Molecular Human Reproduction Vol.10, No.2 pp. 137±142, 2004
DOI: 10.1093/molehr/gah018
Human sperm lipid content is modi®ed after migration into
human cervical mucus
N.Chakroun Feki1,4, P.TheÂrond2,3, M.Couturier2, G.LimeÂa1, A.Legrand2,3, P.Jouannet1 and
J.Auger1,5
1
Service d'Histologie-Embryologie, Biologie de la Reproduction/CECOS, HoÃpital Cochin, 123 Bd de Port-Royal, 75014 Paris,
INSERM unite 347 and 3Laboratoire de Biochimie, HoÃpital de BiceÃtre, 78 Rue du GeÂneÂral Leclerc, 94275 Le Kremlin-BiceÃtre,
France 4Current address: Laboratoire d'Histologie-Embryologie, Faculte de MeÂdecine de Sfax, Avenue Majida Bouleila, BP813,
3029, Tunisia
2
5
To whom correspondence should be addressed: Service de Biologie de la Reproduction, HoÃpital Cochin, 123 Bd de Port Royal,
75014 Paris. E-mail: [email protected]
The effect of the female genital tract on sperm is not well known. To investigate the effect of cervical mucus on the lipid content
of human sperm, we co-incubated sperm and mucus samples in vitro such that the sperm were able to swim in and out of the
mucus samples. High performance liquid chromatography and UV detection were used to measure the lipid contents of the
sperm and cervical mucus before and after migration. The concentrations of cholesterol, vitamin E, sphingomyelin, diacyls and
plasmalogens in sperm were all ~45% lower after migration in cervical mucus and the cervical mucus was found to be enriched
in some of these lipid species after the sperm migration. These results suggest that the cervical mucus selects a subpopulation of
sperm with a lower lipid content. However, a concomitant ef¯ux of various lipid classes from the sperm to the cervical mucus
cannot be ruled out.
Key words: cervical mucus/cholesterol/HPLC/phospholipids/sperm
Introduction
Mammalian sperm undergo several complex modi®cations before
they are able to fertilize ova. For example, their plasma membrane is
altered during their transport in the epididymis and in the female
genital tract (Yanagimachi, 1994; Jones, 1998). Active and highly
synchronized interactions between sperm and female tract ¯uids
appear to be critical for the survival and functions of sperm (Barratt
and Cooke, 1991; Zhu et al., 1994b; Kawakami et al., 2001;
Rodriguez-Martinez et al., 2001). The cervical mucus is the ®rst
selective ¯uid encountered by sperm after entering the female genital
tract. The cervical mucus has several functions. It selects sperm
according to their kinetic ef®ciency and morphology (Jeulin et al.,
1985), it can store sperm for several days before ovulation and it
initiates sperm capacitation (Gould et al., 1984; Lambert et al., 1985;
Katz et al., 1989; Katz, 1991; Eggert-Kruse et al., 1995; Perry et al.,
1996). However, the molecular bases of the interaction between sperm
and the cervical mucus are poorly understood.
The composition, amount and dynamics of the lipids in the sperm
plasma membrane are major determinants of the physiological
processes required for fertilization such as motility, capacitation,
acrosomal exocytosis and fusion with the oocyte membrane (Langlais
et al., 1985; Jones et al., 1998; Baldi et al., 2000). Most membrane
sperm lipids, especially docosahexaenoic acid (DHA) have been
found to decrease during the process of epididymal maturation in the
mouse (Ollero et al., 2000) or when comparing immature and mature
human sperm selected on density gradients (Ollero et al., 2000; Force
et al., 2001). The role of the various micro-environments encountered
by the sperm in the female genital tract on their lipid composition has
rarely been studied (Langlais, 1985; Hamamah et al., 1995). As the
lipid content of the sperm plasma membrane is critical for
capacitation, we used high performance liquid chromatography
(HPLC) to analyse the sperm lipid content before and after migration
into ovulatory cervical mucus.
Materials and methods
Semen sample collection and preparation
Semen samples were collected from healthy donors by masturbation in the
laboratory after 3±5 days of sexual abstinence. Informed consent was obtained
from all donors. The samples were incubated at 37°C for 30 min, then analysed
following the World Health Organization (WHO, 1999) recommendations.
Sperm morphology was analysed by a modi®ed version of a previously
reported method (Auger et al., 2001). Kinematic parameters were assessed by
use of a computer-assisted semen analysis system (IVOS; Hamilton-Thorn
Research, USA). For each experiment, three semen samples from three donors
were pooled. All the sperm pools tested had normal characteristics according to
the WHO (1999) recommendations. Sperm concentration was between 50 and
1203106/ml, progressive motility was >50% and the percentage of
morphologically normal sperm was >25%, which is normal according to
recent studies based on a previously described method (Auger et al., 2001;
Slama et al., 2002). A 1 ml aliquot of each freshly prepared semen pool was
washed twice in Earle's medium (Eurobio, France), centrifuged for 10 min at
600 g, and the pellet was then resuspended to a ®nal concentration of 107/ml in
Earle's medium. This aliquot was incubated at room temperature for 1 h (i.e.
the same period of time as in sperm±mucus interaction experiments; see below)
before lipid extraction.
Molecular Human Reproduction vol. 10 no. 2 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
137
N.C.Feki et al.
Cervical mucus samples were incubated at 37°C for 60 min with 5 mg/ml (1:1,
v/v) of bromelin (Sigma±Aldrich, USA), then centrifuged at 1200 g for 10 min.
The supernatant (i.e. the liqui®ed mucus phase) was gently aspirated, 20 ml
were deposited on a slide covered by a coverslip and the preparation was
carefully checked under a microscope to see if it was free of cells or sperm just
before assessing the mucus lipid content. For the six samples tested, only ®ve
®gured elements have been observed contrasting with 33106 and 53106
spermatozoa counted in the pellet of two samples tested.
Extraction of lipids from sperm and cervical mucus
Aliquots (1 ml) of the pooled sperm suspensions and of sperm suspensions
containing a known number of sperm (5 to 103106 sperm) and of the native and
post-incubation mucus samples were mixed with 3 ml of chloroform±methanol
(2:1, v/v). The mixtures were vortexed and centrifuged immediately at 600 g
for 10 min. The chloroform layer (under the phase containing lipids) was
evaporated under nitrogen. The dried lipid residue was stored at ±80°C for 1±2
weeks, then dissolved in 125 ml of methanol just before being injected into the
HPLC system.
Figure 1. Technique used to allow sperm to migrate into and to swim out
from cervical mucus. (1) Semen samples were pooled. (2) The sperm and the
cervical mucus samples were placed in a clean glass tube. (3) Co-incubation:
the sperm migrated into the cervical mucus. (4) Removal of the sperm that
had not entered the mucus. (5) Rinsing of cervical mucus with Earle's
medium. (6) Incubation of cervical mucus with Earle's medium. (7) At the
end of the incubation period, the sperm had swum out of the mucus. (8)
Removal of cervical mucus, washing and resuspension of the sperm in
Earle's medium. (9) Solubilization of cervical mucus in bromelin before
lipid analysis.
Human cervical mucus samples
Ovulatory cervical mucus samples were collected from healthy volunteers
between the 9th and 14th day of the menstrual cycle and after a 3 day period of
sexual abstinence. Only volunteers who had not received any medication with a
potentially negative effect on mucus properties, for example estrogen- or
progesterone-containing contraceptive pills, in the 3 months preceding the
study were included. The cervix was exposed with a sterile speculum. Excess
debris was removed with a large cotton swab and the mucus was carefully
aspirated from the endocervix by use of a special capillary (Aspiglaire; CCD,
France). Samples contaminated with blood or vaginal secretions were not used.
Cervical mucus samples were scored according to the WHO (1999) criteria.
Only cervical mucus with a high WHO score (>13) were used. Before each
experiment, the absence of sperm in the cervical mucus was checked under the
microscope.
Sperm selection by cervical mucus samples
After migrating into cervical mucus samples, sperm were recovered by an
adapted version of the `swim-out' method (Zhu et al., 1994a). This procedure is
summarized in Figure 1. In brief, 2 ml of the pooled semen samples were placed
in one to three tubes. Cervical mucus samples were weighed to allow the
concentration of each lipid class per gram to be calculated, and then carefully
deposited onto the top of the sperm suspension. The tubes were incubated at
37°C in a 5% CO2 atmosphere for 25 min to allow the sperm to penetrate into
the cervical mucus progressively.
The mucus samples were then carefully aspirated with a Pasteur pipette,
placed in clean glass tubes and gently rinsed twice with Earle's salt solution to
remove any sperm that had not penetrated the mucus or that were loosely bound
to the outer surface of the mucus. The washed mucus samples were placed in
new glass tubes containing 3 ml of Earle's solution and incubated at 37°C in a
5% CO2 atmosphere for 30 min. During this step, a fraction of the sperm that
had penetrated the cervical mucus sample were able to swim out (Zhu et al.,
1994a). The liquid phase containing the sperm was then carefully aspirated.
This sample and the initial sample were assessed for sperm concentration,
vitality, morphology and kinematic parameters.
Preparation of cervical mucus for lipid analysis
Two native samples of cervical mucus and six samples recovered after the
sperm migration experiment were prepared and analysed in the same way.
138
HPLC analysis of lipids
The HPLC equipment included an automatic injector with a 200 ml sample
loop, and a UV±visible light detector (Thermo Finnigan, France). Molecular
species belonging to different phospholipid classes (1-alkenyl-2-acyl called
plasmalogen or plasmenyl, 1-alkyl-2-acyl termed plasmanyl, and diacyl),
vitamin E, cholesterol and sphingomyelin were separated by using two serial
analytical columns: a 25034.6 mm C18 and a 15034.6 mm C8 Kromasil 5 mm
(A.I.T, France). Demosterol, which is present in human sperm (Alvarez and
Storey, 1995) and has a distinct and lower retention time than cholesterol using
HPLC (TheÂrond, personal data), was not quanti®ed in the present study. The
mobile phase consisted of a solution containing 6% of 10 mmol/l ammonium
acetate (pH 5) and 94% methanol (¯ow rate, 1.5 ml/min). Molecular species of
phospholipids were detected at 205 nm. This method has been previously
validated (TheÂrond et al., 1993). Each phospholipid peak separated by HPLC
was collected and the acyl and alkenyl groups were identi®ed by selective
hydrolysis. The aliphatic chain composition of plasmenyl lipid species was
con®rmed by demonstrating the stoichiometric quantities of the dimethylacetal
(corresponding to the sn-1 aliphatic group) and fatty acid methyl ester
(corresponding to the sn-2 aliphatic group) derivatives produced after acidcatalysed methanolysis and capillary gas chromatography (GC) of the
plasmenyl lipid species (DaTorre and Creer, 1991). After acid-catalysed
methanolysis and GC analysis, the composition of diacyl lipid species was
con®rmed by the demonstration of stoichiometric production of fatty acid
methyl ester derivatives corresponding to the sn-1 and sn-2 aliphatic groups and
for alkylacyl lipid species, the production of a single fatty acid methyl ester
derivative corresponding to the sn-2 aliphatic group.
Each peak on a chromatographic pro®le was identi®ed by comparing its
retention time with that of commercial standards. Cholesterol, sphingomyelin
and vitamin E were purchased from Sigma±Aldrich and alkenylacyl and diacyl
phospholipid species from Interchim (Interchim, France). The concentration of
each component was determined by comparing the surface of the peaks to that
of standards.
Statistical analysis
All statistical analyses were done using the BMDP statistical software (Dixon,
1988). The non-parametric Wilcoxon signed-rank test was used to compare
paired variables in native and post-incubation samples. The undetectable levels
of vitamin E found after migration were arbitrarily considered to be zero to
allow us to compare the vitamin E content of sperm before and after migration.
Differences were considered to be statistically signi®cant when P < 0.05.
Results
Sperm characteristics before and after migration in
cervical mucus
Sperm characterisitcs and lipid pro®les were analysed in 11 experiments using six pools of semen and 11 mucus cervical samples. As
expected, the percentages of live sperm, progressively motile sperm
Sperm lipids and cervical mucus
Table I. Sperm characteristics before and after migration through cervical mucus
Characteristics
Before
% live sperm
% normal sperm
Total motility (%)*
Progressive motility (%)*
VSL (mm/s)*
VAP (mm/s)*
VCL (mm/s)*
ALH (mm)*
BCF (Hz)*
STR (%)*
LIN (%)*
75.1
41.8
54.2
35.8
53.0
61.0
86.5
3.62
29.11
84.1
58.2
6
6
6
6
6
6
6
6
6
6
6
After
2.1 (68±84)
3.2 (31±51)
3.2 (44±76)
4.2 (23±58)
4.1 (43±79)
3.6 (52±84)
4.1 (77±121)
0.16 (3.2±4.6)
0.62 (26.8±34.0)
1.7 (80±93)
1.7 (52±67)
81.9
52.4
75.5
51.9
53.5
62.4
101.3
4.24
28.13
83.0
53.4
Difference
6
6
6
6
6
6
6
6
6
6
6
3.2 (70±97)
4.7 (33±71)
4.2 (47±90)
4.9 (21±77)
3.0 (44±68)
3.4 (45±77)
6.8 (74±130)
0.27 (2.9±6.0)
0.85 (25.8±34.8)
1.5 (77±89)
2.0 (44±62)b
6.8
10.5
21.3
16.1
1.4
0.6
14.8
0.62
±0.98
±1.1
±4.8
6
6
6
6
6
6
6
6
6
6
6
1.4b
2.9b
4.5a
4.2a
5.4
4.9
9.8
0.35
1.03
1.2
1.5
Values are mean 6 SEM (range in parentheses); n = 11.
*CASA parameters; aP < 0.005, bP < 0. 01 with the Wilcoxon test.
VSL = progressive velocity; VAP = path velocity; VCL = curvilinear velocity; ALH = amplitude of lateral
head displacement; BCF = beat cross frequency; STR = straightness; LIN = linearity.
chromatograms revealed the presence of sphingomyelin, cholesterol,
vitamin E, and three diacyl molecular species: (i) 1-palmitoyl-2docosahexaenoyl-sn-glycero-3-phospholipid: 16:0/22:6, (ii) 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospholipid: 16:0/20:4, and (iii) 1stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholipid: 18:0/22:6.
They also revealed one plasmenyl molecular species [1-hexadec-1¢enyl-2-docosahexaenoyl-sn-glycero-3-phospholipid (peak 4)] and one
plasmanyl molecular species (1-hexadecyl-2-docosahexaenoyl-sn-3phospholipid).
Figure 2C shows the lipid chromatogram for a sample of ovulatory
human cervical mucus before the co-incubation experiment.
Cholesterol was the predominant lipid (~85% of total cervical
mucus lipids) and no sphingomyelin or diacyls were detected.
Lipid composition of sperm after migration in cervical
mucus
Figure 2. Typical chromatographic lipid pro®les of human sperm and human
cervical mucus: lipid pro®le of a pool of semen samples (A); lipid pro®le of
the sperm from the same pool after migration into and out of the cervical
mucus (B); lipid pro®le of a native cervical mucus free of cells and sperm
(C); lipid pro®le of the same cervical mucus sample after swim in and out of
sperm (D). Peaks identi®ed: (1) vitamin E, (2) cholesterol, (3) 1-palmitoyl-2docosahexaenoyl-sn-glycero-3-phospholipid (16:0/22:6), (3¢) 1-palmitoyl-2arachidonoyl-sn-glycero-3-phospholipid (16:0/20:4), (4) 1 hexadec-1¢-enyl-2docosahexaenoyl-sn-glycero-3-phospholipid (plasmenyl or plasmalogen),
(5) 1-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phospholipid (plasmanyl),
(6) sphingomyelin, and (7) 1-stearoyl-2-docosahexaenoyl-sn-glycero-3phospholipid (18:0/22:6).
and morphologically normal sperm were signi®cantly higher after
migration than before (Table I). In contrast, the kinematic characteristics were not signi®cantly improved after migration (Table I).
Lipid pro®les of sperm and human cervical mucus before
co-incubation experiment
Figure 2A shows a typical chromatogram of human sperm lipids
before the co-incubation experiment. The human sperm lipid
The concentrations of all sperm lipid species decreased signi®cantly
after migration in the cervical mucus (Table II, Figures 2B and 3). The
vitamin E content of sperm was below the detection limit (20 ng/108
sperm) after migration through the cervical mucus in nine out of 11
experiments. The concentrations of the three phospholipid diacyl
species were signi®cantly decreased after migration (37.2 6 14.8
nmol/108 versus 67.1 6 11.2 mmol/108 sperm cells, P < 0.001)
(compare peaks 3, 3¢ and 7 in Figure 2A and B). The experiments
using the same pool of semen samples and different mucus samples
showed different levels of sperm lipid decreases after migration
(Figure 3).
Finally, the cholesterol/diacyl ratio in sperm was not signi®cantly
different before and after migration (Table II).
Lipid enrichment of cervical mucus after sperm migration
The native cervical mucus samples and those recovered after the
sperm migration had quantitatively and qualitatively different
chromatographic pro®les: the concentrations of most of the lipid
species were higher in cervical mucus after sperm recovery (Figure 2C
and D). For example, the two native samples tested contained 12.9 and
22.9 nmol of cholesterol per gram of cervical mucus whereas the six
samples obtained after sperm migration contained 27.0, 54.6, 59.5,
70.1, 73.4 and 89.5 nmol of cholesterol per gram of cervical mucus.
Discussion
This is the ®rst study to report modi®cations of the lipid composition
of human sperm after their in vitro migration into human cervical
mucus. These results were obtained by using the method originally
139
N.C.Feki et al.
Table II. Sperm lipid concentrations before and after migration through cervical mucus
Characteristics
Before
Vitamin E (ng/108 sperm)²
Cholesterol (nmol/108 sperm)
D*22:6/16:0 (nmol/108 sperm)
D22:4/16:0 (nmol/108 sperm)²
D18:2/16:0 (nmol/108 sperm)²
D22:6/18:0 (nmol/108 sperm)²
D20:4/18:0 (nmol/108 sperm)²
Total diacyls (nmol/108 sperm)
Plasmalogen (nmol/108 sperm)
Sphingomyelin (nmol/108 sperm)
Cholesterol/total diacyls ratio
210.9
93.0
33.6
5.5
11.3
9.1
4.8
67.1
19.3
39.0
1.41
6
6
6
6
6
6
6
6
6
6
6
After
25.0 (148.1±394.8)
4.5 (68.8±114.6)
1.5 (38.8±27.2)
0.1 (5.8±4.9)
0.7 (8.8±15.7)
0.6 (8.5±12.8)
0.3 (4.2±5.7)
3.4 (54±80)
2.7 (11.6±35.8)
10.9 (28.9±65.1)
0.08 (1.19±1.66)
26.5
51.1
22.9
2.9
4.0
4.5
2.1
37.2
9.2
18.9
1.55
6
6
6
6
6
6
6
6
6
6
6
Difference
19.2 (0±199.0)
3.3 (32.7±70.5)
2.3 (34.7±13.4)
0.5 (4.9±1.7)
0.8 (9.5±2.0)
0.8 (2.7±9.1)
0.5 (1.4±4.3)
4.5 (22.5±62.5)
1.1 (5.0±14.9)
7.1 (8.0±27.2)
0.19 (0.87±2.94)
±184.3
±41.9
±10.7
±2.6
±7.3
±4.7
±2.7
±30.0
±10.1
±20.1
0.15
6
6
6
6
6
6
6
6
6
6
6
18.7a
5.5a
3.2a
0.5a
0.8a
0.7a
0.5a
5.7a
3.3a
3.8a
0.20
Values are mean 6 SEM (range in parentheses); n = 11.
*D = Diacyl.
²Concentrations under the detection limit were considered to be 0 for statistical purposes; aP < 0.001 with the
Wilcoxon test.
Figure 3. Sperm lipid concentrations before (grey columns) and after migration (hatched columns) into the 11 cervical mucus samples. The same pool of
semen samples could be used for several experiments (bracketed).
described by TheÂrond et al. (1993) for other cell types. This method
shows most of the lipid species present on a single chromatogram and
can be used to detect the lipid species present in human sperm. The
amounts of cholesterol, diacyls, sphingomyelin, plasmalogen and
vitamin E in washed human sperm were consistent with previously
published data (Alvarez and Storey, 1995; TheÂrond et al., 1996; Force
et al., 2001). Similarly, the cholesterol and phospholipid content of
sperm was variable as previously reported (Hamamah et al., 1995). In
140
addition, we detected a plasmanyl lipid species that is not 1-alkyl-2acetyl-sn-glycero-3-phosphocholine (PAF), but contains docosahexaenoic acid at sn-2. This is the ®rst time that this species has been
identi®ed in human sperm. We also used this method to determine the
nature and amount of lipids present in samples of human ovulatory
cervical mucus. Cholesterol was the predominant lipid in cervical
mucus, as previously shown by gas chromatography (Singh and
Twartwout, 1972).
Sperm lipids and cervical mucus
We con®rmed that the `swim-out' technique described by Zhu et al.
(1994a) could be used to select a subpopulation of good quality sperm.
Therefore, it is a useful tool for studying the structure and functional
ability of sperm after their migration in cervical mucus. The adapted
version of the `swim-out' technique demonstrated that the concentrations of most sperm lipids decreased after migration through cervical
mucus in vitro. Two hypotheses can be drawn from these results: (i) it
is possible that the mucus selects a subpopulation of sperm with a lipid
composition different from that of the native sperm population, and/or
(ii) these changes could be the consequence of molecular interactions
between the cervical mucus and sperm. In both cases, it is important to
remember that the experimental conditions used were not identical to
those occurring in vivo as the parallel arrangement of mucus
macromolecules that favours sperm migration is not preserved in the
`swim-out' technique. Therefore, our ®ndings cannot be totally
extrapolated to in vivo conditions.
Considering the ®rst assumption, it is possible that the highly motile
sperm that can swim in and out the cervical mucus have a composition
lipid different from that of immotile or asthenic sperm. It was
previously reported that sperm selected by the swim-up method have
lower cholesterol and phospholipid contents than native sperm (Force
et al., 2001) and another study has shown that the mature sperm
isolated from the 95% fraction of a Percoll density gradient have
signi®cantly lower cholesterol, total fatty acids and docosahexaenoic
acid (DHA) contents than the respective contents found for the
immature sperm fraction isolated from the 50% fraction (Ollero et al.,
2000). Interestingly, the cholesterol content before and after migration
through cervical mucus and the magnitude of the decrease observed
were remarkably similar to those found for the immature and mature
sperm fractions in the study of Ollero et al. (93 versus 85.6 nmol/ 108
sperm, 51.1 versus 39.1 nmol/ 108 sperm, and ±45.1 versus ±54.3%
respectively). In addition, despite the use of different methodologies,
the decrease in diacyl and alkeny-ether phospholipid molecular
species containing DHA after sperm migration through cervical
mucus was consistent with the difference in DHA of immature to
mature sperm in the Ollero et al. study (±44.7 versus ±60.6%
respectively). The similar lipid trends found by Ollero et al. and our
study are highly consistent with the hypothesis of a `®ltering effect' of
the mucus resulting in the selection of a possibly more mature sperm
fraction partly characterized by a lipid composition differing from the
lipid content of the native sperm sample containing mature and
immature sperm.
The assumption that the mucus `®ltering effect' cannot be the only
mechanism to explain the different lipid pattern for the sperm that
swim in and out can be made because the decrease in the various
sperm lipid species paralleled an enrichment of the same lipid species
in the mucus. At ®rst glance, such a result may suggest molecular
exchanges between the sperm and the cervical mucus involving lipid
exchange proteins. Among proteins, the role of albumin can be evoked
because it is a cholesterol acceptor that is involved in sperm
capacitation (de Lamirande et al., 1997b; Therien et al., 1999;
Visconti et al., 2002), it may bind directly to the sperm surface
(Focarelli et al., 1990) and it is the major protein of the human cervical
mucus (Salas Herrera et al., 1991). However, the albumin content of
human ovulatory mucus samples, 72.9 mg/l according to Salas Herrera
et al. (1991), is certainly too low (~0.01%) for provoking an ef¯ux of
cholesterol which requires albumin concentrations of ~1%. Another
explanation for the observed lipid increases in the mucus samples
could be the contamination of the mucus samples with immobilized
sperm, other cells, cellular debris or residual membranes of the sperm
and cells entrapped in the mucus. We believe that the very rarely
found cells in the washed liqui®ed mucus samples (see Material and
methods section) were a negligible contaminant of the lipids found in
the mucus samples. On the other hand, we cannot rule out the
possibility that residual membranes, mainly from defective sperm
which have higher amounts of lipids (Ollero et al., 2000) may have at
least partly contaminated the supernatant of the washed liqui®ed
mucus samples. Further experiments with homogeneous populations
of mature sperm retrieved from 90% fractions of Percoll gradients coincubated with mucus samples using the modi®ed swim-out technique
reported herein will certainly help to answer this question or to
determine whether mature sperm could transfer lipids to cervical
mucus.
The biological signi®cance of our ®ndings, the selection of sperm
with a low lipid content observed after co-incubation with mucus (and/
or an extraction of sperm lipids by mucus components), remains to be
determined. Sperm capacitation is believed to be initiated during the
process of sperm migration into the cervical mucus (Lambert et al.,
1985; Zinaman et al., 1989), the close contact between sperm and
cervical mucus macromolecules leading to the loss of membrane
molecules, such as decapacitation factors (Katz et al., 1989). It is also
considered that one of the earliest steps of capacitation is a change in
the lipid composition of the sperm membrane (Baldi et al., 2000) more
speci®cally involving an ef¯ux of cholesterol that appears to alter the
¯uidity and ionic permeability of the sperm membrane.
In the present study, we did not ®nd kinematic values or changes in
cholesterol to phospholipid diacyl ratios for the selected sperm
reminiscent of the full capacitation process. We found that the sperm
vitamin E content was markedly decreased after migration into the
mucus. It can be hypothesized that this could disrupt the oxidative
balance in situ, which could in turn favour the initiation of capacitation
since capacitation requires low concentrations of free radicals,
especially superoxide (O2±) and hydrogen peroxide (H2O2) (Griveau
et al., 1995; de Lamirande et al., 1997a) and it is blocked by an
addition of free radical scavengers (Delamirande et al., 1998). Studies
are in progress in our laboratory to assess the oxidative status of the
sperm selected by the mucus but further experiments are warranted to
more precisely describe their functional status.
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Submitted on October 22, 2003; accepted on October 27, 2003