45:25:10)
(Mallows
and Bolton, 1987). For two-dimensional
separations, samples
were spotted
on 10 x 10-cm Merck
Silica Ge! 60 F-254 plates. The plates were developed
TURNOVER
PHOSPHOLIPID
135
first in chloroform:methanol:ammonium
hydroxide
(10:
3:0.6)
and then were turned
90#{176}
and developed
in
butanol:acetic
acid:water
(6:1:1)
(Abdel-Latif
et a!.,
1976). When radioactive
lipids were chromatographed,
bands comigraung
with phospholipid
standards
(detected with 12 vapor) were scraped and reextracted in 2
ml of chloroform:methanol:0.2
M HC! (10:20:8)
for 30
mm. One milliliter
each of chloroform
and water was
added; chloroform
fractions were evaporated
to dryness
in scintillation
vials, and scintillation
fluid (Ready Organic, Beckman
Instruments,
Inc., Fullerton,
GA) then
was added for counting.
Phosphatidic
acid mass was measured
by dipping
plates in 10% (w/v) CuSO4:8%
(w/v) phosphoric
acid,
charring
at
185#{176}C
for 25 miii, and scanning
with an
LKB 2202 Ultroscan
(LKB, Rockville,
MD) laser densitometer
(Goppelt
and Resch, 1984). Standard
curves
of 1.0 pg to 8.0 pg of egg yolk phosphatidic
acid were
made on each plate. Lanes were scanned 3 times and
the mean of the densitometer
units was used to estimate
the mass of phosphatidic
acid from the standard
curve.
The mass of phosphatidic
acid in pg was converted to
nmol by the formula 1 pg = 1.29 nmol, based on the
dioleoyl
form of phosphatidic
acid (Na
salt).
Preparation
Sperm
of Isolated
Cell
Heads
and
Flagella
Sea urchin spermatozoa
can be fractionated
to yield
flagella-less
sperm heads that respond to egg jelly with
induction
of an acrosome
reaction (Vacquier,
1979) as
well as increases
in cyclic AMP (Garbers,
1981). The
sperm flagella are capable of binding the egg peptide,
speract (Suzuki
et al., 1987). Isolated sperm cell heads
and flagella
were prepared with a teflon Dounce
homogenizer
as previously
described
(Suzuki et a!., 1987).
Cross-contamination
of head and flage!!ar fractions was
less than 0.5%.
Scanning
Electron
Microscopy
Intact and fractionated
spermatozoa
were prepared
for the scanning
electron
microscope
by fixing allquots
in 10 ml of seawater containing 2% glutaraldehyde for
1 h. Samples were centrifuged
at 1000 x g for 30 mm,
resuspended
in 100 p.1 of seawater,
and an aliquot was
placed on a poly-L-lysine-treated
coverslip
overnight
at
4#{176}C.
The coverslips,
with cells attached,
were then
treated with osmium tetroxide,
dehydrated
in step gradi-
DOMINO
136
1. Effects
TABLE
of fucose-sulfate
glycoconjugate
(FSG) on accumulation
AND
GARBERS
of (3H]phosphoinositidcs
from sea urchin
spermatozoa
labeled
with myo-[3HJinositol.’
Phospholipid
Pt
2
Treatmentt’
(cpm)
BasaIc
FSG(30s)
P50 (2 mm)
4235
3937
used: PI.phosphatidylmnositol;
SE of 3 incubations,
bSea urchin
except
spermatozoa
where indicated;
(200 tI, 125mg
140±
176 ±
PIP, phosphatidylinositol
similar results
wet weightnl)
(cpm)
221 ± 16
± 304
± 390
3888 ± 101
aA%reviatjo
Tune
(cjxn)
4-phosphate;
115 ±
69
26
5
*
± 6
78 ± 8
PIP2, phosphatidylinositol
4,5-bisphosphate.
were obtained in 2 additional
experiments.
radiolabeled
with myo-(3H]inositol
were added to seawater
Data shown are the mean
(800 Iii artificial
±
sea water, pH 7.9, 15’C)
at
zero with or without FSH (10 tg fucosenl).
CCCIla incubated
for 2 mm with no addition.
=2.
ents
1500,
of ethanol,
and critical point-dried
(Model
The Bomar Co., Tacoma, WA). Coverslips
mounted
on studs and sputter-coated with Au/Pd
nics Hummer,
Technics,
Alexandria,
VA).
were viewed
on a Hitachi
S-500
scanning
microscope
at 20 KV.
SPCwere
(Tech-
Samples
electron
RESULTS
Stimulation
of Phosphoinositide
Turnover
by FSG
Spermatozoa
(100 mg, wet weight)
were extracted
with chloroform:methanol:HC1
(100:200:1)
as described
in Materials
and Methods.
When a!iquots of the chloroform phase were separated
by thin-layer
chromatography and visualized
by charring
(Materials
and Methods),
bands
comigrating
with
P1 standards
were
detected, whereas PIP and PIP2 were below the level of
detection.
To increase
the sensitivity
for PIP and PIP2,
spermatozoa
were
radiolabeled
with
[32P]orthophosphate or myo-[3H]inositol.
The phospholipid
extracts
were then separated by thin-layer
chromatography,
and
the plates subsequently
were
autoradiographed
or
scraped
for scintillation
counting.
Radioactive
bands
comigrating
with PIP and PIP2 standards
were detected
by both radiolabeling
procedures.
To study the turnover
of phosphoinositides,
spermatozoa were radiolabeled
with myo-[3H]unositol,
and FSG
was added to induce
an acrosome
reaction. Table 1
shows the radioactivity
in P1, PIP, and PIP2 after 30 s
and after 2 miii of incubation. Counts in PIP and PIP2
were lower in FSG-treated
than in untreated cells by
approximately
35% and 40%, respectively,
after 30 s,
and 20% and 30%, respectively,
after 2 mm. Radioactivity in P1 did not decrease
significantly.
Furthermore,
concentrations
of the Ca2’-channe1
blocker, verapamil
(100 pM), that were previously
shown to block FSGinduced accumulation
of [3Hjinositol
phosphates
(Domino and Garbers,
1988),
inhibited
FSG-induced
decreases
of radioactivity
in PIP and PIP2 (not shown).
lonophores
that cause a primary
(ionomycmn)
and secondary (gramicidin
S) Ca2 influx were also tested for
effects on [3H]phosphoinositide
accumulation.
Gramicidin S initially
appeared
to increase radioactivity
in PIP
and PIP2. However,
when spermatozoa
were radiolabeled
under
pulse-chase
conditions
(Materials
and
Methods),
gramicidin
S (50 pM) and ionomycin
(50
pM)
decreased
radioactivity
in PIP and PIP2 (not
shown).
Elevation
When
of Phosphatidic
extracts
Concentrations
from sea urchin sperFSG were separated
by two-dimensional
(Fig. 1) or one-dimensional
(Fig.
2) thin-layer
chromatography,
charred,
and quantified
by laser densitometry
(Materials
and Methods),
increased
concentrations
of phosphatidic
acid were detected in reaction
mixtures
of cells treated with FSG.
No changes
were detected
in the mass of the major
phospholipids,
phosphatidylcholine,
phosphatidylethanolaxnine,
cardiolipun,
phosphatidylserine,
or P1.
To determine
whether phosphatidic
acid was released
into the seawater or remained associated
with spermatozoa, reaction mixtures
containing
spermatozoa
and FSG
were centrifuged
(100 x g, 20 mm), and the supematant
fractions
and pellets were extracted
with chloroform:
methanol:HC1
(100:200:1).
Phosphatidic
acid in the supennatant
fractions
was below the level of detection.
In
addition,
the amounts
of phosphatidic
acid obtained
matozoa
phospholipid
Acid
treated with and without
COMPARTMENTALIZATION
PA Standard
OF MEMBRANE
Basal
PHOSPHOUPID
TIME
0
TURNOVER
0.5
1.0
,
‘
,‘
X
w
F
2.0
.
w
‘
137
5.0
K
‘.
Z
K
w
K
S.
4:
FSG Treated
4
4
Basal
MIN
F
with PA
FSG
200
4
4:
#{149}
4:
175
O
FIG. 1. Two-dimensional
thin-layer
chromatography
(TLC) separation
of
phosphatidic
acid standards and chloroform
extracts from sea urchin spennatoma treated with fucose-sulfaie
glycoconjugate
(FSG). Spermatozoa
were incubated with (FSG-Treated)
or without (Basal)
P50(10
&g fucose/mi)
for 2 mm
at 15’C. Reactions
were stopped by the addition of chloroformnnethaaol:HC1
(100:200:1),
and the lipids were extracted and chromatograpbed
by two-dimensional TLC as described in Materials andMethods.
Additional samples containing 2 sg of phosphatidic
acid (PA Standard)
alone or samples from untreated
spermatozoa
plus added phosphatidic
acid (Basal with PA) were also
chromatographed
and visualized
by charring.
Phosphatidic
acid in each chromatograph
is marked with a large arrow. The thin arrow shows the direction of
the second dimension
of separation.
a’
150
-
FSG
C)
j125
‘10o
(/)75
Oo
IE
oB
0
50/
Basal
/
-#{149}
p-.-
0
0.0
1.0
2:0
TIME
from the pellets
were the same whether
or not the
supematant
fractions
were removed
(data not shown),
consistent
with
the hypothesis
that the increased
amounts
of phosphatidic
acid obtained
from reaction
mixtures
of spermatozoa
treated with FSG reflect increased concentrations
of the phospholipid
in the cell
plasma
membrane.
Elevations
of phosphatidic
acid were dependent
on
extracellular
Ca2. Verapamil
(100 pM) blocked FSGinduced increases
of phosphatidic
acid (Table 2), and
spermatozoa
incubated
with FSG in Ca2-free
seawater
exhibited
no changes
in phosphatidic
acid (Table 2).
The addition of Ca2 (20 mM) and FSG to cells washed
with Ca2-free
seawater however,
elevated phosphatidic
acid concentrations
(Table 2).
Treatments
other than FSG that induce the acrosome
reaction,
including
the divalent
cation
ionophore,
ionomycin,
the monovalent
cation ionophore,
gramicidin S. and alkaline
(pH 8.8) seawater,
also caused
increases
in phosphatidic
acid concentrations
(Table 3).
3:0
4:0
5:0
(mm)
FIG. 2. One-dimensional
thin-layer
chromatography
(ThC) separation
of
phosphasidic
acid from sea urchin spermatozoa
treated with fucose-sulfate
glycoconjugate
(FSG).
Sea urchin spermatozoa
were washed, and I-mi aliquots
(100mg.
wet weight) were preincubated
for 5 mm at 15’C. At tune zero. FSG
(10 sg flicoselml)
(+) or an equal volume of water (-) was added. Reactions
were stopped at the times indicated by the addition of 3.75 ml of chloroform:
methanol :HCI (100:200:1), and lipid extracts were chromatographed
by one-dimensional TLC, charred, and quantified
according
to Materials
and Methods.
Upper panel shows a sample TLC separation
(phosphatidic
acid is marked with
the arrows); Wwer panel represents
the quantification
of phosphatidic
acid by
laser densitometer
scanning of duplicate reaction mixtures at various times after
addition of FSG (0) or waler (#{149}).
Duplicate detenninations
were within 20% of
each other.
Verapamil
(100 pM) inhibited
gramicidin
S-induced
increases
in phosphatidic
acid (not shown). Monensin,
tested under conditions
previously
reported not to elevate
cyclic
AMP
concentrations
or
induce
[3H]I(1,4,5)P3
accumulation
or an acrosome
reaction
(Garbers,
1981; Domino
and Garbers,
1988), did not
affect phosphatidic
acid concentrations
(Table 3).
Speract, a spermatozoan-activating
peptide purified
from sea urchin egg-conditioned
medium,
causes in-
138
DOMINO
TABLE 2. Calcium
dependence
of fucose -sulfate
duced phosphatidic
acid elevations.
Incubation
buffera
22
135
19
25
seawater
24
P50
FSG
(FSG)-un-
Phosphatidic
acid
(pniol/nig
wet
weight)
Normal seawatert’
Basa1
FSG
P50 + verapamil
(100 pM)
Verapamil
(100 pM)
Ca2+free
BasalC
glycoconjugate
AND
20
(20 mM)
Ca2 (20 mM)
26
158
+
aSea urchin
spermatozoa
(100mg
wet weight)
were washed
TABLE 3. Effects of fucose-sulfate
on pliosphatidic
acid concentrations
Addition
glycoconjugate
(P50) and other treatments
of sea urchin spermatozoa.
Phosphatidic
acid
(pmolftng wet
weight)
to incubation5
Basalt’
P50
26
110
lonomycmn (50 pM)
Gramicidun
S (50 pM)
Monensin
(100 pM)
pH8.Sseawater
Speract (1 pM)
MIX* (0.3 mM)
Speract (1 pM) + MIX (0.3 mM)
124
139
23
125
5Spetozoa(100
and incubated
for 2 miii at 15#{176}C
in normal or Ca2-free
seawater
with or without P50(10
pg
fucose/ml).
Data shown are the means of duplicate reaction mixtures; this experiment was repeated twice with similar results.
mg wet weight)
20
24
78
were
washed
and incubated
for 2 min at
inthe peesence of FSG (10 pg fucoseithl)
orother treatments.
Data shown
are means of duplicate reaction mixtures and are representative
of 3 additional
experiments
15C,
1’No addition.
5MIX, l-methyl-3-isobutylxanthmne.
‘9.6mM
Ca2.
CNO addition.
creased respiration
and transient elevations
of intracellular Ca2 concentrations,
but does not induce an acmsome
reaction
unless
combined
with
1-methyl-3isobutyixanthine
(MIX)
treatment
(Schackmann
and
Chock, 1986). Speract (1 tM) alone did not appreciably
alter phosphatidic
acid concentrations,
but increased
phosphatidic
acid concentrations
when combined
with
0.3 mM MIX (Table 3).
Stimulation
Accumulation
GARBERS
of [3H]Jnositol
Phosphate
in Sperm
Heads
To investigate
the possible
compartmentalizalion
of
phosphoinositide
changes in the head or flagellum,
isolated sperm heads and flagella
were prepared (Materials and Methods).
When spermatozoa
were radiolabeled
with myo-[3H]inositol
and isolated
heads and flagella
subsequently were prepared, chloroform
extracts
from
equal amounts
(wet weight)
contained
approximately
26,500 cpm per sample of sperm heads and approximately 2500 cpm per sample of flagella,
despite the
greater surface
area of the flagella.
Therefore,
membranes from the head either contain
higher concentrations of phosphoinositides
than those from the flagellum, or have higher basal turnover
of phospholipids
leading
to
preferential
incorporation
of
myo[3H]inositol.
Aliquots
of 25 mg (wet weight)
of the head and
flagellar
preparations
were added to 1-mi reaction mixtures containing
seawater
and seawater
plus FSG or
gramicidin
S at 15#{176}C.
In control experiments,
gramici-
din S induced acrosome
reactions
in 80-95%
of isolated
heads compared
with 10-30%
after incubation
at relatively high concentrations
of FSG. Treatment
of intact
cells with FSG at concentrations
of 10-30 .tg (fucose)/
ml resulted in acrosome
reactions
in 90-100%
of the
cells. Gramicidin
S was more effective
and consistent
than FSG in inducing
the acrosome
reaction in isolated
sperm heads. Reactions
were terminated
after 2 mm
with trichioroacetic
acid and the aqueous extracts were
separated
by HPLC
as described
in Materials
and
Methods.
Compared
with controls,
sperm heads incubated with FSG contained
30% and 80% higher radioactivity in 1(1 ,4)P2 and 1(1 ,4,5)P3,
respectively,
whereas
TABLE 4. Effects of fucose-sulfate
[3H]inositol
phosphate accumulation
labeled with myo-[3H]inosito1,
Headsb
BasaIC
P50
GramicidinS
Flagellat
BasaIC
P50
GramicidinS
5Abviati
used:
I(1)P1,
glycoconjugate
(FSG) and gramicidmn Son
(cpm) from sea urchin spermatozoa
radio-
I(l)P1
l(1.4)P2
1823
2350
2698
2442
I(1,4,5)P3
306
3151
552
11795
1700
65
149
46
60
54
137
155
42
48
[3H]mositol
[3H]inositol
1,4-bisphosphate;
I(1,4,,5)P3,
[3H]inositol
b Aliquots of 25 mg, wet weight, of isolated sperm
1-phosphate;
l(1,4)P2),
1,4.5,-trisphosphate.
heads and flagella
were
added to lml of seawater containing
FSG (10 pg fucoseinl)
or gramicidin
S (50
pM) at 15#{176}C
(after 2 min. reactions were terminated).
Data shown are means of
duplicate incubations.
Duplicate determinations
were within 10% of each other.
CNo addition.
COMPARTMENTALIZATION
OF MEMBRANE
gramicidin
S-treated sperm
cell heads contained
3.8fold and 4.5-fold
higher radioactivity
in I(1,4)P2
and
I(1,4,5)P3,
respectively
(Table
4). No changes
in
[3Hjinositol
phosphate
accumulation
were detected
after addition of FSG (10 ig fucose/ml)
or gramicidin
S
(50 tm)
to sperm
flagellar
preparations
(Table 4).
Effects
of FSG
Concentrations
on
Phosphatidic
in Sperm
Heads
Acid
and
Flagella
To examine
whether
phosphatidic
acid concentrations are elevated
in the sperm head region, isolated
sperm heads and flagella
were prepared
according
to
Materials
and Methods.
Isolated
sperm
heads
were
reacted with FSG (10 ig fucose/ml)
or gramicidin
S
(50 pM) for various
times up to 5 mm. As shown
in
Figure
3, phosphatidic
acid concentrations
were elevated up to 2-fold and 5-fold in isolated sperm heads
after incubation
with FSG and gramicidin
5, respectively.
Changes
in phosphatidic
acid concentrations
were
not detected
in isolated
flagella
incubated
with FSG.
However,
when cells were treated with FSG and the
flagella
were subsequently
isolated,
phosphatidic
acid
concentrations
in the flagellum
were increased.
Therefore, FSG either has direct effects on the flagellum
not
observed
in broken cell preparations
or FSG-induced
increases
in phosphatidic
acid in the head of the intact
cell can diffuse
to the flagellar
plasma membrane.
PHOSPHOUPID
TURNOVER
139
phospholipase
C hydrolysis
of polyphosphoinositides.
FSG also caused elevations
of phosphatidic
acid concentrations.
In hormone-stimulated
cells, phosphatidic
acid elevations
may be due either to the sequential
action of phospholipase
C and diacyiglycerol
kinase, or
to phospholipase
D (Bocckino
et al., 1987; Exton,
1988). Experiments
investigating
the potential
activation of phospholipase
D by FSG has been reported
elsewhere
(Domino
et at., 1989).
Human sperm lysates have been reported to contain a
Ca2-dependent
phospholipase
C activity that can hydrolyze
P1 (Ribbes
et al., 1987). In addition,
a phosphatidylcholine-speciflc
phospholipase
C has been purified
from
bull
spermatozoa
(Sheikhnejad
and
Srivastava,
1986). When phospholipase
C localization
was examined
by immunofluorescence
in rabbit and
guinea pig spermatozoa,
immunoreactive
phospholipase
C appeared
predominantly
localized
to the acrosome
(Sheikhnejad
and Srivastava,
1986). Additionally,
the
outer acrosomal
membrane
was the preferential
site of
staining
when neomycin,
which binds anionic
compounds including
phosphoinositides,
was used as a cytochemical
probe to study the polyphosphoinositide
distribution
in boar spermatozoa
(Berruti
and Franchi,
1986). We have examined
the compartmentalization
of
phosphoinositide
turnover
in the sperm heads. Despite
the 3-fold greater membrane
surface area of the flagel-
300
-
DISCUSSION
Sperm cell lipids have been studied during maturation and capacitation
of mammalian
spermatozoa
in
various
other investigations
(Davis,
1981; Langlais
et
at., 1981; O’Rand,
1982; Clegg, 1983; Parks and Hammerstedt,
1985).
Membrane
phospholipids
also have
been examined
as possible
endogenous
energy
sources
in bull (Storey, 1980) and sea urchin spermatozoa
(Mita
and Ueta,
1988). Effects
of external
stimuli
on P1
turnover
in spermatozoa,
however,
have been reported
only recently
(Berruti
and Franchi, 1986; Nikolopoulou
et al., 1986; Bennet et at., 1987; Domino
and Garbers,
1988). The experiments
in this report provide
direct
evidence
of changes in polyphosphoinositides
and phosphatidic
acid stimulated
by the fucose-sulfate
component of sea urchin egg jelly.
FSG significantly
decreased
radioactivity
in [3HIPIP
and [3H]PIP2, but not P1, consistent
with stimulation
of
Gramicidin
,
150
0-E
U,...-
__-o
1/
FSG
IE
O_.
S
Basal
50
0.0
1:0
2.0
3:0
TIME
(mm)
4:0
5.0
FIG. 3. Time course of phosphatidic
acid accumulation
in isolated sperm
heads in response
to fucose-sulfate
glycoconjugate
(FSG)
and granucidin
S.
Isolated sperm heads were prepared
as described
in Materials
and Methods.
One-milliliter
aliquots (100 mg, wet weight) of isolated sperm heads were
preuncubated
for 5 rein at 15#{176}C.
At Time zero, 10 pg of P50 (fucose)/ml
(0), 50
pM gramicidin S (ii), or an equal volume of water (#{149})
were added. At the times
indicated, reactions were terminated
and phosphatidic
acid concentrations
were
measured
as described
in Materials
and Methods.
Data shown represent the
mean of duplicate reaction mixtures and are representative
of 2 additional cxpenmen
140
DOMINO
AND
la, ten times more radioactivity
from myo-[3H]inositol
was incorporated
in lipid from sperm heads. In addition, isolated sperm heads responded
to treatments
that
induce
an acrosome
reaction
with
elevations
in
[3Hjinositol
phosphates
and phosphatidic
acid. Spermatozoa,
then, are one of only a few types of cells,
such as retinal cells (Anderson
et at., 1983), shown to
have
a distinct
compartmentalization
of P1 turnover.
It should be noted, however,
that the number of
counts measured
in [3H]inositol
phosphates
in flagella
were relatively
low, and potential
P1 turnover
in the
flagella may have been below the level of detection.
In
addition, although phosphatidic
acid concentrations
did
not change upon addition of FSG to isolated flagella,
phosphatidic
acid concentrations
were increased
in flagella isolated from intact cells treated with FSG. From
these experiments,
it appears
that phosphatidic
acid
either diffused from the head to flagellum,
or phosphatidic acid was also produced
in the flagellum
of the
intact cell.
The functions,
if any, of phospholipid
turnover
in sea
urchin spermatozoa
are not known. Membrane
fusion
has been postulated
to result from enzymatically
altered
membrane
phospholipid
or protein (Finkeistein et at.,
1986;
Zimmerberg,
1987).
Recently,
a metalloendoprotease
has been reported
to be required for induction of the acrosome
reaction at a step following
influx
of extracellular
Ca2 (Farach et at., 1987). In addition
of protease
activity,
phospholipases
may or may not
contribute
to induction
of an acrosome
reaction.
Alternatively,
phospholipid
changes
in spermatozoa
may promote
extracellular
fusion between
the acmsome-reacted
spermatozoon
and egg (Conway
and
Metz, 1976) or have effects on the egg plasma membrane following
gamete fusion. Membrane
components
of sea urchin spermatozoa
have been shown to be
incorporated
during fertilization
into the egg plasma
membrane,
contributing
in part to development
of the
fertilization
cone,
and are eventually
redistributed
throughout
the egg plasma membrane
(Longo,
1986;
Nishioka
et at., 1987),
ACKNOWLEDGMENTS
We thank Dr. Loren H. Hoffman of the Department
of Cell Biology for use
of the scanning electron microscope
facility, Dr. Stephen B. Boeckino
for advice on separation
of phospholipids,
and Dr. Guy Augert for gifts of neosnycinpurified bovine brain phosphoinositides.
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