BIOLOGY OF REPRODUCTION 74, 1104–1113 (2006)
Published online before print 1 March 2006.
DOI 10.1095/biolreprod.105.049304
Lipid Remodeling of Murine Epididymosomes and Spermatozoa During
Epididymal Maturation1
Hanae Rejraji,3,4 Benoit Sion,5 Gerard Prensier,6 Martine Carreras,7 Claude Motta,8 Jean-Marie Frenoux,4
Evelyne Vericel,7 Genevieve Grizard,5 Patrick Vernet,4 and Joël R. Drevet2,4
Laboratoire Epididyme et Maturation des Gamètes,4 Université Blaise Pascal, CNRS UMR 6547 GEEM,
63177 Aubière, France
Laboratoire de Biologie de la Reproduction,5 EA975, Université d’Auvergne, 63000 Clermont-Ferrand, France
Laboratoire de Biologie des Protistes,6 Universite Blaise Pascal, CNRS UMR 6023, 63177 Aubière, France
Laboratoire de Physiopathologie des Lipides et Membranes,7 INSERM U585, INSA-Lyon, 69622 Villeurbanne, France
Laboratoire de Biochimie,8 CHU, 63000 Clermont-Ferrand, France
ABSTRACT
We have isolated vesicular structures from mouse epididymal
fluid, referred to as epididymosomes. Epididymosomes have a
roughly spherical aspect and a bilayer membrane, and they are
heterogeneous in size and content. They originate from the
epididymal epithelium, notably from the caput region, and are
emitted in the epididymal lumen by way of apocrine secretion.
We characterized their membranous lipid profiles in caput and
cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and
arachidonic acid. The proportion of SM increased markedly
during epididymal transit and represented half the total
phospholipids in cauda epididymidal epididymosomes. The
cholesterol:phospholipid ratio increased from 0.26 in the caput
to 0.48 in the cauda epididymidis. Measures of epididymosomal
membrane anisotropy revealed that epididymosomes became
more rigid during epididymal transit, in agreement with their
lipid composition. In addition, we have characterized the
membrane lipid pattern of murine epididymal spermatozoa
during their maturation. Here, we have shown that mouse
epididymal spermatozoa were distinguished by high percentages
of SM and polyunsaturated membranous fatty acids (PUFAs),
principally represented by arachidonic, docosapentanoic, and
docosahexanoic acids. Both SM and PUFA increased throughout
the epididymal tract. In particular, we observed a threefold rise
in the ratio of docosapentanoic acid. Epididymal spermatozoa
had a constant cholesterol:phospholipid ratio (average, 0.30)
during epididymal transit. These data suggest that in contrast
with epididymosomes, spermatozoal membranes seem to
become more fluid during epididymal maturation.
epididymis, gamete biology, male reproductive tract, sperm
maturation
INTRODUCTION
During their transit from the caput to the cauda epididymidis, mammalian spermatozoa undergo significant morpholog1
Supported by grants from the French ‘‘Centre National de la Recherche
Scientifique’’ and the French ‘‘Ministère de l’Enseignement Supérieur et
de la Recherche.’’
2
Correspondence. FAX: 33 04 73 40 52 45;
e-mail: [email protected]
Received: 16 November 2005.
First decision: 3 December 2005.
Accepted: 14 February 2006.
Ó 2006 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
ical and biochemical modifications. This posttesticular
remodeling leads to the acquisition of their forward motility
and their ability to recognize and fertilize oocytes [1–3]. These
events are mediated mainly by the secretory activities of the
epididymal epithelium [4–6]. Besides the classical secretory
exocrine process, several studies have described epithelial cells
lining the male genital tract also being involved in apocrine
secretion mechanisms [7, 8]. Apocrine secretion results in the
formation of cell blebs protruding from the apex of the cell and
in the release of vesicles into the lumen of the ducts when
apical blebs break down. These vesicles, which are named
aposomes, have been described in the seminal plasma and in
the lumen of male excurrent ducts in several species, including
rabbit, monkey, sheep, dog, human, cat, bovine, rat, horse, and
lately, mouse (for an exhaustive list of references, see [7, 9–
11]). During the past 10 years, efforts have been made to
understand how aposomes interact with sperm cells and what
functions are associated with these structures. Aposomes were
reported to possess fusogenic membranous properties, allowing
the exchange of materials with spermatozoa. In vitro, protein
transfer has been demonstrated between aposomes and
spermatozoa [12–17]. In vivo, few proteins were suggested
to be transported by aposomes onto spermatozoa (for review,
see [9, 10]). Besides proteins, lipids also were shown to be
transported by aposomes [18].
The most-studied aposomes are human vesicles synthesized
by the prostate, the so-called prostasomes [19, 20]. These are
characterized by a multilamellar lipoprotein membrane with a
very high cholesterol:phospholipid (chol:pl) ratio (;2) and an
important sphingomyelin (SM) concentration, representing
approximately 50% of the total phospholipid content [21].
They also are notably rich in complement regulatory proteins,
enzymes, and neuroendocrine markers. Prostasomes were
shown to interact with spermatozoa in semen and have been
proposed to play several roles in sperm physiology, such as
promotion of forward motility and prevention of premature
acrosome reaction. It also was proposed that they have
antibacterial as well as antioxidant and immunosuppressive
actions [9, 10, 22–24]. Although prostasomes have been
studied quite extensively, only recently has interest arisen
regarding aposomes generated by other epithelia of the male
genital tract, such as the vas deferens [25] and the epididymis
[12, 26].
In the present study, we first confirmed that apocrine
secretion processes occur in the mouse epididymis, essentially
from caput epididymidal principal cells. To determine a
potential role for these secretions, we isolated and purified
epididymosomes. We then analyzed their ultrastructure and
1104
EPIDIDYMOSOMAL LIPID ANALYSIS
lipid composition during epididymal transit to see how they
evolve during their transit in the epididymal duct. In the
meantime, to correlate the evolution of epididymosomes with
sperm cell maturation, we isolated caput and cauda epididymidal mouse spermatozoa and analyzed their lipid content.
MATERIALS AND METHODS
Animals and Chemicals
Sexually mature Swiss OR1 (Janvier) mice were used in the present study.
They were housed under constant conditions of temperature (20 6 18C, mean
6 SEM) and light (12L:12D) with food (Usine d’Alimentation Rationnelle)
and water available ad libitum. All investigations on animals were conducted in
accordance with the French Guidelines on the Use of Living Animals in
Scientific Investigations. Procedures described in the present paper were
reviewed and approved by the University Blaise Pascal Animal Care and Use
Committee. Unless otherwise indicated, all chemicals were obtained from
Sigma Chemical Corp. Chloroform was purchased from Prolabo; methanol,
sulfonic-4 acid, and amino-1-hydroxy-naphthalene were from Merck. Hexane
was from BDH Laboratory Supplies (Poole, UK). Phosphatidylethanolamine
(PE), phosphatidylcholine (PC), phosphatidylinositol, and phosphatidylserine
standards were obtained from Interchim. For the analyses of fatty acids bound
to membranous phospholipids, 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine was used for internal standard. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was
provided by Molecular Probes.
Sperm and Epididymal Fluid Preparation
Male mice were killed by cervical dislocation, and epididymides were
excised, defatted, and dissected into caput, corpus, and caudal segments. Caput
and caudal parts were punctured with a needle in 13 PBS (2.7 mM KCl, 1.5
mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4; pH 7.2) plus 1 mM
EDTA and gently agitated to allow sperm dispersion. Epididymal tissues were
removed after centrifugation for 1 min at 100 3 g. Spermatozoa were
concentrated by two centrifugations of 5 min at 500 3 g and washed at least
three times with PBS-EDTA before any experimental treatments. Epididymal
fluids were collected and centrifuged twice (10 min, 10 000 3 g, 48C) to
eliminate cell debris and residual spermatozoa. Each sample was constituted
with five animal epididymides.
Preparation of Epididymal Fluid Vesicles
Epididymosomes were isolated by subjecting epididymal fluid to
ultracentrifugation (1 h, 200 000 3 g, 48C). The supernatant was discarded,
and the epididymosome-containing pellet was resuspended in PBS-EDTA.
Epididymosomes were separated from amorphous material by chromatography
on Superdex 200 prep-grade media packed in an XK 16 column (1.6 3 20 cm;
Amersham Pharmacia Biotech) pre-equilibrated with 30 mM Tris and 130 mM
NaCl buffer (pH 7.6). Epididymosomes were not retained by the column and
were collected in the void volume. Finally, they were concentrated by
ultracentrifugation (1 h, 200 000 3 g, 48C), and the pellet was suspended in
PBS-EDTA and stored at 208C until further processing.
Electron Microscopic Analyses
Four epididymides were fixed by vascular perfusion with 2% (v/v)
glutaraldehyde buffered in 0.2 M sodium cacodylate at pH 7.4. After fixation
for 15 min, epididymides were removed and incubated in 1.2% (v/v)
glutaraldehyde buffered in 0.07 M sodium cacodylate at pH 7.4 containing
0.05% (w/v) ruthenium red for 1 h at room temperature. Organs were divided
into five segments: initial, proximal, and distal caput segments, and corpus and
cauda, according to the definition of Abe et al. [27]. The five segments and
epididymosomes, purified or not, were fixed in the same solution for 2 h at
room temperature. After washing in 0.07 M sodium cacodylate buffer (pH 7.4)
plus 0.05% (w/v) ruthenium red, samples were postfixed with 1% (v/v) osmium
tetroxide in the same buffer devoid of ruthenium red for 1 h. Tissue and
epididymosomes were then dehydrated with ethanol (50%, 70%, 96%, and
100% [all v/v] three times for each concentration for 15 min) and propylene
oxide (three times for 20 min). Epididymal segments and vesicles were
embedded in propylene oxide and Epon epikote resin (1:1, v/v) overnight and
in Epon two times for 3 h. Polymerization was conducted at 608C for 72 h. Thin
sections (thickness, 80 nm) were cut with a diamond knife (Leica Ultracut S);
placed on copper grids; contrasted with a solution of 2% (v/v) uranyl acetate,
50% (v/v) ethanol, and lead citrate; and examined with a Jeol 1200 EX
1105
Transmission Electron Microscope. The preservation of junctional complexes
and organelles in epithelial cells ruled out the possibility of fixation artifacts.
Lipid Analyses
Epididymosomes and sperm lipids were extracted by the Folch method with
modifications [28]. Cholesterol was assayed using Lieberman-Burchard reagent
[29], and total phospholipid concentrations were determined by measuring the
amount of inorganic phosphorus [30]. Six epididymosome samples and five
sperm cell samples were analyzed.
Phospholipid classes were separated by high-performance, thin-layer
chromatography (HPTLC; 10 3 10 cm; Merck) with methyl acetate, propan2-ol, chloroform, methanol, and 0.25% (w/v) aqueous KCl (25:25:25:10:9, v/v)
[28, 31]. Lipid-containing regions of the chromatogram were visualized by
treatment with a 10% (w/v) CuSO4 and 8% (v/v) H3PO4 solution and heated at
1808C. The chromatograms were scanned, and spots were quantified via
densitometry (Quantity One; Bio-Rad) by reference to different concentrations
of phospholipid standards on each HPTLC migration. Seven samples of
epididymosomes and sperm cells were analyzed; each analysis was performed
in duplicate. Standard dosage values gave curves with linear-regression
coefficients (R2) of 0.90 or greater. For phospholipid fatty acid analysis, total
lipids from four samples were extracted twice from either sperm or epididymal
vesicles with ethanol:chloroform (1:2, v/v) containing 5 3 105 M
butylhydroxylated toluene as an antioxidant. The different lipid classes were
separated on silica gel G60 plates (Merck) with the solvent system hexanediethyl ether-acetic acid (80:20:1, v/v). The silica gel areas corresponding to
phospholipids were scraped off the plate and treated for 90 min with 5% H2SO4
in methanol to obtain fatty acid methyl esters [32]. Fatty acid methyl esters
were extracted and analyzed using a Hewlett-Packard HP 6890 gas
chromatograph equipped with an SP 2380 capillary column (30 m 3 0.32
mm; Supelco).
Epididymosome Fluidity Analysis
Membrane fluidity of seven samples of epididymosomes was measured by
fluorescence polarization with a lipophilic fluorescent probe, DPH (Molecular
Probes). Fluorescence measurements were performed on an AMICO SPG500
spectrofluorimeter (American Instrument Company) equipped for fluorescence
polarization. The incubation was conducted for 30 min at room temperature in
the presence of 3 ml of fresh dispersion of 2 lM DPH in 13 PBS (pH 7.4).
Throughout the incubation time, the probe was located mainly in the membrane
[33]. The fluorescence anisotropy (r) was calculated related to the temperature,
from fluorescence intensities, I// (excitation, 360 nm) and I? (emission, 460 nm),
according to the following equation: r ¼ (I// I?)/(I// þ 2I?). According to
Perrin’s law, the anisotropy value is inversely proportional to fluidity.
Statistical Analysis
Data were tested using the Mann-Whitney U-test. When n . 5, the
Wilcoxon t-test applied to paired series was used, in parallel, to compare
evolution of sperm cells and epididymosomes between the caput and cauda
epididymidis. A P value of less than 0.05 was considered to be statistically
significant.
RESULTS
Electron Microscopic Observations of Mouse Epididymal
Fluid Vesicles
We used electron microscopy to study vesicles produced in
the different epididymal segments (Figs. 1–4). Vesicular
structures were observed in the lumen of caput and cauda
epididymal segments, with noticeable differences throughout
the organ. We observed large protrusions leaving the apex of
principal cells of the epididymis, notably from the three first
epididymal segments (segments I–III, according to Abe et al.
[27], in Figs. 1–3). Vesicles were still bound to the cell apex
and reached 2 to 5 lm in height in the first three segments of
the epididymal caput. Their content appeared to be homogenous (Fig. 1), without organelles, whereas they sometimes
included multivesicular bodies with diameters from 20 to 500
nm at the junction between the cell apical cytoplasm and the
protrusions (Figs. 1B and 2B). Protrusions seemed to detach
1106
REJRAJI ET AL.
FIG. 1. Electron photomicrographs showing apocrine secretion activity of caput
epididymidis segment I. Large protrusions
emitted from the apex of principal cells
reach 2 to 5 lm in height (Ap for aposomes).
Vesicles (Ves) are roughly spherical, with a
diameter of between 1 and 10 lm, and also
are observed in the epididymal lumen
surrounded by cell microvilli (Mv). For the
majority, their content appears to be homogenous, without organelles, and their
membrane is bilayered. Smaller particles
are observed either inside (small arrows in
D and E) of these vesicular structures or
aggregated to their inner membrane (small
arrow in C). A cytoskeletal organization also
is observed at the apex of some epithelial
cells during apocrine secretion (long arrows
in B). Note the coated pits on the apical
plasma membrane (arrowheads in A), Ap,
Aposomes; E, endosomes; J. junction; L,
lysosome; m, mitochondria; Mb, lateral cell
membrane.
themselves from the originating cell by fission or by
constriction of the junctional area (Figs. 1B and 2).
Figures 1 and 2 illustrate an apocrine secretion phenomenon
at an early stage, with protrusions still bound to the cell, in
which one can observe a clear cytoskeletal reorganization at the
apex of epithelial cells. The cytoskeleton covers about twothirds of the cell’s apex on both sides, letting small vesicles of
FIG. 2. Reorganization of apical cytoskeleton at the apex of caput segment I
principal cells during apocrine secretion. B
and D correspond to higher magnifications
of A and C, respectively. Long arrows point
the region concerned by the reorganization
of the cortical cytoskeleton. Arrowheads (in
A) and small arrows (in B) point out small
vesicles (diameter, 20–50 nm) localized at
the basis of aposomes. Ap, Aposomes; m,
mitochondria; Mb, membrane; Mv, microvilli; Ves, vesicles.
the cell cytoplasm escape toward the protrusion (Fig. 1B). Cell
cytoskeletal reorganization also was seen during the separation
between a protrusion and a principal cell (Fig. 2). At this stage,
the cytoskeleton completely takes up the cell apex. The
protrusion detached from the cell by rupture of both sides of
the junctional area above the cytoskeleton. We observed small
vesicles (diameter, 20–50 nm) at the base of this protrusion.
EPIDIDYMOSOMAL LIPID ANALYSIS
1107
FIG. 3. Electron photomicrographs showing apocrine secretion activity of caput
epididymidis segment II (A–C) and segment
III (D–F). In caput segment II, vesicles have a
multilayer membrane (arrowheads) and
seem to overlap, forming multivesicular
bodies (MVB; thin arrows in B) with a size
ranging from 20 nm to 2.5 lm. They appear
to be surrounded by cell microvilli (Mv) and
spermatozoa. In caput segments II and III,
aposomes (Ap) in formation are visible at
the apex of principal cells. Smaller vesicles
(diameter, 20–500 nm) can be seen entering
the aposomes (bold arrows in E and F). Long
arrows in F show cell apex cytoskeletal
reorganization occurring in caput segment
III, as is the case in segment I. CD,
Cytoplasmic droplet; F, flagellum; J, junction; m, mitochondria.
Figure 2D illustrates a caput principal cell with a cytoskeletal
structure localized at its apical pole, most likely after the
detachment of a vesicle.
Free vesicles were observed in the luminal compartment of
the first three epididymal segments, surrounded with the cell’s
microvilli. They were roughly spherical, with a diameter
between 1 and 5 lm (Figs. 1–3), resembling the aposomes
previously observed. In some cases, smaller particles (diameter,
20–200 nm) were observed either inside these vesicular
structures or aggregated to their inner membrane. However,
we observed discrepancies in the structure of blebbing cells and
free vesicles between segments II/III and segment I (compare
Figs. 1 and 2 with Fig. 3). Vesicles of segments II/III
overlapped one another in the epididymal lumen (forming
multivesicular bodies with a size ranging from 20 nm to 2.5
lm) presented a multilamellar membrane (Fig. 3, B and C), and
were surrounded by microvilli and spermatozoa. Apocrine
secretion processes, foaming cells, and large vesicles in the
corpus and cauda epididymides were much less abundant (Fig.
4). The cauda epididymidal lumen appeared to have a different
texture, being loaded with electron-dense bodies of 1 lm in
diameter, small vesicles (diameter, 15–20 nm), and membranous fragments.
Figure 5A represents the pellet resulting from ultracentrifugation as observed by electron microscopy. The pellet
consisted in a heterogeneous population of vesicles surrounded
by an amorphous substance. Vesicles vary in size and content.
Their diameter ranged from 50 to 800 nm. They were roughly
spherical and had a bilayer membrane. Most of the smaller
vesicles generally contained an electron-dense, granular
1108
REJRAJI ET AL.
FIG. 4. Electron micrographs of mouse
corpus and cauda epididymides. Very few
or no apical blebs were detected in the
epithelia of corpus and cauda epididymides. As shown in A and B for the corpus and
cauda territories, respectively, the apical
cell border has a uniform aspect full of
microvilli (Mv), whereas the lumen of the
epididymal duct is loaded with very small
vesicles (diameter, 15–20 nm), membranous
fragments, and sperm cells, with some of
them bearing cytoplasmic droplets (CD). m,
Mitochondria of the sperm midpiece.
material that was uniformly distributed all over the structure.
Most of the bigger ones appeared to be empty, but some had
aggregates of granular material in proximity with their inner
membrane. When epididymosomes were purified through a gel
filtration column (Fig. 5B), the surrounding substance was
efficiently eliminated; however, membranes of the vesicles also
were damaged. Epididymosomes isolated from caput and
caudal fluid samples seemed to be rather similar in size and
structure; however, cauda epididymidal fluid contained much
fewer vesicles (data not shown).
Lipid Composition of Caput and Caudal Epididymosomes
and Epididymal Spermatozoa
Phospholipids and cholesterol. As shown in Table 1,
epididymosomal phospholipid composition goes through
various modifications along the epididymal tubule. The major
discrepancy was the overwhelming proportion of SM in caudal
epididymosomes. The SM ratio was twofold higher in caudal
compared to caput vesicles. The SM became the predominant
phospholipid in cauda epididymidal vesicle membranes instead
of PC in caput epididymosomes. Proportions of other
phospholipids decreased in caudal epididymosomes. This was
particularly true for PC and phosphatidylinositol, which were
reduced by 1.6- and 2.0-fold, respectively. Phosphatidylserine
FIG. 5. Electron photomicrographs of
vesicles isolated from mouse epididymal
fluid before (A) and after (B) purification.
Epididymosomes are represented by an
heterogeneous population of vesicles surrounded by amorphous substance (A), and
the latter is eliminated after purification
through a gel filtration column (B).
was as abundant (;3%) in caput and as in caudal
epididymosomes. Cholesterol and total lipid phosphorus
quantities in caudal epididymosomes were, respectively, 2.3and 4.5-fold lower than in caput epididymosomes. The chol:pl
ratio increased twofold during epididymal transit, to reach the
value of 0.5 in caudal epididymosomes. Regarding spermatozoa, not much evolution occurred in the distribution of the
different classes of phospholipids between sperm of the two
epididymal segments studied (Table 2). Both PC and PE
represent the major phospholipids in spermatozoa of both
segments, and their proportions were approximately 60%–70%
of total phospholipids. We observed a slight reduction of PC
and PE proportions in cauda epididymal sperm cells, from
46.7% and 21.8%, respectively, in caput to 41.4% and 18.8%,
respectively, in cauda. The phosphatidylserine ratio also
underwent a slight decrease in membranes of cauda epididymidal sperm cells. The phosphatidylinositol ratio was unchanged, whereas the SM proportion increased during sperm
maturation, from 20.9% in caput to 29.2% in the cauda
epididymidis. No significant difference was noted for the
chol:pl ratio (;0.25) between caput and cauda epididymidal
gametes.
Phospholipid fatty acid analysis. Table 3 presents the
major phospholipid fatty acids of caput and cauda epididymidal
1109
EPIDIDYMOSOMAL LIPID ANALYSIS
TABLE 1. Phospholipids and cholesterol pattern of caput and cauda
epididymosomes.
TABLE 2. Phospholipids and cholesterol pattern of caput and cauda
epididymal spermatozoa (spz).
Phospholipids, lipid phosphorus,
& cholesterol composition
Phospholipids, lipid phosphorus,
& cholesterol composition
Caput sperm
cells
Phospholipid classes (%)
PC
PE
SM
PI
PS
Total lipid phosphorus (lmol 3 106)
Cholesterol (lmol 3 106)
Total lipid phosphorus (1015 mol/spz)
Cholesterol (1015 mol/spz)
Cholesterol/phospholipid ratio
46.7
21.8
20.9
8.9
1.6
1.0
0.24
28.0
6.9
0.24
Phospholipid classes (%)
PC
PE
SM
PI
PS
Total lipid phosphorus
(lmol 3 106)
Cholesterol (lmol 3106)
Cholesterol/phospholipid ratio
Caput
epididymosomes
46.3
17.5
23.4
9.7
3.1
6
6
6
6
6
2.4a
1.4a
1.9a
1.5a
1.8a
Cauda
epididymosomes
28.8
13.1
50.0
4.7
3.3
6
6
6
6
6
3.8*a
2.3*a
5.2*a
1.4*a
0.6a
0.54 6 0.12b
0.12 6 0.04*b
b
0.06 6 0.01*b
0.50 6 0.10*b
0.14 6 0.02
0.26 6 0.05b
* P , 0.05 compared with epididymosomes from caput epididymis.
a
Values are means 6 SD; n ¼ 7.
b
Values are means 6 SD; n ¼ 5.
sperm cells and epididymosomes. Saturated fatty acids in caput
and caudal epididymosomes were essentially represented by
palmitic (16:0) and stearic (18:0) acids and amounted to 40% of
total membranous fatty acids (MFAs). The stearic acid
proportion was unchanged in epididymosomal membranes of
both segments and represented approximately 25% of total
MFA. Major differences lay in the ratio of arachidonic acid
(20:4 [n-6]) and palmitic acid. Arachidonic acid decreased
fourfold, from 23.4% in caput to 6.1% in caudal epididymosomes, whereas palmitic acid doubled in the cauda epididymidis, from 12.5% (in the caput) to 24.2%. We also observed that
docosahexaenoic acid (22:6 [n-3]) became the major polyunsaturated fatty acid (PUFA) in cauda epididymosomal membrane. Linolenic (18:2 [n-6]) and docosapentaenoic acids were
represented in low proportion in epididymosomal membranes
of both segments studied. The monounsaturated fatty acid ratio
was unchanged from caput to cauda epididymidis and
corresponded to 10% of total MFA. The ratio of saturated to
unsaturated fatty acids was almost the same in caput and caudal
epididymosomes. However, we noticed a strong reduction of
the PUFA ratio in cauda epididymosomal membrane.
Regarding spermatozoa, they went through a significant
remodeling of their MFA composition during epididymal
transit. Major fatty acids of the sperm membrane were
classically 16:0 and 18:0, followed by a PUFA, either 20:4
(n-6) for caput sperm cells or 22:5 (n-6) for cauda epididymidal
gametes. Stearic acid was the predominant fatty acid in caput
epididymidal sperm. During epididymal maturation. its proportion decreased from 26.8% to 19.5%, whereas 16:0
increased and became the major MFA of cauda epididymidal
spermatozoa. The MFAs of murine epididymal spermatozoa
were represented principally by unsaturated fatty acids, and
their ratio increased during epididymal maturation. The PUFA
ratio increased significantly, notably involving 22:5 (n-6) and
22:6 (n-3) (from 4.9% and 9.4%, respectively, in caput to
14.7% and 11%, respectively, in cauda epididymidal spermatozoa). Contrary to the other PUFAs, 20:4 (n-6) was found in
lower proportion in cauda epididymidal sperm (from 18.8% to
9.8%). The monounsaturated fatty acids constituted approximately 13% of MFA in the two regions studied. We observed a
significant reduction in the ratio of saturated to unsaturated
fatty acids in sperm membrane during epididymal transit.
Epididymosomal Membrane Anisotropy
To gain more insight regarding epididymosomal membrane
properties, we directly analyzed the fluidity of epididymosomal
6
6
6
6
6
6
6
6
6
6
3.3a
3.7a
4.3a
3.8a
0.7a
0.12b
0.06b
3.1b
1.4c
0.04c
Cauda sperm
cells
41.4
18.8
29.2
9.7
1.0
0.9
0.26
8.3
2.4
0.29
6
6
6
6
6
6
6
6
6
6
3.5*a
2.3*a
3.0*a
5.3*a
0.3 a
0.18*b
0.04*b
1.7*c
0.4*c
0.07*c
* P , 0.05 compared with spermatozoa from caput epididymidis.
a
Values are means 6 SD; n ¼ 7.
b
Values are means 6 SD; n ¼ 5.
c
Values are means 6 SD; n ¼ 6.
membranes by fluorescence polarization. Figure 6 illustrates
anisotropy values (r) relative to temperature for seven samples
of caput and caudal epididymosomes. Anisotropy values were
homogenous for caput and caudal epididymosomes, and
coefficients of regression curves were higher than 0.90 for
epididymosomes of both segments studied. The two regression
curves were linear, without phase transition. The table within
Figure 6 summarizes anisotropy values obtained for three
temperatures: 24, 33, and 378C. Anisotropy values were almost
equivalent for caput and caudal epididymosomes at low
temperature (248C), but regression curves diverged significantly in the 25–408C temperature range. For physiological
temperatures, such as 33 or 378C, anisotropy was statistically
significantly lowest in caput epididymosomes.
DISCUSSION
Apocrine Secretion in Mouse Epididymis
Although they are structurally complete, sperm cells leaving
the testis are unable to fertilize oocytes and must go through the
epididymal duct to acquire the modifications necessary to
reach, recognize, and fuse with the female gamete. These
modifications consist essentially of remodeling the sperm’s
membranous proteins and lipids [2, 34], mediated by the
interactions of spermatozoa with the epididymal luminal
environment, which is loaded with the secretions from
epithelial cells [3–5]. Until recently, merocrine secretion was
considered to be the major form of secretion in the epididymal
duct. In this type of secretion, proteins produced by epithelial
cells are released in epididymal fluid through the fusion of
large protein-containing secretory granules with the cell plasma
membrane [7, 8, 35]. A few authors have described an
alternative way of secretion, referred to as apocrine secretion,
along the male reproductive tract of different species [7, 25,
35–37]. This particular process is defined by protrusions or
bleb emission from the apical cytoplasm of cells, which form
vesicular structures, called aposomes, circulating in the lumen
of the genital tract [38].
We have analyzed apocrine secretion activity of mouse
epididymal epithelium in the five segments described by Abe et
al. [27] and observed the formation of vesicular structures,
confirming the observations reported by Hermo and Jacks [39].
We have observed that principal cells emitted apocrine
protrusions along the entire epididymal tract, but most notably
in caput epididymidis. In agreement with the observations of
Hermo and Jacks, the proximal epididymis (i.e., segments I–III
1110
REJRAJI ET AL.
TABLE 3. Major membranous fatty acids of caput and cauda epididymidal spermatozoa and epididymosomes.
Fatty acids
16:0
18:0
18:1 (n-9 & n-7)
18:2 (n-6)
20:4 (n-6)
22:5 (n-6)
22:6 (n-3)
Saturated
Monounsaturated
Polyunsaturated
Saturated/unsaturated
Caput vesiclesa
12.5
27.6
11.2
3.8
23.4
1.0
8.4
48.4
12.6
38.9
0.94
6
6
6
6
6
6
6
6
6
6
6
0.6
0.9
0.2
0.03
1.0
0.1
0.2
0.9
0.3
1.1
0.03
Cauda vesiclesa
24.5
24.2
9.8
1.3
6.3
1.6
8.0
61.3
11.0
27.6
1.84
6
6
6
6
6
6
6
6
6
6
6
4.52*
6.52
7.65
0.43*
4.47*
0.57
3.72
13.48
8.16
8.05*
0.09
Caput sperm cellsa
15.5
26.8
11.6
3.6
18.8
4.9
9.4
49.0
12.7
38.3
0.96
6
6
6
6
6
6
6
6
6
6
6
1.1
0.5
0.8
0.1
0.8
0.3
0.4
1.5
0.8
0.9
0.06
Cauda sperm cellsa
10.6
19.1
11.6
4.0
9.8
14.7
11.0
46.3
12.6
41.2
0.86
6
6
6
6
6
6
6
6
6
6
6
0.6*
0.2*
0.8
0.3*
0.6*
0.8*
0.2*
0.6*
0.9
0.9*
0.02*
a
Values are means 6 SD; n ¼ 4.
* P , 0.05 compared to the samples from caput epididymidis.
of the caput) was very active in terms of apocrine secretions.
However, contrary to what was reported initially by those
authors, we found that the corpus and cauda epididymides were
very poorly active. Free vesicles also were observed in the
epididymal lumen, certainly resulting from blebs emitted by
principal cells. The content of the protrusions looked very
different from the epithelial cell cytosolic content, even though
a cytoplasmic continuity existed between the two entities
during the mechanism of apocrine secretion. Blebs had no
cytoplasmic organelles, whereas the cell apex contained
numerous mitochondria, lysosomes, and granules. Even more,
and contrary to epithelial cells, vesicle membranes possessed
no coated pits and microvilli. Their structure and content were
different from those of sperm cytoplasmic droplets. Some
protrusions from epithelial cells were limited by a cytoskeletal
structure that resembled a junctional complex. This cytoskeletal structure was not present in the free vesicles from the
epididymal lumen. This structure seemed to close the junction
between epithelial cells and the protrusion, thus allowing
vesicle release while preserving cell integrity. To our
knowledge, this structure was not described in other studies
of epididymal apocrine secretion. However, cytoskeletal
proteins like myosin and b-actin already have been located
during apocrine secretion, in the junctional area between blebs
and epithelial cells of the coagulating gland of male rats [7, 8].
Protrusions did not have the same structure and size throughout
the epididymal tract. In caput segment II, vesicles formed
multivesicular bodies, as if the different vesicles had fused
FIG. 6. Anisotropy of caput and cauda epididymosomal membranes.
Regression curves of anisotropy values (r) relative to temperature (8C) of
seven samples of caput and caudal epididymosomes. The table within the
graph presents the anisotropy values at some arbitrarily chosen
temperatures.
together. Microscopic study of corpus and cauda epididymidal
lumen showed very few free large vesicles. The fluid had a
different texture, loaded with electron-dense bodies and
membranous fragments. Similar observations recently were
reported in rat and mouse epididymides [40, 41]. Our
assumption is that cauda vesicles result principally from the
caput apocrine secretory activity; these vesicles have degenerated and/or fragmented, with eventual release of their
contents into the lumen. We envisage, too, that big vesicles
(diameter, 2–10 lm) emitted by caput epithelial cells could
exchange between themselves or fuse together, and that they
could do the same with sperm cells during epididymal transit,
forming smaller multivesicular entities that, finally, would
degenerate and liberate small vesicles in cauda epididymal
lumen.
Are Epididymosomes Similar to Prostasomes and
Prostasome-Like Vesicles?
Epididymosomes had a roughly spherical aspect and
presented a bilayer membrane. They were heterogeneous in
size (diameter, 50–800 nm) and in content. The smallest
vesicles generally contained an electron-dense, granular
materiel that was distributed uniformly, whereas the larger
ones looked rather empty. A few had granular material
aggregated in the vicinity of their inner membrane. Thus, their
ultrastructure was almost the same as that observed for human
prostasomes or for prostasome-like vesicles in rat epididymis,
stallion semen, or bovine seminal vesicles [22, 42–45]. The
major differences between mouse epididymosomes and human
prostasomes reside in the fact that the membrane of the former
generally is bilayered and the membrane of the latter generally
is trilayered (or more) [22].
One of the main characteristics of prostasomes and
prostasome-like vesicles is their high chol:pl ratio (average,
2) [21, 44]. This is not the case for mouse epididymosomes
extracted from both caput and caudal segments, for which the
chol:pl ratios were 0.26 and 0.48, respectively. Looking at the
different phospholipids, the SM proportion was approximately
0.5 for epididymosomes recovered from mouse cauda
epididymidis, as in human prostasomes and stallion prostasome-like vesicles [21, 44, 46, 47]. For other phospholipids,
the distribution is rather different, because mouse caudal
epididymosomes contained more PC and less phosphatidylserine than human prostasomes or stallion prostasome-likes
structures. Regarding fatty acids, the three vesicular structures
(human prostasomes, stallion prostasome-like vesicles, and
mouse epididymosomes) are comprised of more than 50%
saturated fatty acids [46, 48]. Major discrepancies between the
EPIDIDYMOSOMAL LIPID ANALYSIS
three species were seen in unsaturated fatty acid contents. In
mouse epididymosomes, monounsaturated fatty acids represent
approximately 10% of total phospholipid fatty acids, whereas
the proportion is more than 20% in human prostasomes and
approximately 5% in equine prostasome-like structures. In
these three species, 18:1 is the major monounsaturated fatty
acid. Surprisingly, 20:4 n-6 and 22:6 n-3, which are the most
abundant PUFAs in mouse epididymosomes, are either absent
or weakly represented in human and horse vesicles.
Although the three vesicular structures display close
membranous characteristics, containing a high percentage of
SM and significant amounts of cholesterol and saturated fatty
acids, we have shown that mouse epididymosomes have a lipid
pattern that is rather different from what is reported for human
prostasomes and stallion prostasome-like vesicles. It must be
noted that human prostasomes and horse prostasome-like
structures were isolated from seminal plasma, whereas we
worked with epididymal vesicles. Indeed, the epididymis is the
first secretory organ through which sperm pass after their
production in the testis. Throughout the genital tract, fluid
containing spermatozoa and vesicles undergoes important
modifications, resulting from the epithelia absorptive and
secretory activities [35]. We have observed that vesicles in
cauda epididymis already were different from those isolated
from caput epididymidis. Thus, epididymosomes likely will
undergo other modifications later on, and their lipid and protein
pattern will continuously evolve along the male genital tract.
Thus, because of their structural differences and/or their
various activities, prostasomes and prostasome-like vesicles
constitute a heterogeneous population in seminal plasma.
Therefore, part of the so-called prostasomes detected in
seminal plasma likely does not originate from the prostate
but, rather, is a mixture of vesicles of different origins. The
localization of proteins produced in the epididymis on or in
vesicular structures in seminal plasma favors such exchanges
[49, 50].
Evolution of Mouse Spermatozoa During
Epididymal Transit
More than 60% of mouse epididymidal spermatozoal
membrane phospholipids were PE and PC. In addition, as
reported previously for rabbit sperm cells [51], SM was an
important lipid of mouse epididymal spermatozoa. During
epididymal transit, no change occurred in the PE:PC ratio,
whereas the proportion of SM increased from 20.9% in
spermatozoa from the mouse caput to 29.2% in spermatozoa
from the mouse cauda epididymidis. The chol:pl ratio did not
change in mouse spermatozoa recovered from the caput or the
cauda epididymidis, and its value (0.25) was similar to that
recorded in another rodent [52]. Regarding the fatty acid
content, major fatty acids of mouse sperm membrane were
classically 16:0 and 18:0, followed by a PUFA, either 20:4 n-6
in caput sperm or 22:5 n-6 in cauda sperm. The PUFA
proportion was very important in mouse spermatozoa, and it
increased significantly during epididymal maturation, notably
for 22:5 n-6 and 22:6 n-3. Contrary to the other PUFA, 20:4 n6 was found in lower proportion in mouse cauda epididymidal
sperm. These data also are in agreement with previous reports
that pinpointed these three PUFAs as the major PUFAs in
membrane sperm cells [52–55]. Both the increase in 22:5 n-6
and 22:6 n-3 and the decrease in 20:4 n-6 favor a reduction in
the ratio of saturated to unsaturated fatty acids in sperm
membrane during epididymal transit. Consequently; this could
contribute to increasing the mouse sperm membranous fluidity,
as suggested previously for rat, boar, and human spermatozoa
1111
[56–59]. These data also are in line with those of a recent study
in which the fluorescence recovery after photobleaching
(FRAP) technique was used to demonstrate that lipid diffusion
in the plasma membrane of mouse spermatozoa increased
significantly during passage from caput to cauda epididymidis
[60]. This was logically interpreted as an increase in the
membrane fluidity of spermatozoa during epididymal maturation.
Are Lipids Exchanged Between Epididymosomes and
Mouse Spermatozoa During Epididymal Transit?
The evolution in the lipid content of epididymosomes
during their epididymal transit (i.e., the threefold increase in
SM:PC ratio, twofold increase in chol:pl ratio, and reduction of
PUFA proportions) favors a loss of fluidity of the vesicles
membranes. This is supported by the direct measurement of the
membrane fluidity of mouse epididymosomes by fluorescence
anisotropy (Fig. 6). In parallel, throughout the epididymis,
sperm membranes underwent an increase in sperm membranous fluidity. Therefore, it is interesting to note that the fluidity
of epididymosomes and sperm membranes evolved in opposite
ways during epididymal sperm maturation. Although we have
no direct evidence that any specific lipid is exchanged between
spermatozoa and epididymosomes, this opposite behavior
favors such exchange between the two structures. Interestingly,
it has been proposed elsewhere that aposomes could drive
away cholesterol from sperm cells [61, 62]. Moreover, several
recent studies have demonstrated the presence of particularly
rigid and detergent-resistant microdomains in sperm membrane, notably in mouse spermatozoa [63–65]. These structures, which are called rafts, contain a high proportion of
cholesterol, sphingolipids, and glycosyl phosphatidylinositolbound proteins [66, 67], which remind us of epididymosomal
and prostasomal characteristics. It does not seem too farfetched to imagine that epididymosomes (and aposomes in
general) could exchange lipids and protein materials with
sperm cells, contributing to the formation of structures such as
rafts in sperm cells membrane.
In conclusion, our analysis supports the idea that all along
the epididymal duct, mouse spermatozoa encounter and, most
likely, interact and/or exchange with vesicular structures
originating from the caput epithelium by way of apocrine
secretion. Epididymosomes share some of the characteristics of
other aposomes (prostasomes and prostasome-like vesicles)
found in the seminal fluid. However, they differ in their lipid
content, suggesting that each secretory epithelium lining the
male excurrent duct might produce its own particular type of
vesicles. Analysis of the protein contents of these different
vesicles by way of proteomic approaches should provide
information regarding their role in the process of sperm
maturation. Our survey and comparison of sperm and vesicle
lipid contents in the mouse caput versus the cauda epididymidal compartments show that whereas the vesicle membrane
seems to be more rigid in the cauda, the fluidity of the sperm
membrane conversely increases. Starting with epididymosomes
and continuing with prostasome-like vesicles in the vas
deferens and prostasomes in the semen, spermatozoa seem to
be in permanently close proximity to these structures.
Therefore, these vesicles should be considered as important
actors in the acquisition and maintenance of sperm functions.
ACKNOWLEDGMENTS
The authors would like to thank F. Saez, P. Leclerc, E. Pons, and E.
Grignard for their suggestions during the course of the present study and
for critically reading the manuscript.
1112
REJRAJI ET AL.
REFERENCES
1. Cooper TG. Role of the epididymis in mediating changes in the male
gamete during maturation. Adv Exp Med Biol 1995; 377:87–101.
2. Jones R. Plasma membrane structure and remodelling during sperm
maturation in the epididymis. J Reprod Fertil Suppl 1998; 53:73–84.
3. Toshimori K. Biology of spermatozoa maturation: an overview with an
introduction to this issue. Microsc Res Tech 2003; 61:1–6.
4. Cooper TG. Interactions between epididymal secretions and spermatozoa.
J Reprod Fertil Suppl 1998; 53:119–136.
5. Dacheux JL, Gatti JL, Dacheux F. Contribution of epididymal secretory
proteins for spermatozoa maturation. Microsc Res Tech 2003; 61:7–17.
6. Dacheux JL, Castella S, Gatti JL, Dacheux F. Epididymal cell secretory
activities and the role of proteins in boar sperm maturation. Theriogenology 2005; 63:319–341.
7. Aumuller G, Renneberg H, Schiemann PJ, Wilhelm B, Seitz J, Konrad L,
Wennemuth G. The role of apocrine released proteins in the post-testicular
regulation of human sperm function. Adv Exp Med Biol 1997; 424:193–
219.
8. Aumuller G, Wilhelm B, Seitz J. Apocrine secretion—fact or artifact? Ann
Anat 1999; 181:437–446.
9. Saez F, Frenette G, Sullivan R. Epididymosomes and prostasomes: their
roles in posttesticular maturation of the sperm cells. J Androl 2003; 24:
149–154.
10. Rejraji H, Vernet P, Drevet JR. GPX5 is present in the mouse caput and
cauda epididymis lumen at three different locations. Mol Reprod Dev
2002; 63:96–103.
11. Sullivan R, Saez F, Girouard J, Frenette G. Role of exosomes in sperm
maturation during the transit along the male reproductive tract. Blood
Cells Mol Dis 2005; 35:1–10.
12. Frenette G, Lessard C, Sullivan R. Selected proteins of ‘‘prostasome-like
particles’’ from epididymal cauda fluid are transferred to epididymal caput
spermatozoa in bull. Biol Reprod 2002; 67:308–313.
13. Frenette G, Sullivan R. Prostasome-like particles are involved in the
transfer of P25b from the bovine epididymal fluid to the sperm surface.
Mol Reprod Dev 2001; 59:115–121.
14. Yeung CH, Schroter S, Wagenfeld A, Kirchhoff C, Kliesch S, Poser D,
Weinbauer GF, Nieschlag E, Cooper TG. Interaction of the human
epididymal protein CD52 (HE5) with epididymal spermatozoa from men
and cynomolgus monkeys. Mol Reprod Dev 1997; 48:267–275.
15. Arienti G, Carlini E, Verdacchi R, Cosmi EV, Palmerini CA. Prostasome
to sperm transfer of CD13/aminopeptidase N (EC 3.4.11.2). Biochim
Biophys Acta 1997; 1336:533–538.
16. Arienti G, Polci A, Carlini E, Palmerini CA. Transfer of CD26/dipeptidyl
peptidase IV (E.C. 3.5.4.4) from prostasomes to sperm. FEBS Lett 1997;
410:343–346.
17. Minelli A, Allegrucci C, Mezzasoma I, Ronquist G, Lluis C, Franco R.
CD26 and adenosine deaminase interaction: its role in the fusion between
horse membrane vesicles and spermatozoa. Biol Reprod 1999; 61:802–
808.
18. Cross NL. Role of cholesterol in sperm capacitation. Biol Reprod 1998;
59:7–11.
19. Ronquist G, Brody I, Gottfries A, Stegmayr B. An Mg2 þ- and Ca2 þstimulated adenosine triphosphatase in human prostatic fluid: part II.
Andrologia 1978; 10:427–433.
20. Ronquist G, Brody I, Gottfries A, Stegmayr B. An Mg2 þ- and Ca2 þstimulated adenosine triphosphatase in human prostatic fluid: part I.
Andrologia 1978; 10:261–272.
21. Arvidson G, Ronquist G, Wikander G, Ojteg AC. Human prostasome
membranes exhibit very high cholesterol/phospholipid ratios yielding high
molecular ordering. Biochim Biophys Acta 1989; 984:167–173.
22. Ronquist G, Brody I. The prostasome: its secretion and function in man.
Biochim Biophys Acta 1985; 822:203–218.
23. Fabiani R. Functional and biochemical characteristics of human
prostasomes. Minireview based on a doctoral thesis. Ups J Med Sci
1994; 99:73–111.
24. Arienti G, Carlini E, Saccardi C, Palmerini CA. Role of human
prostasomes in the activation of spermatozoa. J Cell Mol Med 2004; 8:
77–84.
25. Manin M, Lecher P, Martinez A, Tournadre S, Jean C. Exportation of
mouse vas deferens protein, a protein without a signal peptide, from mouse
vas deferens epithelium: a model of apocrine secretion. Biol Reprod 1995;
52:50–62.
26. Fornes MW, De Rosas JC. Interactions between rat epididymal epithelium
and spermatozoa. Anat Rec 1991; 231:193–200.
27. Abe K, Takano H, Ito T. Ultrastructure of the mouse epididymal duct with
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
special reference to the regional differences of the principal cells. Arch
Histol Jpn 1983; 46:51–68.
Force A, Grizard G, Giraud MN, Motta C, Sion B, Boucher D. Membrane
fluidity and lipid content of human spermatozoa selected by swim-up
method. Int J Androl 2001; 24:327–334.
Burke RW, Diamondstone BI, Velapoldi RA, Menis O. Mechanisms of
the Liebermann-Burchard and Zak color reactions for cholesterol. Clin
Chem 1974; 20:794–781.
Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem
1959; 234:466–468.
Vitiello F, Zanetta JP. Thin-layer chromatography of phospholipids. J
Chromatogr 1978; 166:637–640.
Bowyers DE, Leat WM, Howard AN, Gresham GA. The determination of
the fatty acid composition of serum lipids separated by thin-layer
chromatography, and comparison with column chromatography. Biochim
Biophys Acta 1963; 70:423–431.
Shinitzky M, Yuli I. Fluidity parameters of lipid regions determined by
fluorescence polarization. Biochim Biophys Acta 1978; 515:367–394.
Kirchhoff C, Pera I, Derr P, Yeung CH, Cooper T. The molecular biology
of the sperm surface. Posttesticular membrane remodelling. Adv Exp Med
Biol 1997; 424:221–232.
Hermo L, Oko R, Morales CR. Secretion and endocytosis in the male
reproductive tract: a role in sperm maturation. Int Rev Cytol 1994; 154:
106–189.
Agrawal Y, Vanha-Perttula T. Electron microscopic study of the secretion
process in bovine reproductive organs. J Androl 1988; 9:307–316.
Nilsson BO, Jin M, Ronquist G. Immunolocalization of prostasomes in the
human prostate. Ups J Med Sci 1996; 101:149–157.
Aumuller G, Seitz J. Protein secretion and secretory processes in male
accessory sex glands. Int Rev Cytol 1990; 121:127–231.
Hermo L, Jacks D. Nature’s ingenuity: bypassing the classical secretory
route via apocrine secretion. Mol Reprod Dev 2002; 63:394–410.
Fornes MW, Burgos MH. Sperm association in the rat epididymis.
Microsc Electron Biol Cell 1990; 14:115–129.
Asquith KL, Harman AJ, McLaughlin EA, Nixon B, Aitken RJ.
Localization and significance of molecular chaperones, heat shock protein
1, and tumor rejection antigen gp96 in the male reproductive tract and
during capacitation and acrosome reaction. Biol Reprod 2005; 72:328–
337.
Fornes WM, Sosa MA, Bertini F, Burgos MH. Vesicles in rat epididymal
fluid. Existence of two populations differing in ultrastructure and
enzymatic composition. Andrologia 1995; 27:233–237.
Minelli A, Moroni M, Martinez E, Mezzasoma I, Ronquist G. Occurrence
of prostasome-like membrane vesicles in equine seminal plasma. J Reprod
Fertil 1998; 114:237–243.
Arienti G, Carlini E, De Cosmo AM, Di Profio P, Palmerini CA.
Prostasome-like particles in stallion semen. Biol Reprod 1998; 59:309–313.
Agrawal Y, Vanha-Perttula T. Effect of secretory particles in bovine
seminal vesicle secretion on sperm motility and acrosome reaction. J
Reprod Fertil 1987; 79:409–419.
Arienti G, Carlini E, Polci A, Cosmi EV, Palmerini CA. Fatty acid pattern
of human prostasome lipid. Arch Biochem Biophys 1998; 358:391–395.
Carlini E, Palmerini CA, Cosmi EV, Arienti G. Fusion of sperm with
prostasomes: effects on membrane fluidity. Arch Biochem Biophys 1997;
343:6–12.
Arienti G, Polci A, De Cosmo A, Saccardi C, Carlini E, Palmerini CA.
Lipid fatty acid and protein pattern of equine prostasome-like vesicles.
Comp Biochem Physiol B Biochem Mol Biol 2001; 128:661–666.
Rooney IA, Heuser JE, Atkinson JP. GPI-anchored complement
regulatory proteins in seminal plasma. An analysis of their physical
condition and the mechanisms of their binding to exogenous cells. J Clin
Invest 1996; 97:1675–1686.
Kirchhoff C, Hale G. Cell-to-cell transfer of glycosyl phosphatidylinositolanchored membrane proteins during sperm maturation. Mol Hum Reprod
1996; 2:177–184.
Touchstone JC, Alvarez JG, Levin SS, Storey BT. Evidence for
diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine. Lipids 1985; 20:869–875.
Awano M, Kawaguchi A, Mohri H. Lipid composition of hamster
epididymal spermatozoa. J Reprod Fertil 1993; 99:375–383.
Alvarez JG, Storey BT. Differential incorporation of fatty acids into and
peroxidative loss of fatty acids from phospholipids of human spermatozoa.
Mol Reprod Dev 1995; 42:334–346.
Rana AP, Majumder GC, Misra S, Ghosh A. Lipid changes of goat sperm
plasma membrane during epididymal maturation. Biochim Biophys Acta
1991; 1061:185–196.
Nikolopoulou M, Soucek DA, Vary JC. Changes in the lipid content of
EPIDIDYMOSOMAL LIPID ANALYSIS
56.
57.
58.
59.
60.
boar sperm plasma membranes during epididymal maturation. Biochim
Biophys Acta 1985; 815:486–498.
Evans RW, Setchell BP. Lipid changes in boar spermatozoa during
epididymal maturation with some observations on the flow and
composition of boar rete testis fluid. J Reprod Fertil 1979; 57:189–196.
Hall JC, Hadley J, Doman T. Correlation between changes in rat sperm
membrane lipids, protein, and the membrane physical state during
epididymal maturation. J Androl 1991; 12:76–87.
Aveldano MI, Rotstein NP, Vermouth NT. Lipid remodelling during
epididymal maturation of rat spermatozoa. Enrichment in plasmenylcholines containing long-chain polyenoic fatty acids of the n-9 series.
Biochem J 1992; 283:235–241.
Haidl G, Opper C. Changes in lipids and membrane anisotropy in human
spermatozoa during epididymal maturation. Hum Reprod 1997; 12:2720–
2723.
Christova Y, James PS, Cooper TG, Jones R. Lipid diffusion in the plasma
membrane of mouse spermatozoa: changes during epididymal maturation,
effects of pH, osmotic pressure, and knockout of the c-ros gene. J Androl
2002; 23:384–392.
1113
61. Cross NL. Effect of cholesterol and other sterols on human sperm
acrosomal responsiveness. Mol Reprod Dev 1996; 45:212–217.
62. Kirchhoff C, Osterhoff C, Pera I, Schroter S. Function of human
epididymal proteins in sperm maturation. Andrologia 1998; 30:225–232.
63. Sleight SB, Miranda PV, Plaskett NW, Maier B, Lysiak J, Scrable H, Herr
JC, Visconti PE. Isolation and proteomic analysis of mouse sperm
detergent-resistant membrane fractions. Evidence for dissociation of lipid
rafts during capacitation. Biol Reprod 2005; 63:721–729.
64. Ohta K, Sato C, Matsuda T, Toriyama M, Vacquier VD, Hirohashi N,
Lennarz WJ, Kitajima K.Lipid raft on gametic cells as a functional domain
for sperm-egg interaction coupled with signal transduction. Zygote 2000;
8(suppl 1):S63.
65. Cross NL. Reorganization of lipid rafts during capacitation of human
sperm. Biol Reprod 2004; 71:1367–1373.
66. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol
Cell Biol 2000; 1:31–39.
67. Brown DA, London E. Structure and function of sphingolipid- and
cholesterol-rich membrane rafts. J Biol Chem 2000; 275:17221–17224.