Molecular Human Reproduction, Vol. 10, No. 2, pp. 137-142, 2004
© European Society of Human Reproduction and Embryology 2004
Human sperm lipid content is modified after migration into human cervical mucus
1Service d’Histologie-Embryologie, Biologie de la Reproduction/CECOS, Hôpital Cochin, 123 Bd de Port-Royal, 75014 Paris, 2INSERM unité 347 and 3Laboratoire de Biochimie, Hôpital de Bicêtre, 78 Rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France 4Current address: Laboratoire d’Histologie-Embryologie, Faculté de Médecine de Sfax, Avenue Majida Bouleila, BP813, 3029, Tunisia
5 To whom correspondence should be addressed: Service de Biologie de la Reproduction, Hôpital Cochin, 123 Bd de Port Royal, 75014 Paris. e-mail: jacques.auger{at}cch.ap-hop-paris.fr
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ABSTRACT |
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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 efflux of various lipid classes from the sperm to the cervical mucus cannot be ruled out. Key words: Key words: cervical mucus/cholesterol/HPLC/phospholipids/sperm
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Introduction |
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TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Mammalian sperm undergo several complex modifications 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 fluids 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 first selective fluid encountered by sperm after entering the female genital tract. The cervical mucus has several functions. It selects sperm according to their kinetic efficiency 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.
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Materials and methods |
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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 modified 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 120x106/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 final 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.
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.
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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. 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 liquified mucus phase) was gently aspirated, 20 µl 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 five figured elements have been observed contrasting with 3x106 and 5x106 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 10x106 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 µl of methanol just before being injected into the HPLC system.
HPLC analysis of lipids
The HPLC equipment included an automatic injector with a 200 µl 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 250x4.6 mm C18 and a 150x4.6 mm C8 Kromasil 5 µm (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 (Thérond, personal data), was not quantified in the present study. The mobile phase consisted of a solution containing 6% of 10 mmol/l ammonium acetate (pH 5) and 94% methanol (flow rate, 1.5 ml/min). Molecular species of phospholipids were detected at 205 nm. This method has been previously validated (Thérond et al., 1993). Each phospholipid peak separated by HPLC was collected and the acyl and alkenyl groups were identified by selective hydrolysis. The aliphatic chain composition of plasmenyl lipid species was confirmed 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 acid-catalysed 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 confirmed 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 profile was identified 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 significant when P < 0.05.
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Results |
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Sperm characteristics before and after migration in cervical mucus
Sperm characterisitcs and lipid profiles 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 and morphologically normal sperm were significantly higher after migration than before (Table I). In contrast, the kinematic characteristics were not significantly improved after migration (Table I).
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Lipid profiles 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 chromatograms revealed the presence of sphingomyelin, cholesterol, vitamin E, and three diacyl molecular species: (i) 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospholipid: 16:0/22:6, (ii) 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospholipid: 16:0/20:4, and (iii) 1-stearoyl-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-3-phospholipid).
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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
The concentrations of all sperm lipid species decreased significantly 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 significantly decreased after migration (37.2 ± 14.8 nmol/108 versus 67.1 ± 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).
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Finally, the cholesterol/diacyl ratio in sperm was not significantly 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 profiles: 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.
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Discussion |
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This is the first study to report modifications 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 described by Thérond 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; Thérond 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 addition, we detected a plasmanyl lipid species that is not 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF), but contains docosahexaenoic acid at sn-2. This is the first time that this species has been identified 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).
We confirmed 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 findings cannot be totally extrapolated to in vivo conditions.
Considering the first 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 significantly 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 ‘filtering 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 ‘filtering 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 first 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 efflux 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 liquified 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 liquified mucus samples. Further experiments with homogeneous populations of mature sperm retrieved from 90% fractions of Percoll gradients co-incubated with mucus samples using the modified 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 significance of our findings, 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 specifically involving an efflux of cholesterol that appears to alter the fluidity and ionic permeability of the sperm membrane.
In the present study, we did not find 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.
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