MOLECULAR REPRODUCTION AND DEVELOPMENT 47:105–112 (1997)
Phospholipid Metabolism in Boar Spermatozoa
and Role of Diacylglycerol Species in the De Novo
Formation of Phosphatidylcholine
JUAN MARIA VAZQUEZ1,2 AND EDUARDO R.S. ROLDAN1,3*
1Department of Development and Signalling, The Babraham Institute, Cambridge, United Kingdom
2Departamento de Patología Animal (Reproducción y Obstetricia), Universidad de Murcia, Murcia, Spain
3Departamento de Reproducción Animal, Centro de Investigación y Tecnología, INIA, Madrid, Spain
ABSTRACT
We have investigated pathways
of lipid metabolism in boar spermatozoa sperm cells
incubated for up to 3 days with [14C]palmitic acid,
[14C]glycerol, [14C]choline, or [14C]arachidonic acid or
incorporated these precursors into diglycerides and/or
phospholipids. When spermatozoa were incubated with
[14C]palmitic acid or [14C]glycerol, there was first an
incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidylcholine (PC). This indicates that the de novo pathway of
phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of
di-saturated DAG, which represented a considerable
proportion of the high basal levels of DAG, accumulated
the majority of label. Another DAG pool, containing
saturated fatty acids in position 1 and unsaturated fatty
acids in position 2 and representing the remaining basal
DAG, was in equilibrium with PC. When spermatozoa
were incubated with [14C]arachidonic acid, there was a
considerable incorporation of label into PC, which
indicates the presence of an active deacylation/
reacylation cycle. The behaviour of certain lipid pools
varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of
[14C]palmitic acid or [14C]arachidonic acid, there was
more incorporation of label into PC when spermatozoa
were incubated at 257C than when incubated at 177C.
Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may
therefore be possible to design methods to evaluate
the metabolic activity of boar spermatozoa based on
the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997.
r 1997 Wiley-Liss, Inc.
Key Words: phospholipids; diacylglycerol; arachidonic acid; phospholipase C; phospholipase A2; boar;
spermatozoa
INTRODUCTION
Mammalian spermatozoa are highly compartmentalized cells. Two main components are recognized in
these cells: the sperm head bearing the nucleus and the
acrosomal granule and the flagellum containing the
r 1997 WILEY-LISS, INC.
mitochondria and the axoneme. During differentiation
in the testis and maturation in the epididymis, spermatozoa shed a number of organelles, including the endoplasmic reticulum. Spermatozoa lack the ability to
synthesize DNA or proteins, and they have only glycolytic and respiratory capacity, which is mainly related
to the need to sustain motility (Bedford and Hoskins,
1990). It is thought that lipid pools are stable and
turnover is not very active (Terner and Korsh, 1962;
Neill and Masters, 1972, 1973; Hamilton and Olson,
1976).
Lipid composition in spermatozoa shows some peculiar characteristics (Mann and Lutwak-Mann, 1981).
Sperm neutral lipids, especially diacylglycerol (DAG),
are present in unusually high amounts (Selivonchick et
al., 1980; Mann and Lutwak-Mann, 1981; Nikolopoulou
et al., 1985), and the phospholipids also show distinctive features: alkenyl-phospholipids (plasmalogens) represent a high proportion of the choline- and ethanolamine-containing phosphoglycerides, with up to 50% of
the total mass in some species (Glegg and Foote, 1973;
Neill and Masters, 1973; Evans et al., 1980; Selivonchick et al., 1980; Mann and Lutwak-Mann, 1981; Nikolopoulou et al., 1985; Alvarez et al., 1987; Alvedaño et al.,
1992). Fatty acid composition also is very peculiar
because a large proportion of highly unsaturated fatty
acids (C22:5 and C22:6) are present in sperm phospholipids (Neill and Masters, 1972, 1973; Selivonchick et al.,
Abbreviations: PLC, phospholipase C; PLD, phospholipase D; PLA2,
phospholipase A2; DAG, diacylglycerol; 1,2-DS-DAG, DAG with saturated fatty acids in positions 1 and 2 of the glycerol backbone;
1,2-SU-DAG, DAG with a saturated fatty acid in position 1 and an
unsaturated fatty acid in position 2 of the glycerol backbone; MAG,
monoacylglycerol; PC, phosphatidylcholine; PA, phosphatidic acid; PS,
phosphatidylserine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; Cho-P, choline phosphate; CT, CTP:choline phosphate cytidylyltransferase; CPT, DAG:cholinephosphotransferase; t.l.c., thin layer
chromatography.
Supported by the Biotechnology and Biological Sciences Research
Council (UK).
*Correspondence to: Dr. E.R.S. Roldan, Departamento de Reproducción Animal, Centro de Investigación y Tecnología, INIA, Ctra. de La
Coruña km 5.9, 28040-Madrid, Spain.
Received 6 August 1996; Accepted 23 October 1996
106
J.M. VAZQUEZ AND E.R.S. ROLDAN
Fig. 1. Pathways of phospholipid metabolism in somatic cells. Not
all pathways are shown (e.g., the de novo pathway for PE synthesis,
which is analogous to that for PC synthesis). Enzymes involved are: (1)
sn-glycero-3-phosphate acyltransferase; (2) lysoPA acyltransferase; (3)
CDP-DAG synthase; (4) PI synthase; (5) PI kinase and phosphatidylinositol 4-phosphate kinase; (6) PA phosphatase (phosphohydrolase); (7) DAG
kinase; (8) DAG lipase; (9) MAG lipase; (10) MAG acyltransferase; (11)
choline kinase; (12) CTP:choline phosphate cytidylyltransferase; (13)
DAG:cholinephosphotransferase; (14) PE N-methyltransferase; (15)
PE:serine O-phosphatidyltransferase; (16) PS decarboxylase; (17) PC:
serine O-phosphatidyltransferase; (18) phospholipase A2; (19) lysophospholipase; (20) GPC diesterase; (21) GPC acyltransferase; (22) lysophosphatide acyltransferase (Pelech and Vance, 1984, 1989; Bishop and
Bell, 1988; Hjelmstad and Bell, 1991). Abbreviations: Cho, choline;
GPC, glycerophosphocholine, G3P, sn-glycero-3-phosphate, PPIs, polyphosphoinositides.
1980; Mann and Lutwak-Mann, 1981; Nikolopoulou et
al., 1985; Aveldaño et al., 1992).
Lipid metabolism in spermatozoa is poorly understood and since synthetic ability is thought to be
unimportant (Terner and Korsh, 1962; Neill and Masters, 1972, 1973; Hamilton and Olson, 1976), it is far
less characterized than phospholipid metabolism in
somatic cells (Pelech and Vance, 1984, 1989; Bishop and
Bell, 1988; Hjelmstad and Bell, 1991) (Fig. 1). The
majority of the studies carried out in the past concerning sperm lipid metabolism attempted to understand
whether spermatozoa utilize fatty acids as energy
sources (Mann and Lutwak-Mann, 1981; Bedford and
Hoskins, 1990). The ability of sperm to synthesize
phospholipids has received some earlier attention
(Terner and Korsh, 1962; Neill and Masters, 1972), but
studies were carried out under conditions that did not
result in proper characterization of the pathways involved and/or their potential regulation. Due to the
currently recognized central role of (phospho)lipids in
sperm intracellular signalling (Roldan and Harrison,
1993) and in membrane fusion (Roldan and Harrison,
1993; Yanagimachi, 1994), a better understanding of
lipid metabolism in these cells is needed. Mammalian
spermatozoa are, in general terms, short-lived cells
when kept under in vitro conditions and, therefore,
studies of lipid metabolism have been hampered by the
inability to guarantee long-term sperm survival.
In this study we have taken advantage of the ability
of boar spermatozoa to survive in defined media used to
preserve semen for artificial insemination. We have
studied sperm lipid metabolism under these conditions
and have found that, contrary to previous assumptions,
spermatozoa have an active lipid synthetic capacity:
sperm cells were found to use the de novo pathway to
synthesise phospholipids, as revealed by their capacity
to utilize exogenous labelled precursors. Two different
sperm DAG pools were identified under ‘‘resting’’ conditions (i.e., in nonstimulated cells), and it appears that
only one of these pools contributes to phospholipid
synthesis. In addition, unsaturated fatty acids were
found to be incorporated into phospholipids by deacylation/reacylation.
These results, therefore, would allow studies on the
generation of lipid messengers and lipid metabolism
during acrosomal exocytosis in sperm cells whose lipid
pools are labelled to equilibrium. On a more applied
note, our results may lead to the development of
objetive methods to evaluate lipid metabolism of spermatozoa from individual boars.
MATERIALS AND METHODS
Reagents
14
[1- C]Palmitic acid (57 mCi/mmol; toluene solution),
[U-14C]glycerol (159 mCi/mmol; 50% aqueous ethanol
solution), [1-14C]arachidonic acid (54 mCi/mmol, toluene solution), [methyl-14C]choline chloride (54 mCi/
mmol; aqueous solution), and L-[U-14C]serine (160 mCi/
mmol; aqueous solution) were from Amersham International (Amersham, UK). Chemicals were of analytical grade and were purchased from Sigma or BDH (both
of Poole, Dorset, UK). Percoll was from Pharmacia
(Milton Keynes, UK). Organic solvents were of reagent
grade and were purchased from BDH. Neutral lipids
and phospholipids used as standards were from Sigma,
Serdary (Ontario, Canada), or Novabiochem (Nottingham, UK). Choline metabolites were from Sigma. Polyphosphoinositide standards were kindly provided by Dr
R.F. Irvine of The Babraham Institute.
Preparation, Labelling, and Treatment
of Spermatozoa
The medium used for storage and labelling of boar
spermatozoa was the Beltsville extender (BTS) (Johnson
et al., 1988). Its composition was 10 mM KCl, 20.4 mM
trisodium citrate (2H2O), 15 mM NaHCO3, 3.36 mM
EDTA (disodium salt), 205 mM glucose, and 50 µg
kanamycin monosulphate/ml, and once semen has been
added, its pH was 7.0–7.4. Lipid precursors were dissolved in BTS after their solvents (i.e., toluene or ethanol)
were blown dry under nitrogen.
The standard saline medium used consisted of 142
mM NaCl, 2.5 mM KOH, 10 mM glucose, and 20 mM
Hepes, adjusted to pH 7.55 at 20°C with NaOH (Roldan
and Harrison, 1988). This medium also contained 1 mg
of poly(vinyl alcohol)/ml and 1 mg of polyethylene
glycol/ml and had an osmolality of 305 mOsm/kg.
Sperm-rich fractions of semen were collected from
Large White boars, and they were allowed to cool slowly
LIPID METABOLISM IN SPERMATOZOA
to room temperature (,22–24°C) and filtered through
gauze. Spermatozoa were incubated in BTS (,1 3108
cells/ml) at 17°C or 25°C, in the presence of phospholipid precursors for up to 72 hr (see Results for details).
During the labelling period, viability of spermatozoa
remained constant (85–95%) as estimated using propidium iodide (Harrison and Vickers, 1990).
Aliquants of 4 3 108 spermatozoa were washed
through two layers of 35% (4 ml) and 70% (2 ml)
Percoll-saline for 5 min at 200g and 15 min at 900g
(Harrison et al., 1993). After centrifugation, 0.5 ml of
infranatant containing loosely pelleted spermatozoa
was diluted 1:5 in the saline medium and centrifuged
for 5 min at 700g to remove Percoll. At this stage, $90%
viable cells were found.
Lipid Analyses
To measure changes in phospholipids, lipids were
extracted essentially as described previously (Roldan
and Harrison, 1989, 1990) except that reactions were
stopped with 10% (w/v) perchloric acid (Roldan and
Fragio, 1993). To measure changes in DAGs, incubations were stopped by addition of chloroform/methanol
(1:2, v/v) and lipids were then extracted according to
Bligh and Dyer (1959).
Lipids were separated by thin layer chromatography
(t.l.c.) on Silica Gel-60-coated glass plates (0.25 mm
thickness) or plastic sheets (0.2 mm thickness) (E.
Merck, Darmstadt, Germany). Phospholipids were separated on 10 3 10 cm plastic sheets pretreated by
spraying with 1% (w/v) potassium oxalate, activated by
heating at 110°C for 10 min, and developed in a
two-dimensional system where the first solvent was
chloroform/methanol/water/concentrated NH3 (48:40:
7:5, v/v), and the second solvent consisted of chloroform/
methanol/formic acid (11:5:1, v/v) (Roldan and Harrison, 1989; Roldan and Fragio, 1993). The plates were
air-dried briefly, and the various spots were detected by
autoradiography using Fuji RX film. Using the autoradiographs as templates, the individual spots were
scraped off and the radioactivity in each determined by
liquid scintillation counting.
For DAG mass quantification, neutral lipids were
separated in the solvent chloroform/methanol/acetic
acid (98:2:1, v/v) (Agwu et al., 1989). This solvent
system resolves 1,2- from 1,3-DAGs and among 1,2DAGs, those with saturated fatty acids in positions 1
and 2 of the glycerol backbone (1,2-DS-DAG) from
DAGs with saturated fatty acids in position 1 and
unsaturated fatty acids in position 2 (1,2-SU-DAG)
(Roldan and Murase, 1994). DAGs were quantified by
Coomassie Blue staining (Nakamura and Handa, 1984)
and densitometry using 1,2-dioleoyl-sn-glycerol to construct standard curves for each plate (Bocckino et al.,
1987; Roldan and Harrison, 1990, 1992; Roldan and
Murase, 1994). The plates were scanned with a Chromoscan-3 UV densitometer (Joyce-Loebl, Gateshead,
Tyne and Wear, UK).
Labelled DAGs and other neutral lipids were separated by developing plates twice in one of the following
107
solvent systems: (1) chloroform/methanol/acetic acid
(98:2:1, v/v) (Agwu et al., 1989; Roldan and Murase,
1994), with 1,2-sn- and 1,3-dimyristoylglycerol, and
1,2-sn- and 1,3-dioleoylglycerol used as internal lipid
standards, or (2) n-hexane/diethyl ether/acetic acid
(70:30:1, v/v), with 1,2-dioleoyl-rac-glycerol, 1,3-dioleoylglycerol, arachidonic acid, and 1-monooleoylglycerol
used as internal standards. The former solvent system
was used when cells were labelled with [14C]palmitic
acid or [14C]glycerol, whereas the latter was employed
for [14C]arachidonic acid-labelled cells. After development in the appropriate solvent system, plates were
allowed to dry and lipid spots were visualized by
staining with iodine vapours or Coomassie Blue, scraped
off, and the radioactivity in each determined by liquid
scintillation counting.
[14C]Choline-labelled lipids were separated on glass
plates pretreated by spraying with 1% (w/v) EGTA pH
5.5, activated by heating at 110°C for 60 min and
developed in the solvent chloroform/methanol/water/
acetic acid (65:50:5:2, v/v). Lipid spots were detected by
iodine staining, identified by comparison with internal
standards of phosphatidylcholine, lysophosphatidylcholine, and sphinglomyelin, scraped off, and radioactivity
in each determined by liquid scintillation counting.
Quantitation of Choline Metabolites
The aqueous fractions of a Bligh and Dyer extraction
were used for measurement of choline metabolites, with
authentic choline, choline phosphate, glycerophosphocholine, and CDP-choline standards being added before
extraction. Samples were dried under nitrogen and
resuspended in water/methanol (1:1, v/v) and resolved
by t.l.c. using the system methanol/0.9% (w/v) NaCl/
NH3 (50:50:5, v/v) (Yavin, 1976; Bonser and Thompson,
1992). Plates were air-dried, and spots were identified
by staining with iodine vapours or the Dragendorff
stain (Kates, 1986), scraped off, and counted.
RESULTS
Palmitic Acid as Lipid Precursor
Incubation of boar spermatozoa in BTS with 0.1 µCi
[1-14C]palmitic acid/ml resulted in a rapid initial uptake of radioactive fatty acid, as seen by a rise in the
labelled free fatty acid pool; levels declined after 6 hr
(Fig. 2a). The early increase in the fatty acid pool was
accompanied by a parallel rise in labelled phosphatidic
acid (PA) (Fig. 2b), although the levels of label in PA
were considerably higher. Labelling of PA preceded
labelling of 1,2-DAG (Fig. 2c) and this, in turn, preceded incorporation into PC (Fig. 2d); levels of labelled
PC reached a plateau after 24 hr. There was very little
incorporation of label into phosphatidylserine (PS),
phosphatidylethanolamine (PE), phosphatidylinositol
(PI), and also little labelled lysoPC (data not shown).
These results suggest a very rapid acylation of [14C]palmitic acid to form PA, a considerable PA phosphohydrolase activity, and that the forward reaction to generate
DAG was more active than the back reaction catabo-
108
J.M. VAZQUEZ AND E.R.S. ROLDAN
Fig. 2. Labelling of boar sperm lipids with [14C]palmitic acid.
Spermatozoa in BTS medium with 0.1 µCi [1-14C]palmitic acid/ml
were incubated at two different temperatures. At various times, lipids
were extracted and resolved as indicated in Materials and Methods.
Results (means 6 S.E.M. of three experiments) show incorporation of
[14C]palmitic acid into different lipid pools when spermatozoa were
incubated at (X) 17°C or (W) 25°C.
lized by DAG kinase. More importantly, these results
are good evidence that de novo PC synthesis (sensu
Dennis, 1992) via the Kennedy pathway (Pelech and
Vance, 1984; Bishop and Bell, 1988) is active in spermatozoa.
When cells were incubated with [14C]palmitic acid, a
very early incorporation of label into 1,3-DAG was also
seen (Fig. 2e), which paralleled the incorporation of
label into PA. Since the monoacylglycerol (MAG) pool
was poorly labelled throughout (Fig. 2f), it may be that
1,3-DAG does not originate by acylation of MAG and,
therefore, labelling of 1,3-DAG may occur via a different mechanism.
Labelling of spermatozoa (i.e., incorporation of the
lipid precursor) took place at both temperatures used
for incubation (17°C and 25°C). However, the behaviour
of various lipid pools varied depending on the temperature at which spermatozoa were incubated. At 17°C,
there was a rapid early incorporation of [14C]palmitic
acid into PA, but a lower incorporation into 1,3-DAG, as
opposed to the results obtained at 25°C. In addition,
more labelled 1,2-DAG substrate seemed to be converted to PC at 25°C, as suggested by the levels of these
metabolites.
after shorter incubation times with similar precursors
(Neill and Masters, 1972, 1973; Hamilton and Olson,
1976) but that has remained unexplained. We reasoned
that perhaps not all the 1,2-DAG was used by DAG:
cholinephosphotransferase (CPT), the enzyme that catalyzes PC synthesis via the Kennedy pathway (Pelech
and Vance, 1984, 1989; Bishop and Bell, 1988; Hjelmstad and Bell, 1991), and we have therefore examined
whether different types of DAGs could be distinguished. Previous work has revealed that ram spermatozoa under resting conditions have two distinct DAG
pools, one with saturated fatty acids in positions 1 and
2 (1,2-DS-DAG) and another with saturated fatty acids
in position 1 but unsaturated fatty acids in position 2
(1,2-SU-DAG) (Roldan and Murase, 1994). When mass
of boar sperm DAG was resolved, we found that the
majority of DAG at resting conditions was in fact
1,2-DS-DAG (about 90%) (Table 1). We therefore examined which proportion of the DAGs labelled with
[14C]palmitic acid corresponded to each type of DAG
when cells were incubated with precursors at 25°C.
Labelled 1,2-SU-DAG represented ,35% of total labelled DAG, with 1,2-DS-DAG representing the remaining 65% of total DAG (Table 1). The level of labelling
seen in 1,2-SU-DAG (,6300 cpm/108 cells) was similar
to that seen in PC (,6,200 cpm/108 cells) when cells
were incubated at 25°C (Table 1, Fig. 2d), which agrees
with the known substrate preference of CPT for 1,2-SUDAG (Ansell and Spanner, 1982) and with the observa-
Types of DAG
Although a high level of [14C]palmitic acid was incorporated into 1,2-DAG, conversion of this pool to PC
seemed limited (Fig. 2), an observation made previously
LIPID METABOLISM IN SPERMATOZOA
109
TABLE 1. Proportion of DAG Types in Unlabelled
Spermatozoa and After Labelling With [14C]Palmitic
Acid or [14C]Glycerol*
1,2-DAG type
1,2-SU-DAG
1,2-DS-DAG
Mass
µg/108 cells
(% of total)
[14C]palmitic
acid-labelling
cpm/108 cells
(% of total)
[14C]glycerollabelling
cpm/108 cells
(% of total)
0.590 6 0.064
(11%)
4.667 6 0.132
(89%)
6329 6 181
(33%)
12851 6 540
(67%)
1810 6 155
(43%)
2399 6 123
(57%)
*Spermatozoa were left unlabelled or were incubated in BTS
with 0.1 µCi [14C]palmitic acid/ml or 1.5 µCi [14C]glycerol/ml
for 24 hr at 25°C. Lipids were extracted and neutral lipids
were separated by t.l.c. using the solvent system chlorofom/
methanol/acetic acid (98:2:1, v/v). Unlabelled DAGs were
quantified by staining with Coomassie Blue and densitometry.
Labelled DAGs were scrapped off and radioactivity determined by liquid scintillation counting (see Materials and
Methods for details). Results are averages 6 S.E.M. of two
different experiments.
Fig. 3. Labelling of boar sperm lipids with [14C]glycerol. Spermatozoa in BTS medium with 1.5 µCi [U-14C]glycerol/ml were incubated at
25°C. At different times, lipids were extracted and resolved as
indicated in Materials and Methods. Results (means 6 S.E.M. of three
experiments) show incorporation of label into (X) PA, (S) 1,2-DAG,
and (N) PC.
tion that this enzyme is near equilibrium (Pelech and
Vance, 1984; Shears, 1993).
Glycerol as Precursor
Incubation of spermatozoa with 1.5 µCi [U-14C]glycerol/ml at 25°C resulted in a very rapid incorporation of
label into PA (Fig. 3). Incorporation of glycerol could
take place directly via phosphorylation by glycerol
kinase to form glycerol-3-phosphate, but the activity of
this enzyme is not detectable in spermatozoa from
various species, including boar (Mohri and Masaki,
1976; Jones et al., 1992). Spermatozoa convert glycerol
to dihydroxyacetone phosphate (Jones et al., 1992), and
this might be the origin of labelled PA (Bishop and Bell,
1988; Hjelmstad and Bell, 1991). The rise in labelled PA
was followed by incorporation of label mainly into
1,2-DAG (Fig. 3). The labelling of PC reached a plateau
at 24 hr, but the level of labelling was lower than that
seen in 1,2-DAG indicating, in agreement with results
obtained with [14C]palmitic acid that poor conversion of
the whole DAG pool to PC could be taking place.
Resolution of the two types of DAGs revealed different
levels of labelling for 1,2-DS-DAG and 1,2-SU-DAG
(Table 1) and again in agreement with results obtained
with [14C]palmitic acid, a similar level of labelling
between 1,2-SU-DAG and PC, reinforcing the idea that
this DAG pool may be the one giving rise to PC.
Attempts to increase the labelling of the PC pool by
incubating sperm with 5 µCi [U-14C]glycerol/ml at 25°C
led to a much higher incorporation of label into the
1,2-DAG pool, but not to a substantial increase of label
in PC (not shown), therefore proving unsatisfactory for
further studies. The lack of substantial labelling with
[14C]glycerol probably was not due to rapid oxidation of
glycerol by spermatozoa (Mohri and Masaki, 1967;
Jones et al., 1992), because amounts of labelled precursor in the incubation medium did not decrease significantly over time (not shown).
Fig. 4. Labelling of boar sperm lipids with [14C]choline. Spermatozoa in BTS medium with 2 µCi [methyl -14C]choline chloride/ml were
incubated at 25°C. At different times, lipids were extracted and
resolved as indicated in Materials and Methods. Results
(means 6 S.E.M. of two experiments) show incorporation of label into
(X) PC, (N) LPC, and (S) sphingomyelin.
Choline as Precursor
Incubation with 2 µCi [methyl-14C]choline chloride/ml at 25°C revealed that a sperm PC pool can be
clearly labelled using this precursor (Fig. 4). After 72
hr, considerable labelling of PC was observed, with
some incorporation of label into lysoPC and somewhat
less into sphingomyelin. Incorporation into PC appears
to take place via the de novo pathway because high
levels of labelled choline phosphate (Cho-P) and CDPcholine were concomitantly found (data not shown).
Attempts to label cells using a different polar head
group were not successful. Incubation with 2 µCi L-
110
J.M. VAZQUEZ AND E.R.S. ROLDAN
Fig. 5. Labelling of boar sperm lipids with [14C]arachidonic acid.
Spermatozoa in BTS medium with 0.1 µCi [1-14C]arachidonic acid/ml
were incubated at different temperatures. At various times, lipids
were extracted and resolved as indicated in Materials and Methods.
Results (means 6 S.E.M. of three experiments) show incorporation of
[14C]arachidonic acid into different lipid pools when spermatozoa were
incubated at (X) 17°C or (W) 25°C.
[U-14C]serine/ml at 25°C for 72 hr did not result in
labelling of PS, which suggests that a base-exchange
mechanism (with PC) is not very active in spermatozoa
and thus indicating that labelling of PS observed with
other precursors takes place via alternative mechanisms.
plateau after 24 hr of incubation at 25°C. When cells
were incubated at 17°C, there was less labelling of PC,
which reached a lower plateau at 48 hr. Some incorporation of label was seen in PE and PS, but values were
never high (Fig. 5e,f). There was also some label in PI
and phosphatidylinositol 4-phosphate. Phosphatidylinositol 4,5-bisphosphate was not labelled under these
conditions (data not shown).
Arachidonic Acid as Lipid Precursor
When boar spermatozoa were incubated with 0.1 µCi
[1-14C]arachidonic acid/ml, there was an extremely
rapid incorporation into the fatty acid pool, followed by
a fast decline (Fig. 5a). There was some early incorporation of [14C]arachidonic acid into PA (Fig. 5b) and, in
parallel, into 1,2-DAG (Fig. 5c), perhaps via acylation,
and PA phosphohydrolase action, respectively, as previously seen with [14C]palmitic acid. By 24 hr, levels of
1,2-DAG decreased and levels of PC increased (Fig. 5d),
suggesting synthesis of PC via the de novo pathway.
However, despite some contribution of the de novo
pathway to PC synthesis, a direct incorporation of
[14C]arachidonic acid label via lysophosphatide acyltransferase was probably the major route for PC labelling. The considerable and rapid increase in radioactivity in [14C]arachidonic acid-labelled PC (Fig. 5d)
outweighed the potential contribution of the de novo
pathway (the latter appears to contribute about onethird of the total PC, as deduced when the level of
labelling in 1,2-DAG was compared to the final amount
of label seen in PC). The labelling of PC reached a
Sperm Washing Does Not Cause Loss of Label
Spermatozoa were incubated for 48 or 72 hr with
each of the precursors indicated above, washed through
two layers of Percoll, and the infranatant resuspended
in the saline medium and centrifuged again to remove
the Percoll, as indicated in Materials and Methods.
Separation of lipids by mono- or bidmensional t.l.c. and
quantitation revealed that there was no appreciable
loss of label or changes in the labelling pattern after
such procedure (data not shown). Hence, these labelling
and washing protocols can be used for experiments in
which prelabelled cells would be stimulated to undergo
exoctyosis.
DISCUSSION
This is the first study to characterize pathways for
phospholipid biosynthesis in mammalian spermatozoa,
a highly differentiated cell with no other synthetic
capacity (Bedford and Hoskins, 1990). Using various
precursors, we have found that these cells synthesize
LIPID METABOLISM IN SPERMATOZOA
phospholipids de novo and that various lipid pools can
be labelled to equilibrium.
Unlike many somatic cells, spermatozoa do not survive long-term incubations in vitro. Therefore, previous
studies have only managed to label and study sperm
cells for a few hours in attempts to understand whether
spermatozoa use fatty acids as energy sources (Neill
and Masters, 1972, 1973; Darin-Bennett et al., 1973;
Bedford and Hoskins, 1990). It has been concluded that
the major phospholipid fractions are metabolically stable
and that pathways for de novo synthesis are not very
active in spermatozoa (Neill and Masters, 1972). We
have used a different approach to examine sperm
phospholipid synthesis and to achieve labelling of lipid
pools to equilibrium in order to follow changes in
metabolites relevant to signal transduction during exocytosis. We have taken advantage of the ability of
sperm to survive at, or below ambient temperature, in
so-called semen entenders, i.e., defined media used to
preserve spermatozoa for artificial insemination. The
BTS extender is one of these storage media routinely
used to preserve boar spermatozoa; no or little decline
in viability/fertility takes place if spermatozoa are kept
in this medium for up to 4 days (Johnson et al., 1988).
Two main metabolic pathways were found to be very
active in spermatozoa. Incubation with [14C]palmitic
acid, [14C]glycerol, and [14C]choline revealed that sperm
can synthesise phospholipids de novo via the Kennedy
pathway, whereas the use of [14C]arachidonic acid
showed that phospholipid pools could be deacylated/
reacylated to a considerable extent; the phospholipid
that accumulated most label was PC. It is not clear at
this stage whether the pools labelled by the different
precursors are identical.
Other pathways for phospholipid synthesis did not
appear to be very important in spermatozoa. For instance, generation of PC cannot be explained by synthesis via the sequential acylation of glycerophosphocholine and lysoPC (Pelech and Vance, 1984; Bishop and
Bell, 1988) because no major labelling of lysoPC was
found to precede labelling of PC, as noted with various
precursors. Pathways involving methylation, base exchanges, or lysophospholipid transacylation (Pelech
and Vance, 1984; Bishop and Bell, 1988; Dennis, 1992)
also seemed negligible or only of minor importance for
sperm phospholipid synthesis. Several major points
emerge from or relate to these observations.
First, different phospholipids appeared to turn over
at different rates. For instance, the mass of diacyl-PC is
similar to the mass of diacyl-PE in boar spermatozoa
(Nikolopoulou et al., 1985). However, the amount of
palmitic acid label in PC was much higher than that
found in PE. Similarly, the incorporation of [14C]arachidonic acid into PC was ,10 times higher than its
incorporation into PE, in spite of the fact that the PC
pool capable of labelling with this precursor is twice the
size of the analogous PE pool (Nikolopoulou et al.,
1985). One possible explanation for a more prominent
PC turnover is that this phospholipid would be more
involved in messenger generation prior to and during
111
exocytosis (Roldan and Fragio, 1993; Roldan and Murase, 1994).
Second, some precursors incorporated via the de novo
pathway ([14C]palmitic acid, [14C]glycerol) resulted in a
much higher level of label in 1,2-DAG than in PC.
Previous work has noted this discrepancy (Neill and
Masters, 1972, 1973; Hamilton and Olson, 1976), but
conditions in those studies involved incubation with
precursors for only 2–3 hr (Neill and Masters, 1972,
1973; Selivonchick et al., 1980), and perhaps equilibrium of pools was not achieved. In the present study,
levels of labelling reached a plateau at 24 hr, but, still,
1,2-DAG labelling was considerably higher than labelling of PC. The total mass of PC that could be potentially labelled in boar spermatozoa is much bigger than
that of 1,2-DAG (Nikolopoulou et al., 1985), so it is
unclear why this peculiar pattern of labelling develops.
A possible explanation is discussed below in the following point.
Third, we could resolve two 1,2-DAG types differing
in fatty acid composition. The total mass of sperm
1,2-DAG is very high under resting conditions (Selivonchick et al., 1980; Mann and Lutwak-Mann, 1981;
Roldan and Harrison, 1990, 1992), and the 1,2-DAG
type with saturated fatty acids in positions 1 and 2
(1,2-DS-DAG) represented 90% of the total mass of this
metabolite. However, this 1,2-DS-DAG seemed less
active than the 1,2-DAG with a saturated fatty acid in
position 1 and an unsaturated fatty acid in position 2
(1,2-SU-DAG): The 1,2-DS-DAG pool accumulated a
proportion of label (57–67%, depending on the precursor employed) that was much lower than its mass
(90%). Thus it is important that levels of labelled
1,2-SU-DAG were similar to levels of labelled PC, as
seen with two different precursors ([14C]palmitic acid,
[14C]glycerol), suggesting that both pools were in equilibrium. Since CPT, the enzyme that catalyzes the
conversion of DAG to PC, is believed to be close to
equilibrium (Pelech and Vance, 1984; Shears, 1993), it
is possible that only SU-DAG is converted to PC. The
role of high resting levels of 1,2-DS-DAG is unclear
(Roldan and Harrison, 1990), although they could be
related to the generation of other metabolites during
signal transduction.
Fourth, in somatic cells, enzymes that utilize DAG
for de novo synthesis, such as CTP:choline phosphate
cytidylyltransferase (CT) and CPT, are located in the
endoplasmic reticulum. Spermatozoa do not have an
endoplasmic reticulum, so the location of these enzymes is uncertain. Potential sites include the plasma
membrane, the cytosol, and the Golgi-derived outer
membrane of the acrosomal granule. Unfortunately,
there is no information whatsoever on enzymes of the
de novo pathway in spermatozoa.
Finally, our findings suggest that synthesis of lipids
from exogenous precursors may be important for mammalian sperm function during their residence in the
female genital tract. This is particularly relevant for
species such as humans and domesticated animals
where spermatozoa have to survive many hours be-
112
J.M. VAZQUEZ AND E.R.S. ROLDAN
tween sperm deposition and fertilization (Bedford and
Hoskins, 1990; Yanagimachi, 1994). Although spermatozoa are kept in a quiescent state in the female tract
during most of this period (Yanagimachi, 1994), metabolic activity may not be completely repressed. Essential questions raised by our findings thus relate to how
active sperm phospholipid synthesis is and how is it
regulated during transit or residence in the female
tract, or, conversely, whether quiescence in certain
portions of the female tract involves repression of this
synthetic capacity.
In summary, our results have clearly shown that the
long-held view that spermatozoa lack lipid synthetic
ability is no longer tenable and have provided an
explanation for the long-standing paradox of high DAG
labelling and poor concomitant PC labelling seen with
various precursors. Further knowledge regarding the
regulation of the sperm de novo pathway of PC synthesis is now needed. This would lead to a clearer understanding of lipid metabolism during important processes underlying sperm function (e.g., capacitation
and acrosomal exocytosis) and would perhaps allow the
development of an objective test to assess sperm lipid
metabolic activity in the context of semen evaluation.
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