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J. Biol. Chem., Vol. 276, Issue 29, 26962-26968, July 20, 2001

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Evidence for the Autocrine Induction of Capacitation of Mammalian Spermatozoa^*

Cuigi Wu^§¶, Tomas Stojanov^§, Omar Chami, Santoshi Ishii, Takao Shimuzu, Aiging Li, and Chris O'Neill^**

From the  Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia,  CREST of Japan Science and Technology Corporation, Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, April 9, 2001, and in revised form, May 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilization. This process, known as capacitation, occurs spontaneously in simple defined medium implicating a potential role of autocrine induction. This study shows that the ether phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF) meets the criteria for an autocrine mediator of capacitation. Sperm released PAF after their dilution into capacitation medium and expressed a receptor for PAF on their membranes. PAF stimulated changes in the motility of sperm and enhanced fertilization in vitro. These actions were inhibited by a PAF receptor antagonist (UR-12519) and by extracellular recombinant PAF:acetylhydrolase (an enzyme that degrades PAF to a biologically inert form). Seminal plasma contained an acid-labile PAF:acetylhydrolase, whereas capacitation was inhibited by an acid-labile factor within seminal plasma, implicating this factor as a potential decapacitation factor within seminal plasma. Sperm from a PAF receptor knock-out mouse strain failed to express the receptor and displayed a significantly (p < 0.01) reduced rate of capacitation, as assessed by the spontaneous onset of the acrosome reaction in vitro. When used for in vitro fertilization, sperm from PAF receptor knock-out mice gave a significantly lower rate of fertilization (21.5%) than did wild-type sperm (66.7%). The study shows for the first time the operation of an autocrine loop that induces capacitation in sperm in vitro and shows that this loop acts in concert with other mediators of capacitation to promote efficient fertilization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mammalian sperm function is characterized by the requirement for a process of extra testicular maturation following ejaculation, known as capacitation. Capacitation allows sperm to acquire the capacity to undergo the acrosome reaction and fertilization. Capacitation is correlated with an increase in tyrosine phosphorylation of multiple proteins within sperm (1, 2). Two of these proteins are isoforms of extracellular signal-regulated kinase-1 and -2 (3). Capacitation is associated with profound changes in the membrane properties of sperm, including the efflux of cholesterol from the plasma membrane (4).

The activation of extracellular signal-regulated kinases during capacitation (3) suggests that capacitation requires an extracellular signal. However, an intriguing aspect of the process is that it occurs spontaneously in vitro without a requirement for exogenous mediators. This spontaneous response requires that ejaculated sperm be removed from the components of seminal plasma and their dilution in simple defined medium, containing metabolic energy substrates, Ca²⁺, and HCO (5). There is, however, a requirement for exogenous protein in the form of albumin (5). Since albumin is not generally recognized as a signaling molecule, alternative explanations for its role have been sought.

A model to explain this "bootstrapping" of the capacitation process in defined medium has been proposed and involves extracellular albumin acting to remove sperm membrane cholesterol (6). Seminal plasma contains decapacitating activity (7), and removal of seminal plasma is required for capacitation. It was proposed that lipid-dense vesicles within seminal plasma donate cholesterol to the sperm membrane, preventing capacitation (8). The removal of seminal plasma and the presence of albumin reverse this process resulting in a net efflux of cholesterol from the sperm membrane. It was postulated that the resulting change in membrane properties leads to the intracellular signaling events that comprise capacitation. This proposed form of signal induction seems to be unique to the spermatozoon.

As well as acting as a sink for cholesterol, albumin may also act as an acceptor for membrane phospholipids. The phospholipid component of the plasma membrane of most cell types is composed mainly of esterified phospholipids (the acyl chains are linked to the glycerol backbone by ester bonds). A characteristic feature of the spermatozoon is that it has, compared with somatic cells, a high (40%) ether phospholipid component (9). Ether phospholipids have the acyl chain replaced by long chain alcohols linked by ether bonds, and these are primarily 1-O-alkyl phospholipids. One ether phospholipid of particular interest is the potent signaling molecule, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF)¹ (10). The release of PAF by most cells is dependent upon extracellular albumin (11-14).

PAF acts via a high affinity, selective G-protein-linked receptor to induce cellular activation (15). PAF binds to spermatozoa (16), and the PAF receptor was detected in sperm by immunofluorescence (17). PAF is present in the spermatozoa of the mammalian species studied to date, including man (18) and rabbit (19), although it is not well established whether sperm release PAF. The addition of exogenous PAF can induce capacitation in sperm (20), and this is blocked by PAF receptor antagonists (21). The addition of PAF to IVF medium enhanced fertilization rates in vitro (22, 23). PAF is degraded to inactive lyso-PAF by PAF:acetylhydrolase (24), and this enzyme is detected in the seminal plasma (25) and associates with sperm (26).

An hypothesis that may link these observations is that the dilution of sperm away from seminal plasma limits PAF:acetylhydrolase activity allowing sperm-derived PAF to be released onto albumin and accumulate without degradation by PAF:acetylhydrolase. Upon achieving threshold concentrations, PAF acts on specific receptors on sperm to induce signal transduction and the molecular changes of capacitation.

To test this hypothesis, this study determined whether (i) sperm release PAF upon dilution in capacitation medium containing albumin; (ii) sperm possess a specific and functional PAF receptor that is required for capacitation; and (iii) the deletion of this PAF receptor (by homologous recombination) affects capacitation and fertilization. The results support a role of sperm-derived PAF as an autocrine pathway for the induction of capacitation of mammalian sperm.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Media and Reagents-- Preparations of sperm for the assessment of hyperactivated motility was in BWW medium (20 mM sodium lactate, 5 mM glucose, 0.25 mM sodium pyruvate, 95 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, pH 7.4). For all other procedures sperm were prepared in HTF medium (101.6 mM NaCl, 4.69 mM KCl, 0.20 mM MgSO₄, 0.37 mM KH₂PO₄, 21.4 mM sodium lactate, 2.78 mM glucose, 2.04 mM CaCl₂, 25 mM NaHCO₃, 0.33 mM sodium pyruvate) (27). Hepes-buffered HTF medium (HTF media with NaHCO₃ reduced to 5 mM and replaced by equimolar Hepes buffer) (27) was used for collecting and washing oocytes, zygotes, and embyros. Embryos were cultured in modified HTF medium (HTF with 1 mM glutamine and 0.11 mM EDTA added) (28). All media contained 3 mg of bovine serum albumin/ml (Pentex crystallized BSA, Miles Inc., Kankakee, IL) unless otherwise indicated. Recombinant PAF:acetylhydrolase (rPAF:acetylhydrolase) was a generous gift of ICOS Corp. (Bothell, WA); UR-12519 was a generous gift of J. Uriach and Cia, SA (Barcelona, Spain). A monoclonal PAF receptor antibody was from Alexis Corp. (San Diego, CA). PAF (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine; an approximately equimolar mixture of hexadecyl and octadecyl form of PAF, Sigma) was stored as a stock solution of 10 mg/ml in chloroform at 20 °C. Aliquots were placed in sterile siliconized glass tubes and dried under N₂. The PAF was solubilized by the addition of medium, followed by vigorous vortexing for 3 min, and then allowed to stand for 1 h at 37 °C with gentle mixing.

Mice-- Mice were Swiss outbred (Laboratory Animal Services, University of Sydney), C57BL/6 (Laboratory Animal Services), or PAF receptor knock-out (KO) (Department of Biochemistry and Molecular Biology, University of Tokyo (29)). The KO strain had been backcrossed to wild-type C57BL/6 mice 10-12 times. All animals were housed and bred in the Gore Hill Research Laboratory, St. Leonards, New South Wales, Australia. The use of animals was approved by the Royal North Shore Hospital Animal Care and Ethics Committee, according to the Australian Code of Practice for Use of Animals in Research.

Sperm Collection-- Mouse sperm was collected from the epididymides of male mice (14-35 weeks old) of proven fertility and immediately placed into 1 ml of medium. Sperm were allowed to disperse for 10 min at 37 °C. Human sperm was obtained by masturbation from donors of proven fertility. Following incubation at 37 °C for 15 min, the ejaculate was centrifuged at 500 × g for 5 min. The seminal plasma was removed and frozen, and the spermatozoal pellet was washed 3 times. For mouse and human preparations, the sperm concentration and motility were assessed by hemocytometer, and the sperm suspension was diluted as required for each experiment.

PAF Extraction and Assay-- PAF was extracted by a modified version of the Bligh-Dyer organic extraction method, followed by partial purification by TLC (30). Quantification was performed using a PAF-specific scintillation proximity immunoassay (Amersham Pharmacia Biotech).

PAF:Acetylhydrolase Assay-- The assay used to measure PAF:acetylhydrolase was as described previously (31). 1-Hexadecyl-2-[³H]acetyl-sn-glycero-3-phosphocholine (262.7 GBq mmol¹; [³H]PAF) was purchased from PerkinElmer Life Sciences, and 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (unlabeled PAF) was from NOVA Biochem AG (Laufelfingen, Switzerland). Reactions were performed in a final volume of 500 µl containing a final concentration of 5 µmol/liter [³H]PAF (5.25 GBq mmol¹), 60 µg of BSA. The reaction was at 37 °C for 15 min and stopped by the addition of 170 µg of BSA, followed by trichloroacetic acid. The free [³H]acetate was counted on a Packard Tricarb model 1500 scintillation counter (Packard Instrument Co.).

Detection of Gene Expression-- Evidence for the expression of mRNA for the G-protein-linked PAF receptor, the subunits of the intracellular form of PAF:acetylhydrolase 1b (₂- and -subunits), and the plasma/macrophage form of PAF:acetylhydrolase was sought using RT-PCR. Control tissues (liver and brain) were examined in parallel. Sperm were prepared as above and allowed to swim up into a column of medium. The motile sperm were collected, and several hundred were picked off under a dissecting microscope. This method ensured that a pure sample of motile sperm, without contamination by other cells, was tested.

For all RT-PCR assays the following controls were always undertaken. (i) Mouse -actin was used as a positive control for the effectiveness of all RNA extractions, and RT-PCRs were performed on those samples (the -actin primer pair was designed to span the first intron (87 base pairs in length) of the rodent -actin gene; thus contaminating genomic DNA could be detected using these primers). (ii) To control for false-positive PCR amplification of contaminating genomic DNA, some samples did not include reverse transcriptase. (iii) Water was added instead of sample to test for contamination with extraneous DNA. (iv) Some samples were randomly treated with RNase I (Promega Corp., Madison, WI) prior to RT, confirming the RNA origin of positive RT-PCRs.

PCR products were analyzed by electrophoresis on 4% agarose gel, stained with ethidium bromide to visualize PCR product on an UV transilluminator. Fragments were verified by size, and the product was extracted and the sequence analyzed to confirm they were from the target gene (ABI PRISM Dye terminator Cycle Sequencing Ready Reaction Kit from PerkinElmer Life Sciences, performed by SUPAMAC, Redfern, New South Wales, Australia). Primers were obtained from Fisher Biotech (Perth, Australia). The primers are as follows: PAF-R (5'-GAT GGC TCA GGC AAC ATC AC-3' and 5'-TGA TGA ATA CCG CCA AGA CC-3') (32); plasma PAF:acetylhydrolase (5'-GAT GGC TCA GGC AAC ATC ATC AC-3' and 5'-TGA TGA ATA CCG CCA AGA CC-3') (24); PAF:acetylhydrolase 1b, -subunit (5'-GAT GAC AGG ACC CTC CGT GT-3' and 5'-ACC AAT GGG TAA ACT CGA G-3') (33); PAF:acetylhydrolase 1b, ₂-subunit (5'-CTCGAACCCAGCAGCTATTC-3' and 5'-ACCTTAACCCCCTCTATGTT-3') (33, 34); and -actin (5'-CGT GGG CCG CCC TAG GCA CCA-3' and 5'-GGG GGA CTT GGG ATT CCG GTT-3') (35).

RNA was extracted with TRIzol^TM Reagent (Life Technologies, Inc.) containing 50 µg of carrier RNA (yeast transfer RNA, Sigma) as described previously (35). Isolated RNA was treated with DNase to eliminate possible contamination with genomic DNA, by resuspending the RNA pellet in 20 µl of resuspension solution (RS, 40 mM Tris-HCl, pH 7.9, 10 mM NaCl, and 6 mM MgCl₂) (36) containing 2 units of RQ1 DNase (Promega) and incubating at 37 °C for 30 min. Following the addition of a second equal volume of RS, RNA was phenol-chloroform re-extracted. The RNA pellet was dissolved in double-autoclaved Milli-Q water in the presence of RNase Inhibitor (Promega) (final concentration 1 unit/ml).

RNA was reverse-transcribed at 42 °C for 30 min with 1.5 units of murine leukemia virus reverse transcriptase primed with 0.25 µM oligo(dT) in 20 µl of reaction mix containing 3 mM MgCl₂, 60 mM KCl, 50 mM Tris-HCl, pH 8.3, 1 mM each dNTP, and 1 unit of RNase Inhibitor (all reagents supplied by PerkinElmer Life Sciences). The RT reaction was then terminated by heating at 98 °C for 5 min and cooling to 5 °C.

Twenty (20) µl of RT reaction volume were used for test sample in a final PCR volume of 50 µl containing 2 mM MgCl₂, 10 mM KCl, 50 mM Tris-HCl, pH 8.3, 0.2 mmol each dNTP, 2.5 units of AmpliTaq DNA polymerase, and 0.4 µM each of a specific primer pair were subjected to 35 rounds of amplification in a Corbett Thermal Reactor. PCR products were analyzed by electrophoresis on 4% agarose gel stained with ethidium bromide to visualize PCR product on a UV transilluminator.

Immunofluorescence-- A sperm suspension was fixed with 4% formaldehyde. Sheep heat-inactivated serum in phosphate-buffered saline (30% v/v) with 2% BSA at 25 °C for 30 min was used to block nonspecific binding. Sperm were incubated in primary antibody 1:500 monoclonal anti-PAF receptor (Alexis Corp., San Diego, CA) at 25 °C for 24 h, followed by fluorescein isothiocyanate-labeled anti-mouse IgG (Zymed Laboratories Inc. Laboratories, San Francisco, CA) at 25 °C for 1 h. Embryos were viewed with an epifluorescent microscope (Nikon). Photographs were taken with Kodak Tri-X pan 400 print film (Eastman Kodak Co.), using the same exposure conditions for all images. Each experiment incorporated several negative control treatments as follows: incubation of sperm in non-immune IgG (Southern Biotechnology Associates, Birmingham, Alabama); no primary antibody; no secondary antibody; and non-fluorescent secondary antibody.

Assessment of Sperm-hyperactivated Motility Using a Chemotaxis Assay-- Changes in the motility of sperm were assessed by their ability to migrate through a polycarbonate filter containing 8-µm pores (Nucleopore) as described previously (37). The lower wells of a 48-well microchemotaxis chamber (Neuroprobe AP48, Neuroprobe, Cabin John, MD) were filled with a swim-up preparation of mouse or human sperm. The concentration of sperm used is indicated for each experiment. Sperm were treated with putative capacitation factors and/or inhibitors. The upper wells were separated from the lower ones by the polycarbonate filter and were filled with medium containing the desired treatments. The chambers were incubated for 15-30 min at 37 °C, and the sperm accumulating in the upper chamber were assessed by direct counting. This was achieved by placing over the top of the upper well a slide that was pre-coated with a solution of 0.01% (w/v) polylysine and 0.5% (w/v) spermidine. The chamber was then centrifuged upside down at 100 × g for 10 min at room temperature. The sperm in the upper chambers adhering to the coated slide were counted by light microscopy.

Assessment of Acrosome Reaction-- The status of the acrosome was assessed using the Coomassie Blue G-250 staining method (38). Briefly, sperm were fixed with 4% (w/v) formaldehyde for 10 min, washed three times with 100 mM ammonium acetate buffer, pH 9.0, and stained with 0.2% (w/v) Coomassie Blue (Bio-Rad). The intact acrosome stained an intense dark blue. A minimum of 100 sperm was scored per sperm smear.

In Vitro Fertilization (IVF)-- IVF was performed as described previously (39). Females were superovulated by pregnant mare serum gonadotrophin followed 48 h later by human chorionic gonadotrophin (for each gonadotrophin the following doses were given: 10 IU outbred, 5 IU C57b1/6, and PAF receptor KO). Oocytes were collected 13-15 h after human chorionic gonadotrophin and washed. Epididymal sperm were added according to the description for each experiment. Fertilization was performed in a final volume of 1 ml of medium. Putative capacitation factors and/or inhibitors were added to the fertilization medium as indicated in the relevant experiments.

Oocytes and sperm were cultured together for 5 h at 37 °C in 5% CO₂ in air. The oocytes were then retrieved, washed in Hepes-buffered HTF, and their fertilization status assessed by microscopic detection of pronuclei and polar bodies. Fertilized oocytes were transferred to 10-µl drops of modified HTF medium under mineral oil, and their development status was assessed each 24 h for 120 h.

Statistical Analysis-- All analyses were performed using SPSS version 9.0. Differences in the PAF release, enzyme activity, and sperm motility were assessed by analysis of variance. Differences in fertilization rate and the acrosome reaction were assessed using logistic regression analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sperm Release PAF after Incubation in Capacitation Medium-- Freshly collected mouse epididymal sperm contained significant quantities of PAF (74.1 + 26.1 pmol PAF/10⁵ sperm). Following incubation in culture medium for 1 h, the amount of PAF associated with sperm was similar, 77.0 ± 34.8 pmol of PAF/10⁵ sperm, whereas an additional 117.2 ± 13.0 pmol of PAF/10⁵ sperm was found released into the culture medium. After 100 min in capacitation media, the concentration of PAF in both sperm and the culture media increased to 152.3 ± 47.4 pmol/10⁵ sperm and 103.0 ± 20.5 pmol/ml media, respectively. In the nominal absence of albumin from medium, the amount of PAF found in capacitation medium following 60 min of incubation was significantly less than in the presence of albumin (p < 0.01), being 34.0 ± 17.9 pmol released from 10⁵ sperm. The results are the mean ± S.E. of three separate replicates.

Sperm Possess a PAF Receptor and PAF Processing Enzymes-- RT-PCR demonstrated the presence of mRNA within human and mouse sperm that coded for the G-protein-linked PAF receptor (Fig. 1). There was also present mRNA coding for the ₂- and -subunits of intracellular PAF:acetylhydrolase 1b enzyme and the macrophage/plasma-type PAF:acetylhydrolase (Fig. 1). In all cases the homology of the RT-PCR product with the genes of interest was confirmed by sequence analysis. Since sperm were individually picked off from a swim-up preparation, it can be concluded that the mRNA was unequivocally from sperm.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Representative expression pattern of mRNA transcripts detected by RT-PCR for PAF receptor, PAF:acetylhydrolase 1b (Pafah) 2- and -subunit, and plasma (MP) form of PAF:acetylhydrolase in human and mouse sperm. M, molecular weight size markers (Phil × 174 DNA/Hea III); mo, mouse sperm; hu, human sperm; tis, tissue-positive control; neg, negative control.

Labeling of sperm with a specific antibody for the G-protein-linked PAF receptor clearly showed the presence of this receptor on both human and mouse spermatozoa (Fig. 2, i and ii), whereas mouse sperm from a PAF receptor knock-out strain lacked the receptor (Fig. 2iii). For both species, staining was heaviest in the post acrosomal region of the sperm head and in the mid-piece, being particularly evident in the mid-piece of mouse sperm.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Expression of PAF receptor protein in human and mouse spermatozoa. Sperm were stained by indirect immunofluorescence using an antibody to PAF receptor. i, human sperm; ii, mouse sperm, wild-type; and iii, sperm from PAF receptor KO mouse.

Treatment of Sperm with Exogenous PAF-changed Sperm Motility-- The treatment of sperm with PAF caused an increase in the migration of both human and mouse sperm through a membrane with 8-µm pores (Fig. 3). Human sperm showed a significant quadratic dose-response (p < 0.0001) with a significant enhancement reaching a peak of 18 nmol PAF/liter but declining at 18 µmol/liter PAF (p < 0.001). In mice, PAF enhanced migration across the concentration range 18 nmol/liter to 18 µmol/liter (p < 0.001).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The effect of increasing concentrations of PAF on the migration of human (A) and mouse (B) sperm through an 8-µm pore membrane. The results are the mean ± S.E. of five independent replicates. Sperm were at a concentration of 105/ml.

The specificity of the actions of PAF was assessed by determining whether a PAF receptor antagonist (Fig. 4) could inhibit the effect on sperm migration. The sperm were treated with PAF (1.8 µmol/liter) and increasing concentrations of the selective PAF receptor antagonist, UR-12519. For both mouse and human sperm, PAF induced a significant increase in sperm migration confirming the results of the previous experiment. UR-12519 caused a dose-dependent inhibition of this enhanced migration. At a concentration of 0.23-2.3 µmol/liter UR-12519, the migration was not different to that of the negative control (p > 0.05). The results confirm that exogenous PAF acts in a specific fashion to induce sperm motility changes associated with capacitation.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The specificity of the action of PAF on sperm migration was assessed by performing a dose-response of the PAF receptor antagonist UR-12519 for human (A) and mouse (B) sperm. Control sperm were not treated with either PAF or UR-12519. The lines are the first-order regression line.

Endogenous PAF Enhanced Sperm Motility/Migration-- The presence on sperm of PAF receptors and the release of PAF by sperm creates the conditions for an autocrine PAF loop. To assess whether there is a role for endogenous PAF, the effects of UR-12519 and exogenous rPAF:acetylhydrolase on sperm motility in the chemotaxis device were measured. A sperm concentration 10-fold higher was used to accentuate any effect of endogenous PAF. In both mouse (p < 0.01) and human sperm (p < 0.0001), UR-12519 cause dose-dependent inhibition of sperm migration in the absence of exogenous PAF (Fig. 5). There was no adverse effect of this drug on human sperm viability assessed by the vital staining with Eosin-Nigrosin or the proportion of sperm that were motile (76% control compared with 71% UR-12519).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   To determine whether the PAF antagonist UR-12519 exerted an effect on sperm-derived PAF, the effect of increasing doses of UR-12519 on the spontaneous rate of sperm migration across an 8-µm membrane was assessed. The results are the mean ± S.E. of five independent experiments at a sperm concentration of 106/ml.

The addition of rPAF:acetylhydrolase also caused a significant (p < 0.0001) dose-dependent inhibition of spontaneous migration of human and mouse sperm across the membrane (Fig. 6). These effects occurred in the absence of exogenous PAF, and it is therefore assumed that the enzyme blocked the actions of endogenous sperm-derived PAF by degrading it to inactive lyso-PAF.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   The effect of increasing concentrations of rPAF:acetylhydrolase on the migration of human and mouse sperm across an 8-µm membrane. The results are the mean ± S.E. of five independent experiments at a sperm concentration of 106/ml.

Sperm Lacking the PAF Receptor Had a Reduced Incidence of Spontaneous Acrosome Reaction-- Following the dilution of mouse sperm in defined medium in the presence of albumin, capacitation occurs in a time-dependent manner resulting in a spontaneous onset of the acrosome reaction. The role of PAF in this phenomenon was assessed by determining the rate of onset of the acrosome reaction in sperm collected from PAF receptor KO mice compared with wild-type controls. Fig. 7 shows that from a similar base line the rate and extent of onset of the acrosome reaction was less (p < 0.001) in KO mice compared with C57BL/6 controls. The results show that sperm-derived PAF causes the induction of the spontaneous capacitation and acrosome reaction via the G-protein-linked PAF receptor, and the absence of this receptor results in a marked delay in the spontaneous onset of the acrosome reaction.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   The incidence of spontaneous acrosome reaction in sperm from PAF receptor KO mice (squares) compared with control wild-type (circles) sperm. The acrosomal status was assessed at various times after dilution of sperm in HTF medium containing 3 mg of BSA/ml. The results are the mean ± S.E. of three independent experiments at a sperm concentration of 5 × 105/ml.

PAF Activity Was Required for Normal Fertilization-- The definitive assessment of capacitation is the capability of sperm to fertilize the egg. Fertilization was performed in vitro in the presence of exogenous rPAF:acetylhydrolase (Table I). Low concentrations (1 µg/ml) of the enzyme had no significant effect on fertilization, but 100 µg/ml PAF:acetylhydrolase significantly reduced the fertilization rate (p < 0.01), and it was further reduced at 200 µg/ml (p < 0.01). When rPAF:acetylhydrolase was maintained in the subsequent embryo culture medium, it also had an adverse impact on embryo development so that even in embryos that had fertilized, there was a significant further reduction in the rates of embryo development. At 200 µg of rPAF:acetylhydrolase/ml, significantly fewer fertilized zygotes cleaved to the 2-cell stage, and only a small number of these (8%) developed enough to be morphologically normal blastocysts (Table I). The inhibitory action of the enzyme on the fertilization rate was independent of sperm concentration (Fig. 8A), being consistent with a catalytic rather than competitive mode of action.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   The effect of rPAF:acetylhydrolase (100 µg/ml) (A) and UR-12519 (23 µM) (B) on the rate of fertilization in vitro at increasing concentrations of sperm. Each column represents the fertilization rate of a minimum of 100 oocytes.

The effect of PAF:acetylhydrolase was dependent upon normal enzymatic activity of the protein. Boiling the enzyme preparation for 5 min caused greater than 90% loss of enzyme activity. Boiling the enzyme caused the fertilization rate to increase to 59% (n = 92) from 38% (n = 94) (p < 0.01) for the unboiled enzyme. Following boiling the fertilization rate was not different (p > 0.05) from vehicle control (n = 96).

The PAF receptor antagonist UR-12519 also inhibited fertilization (p < 0.01) at a concentration of 23 µmol/liter (Table II) but had no effect at 0.23 µmol/liter. Continued exposure of embryos to UR-12519 also reduced the rate of embryo development to the blastocyst stage. There was a significant (p < 0.01) inverse effect of sperm concentration on the inhibitory action of UR-12519; at higher sperm concentrations the antagonist was less effective (Fig. 8B). This result is consistent with the drug acting as a competitive antagonist.

PAF:Acetylhydrolase in Seminal Plasma Acts as a Decapacitation Factor-- Human seminal plasma was collected by allowing the fresh ejaculate of a fertile male to liquefy for 40 min at 37 °C. Cells were removed by centrifuging at 2000 × g for 5 min, and the supernatant was collected and assayed for PAF:acetylhydrolase activity. Activity was 53.8 ± 7.3 pmol released acetate/mg of protein/min (n = 4), compared with a level of 383.0 ± 18.7 in blood plasma from the same subject. To confirm the acid lability of PAF:acetylhydrolase (40), seminal plasma was treated at pH 3.0 for 30 min at 37 °C (by titration with 1 N HCl), and this degraded PAF:acetylhydrolase activity to undetectable levels. Acid-treated seminal plasma was neutralized by treatment with an equimolar volume of 1 N NaOH.

Seminal plasma and acid-treated/neutralized seminal plasma was diluted in HTF medium to a concentration of 5% (v/v). These media were used for sperm preparation and IVF in outbred mice. The presence of untreated seminal plasma (5% v/v) reduced the fertilization rate from 71% (n = 100) in control to 45% (n = 95) (p < 0.001). The acid-treated seminal fluid gave significantly greater fertilization rates, 63% (n = 97) (p < 0.05), than did the untreated seminal fluid and was not different (p > 0.05) from untreated controls. The results show that PAF:acetylhydrolase in seminal plasma was acid-labile and that an acid-labile activity within seminal fluid accounts for much of the decapacitation factor activity of that fluid.

Capacitation of Sperm Is Defective in Mouse Sperm Lacking the PAF Receptor-- The ability of sperm lacking the PAF receptor to fertilize oocytes was tested by IVF. Wild-type and PAF receptor KO females had ovulation induced with gonadotrophins, and both gave similar rates of oocyte recovery (12.1 ± 3.1 oocytes/female PAF receptor KO compared with 14.1 ± 3.0 for wild-type; mean ± S.E., p > 0.05). When wild-type oocytes were fertilized with wild-type sperm the fertilization rate was 66.7% (n = 60; 3 replicates) compared with 21.5% (n = 65; 3 replicates) for a cross between PAF receptor KO male and female mice (p < 0.0001).

To determine if this poor fertilization rate was due to the failure of PAF signaling in sperm or in oocytes, sperm from KO males were used to fertilize oocytes from either wild-type or KO females. An equivalent reduction in fertilization rate of both wild-type, 40% (n = 60), and PAF receptor KO oocytes, 31% (n = 35) (p > 0.05), was observed. By contrast, PAF receptor KO oocytes showed a normal incidence of fertilization by wild-type sperm, 74% (n = 35). It was also shown that sperm from outbred mice gave normal rates of IVF with PAF receptor KO oocytes (results not shown). The results are consistent with the hypothesis that PAF released from sperm acts in an autocrine manner to induce fertilization in vitro.

In contrast with these results for in vitro fertilization, there was no apparent defect in fertilization for a cross between PAF receptor KO male and female mice when fertilization occurred in the reproductive tract. Following natural mating of females the fertilization rate of eggs collected from the oviduct 12-15 h after mating was 83% (n = 55) for KO × KO crosses and 89% (n = 43) for wild-type × wild-type crosses (p > 0.05). When eggs were collected from naturally mated females that had been superovulated with gonadotrophins, there was also no significant difference (p > 0.05) in the fertilization rate for KO × KO crosses (80%, n = 91) compared with wild-type crosses (84%, n = 102).

These contradictory results might be explicable if there are, within the environment of the reproductive tract, alternative capacitation factors of maternal origin that act in a paracrine or endocrine manner and that are redundant to the autocrine actions of PAF. Many putative capacitation factors of maternal origin have been proposed, and several sources of complex biological fluids are known to act as sources of capacitation factors. To test for the potential redundant actions of paracrine capacitation factors, fertilization medium was supplemented with increasing concentrations of fetal calf serum, and a KO × KO cross by IVF was performed in this medium. There was no significant effect (p > 0.05) at a serum concentration of 0.01% (v/v), but the fertilization rate increased from 25% (n = 200) for controls to 38% (n = 159; p < 0.005) at 0.1% (v/v) and 47% (n = 143; p < 0.0001) at 1% (v/v) fetal calf serum. The results are from three independent replicate experiments and show that within fetal calf serum there are capacitation factor(s) for sperm that acted independently of the G-protein-linked PAF receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mechanisms of induction of sperm capacitation have remained enigmatic. The activation of extracellular signal-regulated kinases in sperm during capacitation (3) infers a role for an extracellular signal in the induction of capacitation. Many paracrine or endocrine candidate capacitation factors have been proposed, yet the characteristic feature of the process is that it can occur spontaneously in the absence of exogenous factors. A current hypothesis is that loss of membrane cholesterol to extracellular albumin leads to the induction of capacitation (2, 4). However, albumin acts as an acceptor for other membrane lipids including PAF (11, 13, 14, 41).

The study confirms that PAF is a product of the sperm cell and shows that it is also released in significant quantities following dilution of sperm in capacitation media, in the presence of extracellular albumin. The amount of PAF produced by sperm is high and may reflect the high concentrations of precursor ether phospholipid within sperm (9). This sperm-derived PAF acted in a receptor-dependent manner to induce capacitation in a fashion analogous to the capacitation induced by exogenous PAF. The absence of the PAF receptor gene from sperm resulted in a marked reduction in the rate of fertilization. It was noteworthy that in the absence of the PAF receptor, capacitation (as assessed by the onset of the spontaneous acrosome reaction) was markedly delayed but did occur. Likewise, the fertilization rate by PAF receptor KO sperm was markedly reduced, but some fertilization did still occur. The occurrence of some capacitation and fertilization by sperm from PAF receptor KO mice in defined medium suggests a role for other endogenous sperm-derived factors that act independently of an autocrine PAF loop. An obvious candidate is the efflux of cholesterol from the sperm membrane in capacitation medium (4). However, the observation that there was still a significant inhibition of fertilization following inhibition of the autocrine PAF loop under conditions that are expected to support membrane cholesterol efflux suggests that the actions of the two pathways are independent, and under these experimental conditions modulation of membrane cholesterol by extracellular albumin does not by itself completely compensate for the absence of the autocrine PAF loop. By contrast, the addition of a complex biological fluid in the form of fetal calf serum could compensate for the absence of a functional autocrine PAF loop for induction of capacitation. This result is consistent with the observations that the fertility (29) and fertilization rate in the reproductive tract in PAF receptor KO mice were not adversely affected. That observation might be explained by the actions of maternally derived paracrine capacitation factors acting within the reproductive tract, with the actions of fetal calf serum mimicking the effect. The fact that the fetal calf serum could partially compensate for the absence of the autocrine PAF loop argues that the actions of the autocrine and paracrine stimulation of capacitation are largely redundant, but shows that stimulation by capacitation factors of either source is required to achieve efficient fertilization. Although rPAF:acetylhydrolase and a PAF antagonist were relatively effective anti-capacitation factors in vitro, they have not been shown to exert such effects when administered to mice around the time of fertilization.² The redundant actions of alternative pathways of capacitation may explain this result.

It was recently demonstrated that exogenous rPAF:acetylhydrolase could block the autocrine action of embryo-derived PAF (42). This required a similar concentration of enzyme as was required to inhibit the autocrine induction of capacitation by PAF. The high concentration of enzyme required to cause the inhibition of autocrine signaling in both sperm and embryos may suggest that the enzyme acts at the lipid interface rather than in the soluble phase. The ability of rPAF:acetylhydrolase to inhibit fertilization implicates this enzyme as a candidate decapacitation factor. Its presence in seminal plasma may be part of the decapacitating activity for that fluid, and it is the major phospholipase A₂ activity in seminal plasma (43). The decapacitation factor activity within seminal plasma has been shown to be associated with a large lipid-dense fraction of seminal plasma that is consistent with the observation that PAF:acetylhydrolase is found associated with the high molecular weight lipoprotein fraction. However, the relative contribution of PAF:acetylhydrolase as a decapacitation factor in seminal plasma requires further characterization. PAF:acetylhydrolase is a ubiquitous enzyme; however, its activity within the reproductive tract is highly dynamic and under the control of estrogen and progesterone (31). The activity in the mouse uterus declines dramatically after ovulation (31). This declining uterine enzyme activity may facilitate the action of autocrine PAF at the site of capacitation.

Thus, a model for the capacitation of sperm would involve the movement of sperm out of seminal plasma via its migration up the reproductive tract. In doing so, sperm leave a cholesterol particle and PAF:acetylhydrolase-rich environment. Albumin is a major protein of the reproductive tract (44); hence sperm migration into this environment will allow sperm to release PAF and lose membrane cholesterol, promoting capacitation. The presence of capacitation factors of maternal origin in the reproductive tract would act in concert with the sperm's autocrine induction of capacitation. Deficiencies in the production of PAF by sperm or the response of sperm to PAF may cause reduced fertility, particularly in in vitro fertilization programs.

	ACKNOWLEDGEMENTS

We thank Brigitte Hermann for technical assistance and Kathryn O'Neill for assistance in preparation of the manuscript. We thank Dr. L. Tjoekler for the generous gift of rRAF:acetylhydrolase and Dr. Manuel Merlos for the kind gift of UR-12519.

	FOOTNOTES

^* This work was supported by a grant from the Australian National Health and Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

^¶ Current address: Dept. of Histology and Embryology, Medical College, Qingdao University, China.

^** To whom all correspondence should be addressed: Human Reproduction Unit, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. Tel.: 61 2 9926 7148; Fax: 61 2 9926 6343; E-mail chriso@med.usyd.edu.au.

Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M103107200

² C. O'Neill, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PAF, phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (platelet-activating factor); KO, knock-out; BSA, bovine serum albumin; RT-PCR, reverse transcriptase-polymerase chain reaction; IVF, in vitro fertilization; rPAF, recombinant PAF.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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