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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022533
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BIOLOGY OF REPRODUCTION 70, 768–774 (2004)
DOI: 10.1095/biolreprod.103.022533
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Involvement of Semenogelin-Derived Peptides in the Antibacterial Activity of Human Seminal Plasma1

Frédéric Bourgeon3, Bertrand Evrard3, Michèle Brillard-Bourdet4, Daniel Colleu5, Bernard Jégou3, and Charles Pineau2,3

GERM-INSERM U.435,3 Campus de Beaulieu, Université de Rennes I, 35042 Rennes Cedex, Bretagne, France INSERM EMI-U 0010,4 Protéases et Vectorisation, Université Francois Rabelais, 2bis Boulevard Tonnelle, 37032 Tours Cedex, France Centre de Fécondation In Vitro,5 Clinique de la Sagesse, 35000 Rennes, Bretagne, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanisms for protecting spermatozoa, and the testes that produce them, from infection are essential, given the importance of these cells and organs for the fertility of the individual and perpetuation of the species. This is borne out by the publication of numerous papers on this subject over the last 50 years. We extended our work and that of others on the anti-infectious defense system of the male genital tract, using a new strategy for the direct identification of antibacterial molecules in human seminal plasma. We subjected a liquefied seminal plasma cationic fraction to reversed-phase HPLC, monitored microbicidal activity by gel overlay and radial diffusion assays, and identified the proteins and/or peptides present in each active fraction by mass spectrometry. In addition to proteins with known potent microbicidal activity—phospholipase A2, lactoferrin, and lysozyme—we also found that peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity.

immunology, male reproductive tract, prostate, seminal vesicles, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of studies carried out over many years have described the antibacterial activities of seminal plasma [1–3]. Molecules with anti-infectious properties, such as spermine [4], lysozyme [5], lactoferrin [6], and protease inhibitors, including secretory leukocyte protease inhibitor (SLPI) [7] and cystatin 11 [8], have been identified. More recent studies have focused on several cationic antimicrobial peptides. The specific production of several members of the ß-defensin family [9–11] and of the cathelicidin family, including human cathelicidin antimicrobial peptide (hCAP-18) [12], has been demonstrated in the male genital tract. It has also been suggested that prostatic fluid contains antibacterial substances [13, 14], and it is possible that other accessory glands, such as the epididymis and seminal vesicles, help to protect the spermatozoa in ejaculated sperm. We have also elucidated major features of the antiviral defense system in the testis (for review, see [15]). The male urogenital tract has an unusual organization and is essential not only for the fertility of individuals, but also for perpetuation of the species. We therefore hypothesized that this tract is likely to have developed a highly efficient defense system involving the production of microbicidal agents, probably either peptides or proteins. Given this likelihood, and the need to identify anti-infectious molecules in seminal plasma in the current context of increases in the incidence of sexually transmitted diseases [16, 17], we aimed to identify new antimicrobial molecules in the cationic fraction of human seminal plasma.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Semen Collection and Preparation

Semen was obtained by masturbation after three days of abstinence from patients with normal sperm characteristics undergoing in vitro fertilization (Center de Fécondation In Vitro, Clinique de la Sagesse, Rennes, Bretagne, France). Informed consent was obtained from all patients and the investigations were approved by the Ethical Committee of the city of Rennes, France, and conducted in accordance with the Institut National de la Santé et de la Recherche Médicale (INSERM) guidelines for the use of human samples. Semen samples were allowed to liquefy at 37°C for 1 h, and were then centrifuged at 1000 x g for 10 min to separate spermatozoa from seminal plasma. Seminal plasma samples were frozen and stored at -80°C until cationic peptide purification.

Extraction of Cationic Peptides from Seminal Plasma

Cationic peptides were extracted from seminal plasma using the weak cation exchange matrix Macro-Prep CM (Bio-Rad, Ivry-sur-Seine, France). Briefly, Macro-Prep CM beads were added to a mixture of seven seminal plasma samples (diluted 1:30 in PBS) at a ratio of 1:60 (v/v). The mixture was incubated overnight at 4°C with gentle stirring, then centrifuged at 1000 x g for 5 min and washed 3 times, for 5 min each time, with 80 matrix volumes of 25 mM ammonium acetate (pH 7.5). Peptides bound to the matrix beads were eluted once with 4 matrix volumes of 10% acetic acid in water for 30 min, and twice with 5% acetic acid in water. Eluates were pooled, lyophilized, and resuspended in 0.01% acetic acid for immediate use for reversed-phase (RP)-HPLC or direct antimicrobial activity assays.

HPLC Fractionation of the Cationic Fraction of Seminal Plasma

The cationic fraction of seminal plasma was further purified by RP-HPLC. Samples (1 mg), resuspended in 500 µl of solvent A (0.1% trifluoroacetic acid [TFA] in water, v/v), were loaded onto a C18 RP-HPLC column (Vydac 218TP54; 4.6 x 250 mm, i.d.; particle size, 5 µm; Interchim, Montluçon, France) at a flow rate of 1.5 ml/min. Bound proteins and peptides were eluted with a water/acetonitrile (ACN)/TFA (0.1%, v/v) step gradient, using 10% ACN during the first 10 min, then increasing ACN concentration by 1.4% per min for 5 min, by 0.22% per min for 60 min, and by 4% per min for the last 15 min. We monitored the UV absorbance of the eluent at 210 nm. We collected 1.5 ml fractions individually, lyophilized them, and resuspended the resulting powder in 40 µl of 0.01% acetic acid for use in radial diffusion assay (RDA), Tris-tricine gel electrophoresis, or nondenaturing acid urea (AU)-PAGE.

Tris-Tricine Gel Electrophoresis

Tris-tricine SDS-PAGE was performed as described by Schaegger and von Jagow [18] for selected RP-HPLC fractions. The Tris-tricine gel was silver-stained, using a protocol compatible with mass spectrometry identification [19]. Briefly, gels were fixed in 50% methanol:5% acetic acid for 20 min, washed for 10 min in 50% methanol, and then thoroughly washed in MilliQ (Millipore, Guyancourt, France) water for 2 h. Gels were sensitized by incubation for 1 min in 0.02% sodium thiosulfate and washed twice for 1 min in MilliQ water. They were then silver-stained in ultragrade silver nitrate for 20 min at 4°C, developed in 0.04% formaldehyde:2% sodium carbonate and incubated in 5% acetic acid to stop the staining reaction.

Antimicrobial Activity Assay

Crude seminal plasma, cationic fraction of seminal plasma, and RP-HPLC fractions were tested in antibacterial RDA as previously described [20] against Escherichia coli 363 and Bacillus megaterium MA strains, kindly provided by Dr. M.H. Metz-Boutigue (INSERM U.338, Strasbourg, France). Briefly, 4 x 106 bacteria were mixed with 11.5 ml gel-underlay solution kept molten at 37°C and poured into 70-cm2 Petri dishes. The underlay solution consisted of 1% low-melting point agarose (Life Technologies, Cergy Pontoise, France) and a 1:100 dilution of tryptone soya broth (TSB; AES Laboratoire, Combourg, France) in 10 mM sodium phosphate, a weak ionic buffer (pH 7.4). A series of wells, each 3 mm in diameter, were punched into the solidified agarose layer, and 5 µl of test solution or standard solution with the classic antibacterial cationic peptide cecropin P1 [21] (concentration of 0.25–250 µg/ml; Sigma-Aldrich, L'Isle d'Abeau Chesnes, Saint-Quentin Fallavier, France) was added to designated wells. Plates were incubated at 37°C for 3 h to allow the peptide to diffuse. The microbe-laden underlay was then covered with 11.5 ml of molten overlay. The overlay solution consisted of 6% TSB and 1% agarose in PBS for all assays. The overlay was allowed to cool and solidify and the plates were incubated overnight at 37°C. The diameters of clear zones around the 3-mm holes, indicating antibacterial activity, were measured to the nearest 0.1 mm. The diameter of the well was subtracted and the resulting size of the clear zone, expressed in units (1 unit = 0.1 mm), was plotted against log peptide concentration. Experiments were carried out in triplicate for the seminal plasma and cationic fraction tests and mean values were used to determine minimal inhibitory concentration (MIC). MIC was estimated as the x intercept calculated from the semilogarithmic plot by a least mean squares method.

We used a similar protocol to test the hypothetical antibacterial activity of semenogelin I (SgI), kindly provided by Dr. F. Gauthier (INSERM EMI-U 00-10, Tours, France), and the products of its digestion by PSA (prostate-specific antigen) and PAP (prostatic acid phosphatase). Purified SgI (68, 45, and 34 µg/ml in 20 mM Tris/HCl pH 9.0 containing 0.1% NP40, corresponding to final concentrations of 1.4, 0.9, and 0.7 µM, respectively) was tested. SgI was incubated with purified PSA (4.5 µM) or PAP (8.5 µM) at 37°C for 3 h and 18 h, respectively. A negative control was performed without SgI and the resulting digestion products were tested in the same manner. SgI, PAP, and PSA were purified from human seminal plasma as previously described by Brillard-Bourdet et al. [22] and Rehault et al. [23], respectively.

Gel Overlay Assay (GOA)

Samples (10 µl) were subjected to nondenaturing AU-PAGE in duplicate as previously described [20]. The gels were then cut into two identical halves. One half was rinsed briefly, to decrease the acetic acid and urea content, and was then placed on top of a bacterial indicator gel (the underlay gel described above). The gels were left to stand for 3 h, to allow the proteins and peptides to diffuse into the indicator gel. The PAGE gel was then removed and replaced by an overlay gel, as described above. The chimeric gel was incubated overnight, to enable the surviving indicator organisms to form microcolonies. Zones into which antimicrobial proteins or peptides had diffused were identified as clear colony-free regions. The second half of the gel, an exact replica of the first half, was stored in 1% acetic acid in MilliQ water, and was used for the excision of antimicrobial zones.

Reduction, Alkylation, and In-Gel Digestion

Proteins/peptides were excised from the AU-PAGE replica and digested as previously described [24] with minor modifications. Briefly, gel pieces were washed twice in MilliQ water, dehydrated in ACN, and dried in a vacuum centrifuge. Gel pieces were reduced by incubation in 10 mM dithiothreitol:0.1 M ammonium bicarbonate (NH4HCO3) at 56°C for 30 min, followed by 55 mM iodoacetamide:0.1 M NH4HCO3 at room temperature for 30 min. Gel pieces were then washed in 0.1 M NH4HCO3, dehydrated in ACN, and dried in a vacuum centrifuge. Gel pieces were finally rehydrated by incubation at 4°C for 45 min in a digestion buffer containing 50 mM NH4HCO3 and 6.7 ng/µl trypsin (modified, sequencing grade, Promega, Charbonnières, France). The supernatants were then replaced by 35 µl of 50 mM NH4HCO3 and samples were incubated overnight at 37°C.

Protein Identification by Mass Fingerprinting

The digested peptides in the supernatants were purified on an in-house manufactured micropurification column containing 0.1 µl of a slurry consisting of 20R2 reversed-phase material (PerSeptive Biosytems, Framingham, MA, USA) packed into a GeLoader tip (Eppendorf, Hamburg, Germany) and equilibrated with 1% TFA, as previously described [25]. We loaded 10 µl of the mixture onto the column, which was washed with 1% TFA. The adsorbed peptides were eluted directly onto the matrix-assisted laser desorption/ionization (MALDI) target (AnchorChip target 600 µm, Bruker Daltonics, Bremen, Germany) with 0.8 µl of 70% ACN:0.1% TFA containing saturated {alpha}-cyano-4-hydroxycinnamic acid (Bruker Daltonics). MALDI-time of flight (-TOF) mass spectra were acquired using an AutoFlex instrument (Bruker Daltonics) internally calibrated with trypsin autodigestion peptides. Peptide mass fingerprints were analyzed with Mascott (http://www.matrixscience.com), as previously described [26].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antimicrobial Activity of a Human Seminal Plasma Pool and Its Corresponding Cationic Fraction

A crude human seminal plasma preparation and its extracted cationic fraction were tested for activity against the Gram-negative bacteria E. coli and the Gram-positive bacteria B. megaterium in RDA, carried out at pH 7.4 (Fig. 1). The known antimicrobial peptide from pig intestine, cecropin P1, was used as a positive control in similar conditions. All samples tested were active against both bacteria. Cecropin P1 was more potent against E. coli than the crude fraction of seminal plasma or its cationic fraction, with MICs of 1.4, 46.0, and 11.8 µg/ml, respectively (Fig. 1A). In contrast, the cationic fraction of seminal plasma was more effective against B. megaterium than cecropin P1 or crude seminal plasma, with MICs of 1.6, 3.4, and 15.5 µg/ml, respectively (Fig. 1B).



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  		FIG. 1. Antibacterial activities of human seminal plasma, its corresponding cationic fraction, and the antibacterial peptide cecropin P1 against E. coli (A) and B. megaterium (B). A unit corresponds to the size of the clear zone (mm) measured for each sample concentration minus the size of the central well (1 unit = 0.1 mm). The data presented are representative of three independent experiments. Results are the means ± SD of three replicates

Fractionation of Proteins and Peptides in the Human Seminal Plasma Cationic Fraction by RP-HPLC

The human seminal plasma cationic fraction was fractionated by RP-HPLC into 42 fractions. About 24 major protein peaks were resolved from the sample (Fig. 2A). We avoided problems associated with interindividual variability of seminal plasma samples by using pooled samples. RP-HPLC fractions for 13 seminal plasma pools of cationic extracts, corresponding to over 50 independent runs, were highly reproducible. This suggests that the samples were not degraded during fractionation, which might have reduced the validity and reproducibility of subsequent biological analysis.



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  		FIG. 2. A) Fractionation of proteins and peptides contained in cationic fraction of human seminal plasma by RP-HPLC. The chromatogram presented is representative of about 50 independent runs performed on various cationic extracts from 13 seminal plasma pools. B) An aliquot (5µl) from each of the fractions of the experiment represented in Figure 1A was bioassayed to assess its inhibitory effects on E. coli or B. megaterium growth by means of the RDA. Negative and positive controls were, respectively, 0.01% acetic acid (*) and cecropin P1 at a concentration of 25 µg/ml (**). This figure is representative of 10 independent experiments

We used RDA to assess the microbicidal potency of fractions against two classical bacterial strains, E. coli and B. megaterium (Fig. 2B). No activity against E. coli was identified in fractions 1–13, whereas fractions 14–38 and 41 consistently inhibited the growth of this bacteria. In addition, all chromatographic fractions inhibited the growth of B. megaterium. Fractions 41 and 42 presented high levels of activity, despite low protein concentrations (Fig. 2B).

Electrophoresis of Selected Chromatography Fractions

The major active fractions that affected the growth of E. coli or B. megaterium (fractions 1–2, 22, and 41) as shown in Figure 2B were resolved by Tris-tricine gel electrophoresis (Fig. 3A). Fractions 1–2 and 22 were found to be quite complex, whereas fraction 41 displayed only two protein bands, at approximately 14 kDa and 6 kDa. Chromatography fractions 1–2, 22, and 41 were also resolved by nondenaturing AU-PAGE, and the microbicidal activities of these fractions were monitored by GOA. This approach made it possible to visualize several clear zones corresponding to the microbicidal activities of the fractions tested (bands 1 to 9; Fig. 3B). As expected, fractions 1–2 were not active against E. coli. Zones of E. coli growth inhibition were observed that corresponded to bands 2, 3, 4, and 8 (Fig. 3B). Similarly, zones of B. megaterium growth inhibition, generating bands 1, 5, 6, 7, and 9, were also observed.



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  		FIG. 3. A) Tris-tricine gel electrophoresis of chromatography fractions 1–2, 22, and 41 from human seminal plasma cationic extract. M, molecular weight standards. B) Evaluation of the microbicidal activity of selected RP-HPLC fractions against E. coli and B. megaterium by GOA. An aliquot (10 µl) of each of fractions 1–2, 22, and 41 was used. Bands corresponding to proteins or peptides inhibiting bacterial growth are numbered (1 to 9). These results are representative of three independent experiments

MALDI-TOF Analysis of Antibacterial Protein Bands

We identified potential microbicidal protein candidates by carrying out tryptic digestions of bands excised from AU-PAGE gels. The digested peptides were analyzed by mass fingerprinting in a MALDI-TOF mass spectrometer. Mass fingerprints showed that bands 8 and 9 corresponded to the group IIA phospholipase A2 (PLA2; molecular mass (MW), 14.7 kDa; pI, 9.38; Fig. 4). Bands 2 and 5 corresponded to the N-terminal part of SgI (MW, 52 kDa; pI, 9.30; Fig. 5A). We were unable to analyze band 1, and no digestion peptides were observed during MALDI analysis for bands 3, 4, 6, and 7. The distribution of the digestion peptides with respect to the sequence of SgI (Fig. 5B) was consistent with the known physiological cleavage of this protein during semen liquefaction by PSA or PAP [22, 27] (Fig. 5C).



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  		FIG. 4. A) MALDI/TOF-MS spectrum of tryptic peptides from bioactive band 9. PLA2 (gi|442665) was identified on the basis of peptide mass fingerprinting and from the NCBI database, using the online Mascott program. *Experimental peptides matching sequences in databases. B) Database sequence of PLA2, with experimental/theoretical matched peptides (bold type). The sequence coverage by identified peptides is presented as a percentage and the putative physicochemical properties of the peptides are also indicated



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  		FIG. 5. A) MALDI/TOF-MS spectrum of tryptic peptides from bioactive band 5. SgI (gi|13937967) was identified on the basis of peptide mass fingerprinting and from the NCBI database, using the Mascott online program. *Experimental peptides matching sequences in databases. B) Database sequence of SgI with its signal peptide (underlined) and experimental/theoretical matched peptides (bold type). C) Physiologically known peptides generated by N-terminal digestion by PSA or PAP enzymes and proposed physicochemical properties. The identified peptide sequences were included in both sequences indicated (bold type), and the sequence coverage is expressed as a percentage

Antibacterial Activity of SgI and SgI-Derived Peptides

We assessed the activity of SgI and SgI-derived peptides against E. coli and B. megaterium by RDA (Fig. 6). SgI, dispensed into wells at a concentration of 68 (data not shown), 45, and 34 µg/ml, gave activities of 38.6, 26.2, and 17.8 units, respectively, for E. coli and 29.8, 15.8, and 6.3 units, respectively, for B. megaterium. We calculated MIC values of 19 µg/ml for E. coli and 28 µg/ml for B. megaterium. The SgI/PSA digest was inactive against E. coli but had activity of 40.7 units against B. megaterium. It should, however, be noted that PSA alone had activity of 30.5 units against B. megaterium. The SgI/PAP digest was highly active (53 units) against E. coli, whereas PAP alone had activity of 5.8 units. The SgI/PAP digest had activity of 22.7 units against B. megaterium whereas PAP alone had activity of 11.8 units.



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  		FIG. 6. Assessment of the antibacterial activity of purified SgI and SgI/PSA or SgI/PAP digest against E. coli and B. megaterium by RDA. Left panel: graphical representation of microbicidal activity. Right panel: experimentally obtained clearing zones. SgI was used at a concentration of 34 µg/ml unless otherwise stated (*: 45 µg/ml). Buffer: Tris 20 mM pH 9, 0.1% NP40. These results are representative of two independent experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of antibacterial molecules in seminal plasma has been suspected for more than five decades [1]. In that time, a regular trickle of publications has provided support for the notion that the male genital tract creates an environment suitable for the protection of spermatozoa production and integrity [2, 3, 28, 29]. In the last 4 yr, a major reawakening of interest in this area has resulted in the publication of a number of papers suggesting that the male genital tract is a potential source of original and specific microbicidal molecules involved in host defense [30]. Two major families of cationic antimicrobial peptides involved in the innate immunity system have been characterized in mammals—defensins and cathelicidins—and both are significantly or specifically produced in the male genital tract. The proteins studied to date include cryptdins [31], human ß-defensin (HBD)-4 [9], HBD-5 and HBD-6 [10], human epididymal protein HE2 [32], Bin1B [33], testis defensin-like protein [34], defensin-related gene Defr1 [35], and hCAP-18 [12]. All of these antibacterial molecules are cationic, with spatially separated hydrophobic and charged regions, which is important for the microbicidal mechanisms of action on pathogens [36].

This study focused specifically on the cationic fraction of the seminal plasma, which we have demonstrated to be responsible for most of the microbicidal activity of this fluid [11]. The methods used were based on those of Hein et al. (2002) for the identification of antimicrobial factors in cervical mucus [37]. Our methods were original in that they involved the direct identification of molecules responsible for the observed antimicrobial effects. The totally integrated technical approach described here is of great potential value for the identification of novel microbicidal molecules in other immunoprivileged biological fluids and tissues.

PLA2 was identified by mass spectrometry in chromatography fraction number 41, giving two major bands at 14 and 6 kDa. This identification was confirmed by Western blotting with a specific antibody raised against PLA2 (data not shown). PLA2 has been shown to exert antimicrobial activity, principally against Gram-positive bacterial species [38]. PLA2 is constitutively present in large amounts in the seminal plasma [39, 40], resulting in seminal plasma concentrations similar to the extracellular levels of this enzyme at inflammation sites (0.1–1.0 µg/ml) [41]. Given the high levels of this enzyme in human seminal plasma and the antimicrobial action of PLA2 confirmed here, we suggest that this enzyme probably makes a significant contribution to the overall activity of this fluid against bacteria.

Analysis of the antibacterial activity of chromatography fraction 22 by Tris-tricine gel electrophoresis coupled to GOA showed that only three major bands corresponded to proteins or peptides that inhibited bacterial growth. Unexpectedly, the most active band was identified as SgI. SgI is one of the most abundant proteins in human seminal plasma, and is produced in the seminal vesicles [42]. It is 439 amino acids in size and has an apparent MW of 49.6 kDa; it acts in sperm coagulation, together with semenogelin II and fibronectin [43]. In addition to this well-known role in sperm coagulation and therefore spermatozoa immobilization, SgI and/or its proteolytic fragments are thought to be involved in regulating spermatozoon motility, capacitation [44, 45], and hyaluronidase activation [46], or to have {alpha}-inhibin-like activity [47, 48]. Our results establish a new role for SgI, the N-terminal fragments of which are antibacterial. The concentration of SgI in seminal plasma is estimated by us to be about 10 mg/ml as it represents 20%–40% of the total seminal plasma protein fraction [45] that corresponds to about 35 mg/ml (our results). As antibacterial activities observed here were detected in the µg/ml range, it is very likely that concentrations of SgI are relevant to antibacterial activities in a physiological context. The SgI-derived peptide present in chromatography fraction 22 that displayed strong antimicrobial activity may be generated by PAP cleavage because high levels of antibacterial activity were detected against both bacterial strains used for the SgI/PAP mixture (Fig. 6). This peptide would thus correspond to the known PAP degradation peptide, with a theoretical size of about 18 kDa (Fig. 5C) detected in Figure 3A. Conversely, the lack of activity of the first 10 chromatography fractions against E. coli may be due to the abundance of a SgI-derived fragment originating from PSA cleavage (Fig. 2B), as the SgI/PSA mixture was not active against E. coli (Fig. 6). Support for this hypothesis is provided by the detection of a major peptide in chromatography fractions 1 and 2, migrating at about 15 kDa (Fig. 3A), corresponding to the predicted size of the theoretical SgI-derived peptide generated by PSA cleavage (Fig. 5C). Thus, our data and those of previous experiments [22, 49, 50] suggest that the combined action of PAP and PSA, which are essential for sperm liquefaction, leads to the formation of a number of SgI cleavage peptides with pleiotropic activities. Like SgI, several other molecules known to inhibit spermatozoa motility, such as the human antimicrobial peptide CAP37 [51] and the chicken spermatozoa motility inhibiting factor [52], have been shown to display antibacterial properties.

Western blotting confirmed the presence in cationic fractions of molecules with antibacterial activity other than PLA2 and the SgI-derived peptides identified here. These molecules included lactoferrin and lysozyme (data not shown), which have previously been detected in seminal plasma. The identification of a diversity of antimicrobial molecules in this and other studies [5, 6, 40] suggests that the various microbicidal molecules in the male genital tract may act in synergy or may have additive effects. Such synergic and additive microbicidal effects have been demonstrated in vitro for HBD-4 and lysozyme [9], and in vivo for more complex combinations of antimicrobial factors (LL-37, ß-defensins, lactoferrin, lysozyme, and SLPI) in bronchoalveolar lavage fluid [53]. These findings suggest that, following ejaculation and sperm coagulation, certain soluble factors in the seminal plasma may protect immobilized spermatozoa against pathogens present in the female genital tract [54, 55]. Upon degradation of the semenogelin-derived coagulum, the spermatozoa would then be progressively released, along with semenogelin-degradation peptides with potential microbicidal properties. Spermatozoa may also protect themselves by transporting protective molecules, such as PLA2 [56], SgI and its N-terminal fragment [42, 57, 58], lactoferrin [59], HBD-1 [11], HE21/HE2E [32], or hCAP-18 [12] to the site of fecundation. In conclusion, our results provide strong evidence that human seminal plasma is an anti-infectious medium. The identification of further low-molecular-mass antimicrobial peptides (MW < 6 kDa) in the cationic fraction of human seminal plasma is currently underway in our laboratory.


   ACKNOWLEDGMENTS
 
We thank Dr. F. Gauthier (INSERM EMI-U 00-10, Tours, France) for generously providing purified SgI, PSA and PAP, and Dr. M.H. Metz-Boutigue (INSERM U.338, Strasbourg France) for kindly supplying bacteria. We also thank Dr. J. Vinh (ESPCI, Paris) for assistance with protein identification by mass spectrometry.


   FOOTNOTES
 
1 This work was supported by grants from INSERM, Conseil Régional de Bretagne, Rennes Métropole and Conseil général d'Ille et Vilaine. F.B. is a recipient of a fellowship from the Ministère de l'Education et de la Recherche. Back

2 Correspondence: Charles Pineau, GERM-INSERM U.435, Campus de Beaulieu, Université de Rennes I, 35042 Rennes cedex, Bretagne, France. FAX: +33 (0)2 23 23 50 55; charles.pineau{at}rennes.inserm.fr Back

Received: 21 August 2003.

First decision: 10 September 2003.

Accepted: 14 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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