shown that trans- methylation inhibitors will inhibit the hamster sperm acrosome ..... Hey. After centri- fugation at 2000. X g for 5 mm at room temperature,.
BIOLOGY
OF
REPRODUCTION
28,
Phospholipid
1043
-1051
(1983)
Methylation of Golden
MIGUEL
Increases
Hamster
N. LLANOS’
and
Department
of School
University
Capacitation
In Vitro
STANLEY
Human of
of
Davis,
During
Sperm
MEIZEL2
Anatomy
Medicine
California,
Davis
California
95616
ABSTRACT The present report describes in vitro experiments with golden hamster sperm designed to determine whether there is any relationship between sperm phospholipid methylation and capacitation and/or the acrosome reaction. Washed cauda epididymal hamster sperm were incubated in a capacitation medium containing lmethyl-3Hlmethionine. After 0.5, 1.5, 2.5 and 3.5 h of incubation, sperm were extracted with a chloroform:methanol:2 N HCI mixture to extract total phospholipids. Liquid scintillation counting revealed that the methyl-3 H-group was incorporated into phospholipids with maximum incorporation at 3.5 h and an increase of 50% between 2.5 and 3.5 h. Uptake of labeled methionine by sperm reached its plateau by 1.5 h of incubation. Some sperm were eapacitated by 3.5 h because that is the time at which the rate of acrosome reactions began to increase and because at least 50% of them were able to undergo the acrosome reaction 10 mm after the addition of the fusogen Iysophophatidylcholine (LPC) at 3.5 h but not at 2.5 h. Homocysteine thiolactone and 3-deazadenosine, inhibitors of transmethylation, inhibited incorporation of methyl-3H into phospholipids at 3.5 h by approximately 90% and also inhibited LPC-induced acrosome reactions by 60%. Separation of methylated sperm phospholipid by thin-layer chromatography demonstrated the presence of 3H-Iabeled phosphatidyl-N-monomethylethanolamine. phosphatidyl-N,N-dimethylethanolamine, and to a lesser extent phosphatidylcholine. In addition, an unidentified lipid was also highly labeled. These results strongly suggest a positive correlation between phospholipid methylation and capacitation and/or the acrosome reaction of hamster sperm in vitro. Possible mechanisms for phospholipid methylation involvement in these events are discussed.
Vanagimachi,
INTRODUCTION
Phospholipid phosphatidyleholine
methylation, (PC)
ethanolamine (PE) via phatidyl-N-monomethylethanolamine and
formation phosphatidyl-
synthesis
of
of
phos(PN E)
phosphatidyl-N,N-dimethylethanolamine
(PNNE),
increases
mediated
events
including mast cell 1980).
the from
during in
the membrane histamine release The
acrosome
vesiculation of branes essential
several
mammalian fusion (Hirata reaction,
mammalian to fertilization
receptor-
somatic
sperm
required for and Axelrod, a fusion head (Meizel,
and mem1978;
prerequisite,
the
be receptor-mediated amines (Cornett and Working,
vitro to biogenic Meizel
et al., 1979; Meizel (1981)
and 1980;
Leibfried and Bavister, has shown that trans-
methylation inhibitors will inhibit sperm acrosome reaction and/or
the hamster capacitation.
Both phospholipid carboxymethylation
and
methylation systems
sperm
Bouchard
et
describes
studies
whether during if used tion
so,
al.,
(Janson
whether
are
and
1980).
The
undertaken
phospholipid hamster
the
Sastry, to
report determine increases
capacitation
in vitro,
transmethylation
methylation.
Part
in
1981;
present
methylation
sperm
protein
present
and
inhibitors
previously to inhibit the acrosome and/or capacitation also inhibit that
phospholipid
1043
its
ster sperm in events involving Meizel, 1978;
mammalian
Accepted December 27, 1982. Received August 13, 1982. ‘Present address: Universidad de Chile, Facultad de Medicina Sur e, Instituto de Nutricion V Technologia de los Alimentos, Santiago, Chile. 2 Reprint requests.
and
series of cellular and as capacitation (Bed1981), appear in ham-
Cornett 1982).
cells,
1981),
incompletely understood molecular changes known ford, 1970; Yanagimachi,
of
this
reacsperm work
1044
LLANOS
was
presented
(Llanos
and
elsewhere Meizel,
preliminary
form
1982).
MATERIALS Materials
in
AND
AND
METHODS
Used
The sources of biochemicals were: L-Imethyi3H] methionine (12-15 Ci/mmol) and Aquassure scintillation fluid, New England Nuclear; L-homocysteine thiolactone (Hey), L--phosphatidylcholine (Type VE, egg yolk), L-s-lysophosphatidylcholmne (LPC) (Type I, egg yolk), L-cs-phosphatidylethanoiamine, N-methyl, a--d ipalmitoyl, L-n-phosphatidylethanolamine, N,Ndimethyl, and 3-y-dipalmitoyl from Calbiochem; 3deazadenosine (3-DZA) from Southern Research Institute; L-methionine, benzamidine, mepacrine, L-ophosphatidylethanolamine dipalmitoyl, lipids standard #189-1 (mixture of methyl esters of linoleic acid, linolenic acid, oleic acid, palmititc acid and stearic acid), sphingomyelin. arachidonic acid methyl ester and penicillin G (sodium salt) from Sigma Chemical Co.; phosphatidylcholine plasmalogen from P. L. Biochemicals; and silica gel G plates (250 pm thickness) from Analtech. The sources of other biochemicals, reagent grade chemicals and other materials used, were the same as those described previously (Llanos et al., 1982). Deionized, glass-distilled F120, further purified by passage through a Nanopure-A system (Barnstead), was utilized throughout all the experiments and always had a specific conductivity of 1 X 10 Ohms’-Cm’ or less. Sperm
Preparation
for
the
incubations
epididymal sperm were removed from sexually mature golden hamsters (Cornett and Meizel, 1978) which had been maintained for at least 3 weeks on a 14L:1OD cycle. Sperm suspensions of 90-94% viability were obtained by washing the sperm in phosphate-buffered saline (PBS)-sucrose medium, pH 7.3, containing penicillin G, passing sperm through a glass bead column and finally eluting them with a modified Tyrode’s solution (TAM) containing 25 mM bicarbonate, 1.5 mg/mi TCA-BSA (bovine serum albumin purified by trichloroacetic acid precipitation and ethanol resolubilization), glucose, lactate, pyruvate and penicillin G. All the procedures were carried out as described previously (Meizel et al., 1980; Llanos et ai., 1982) except for the following modifications: PBS was composed of NaCI (138.6 mM), KCI (8.5 mM), Na2HPO4 (6.45 mM), KH2PO4 (1.48 mM), CaCI22H2O (0.93 mM) and MgCI2’6H2O (0.49mM); Cauda
and
the
TAM
was
composed
of
NaCI
(121
mM),
KCI
(5 mM), NaH2PO4H2O (0.3 mM), NaHCO3 (25 mM), CaCI22H2O (2.4 mM), MgCI2’6H20 (0.49 mM), glucose (5 mM), lactate (12.5 mM) and pyruvate (0.25 mM). Penicillin G concentrations in the PBS-sucrose washing solution and in the TAM were 16 and 100 pg/mI, respectively. Before use, all solutions were passed through a 0.45-pm millipore or nucleopore filters.
Sperm Incubation of Phospholipid
for Determination Methylation During
Column-eluted tained from three
Capacitation
sperm suspensions were usually obdifferent males and pooled. Sperm
MEIZEL
were incubated in 2-mi volumes in 38-mi round bottom polypropylene centrifuge tubes (Nalgene #3110.0380) capped with a plastic cap (punched 24 times with an 18-gauge needle). lncubations were carried out at 37#{176}C in a humidified atmosphere of 5% C02, 95% air. The pH of the sperm suspensions was 7.4-7.6 throughout the incubation. Usually, ten tubes containing 2 ml each of incubation suspension were used in an experiment (6 control and 4 experimental tubes). Each 2-mi incubation suspension contained 480 p1 of TCA-BSA, (60 mg/mI in PBS, pH 7.3), 120 p1 taurine (1 X 102 M in TAM without metabolites or BSA), 100 p1 of (-)-epinephnine (1.4 X iO M in PBS), 1.3X ml of TAM solution and X ml of sperm suspension (X=suspension volume calculated so as to yield a final of 2 X 106 sperm/mI of suspension). Immediately before use, 600 p1 of L-Imethyl-3Hl methionine were dried under a stream of nitrogen and redissolved in 200 p1 of PBS, pH 7.3. Aliquots of 25 p1 containing 75 pCi were added to each 2-mi experimental incubation suspension (final L-lmethyl-3H1 methionine concentration was 2.5-3.1 pM). Control tubes were prepared in the same manner, but instead of labeled methionine they contained the same final concentration of unlabeled methionine. Experiments were designed to follow phospholipid methylation during a time course of capacitation. At 0.5, 1.5, 2.5 and 3.5 h, 5O-pl aliquots of control incubation suspensions were assayed for motility, hyperactivation and acrosome reactions (see below). Two of the six control tubes were used only for assaying the effect of LPC on the acrosome reaction, motility and hyperactivation of 2.5 and 3.5 h of incubation. Aliquots from these tubes were assayed before and 10 mm after the addition of 75 p1 of LPC, 2 mg/mi in TAM, pH 7.4, without BSA or metabolites (final concentration of LPC in incubation suspensions was 75 pg/mI). Also, at each time point, experimental tubes containing labeled methionine and the control tubes containing unlabeled methionine were removed from the incubator. To these tubes were added 50 p1 of a solution containing the transmethylation inhibitors 3-DZA (8 mM) and Hey (10 mM), unlabeled methionine (16 mM), the trypsin inhibitor benzamidine (40 mM) and the phospholipase inhibitor mepacrine (5 mM). All of those compounds were dissolved together in PBS-sucrose, p1-1 7.3, and kept at 37#{176}C before addition to sperm suspension. Also, 75 pCi of L-Fmethyl-3H1 methionine were added to the control sperm suspension in order to correct for any uptake or contamination occurring during the remaining procedures. At each time point, one incubation suspension each from control and experimental tubes were centrifuged at 2000 X g for 4 mm, the supernatants discarded and sperm pellets resuspended in 3 ml of the PBS-sucrose solution containing 1 mM benzamidine, 0.125 mM mepacrine, 0.4 mM unlabeled methionine, 0.2 mM 3-DZA and 0.25 mM Hey. After centrifugation at 2000 X g for 5 mm at room temperature, supernatants were discarded, and sperm pellets were immediately frozen in a mixture of ethanol-dry ice and kept at -80#{176}C until phospholipid extraction. concentration
Assay for Sperm Hyperactivation The
percentage
Motility, and Acrosome of
Reactions
acrosome-reacted
sperm
was
PHOSPHOLIPID
determined
by
counting
the
number
METHYLATION
of
such
sperm
DURING
extracted
in
Incubation
with
Transmethylase
For form
Inhibitors
these studies, incubation and subsequent procedures were carried out as described above except that all tubes contained labeled methionine from the beginning of the incubation. Experimental sperm suspension tubes contained 50 p1 of a solution composed of 8 mM 3-DZA and 10 mM Hey in PBS, pH 7.3, and control tubes contained 50 p1 of PBS, pH 7.3. For
Phospholipid to Determine
Extraction from Total Phospholipid
Hamster Sperm Methylation
Six time course incubations were carried out for these experiments. For each time point, sperm pellets from two of these incubations were pooled (resulting in three replications), extracted and assayed for total phospholipid methylation. The pooled sperm pellets were thawed, resuspended in 0.3 ml of H20, and immediately extracted for 30 sec (with the aid of a vortex mixer) in 3 ml of a mixture of chloroform: methanol:2 N HCI (12:6:1 v/v: Hirata et al., 1978). After a second 30-sec agitation, the suspensions were centrifuged (800 X g, 2 mm) to separate the aqueous and organic phases. The aqueous phase was discarded, and the organic phase was washed 3 times with 2 ml of 0.5 M KCI in 50% methanol (Castano et al., 1980; Hotchkiss et al., 1981). The first washing was performed by agitating for 30 see and the last two by 15 see agitation. In each case, agitation was followed by centrifugation at 800 X g for 2 mm at room temperature and the aqueous phase was discarded. After these extraction and washing procedures were completed, the final chloroform phase volume was measured, and 1 ml was pipetted into a glass counting vial, dried at 60#{176}Cunder N2 for approximately 45 mm, and finally dissolved in 10 ml of Aquassure scintillation fluid. Tritium was counted for estimation
of
equilibration
total
as
phospholipid
described
methylation
in
Liquid
(after
Scintillation
Counting). Total phospholipid methylation was expressed as femtomoles of methyl-3 H-group incorporation into phospholipid per 10’ sperm incubated. Separation of Methylated Sperm
and
Analysis Phospholipids
3.5 h under the same Incubation for Determination of Phospholipid Methylation During Capacitation except that all tubes contained L-(methyl-3 Ill methionine from the beginning of the incubation (n=2, total of 15 tubes). Extraction of the sperm pellet from 15 pooled tubes was carried Out exactly as described in Phospholipid Extraction from Hamster Sperm to Determine Total Phospholipid Methylation (including the use of 3 ml of extraction mixture). The conditions
were
described
incubated in
for
Sperm
-
1045
phospholipids
by thin-layer
100 strongly motile sperm using phase-contrast microscopy (Meizel and Lui, 1976). The percentage of hyperactivated sperm at 37#{176}C(whiplash-like flagellar movement characteristic of the capacitated hamster sperm) was estimated using dark-field microscopy at 125X magnification. The percentage of motile sperm was determined by phase-contrast microscopy. Counts were made of 200 total sperm at 400X magnification and room temperature. Sperm
CAPACITATION
were
separated
and
identified
chromatography.
thin-layer chromatography, phase (approx. 6 ml in total)
the washed was dried
chlorounder a
stream of nitrogen at room temperature and redissolved in 0.4 ml of chloroform:methanol mixture (2:1 v/v). Subsequently, 20 pg each of LPC, PC, PNNE, PNE and PE were added to a 0.2-mi aliquot of the extracted sperm phospholipids. Next, the sample containing the methylated phospholipids and the unlabeled carriers was applied to a prescored silica gel G plate. Also a mixture of carrier phospholipids and single phospholipids was applied to the same plate. The ascending chromatograph was developed with a mixture of propionic acid/n-propyl alcohol/chloroform/water (2:2:1:1 v/v) for phospholipid separation (Hirata et al., 1978). After the solvent front had migrated approximately 14 cm, the plate was dried, standard phospholipids were visualized with iodine vapors, and their chromatographic mobilities determined. The prescored section of the plate corresponding to the sample was divided into 5-mM bands. Each band was then scraped into a 5-mi glass culture tube and eluted with 0.6 ml of freshly made chromatography solvent for 6 h at room temperature in the dark (during the first hour, tubes were continuously shaken). After elution, tubes were centrifuged at 800 X g for 3 mm, and 0.4 ml from each tube were pipetted into a glass counting vial, dried at 60#{176}C during 40-50 mm and finally dissolved in 10 ml of Aquassure
scintillation
equilibration
fluid
as described
Determination
of the
intracellular During Time
Methyl-3 Course
for
counting
(after
below).
Concentration
of TCA
-Soluble
H-Labeled Compounds of Capacitation
Three experiments were carried out and analyzed separately. The procedures followed here were identical to those used for experimental and control tubes described in Sperm incubation for Determination of Phospholipid Mexhylation During Capacitation up to and including the freezing and storage of the sperm pellet. In other to measure total intracellular freelabeled methionine and related labeled compounds (for instance, S-adenosylmethionine), sperm pellets from each time point were thawed, resuspended in 0.25 ml of distilled water, and immediately added to 0.3 ml of 10% trichloroacetie acid (TCA). Suspensions were then centrifuged at 6000 X g for 5 mm, supernatants saved and the pellet washed twice with 0.5 ml of 5% TCA. After this treatment, pellets were discarded, all the supernatants pooled and then recentrifuged at 27,000 X g for 10 mm (all these procedures were carried out at 0-4#{176}C). After centrifugation, supernatant volumes were measured, a 0.5-mI aliquot pipetted into a glass counting vial, mixed with 10 ml of Aquassure scintillation fluid and counted after equilibration as described in Liquid Scintillation Counting. Results were expressed as pmoles of methyl-3 Hgroups in intracellular TCA-soiuble compounds per 10’ sperm incubated. Liquid
Scintillation
Samples librate
for
Counting
in scintillation I
h
at
4#{176}Cin
fluid the
were dark
allowed prior
to
to equicounting.
1046
LLANOS
Scintillation Analytic 92 1% precision minute dard
counting scintillation with 95%
(dpm) ratio
were
AND
MEIZEL
was carried out in a Searle counter set so as to obtain confidence. Disintegrations per
obtained
using
the
external
400
0 Li I-
