Vol. 69, No. 4, 1976
BIOCHEMICAL
EFFECT OF MODIFIED
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MEMBRANE VESICLES
ON THE FERTILIZING
CAPACITY
B. K. Davis Worcester
Received
February
27,
OF RABBIT
and B. J.
Foundation Shrewsbury,
FROM SEMINAL
for
PLASMA
SPERMATOZOA1
Hungund'
Experimental
Massachusetts
Biology 01545
1976
extraction of cholesterol and phosphalipid from SUMMARY: Partial membrane vesicles in rabbit seminal plasma decreased their inhibitory effect on fertilizing capacity in rabbit spermatozoa. Pronase digestion, to remove surface proteins, had no pronounced effect on vesicle decapacitation activity. Evidence of fusion between these vesicles and speE,iatozoa was obtained using [%!]galactose labelled vesicles. The results are consistent with addition of vesicle lipid (cholesterol) to the sperm plasma membrane causing an inhibition of fertilizing capacity, Spermatozoa found
from
to require
gaining
the
seminal
plasma
the
sperm
obtained
capacity
for
capacitation
(1).
indicates
sperm
the
Membrane
by cholesterol
(5,6).
seminal
therefore
cholesterol undertaken
plasma
to the
plasma
in
its
membrane fusion
fusion
to;
membrane.
(a.) broadly
the
1004
been
cell
outer
reported
addition
influenced (2,3). changes
in
acrosomal
to be inhibited vesicles
in
of vesicle
The present characterize
1. Supported by N.i.H. Contract No l-HD-J-2781. 2. Present address: Psychiatric Institute, Dept. 722 kiest 16e Street, New York, N.Y. 10032. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
has also
membrane
by membrane
involve
plasma
in
is
of this
with
has been
could
an attempt
plasma
in
occurring
capacity
Decapacitation
sperm
Evidence
cell
before
vesicles
changes
fertilizing sperm
have been tract
Membrane
during
in the
of mammals
reproductive
fertilization.
facilitates
(4).
female
molecular
capacitation,
membrane
the
to reverse
levels
some way that
in
species
appear
cell that
divergent
a period
by cholesterol During
several
study the
Mental
was lipid
Health,
Vol. 69, No. 4, 1976
and protein (b)
determine
with
pronase
nature
components
of the
experiments
BIOCHEMICAL
AND BIOPHYSICAL
of membrane
decapacitation
activity
and partial
extraction
interaction
between
described
vesicles
RESEARCH COMMUNICATIONS
from
in vesicles of lipid, vesicles
were conducted
in
seminal
plasma,
after
digestion
and (c)
assess
and spermatozoa. the
the The
rabbit.
MATERIALS AND METHODS Rabbits, of mixed strain, were purchased from a local breeder for use in these experiments. Seminal plasma was obtained by centrifugation of fresh semen, collected with an artificial vagina from fertile bucks, at 10,000 g for 30 min. Membrane vesicles were isolated from seminal plasma by sedimentation on discontinuous sucrose density gradients (1). Rabbit seminal plasma contains vesicles with densities of 1.20 and 1.16 g/cc and they are referred to as heavy and light vesicles, respectively. Vesicle protein was estimated calorimetrically (7). Lipids were extracted with chloroform and methanol (281) and the dry extract was weighed, The crude lipid extract was acetylated and then fractionated into neutral lipids, glycolipids and phospholipids using a Florisil (Fisher) column (8). Cholesterol in the neutral lipid fraction was measured with o-phthalaldehyde (3). Glycolipids were identified, after deacetylation, by co-chromatography with standard glycolipids on silica gel plates (Brinkman) using chloroform, methanol and water (100142:6) as the developing solvent. Gangliosides were q-uantitated by multiplying by 2.8 the amount of sialic acid measured (10). Phospholipids were identified by comparing Rf values following chromatography on silica gel plates developed with chloroform, methanol, acetic acid and water (75r25: 1518). The amount of phospholipid was estimated by multiplying by 25 the inorganic phosphorus level (11). Dry weight was determined after placing vesicle preparations in an oven at 54'C for 24 hrs. Electrophoresis of vesicle roteins sodium was performed usin dodecyl sulfate and 7.5 (wPv)$ polyacrylamide gels (12 7 . A gel scanner (Helena) determined the distribution of pol peptide bands in gels stained with Coomassie Brilliant Blue (Mann 7 . Periodic acidSchiff stain located bands containing sugar. Vesicles suspended in 0.1 M KC1 and 0.01 ill Tris, pW 7.0, were digested with 10 (w/w)% pronase (Nutritional Biochemicals) for 4 hrs. at 37OC. They were subsequently isolated by sedimentation on 5 (w/v,% sucrose at 105,000 g for 3 hrs. Li.pi.ds were partially depleted from the vesicle preparations by extraction with dry ethyl ether (13) and with acetone, ethyl ether and water (30rlOtl). The extracted vesicles were lyophilized and assayed for protein, Samples of these preparations were phospholipid and cholesterol. negatively stained with 1 (w/v)% uranyl acetate and examined under an electron microscope (Zeiss) to assess vesicle structure. The effect of these vesicles on sperm fertilizing Capacity was established from the rate of fertilization observed after oviductal insemination with treated sperm cells (1). Capacitated sperm were obtained by flushing the uterus of a doe 12 hrs. after mating. Ovulation was induced by injection of 70 I.U. human chorionic gonadotrophin and inseminated females were autopsied after 24 hrs. Fertilized eggs were at the 4 or P cell stage when recovered from the oviducts.
1005
Vol. 69, No. 4, 1976
BIOCHEMICAL
HEAVY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LIGHT
VESICLES
VESICLES
IO e 6
125
100
80
60
50
40 30 MOLECULAR
125 WEIGHT
loo I lO-3
80
60
50
vesicle polypeptide patterns Fig. 1 ,- Heavy and light effects of pronase digestion. The scans were obtained electrophoresis using sodium dodecyl sulfate and 7.59 Stain, Coomassie Brilliant Blue. Numbers in parenthesis amounts (%).
40
30
showing after polyacrylamide. are relative
II Galactose labelled vesicles, containing 6.5 x 105 cpm/mg PIwere protein, prepared by incubation with galactose oxidase (I;abi) and [3H]NaBw according to the ,method of Saito and Hakomori (14). Wa hed ejaculated rabbit spermatozo at a concentration of 16 x 10 i? sperm/ml, were incubated with [3'Hlgalactose labelled vesicles at 20°C in Hanks solution (Difco). Following incubation for 30 min. sperm cells were recovered by sedimentation through 2 (w/v)% albumin in Hanks. Radioactivity in the cells was assayed in a liquid scintillation counter (Packard) using a water miscible phosphor (New England Nuclear), after dissolution of the sperm in 50 (v/v)% Biosolv (Beckman). The fraction of sperm radioactivity in glycolipid was estimated following extraction with cholorform and methanol, and it has been compared with the fraction of radioactivity in vesicle glycolipid. RESULTS AND DISCUSSION Fig.
1 to have
both
possess
weight
of
significant it
is
differ proteins. vesicle
Heavy
distinct
and complex
a major
polypeptide
x 10'.
Component
9.5
a glycoprotein.
Since
electrophoretic These
surface,
b,
as they
compositions. a) with
quantitatively
they
clearly
appear
to be located
attacked
by pronase
1006
a molecular
sugar
vesicle are
They
the most for
and light
mobility,
were
can be seen from
polypeptide
positively
heavy
glycoproteins
vesicles
(component
reacted
polypeptide,
in
and light
indicating component
b
different on the (Pig.
1).
outer In
Vol. 69, No. 4, 1976
BIOCHEMICAL
pronase-digested is
light
a cleavage
accounted
product
for
nearly
heavy
one fifth
and light
fourths
vesicles
have
been
and phospholipid acetone,
Vesicle
ether
of
the
remaining
did
vesicles
cells
ductal
persist
insemination
ether
with
eggs
recovered
from
the
in
probably
important. this
containing
these
indicate not This
.(Table
2).
With
approximately
phospholipid
this
was eluted.
treatment,
vesicles, gave
however,
of
dry
the
extracted
that
surface
in
in
decapacitation,
in
agreement
that
synthetic
reversibly
inhibit
showing
(3). 1007
ovi-
3).
By
with
with
other
three of
vesicle a recent
phospholipid sperm
untreated
= 43.42,
proteins
with
the
CXZ = 1.45)
achieved
the
acetone,
of
different
(I$
of
after (Table
significantly
than
is
rates
64% (32/50)
76% (48/63)
extracted
fertilization
to vesicles
while
ether
respectively,
two groups
finding
cholesterol
cholesterol
was significantly
implicated
laboratory
decreased
of fertilizing
is not rate
more
results
light activity
post-ovulation
exposed
and this
Fertilization
These
3 hrs.
were capable
spermatozoa. frequent
also
decapacitation
the
and 36% (8/22),
cells
fertilization
native vesicles
about
and water
They
as solvent,
after
vesicles
was stable. treated
sperm
light
that
amounts :I)
and
comprise
vesicles.
(13)
and half
not
26% (y/34)
comparison,
ether
(30110
and pronase-digested
237; (14/61),
higher
it
respectively.
to note
small
and water
suspension Sperm
ethyl
cholesterol
structure
as heavy
with
dry
weight,
As expected,
interesting
by comparatively
ethyl
two thirds
is
wet weight
and cholesterol
as much lipid
with
dry
indicating
proteins
of vesicle
lipid.
attributed
Extraction
c increased
phospholipids
It
RESEARCH COMMUNICATIONS
Vesicle
vesicle
of vesicle
twice
cholesterol.
in
protein.
three
had more
are
of another
1 shows that
about
l
component
Table
contained
(15)
vesicles
approximately
70% and 54% of
AND BIOPHYSICAL
fertilizing
the
O.Ol>>P) groups. vesicles
lipids
are
observation vesicles ability
Vol. 69, No. 4,1976
BIOCHEMICAL
AND BlOPHYStCAL
RESEARCH COMMUNICATIONS
TABLE 1 LIPID
COMPOSITION
OF MEMBRANE VESICLES
Lipid
Heavy
Total lipids (pg/mg protein)
vesicles
Light
vesicles
cent
41
41
i2$
it:; ( 5) ( 3)
1%
(2 gal, 1 gal)
1 glu;
7
5
8
13
33 10
lipids
PLASMA
865 Per
Ganglioside
Cholesterol Other neutral
SEMINAL
422
Phospholipid Sphingomyelin Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine
Cerebroside
FROM RABBIT
:;
TABLE 2 CHOLESTEROL AND PHOSPHOLlPID LEVELS IN EXTRACTED MEMBRANE VESICLES FROM SEMINAL PLASMA
Vesicles
Heavy Phospholipid
vesicles Cholesterol
Light Phospholipid
(pg/mg
vesicles Cholesterol
protein)
Native
173
139
354
260
Extracted Dry ether Acetone, ether and water
156 91
118 58
243 177
225 106
Rabbit incubation appeared
sperm
with to
cells
[ 3 H]galactose
represent
similar
became
radioactively
containing fractions
1008
labelled
vesicles. of
sperm
during
Glycolipid and vesicle
Vol. 69, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 3 EFFECT OF PRONASE-DIGESTED AND EXTRACTED MEi$BRANE VESICLES FROM SEMINAL PLASXA ON THE FERTILIZING CAPACITY OF RABBIT SPERM CELLS
Vesicles+
No. of insemnt*
Nil Native Pronase-digested Extracted Dry ether Acetone, ether and water
No. of eggs
Fertilized No.
eggs $
13 13 7
63 61 22
48
76
14
a
23 36
a
34 50
9 32
26 64
12
* Vesicle concentration was 1 mg protein/ml heavy and light vesicles). **There were 1.25 to 5.0 x 10' sperm cells
(0.5
mg protein/ml
deposited
in
from
each oviduct,
TABLE 4 FRACTION OF [3H]GALACT@SE IN GLYCOLIPID FROM VESICLES AED SPERM CELLS INCUBATED WITH THE VESICLES
Total radioactivity (cpm)
Source
Vesicles Sperm
radioactivity 1 glucose vesicles
cells
(Table residue have
Non-extractable
in
4).
450
40
214
48
A cerebroside
was found
Glycolipid fraction
in both
addition
a cerebroside
radioactivity
in
containing types
these
1009
2 galactose
of vesicles. with
a single
preparations
($)
Heavy galactose. can be
and
Vol. 69, No. 4, 1976
attributed results the
BIOCHEMICAL
largely imply
sperm
that
to labelled
glycoproteins
decapaciation
results
(8). from
Hence,
vesicle
these
fusion
with
cell.
ACKNOWLEDGEMENT We are assistance
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in performing
indebted these
to Mr R. Byrne
for
his
capable
experiments.
REFERENCES B. K. (1974) 5. Reprod. Fert. 41, 241-244. Med. FJ, 240-243. 2. Davis, B. K. (1976a) Proc. Sot. Exp. sol. Davis, B. K. (1976b) Proc. Sot. Exp. Biol. Med. in press. 2: Barros, L. E. and Austin, C. R. C., Bedford, J. M., Franklin, (1967) J. Cell Biol. 2, Cl-C5. C. and Pette, D. (1973) Exp. Cell Res. 5. Van der Bosch, J., Schudt, Et, 433-438. D., Poste, G., Schaeffer, B. E. and Vail, W. J. 6, Papahadjopoulos, (1974) Biochim. Biophys. Acta z, 10-28. N. J., Farr, A. L. and Randall, R. J. 7. Lowry, 0. H., Rosebrough, (1951) J. Biol. Chem. w, 265-275. T. and Hakomori, S. (1971) J. L,ipid Res. 12, 257-259. 8. Saito, Rudel, L. L. and Morris, M. D. (1973) J. Lipid Res. 2, 364-366. 1:: Warren, L. (1959) J. Biol. Chem. e, 1971-1975. 11. Bartlett, G. R. (1959) J. Biol. Chem. 2 4, 466-468. 12. Laemmli, U. K. (1970) Nature a, 680- d- 5. W., Sarzala, M. G., Wroniszewska, A., Lagwinska, E. 13. Drabikowski, and Drzewiecka, B. (1972) Biochim. Biophys. Acta m, 158-170. 14, Gahmberg, C. G. and Hakomori, S. (1973) J. Biol. Chem. &?, 4311-4317. B. K. and Niwa, K. (1974) Proc. Sot. Exp. Biol. led. 146, 15. Davis, 11-16. 1. Davis,
1010
BIOCHEMICAL
EFFECT OF MODIFIED
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MEMBRANE VESICLES
ON THE FERTILIZING
CAPACITY
B. K. Davis Worcester
Received
February
27,
OF RABBIT
and B. J.
Foundation Shrewsbury,
FROM SEMINAL
for
PLASMA
SPERMATOZOA1
Hungund'
Experimental
Massachusetts
Biology 01545
1976
extraction of cholesterol and phosphalipid from SUMMARY: Partial membrane vesicles in rabbit seminal plasma decreased their inhibitory effect on fertilizing capacity in rabbit spermatozoa. Pronase digestion, to remove surface proteins, had no pronounced effect on vesicle decapacitation activity. Evidence of fusion between these vesicles and speE,iatozoa was obtained using [%!]galactose labelled vesicles. The results are consistent with addition of vesicle lipid (cholesterol) to the sperm plasma membrane causing an inhibition of fertilizing capacity, Spermatozoa found
from
to require
gaining
the
seminal
plasma
the
sperm
obtained
capacity
for
capacitation
(1).
indicates
sperm
the
Membrane
by cholesterol
(5,6).
seminal
therefore
cholesterol undertaken
plasma
to the
plasma
in
its
membrane fusion
fusion
to;
membrane.
(a.) broadly
the
1004
been
cell
outer
reported
addition
influenced (2,3). changes
in
acrosomal
to be inhibited vesicles
in
of vesicle
The present characterize
1. Supported by N.i.H. Contract No l-HD-J-2781. 2. Present address: Psychiatric Institute, Dept. 722 kiest 16e Street, New York, N.Y. 10032. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
has also
membrane
by membrane
involve
plasma
in
is
of this
with
has been
could
an attempt
plasma
in
occurring
capacity
Decapacitation
sperm
Evidence
cell
before
vesicles
changes
fertilizing sperm
have been tract
Membrane
during
in the
of mammals
reproductive
fertilization.
facilitates
(4).
female
molecular
capacitation,
membrane
the
to reverse
levels
some way that
in
species
appear
cell that
divergent
a period
by cholesterol During
several
study the
Mental
was lipid
Health,
Vol. 69, No. 4, 1976
and protein (b)
determine
with
pronase
nature
components
of the
experiments
BIOCHEMICAL
AND BIOPHYSICAL
of membrane
decapacitation
activity
and partial
extraction
interaction
between
described
vesicles
RESEARCH COMMUNICATIONS
from
in vesicles of lipid, vesicles
were conducted
in
seminal
plasma,
after
digestion
and (c)
assess
and spermatozoa. the
the The
rabbit.
MATERIALS AND METHODS Rabbits, of mixed strain, were purchased from a local breeder for use in these experiments. Seminal plasma was obtained by centrifugation of fresh semen, collected with an artificial vagina from fertile bucks, at 10,000 g for 30 min. Membrane vesicles were isolated from seminal plasma by sedimentation on discontinuous sucrose density gradients (1). Rabbit seminal plasma contains vesicles with densities of 1.20 and 1.16 g/cc and they are referred to as heavy and light vesicles, respectively. Vesicle protein was estimated calorimetrically (7). Lipids were extracted with chloroform and methanol (281) and the dry extract was weighed, The crude lipid extract was acetylated and then fractionated into neutral lipids, glycolipids and phospholipids using a Florisil (Fisher) column (8). Cholesterol in the neutral lipid fraction was measured with o-phthalaldehyde (3). Glycolipids were identified, after deacetylation, by co-chromatography with standard glycolipids on silica gel plates (Brinkman) using chloroform, methanol and water (100142:6) as the developing solvent. Gangliosides were q-uantitated by multiplying by 2.8 the amount of sialic acid measured (10). Phospholipids were identified by comparing Rf values following chromatography on silica gel plates developed with chloroform, methanol, acetic acid and water (75r25: 1518). The amount of phospholipid was estimated by multiplying by 25 the inorganic phosphorus level (11). Dry weight was determined after placing vesicle preparations in an oven at 54'C for 24 hrs. Electrophoresis of vesicle roteins sodium was performed usin dodecyl sulfate and 7.5 (wPv)$ polyacrylamide gels (12 7 . A gel scanner (Helena) determined the distribution of pol peptide bands in gels stained with Coomassie Brilliant Blue (Mann 7 . Periodic acidSchiff stain located bands containing sugar. Vesicles suspended in 0.1 M KC1 and 0.01 ill Tris, pW 7.0, were digested with 10 (w/w)% pronase (Nutritional Biochemicals) for 4 hrs. at 37OC. They were subsequently isolated by sedimentation on 5 (w/v,% sucrose at 105,000 g for 3 hrs. Li.pi.ds were partially depleted from the vesicle preparations by extraction with dry ethyl ether (13) and with acetone, ethyl ether and water (30rlOtl). The extracted vesicles were lyophilized and assayed for protein, Samples of these preparations were phospholipid and cholesterol. negatively stained with 1 (w/v)% uranyl acetate and examined under an electron microscope (Zeiss) to assess vesicle structure. The effect of these vesicles on sperm fertilizing Capacity was established from the rate of fertilization observed after oviductal insemination with treated sperm cells (1). Capacitated sperm were obtained by flushing the uterus of a doe 12 hrs. after mating. Ovulation was induced by injection of 70 I.U. human chorionic gonadotrophin and inseminated females were autopsied after 24 hrs. Fertilized eggs were at the 4 or P cell stage when recovered from the oviducts.
1005
Vol. 69, No. 4, 1976
BIOCHEMICAL
HEAVY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LIGHT
VESICLES
VESICLES
IO e 6
125
100
80
60
50
40 30 MOLECULAR
125 WEIGHT
loo I lO-3
80
60
50
vesicle polypeptide patterns Fig. 1 ,- Heavy and light effects of pronase digestion. The scans were obtained electrophoresis using sodium dodecyl sulfate and 7.59 Stain, Coomassie Brilliant Blue. Numbers in parenthesis amounts (%).
40
30
showing after polyacrylamide. are relative
II Galactose labelled vesicles, containing 6.5 x 105 cpm/mg PIwere protein, prepared by incubation with galactose oxidase (I;abi) and [3H]NaBw according to the ,method of Saito and Hakomori (14). Wa hed ejaculated rabbit spermatozo at a concentration of 16 x 10 i? sperm/ml, were incubated with [3'Hlgalactose labelled vesicles at 20°C in Hanks solution (Difco). Following incubation for 30 min. sperm cells were recovered by sedimentation through 2 (w/v)% albumin in Hanks. Radioactivity in the cells was assayed in a liquid scintillation counter (Packard) using a water miscible phosphor (New England Nuclear), after dissolution of the sperm in 50 (v/v)% Biosolv (Beckman). The fraction of sperm radioactivity in glycolipid was estimated following extraction with cholorform and methanol, and it has been compared with the fraction of radioactivity in vesicle glycolipid. RESULTS AND DISCUSSION Fig.
1 to have
both
possess
weight
of
significant it
is
differ proteins. vesicle
Heavy
distinct
and complex
a major
polypeptide
x 10'.
Component
9.5
a glycoprotein.
Since
electrophoretic These
surface,
b,
as they
compositions. a) with
quantitatively
they
clearly
appear
to be located
attacked
by pronase
1006
a molecular
sugar
vesicle are
They
the most for
and light
mobility,
were
can be seen from
polypeptide
positively
heavy
glycoproteins
vesicles
(component
reacted
polypeptide,
in
and light
indicating component
b
different on the (Pig.
1).
outer In
Vol. 69, No. 4, 1976
BIOCHEMICAL
pronase-digested is
light
a cleavage
accounted
product
for
nearly
heavy
one fifth
and light
fourths
vesicles
have
been
and phospholipid acetone,
Vesicle
ether
of
the
remaining
did
vesicles
cells
ductal
persist
insemination
ether
with
eggs
recovered
from
the
in
probably
important. this
containing
these
indicate not This
.(Table
2).
With
approximately
phospholipid
this
was eluted.
treatment,
vesicles, gave
however,
of
dry
the
extracted
that
surface
in
in
decapacitation,
in
agreement
that
synthetic
reversibly
inhibit
showing
(3). 1007
ovi-
3).
By
with
with
other
three of
vesicle a recent
phospholipid sperm
untreated
= 43.42,
proteins
with
the
CXZ = 1.45)
achieved
the
acetone,
of
different
(I$
of
after (Table
significantly
than
is
rates
64% (32/50)
76% (48/63)
extracted
fertilization
to vesicles
while
ether
respectively,
two groups
finding
cholesterol
cholesterol
was significantly
implicated
laboratory
decreased
of fertilizing
is not rate
more
results
light activity
post-ovulation
exposed
and this
Fertilization
These
3 hrs.
were capable
spermatozoa. frequent
also
decapacitation
the
and 36% (8/22),
cells
fertilization
native vesicles
about
and water
They
as solvent,
after
vesicles
was stable. treated
sperm
light
that
amounts :I)
and
comprise
vesicles.
(13)
and half
not
26% (y/34)
comparison,
ether
(30110
and pronase-digested
237; (14/61),
higher
it
respectively.
to note
small
and water
suspension Sperm
ethyl
cholesterol
structure
as heavy
with
dry
weight,
As expected,
interesting
by comparatively
ethyl
two thirds
is
wet weight
and cholesterol
as much lipid
with
dry
indicating
proteins
of vesicle
lipid.
attributed
Extraction
c increased
phospholipids
It
RESEARCH COMMUNICATIONS
Vesicle
vesicle
of vesicle
twice
cholesterol.
in
protein.
three
had more
are
of another
1 shows that
about
l
component
Table
contained
(15)
vesicles
approximately
70% and 54% of
AND BIOPHYSICAL
fertilizing
the
O.Ol>>P) groups. vesicles
lipids
are
observation vesicles ability
Vol. 69, No. 4,1976
BIOCHEMICAL
AND BlOPHYStCAL
RESEARCH COMMUNICATIONS
TABLE 1 LIPID
COMPOSITION
OF MEMBRANE VESICLES
Lipid
Heavy
Total lipids (pg/mg protein)
vesicles
Light
vesicles
cent
41
41
i2$
it:; ( 5) ( 3)
1%
(2 gal, 1 gal)
1 glu;
7
5
8
13
33 10
lipids
PLASMA
865 Per
Ganglioside
Cholesterol Other neutral
SEMINAL
422
Phospholipid Sphingomyelin Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine
Cerebroside
FROM RABBIT
:;
TABLE 2 CHOLESTEROL AND PHOSPHOLlPID LEVELS IN EXTRACTED MEMBRANE VESICLES FROM SEMINAL PLASMA
Vesicles
Heavy Phospholipid
vesicles Cholesterol
Light Phospholipid
(pg/mg
vesicles Cholesterol
protein)
Native
173
139
354
260
Extracted Dry ether Acetone, ether and water
156 91
118 58
243 177
225 106
Rabbit incubation appeared
sperm
with to
cells
[ 3 H]galactose
represent
similar
became
radioactively
containing fractions
1008
labelled
vesicles. of
sperm
during
Glycolipid and vesicle
Vol. 69, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 3 EFFECT OF PRONASE-DIGESTED AND EXTRACTED MEi$BRANE VESICLES FROM SEMINAL PLASXA ON THE FERTILIZING CAPACITY OF RABBIT SPERM CELLS
Vesicles+
No. of insemnt*
Nil Native Pronase-digested Extracted Dry ether Acetone, ether and water
No. of eggs
Fertilized No.
eggs $
13 13 7
63 61 22
48
76
14
a
23 36
a
34 50
9 32
26 64
12
* Vesicle concentration was 1 mg protein/ml heavy and light vesicles). **There were 1.25 to 5.0 x 10' sperm cells
(0.5
mg protein/ml
deposited
in
from
each oviduct,
TABLE 4 FRACTION OF [3H]GALACT@SE IN GLYCOLIPID FROM VESICLES AED SPERM CELLS INCUBATED WITH THE VESICLES
Total radioactivity (cpm)
Source
Vesicles Sperm
radioactivity 1 glucose vesicles
cells
(Table residue have
Non-extractable
in
4).
450
40
214
48
A cerebroside
was found
Glycolipid fraction
in both
addition
a cerebroside
radioactivity
in
containing types
these
1009
2 galactose
of vesicles. with
a single
preparations
($)
Heavy galactose. can be
and
Vol. 69, No. 4, 1976
attributed results the
BIOCHEMICAL
largely imply
sperm
that
to labelled
glycoproteins
decapaciation
results
(8). from
Hence,
vesicle
these
fusion
with
cell.
ACKNOWLEDGEMENT We are assistance
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in performing
indebted these
to Mr R. Byrne
for
his
capable
experiments.
REFERENCES B. K. (1974) 5. Reprod. Fert. 41, 241-244. Med. FJ, 240-243. 2. Davis, B. K. (1976a) Proc. Sot. Exp. sol. Davis, B. K. (1976b) Proc. Sot. Exp. Biol. Med. in press. 2: Barros, L. E. and Austin, C. R. C., Bedford, J. M., Franklin, (1967) J. Cell Biol. 2, Cl-C5. C. and Pette, D. (1973) Exp. Cell Res. 5. Van der Bosch, J., Schudt, Et, 433-438. D., Poste, G., Schaeffer, B. E. and Vail, W. J. 6, Papahadjopoulos, (1974) Biochim. Biophys. Acta z, 10-28. N. J., Farr, A. L. and Randall, R. J. 7. Lowry, 0. H., Rosebrough, (1951) J. Biol. Chem. w, 265-275. T. and Hakomori, S. (1971) J. L,ipid Res. 12, 257-259. 8. Saito, Rudel, L. L. and Morris, M. D. (1973) J. Lipid Res. 2, 364-366. 1:: Warren, L. (1959) J. Biol. Chem. e, 1971-1975. 11. Bartlett, G. R. (1959) J. Biol. Chem. 2 4, 466-468. 12. Laemmli, U. K. (1970) Nature a, 680- d- 5. W., Sarzala, M. G., Wroniszewska, A., Lagwinska, E. 13. Drabikowski, and Drzewiecka, B. (1972) Biochim. Biophys. Acta m, 158-170. 14, Gahmberg, C. G. and Hakomori, S. (1973) J. Biol. Chem. &?, 4311-4317. B. K. and Niwa, K. (1974) Proc. Sot. Exp. Biol. led. 146, 15. Davis, 11-16. 1. Davis,
1010
