Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 3, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.036095
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
72/3/574    most recent
biolreprod.104.036095v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Furimsky, A.
Right arrow Articles by Tanphaichitr, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Furimsky, A.
Right arrow Articles by Tanphaichitr, N.
Agricola
Right arrow Articles by Furimsky, A.
Right arrow Articles by Tanphaichitr, N.
BIOLOGY OF REPRODUCTION 72, 574–583 (2005)
DOI: 10.1095/biolreprod.104.036095
© 2005 by the Society for the Study of Reproduction, Inc.

Percoll Gradient-Centrifuged Capacitated Mouse Sperm Have Increased Fertilizing Ability and Higher Contents of Sulfogalactosylglycerolipid and Docosahexaenoic Acid-Containing Phosphatidylcholine Compared to Washed Capacitated Mouse Sperm1

Anna Furimsky3,4, Ngoc Vuong3,4, Hongbin Xu3,4, Premkumari Kumarathasan5, Min Xu3, Wattana Weerachatyanukul3, Maroun Bou Khalil3,4, Morris Kates4, and Nongnuj Tanphaichitr2,3,4,6

Hormones/Growth/Development Research Group,3 Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada Department of Biochemistry/Microbiology/Immunology,4 University of Ottawa, Ottawa, Ontario K1H 8M5, Canada Inhalation Toxicology and Aerobiology Section,5 Environment and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada Reproductive Medicine Division,6 Department of Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.

fertilization, gamete biology, in vitro fertilization, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Centrifugation through a gradient of Percoll has been used for more than a decade to prepare motile sperm from human ejaculates, which contain a significant number of immotile sperm cells, for intrauterine insemination and in vitro fertilization [1–4]. On resuspension in capacitating medium containing albumin, 90% or more of Percoll gradient-centrifuged (PGC) sperm possess hyperactivated motility patterns. These PGC capacitated human sperm also are implied to have higher fertilizing ability than the whole population of sperm that simply are washed by centrifugation and capacitated in the same incubation medium [3, 5]. Electron microscopy and atomic force microscopy reveal that PGC sperm uniformly possess normal morphology with complete chromatin condensation and mitochondria formation as well as the "clean" surface, whereas washed human sperm from the whole ejaculate have heterogeneous morphology, with a significant number showing abnormal chromatin and mitochondrial structure as well as membrane vesicles on their surface [5, 6]. Percoll gradient centrifugation also has been used to prepare motile sperm from rodents [7–10], although its application in these species is at a lesser extent than that in humans. However, the superiority of PGC sperm to washed sperm in fertilization has not been demonstrated in rodents. In mice, the difference in the ultrastructure between PGC capacitated sperm and washed capacitated sperm is the same as that observed in humans [9]. Therefore, PGC capacitated mouse sperm likely have higher fertilizing ability than washed capacitated mouse sperm. If this is the case, then PGC capacitated sperm should be used for fertilization-related studies/work in rodents. Because mice are widely used experimental models for fertilization research and in vivo transgenesis, the question of whether PGC capacitated mouse sperm have higher fertilizing ability than washed capacitated sperm should be answered immediately, and this was one of the objectives of the present study.

To date, information regarding differences in biochemical properties between PGC capacitated sperm and washed capacitated sperm has been limited. This information would shed light on how sperm gain higher fertilizing ability. Because PGC sperm differ from washed sperm in their subcellular structure, as evidenced by the absence of coating membrane vesicles in the former [5, 6, 9], we decided to direct our efforts at characterizing lipid profiles in both sperm types. Accumulated evidence reveals that composition and changes of sperm lipids can regulate sperm quality and functions [11]. Contents of major lipids, cholesterol, and phospholipids are much lower in PGC sperm than in washed sperm in both human and mouse species [12–14]. These results corroborate the absence of membrane vesicles on the PGC sperm surface. However, other lipid classes and subclasses also may differ between PGC sperm and washed sperm. Unique to mammalian sperm and testicular germ cells is the selective existence of the phospholipid molecular species with C22-highly unsaturated fatty acyl chains [15–24]. The contents of C22-highly unsaturated fatty acids (HUFAs)-containing phospholipids appear to correlate with mammalian sperm maturity and motility [15, 19, 21, 25, 26]. These phospholipids have low phase-transition temperatures [15] and contribute to high membrane fluidity [27], which is required for hyperactivated motility and fusion-related events during gamete interaction (including the zona pellucida [ZP]-induced acrosome reaction and gamete plasma membrane fusion). Direct correlation between the contents of C22-HUFA-containing phospholipids and fertilizing ability of PGC sperm, however, has yet to be demonstrated.

Sulfogalactosylglycerolipid (SGG; also known as seminolipid) is another lipid that exists selectively in mammalian sperm and male germ cells [28–30]. In contrast to HUFA, SGG is an ordered lipid [3, 31]. It possesses ZP-binding ability [32, 33], and the majority of SGG on the sperm surface is present in the sperm lipid rafts, which have ZP-binding ability (unpublished results). It therefore is possible that PGC capacitated sperm also have a higher content of SGG than washed capacitated sperm have. This would allow PGC capacitated sperm to bind to the egg ZP with a higher efficiency. In the present study, we have shown, to our knowledge for the first time, that the contents of both SGG and docosahexaenoic acid (DHA; C22:6n-3)-containing phospholipids are higher in mouse PGC capacitated sperm than in washed capacitated sperm. These findings would explain, in part, the higher fertilizing ability of PGC capacitated sperm, which also is reported herein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Washed Capacitated and PGC Capacitated Mouse Sperm

Male CD-1 mice (age, 10–12 wk; Charles River Canada, St-Constant, QC) were boarded in a temperature-controlled room (22°C) with a 12L: 12D photoperiod. They were fed ad libitum with Purina 5075 Rodent Chow (Charles River Canada) and water. In all experiments described herein, these mice were fasted overnight and killed the following day by cervical dislocation. Krebs Ringer bicarbonate medium buffered with Hepes (KRB-Hepes) or with NaHCO3 (KRB) was used. The KRB-Hepes consisted of 119.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 4 mM NaHCO3, 21 mM Hepes, 1 mM sodium pyruvate, 25 mM sodium lactate, 5.6 mM glucose, 1 U/ml of penicillin G, 1 µg/ml of streptomycin sulfate, and 28 µM phenol red at pH 7.4. The KRB consisted of the same components as in KRB-Hepes, except that 25 mM NaHCO3 was used in place of 21 mM Hepes and 4 mM NaHCO3. One of these media was employed in all sperm preparations except when sperm were subjected to in vitro fertilization experiments, in which case KSOM medium was used. The KSOM medium consisted of 95.0 mM NaCl, 2.5 mM KCl, 1.7 mM CaCl2, 0.35 mM KH2PO4, 0.2 mM Mg2SO4, 25 mM NaHCO3, 0.2 mM sodium pyruvate, 10 mM sodium lactate, 0.2 mM glucose, 0.01 mM sodium EDTA, 1.0 mM L-glutamine, 1 U/ml of penicillin G, 1 µg/ml of streptomycin sulfate, and 28 µM phenol red at pH 7.4. Following our previously described method [30], sperm were collected from the cauda epididymis and vas deferens into 1 ml of KRB-Hepes (prewarmed to 37°C), with the precaution of removing of fat pads surrounding the tissues before sperm collection. Sperm from the same mice were divided into two halves, each for the preparation of washed capacitated sperm and PGC capacitated sperm samples.

To prepare washed capacitated sperm, the collected sperm were centrifuged (430 x g, 10 min, 25°C), and the sperm pellet was resuspended in KRB supplemented with 0.3% BSA (KRB-BSA; fraction V, embryo tested, catalog no. A-3311; Sigma, St. Louis, MO) to a concentration of approximately 10 million sperm/ml. Following incubation (30 min, 37°C, 5% CO2), the sperm suspension was centrifuged (430 x g, 10 min, 25°C). The BSA-containing supernatant devoid of sperm cells was removed, and the sperm pellet was resuspended in KRB to a concentration of 5–10 million sperm/ml.

To prepare PGC capacitated sperm, the sperm suspension, collected as described above, was diluted to 2 ml with KRB-Hepes and loaded onto a two-step Percoll gradient (45% and 90% Percoll [Amersham Biosciences, Uppsala, Sweden] in KRB-Hepes) [9]. The gradient was centrifuged (650 x g, 30 min, 25°C), allowing motile sperm to sediment as a pellet and immotile sperm to interface between the two Percoll layers. After removing the interfaced immotile sperm and 45% and 90% Percoll solutions, the PGC motile sperm pellet was washed once in KRB-Hepes (430 x g, 10 min, 25°C) and then capacitated in KRB-BSA (30 min, 37°C, 5% CO2) at a concentration of approximately 10 million sperm/ml. The capacitated sperm were then centrifuged (430 x g, 10 min, 25°C), and the PGC capacitated sperm pellet was resuspended in KRB to a concentration of 5– 10 million sperm/ml.

Two 200-µl aliquots were removed from both washed capacitated sperm and PGC capacitated sperm for the DNA assay and sperm counting. The remainder was placed in a glass vial, flushed with a stream of N2, and capped tightly. Alternatively, antioxidants (0.02 M diethylenetriaminepentaacetic acid and 0.06 M butylatedhydroxytoluene) were added to the sperm suspension in the vial before capping. These sperm suspension vials were stored at –20°C for subsequent sperm lipid extraction (see below). For cholesterol and phospholipid quantification, lipids extracted from the sperm vial that was flushed with N2 were used, whereas for other lipid analyses, including gas chromatography/mass spectrometry (GC/MS) of phospholipid fatty acyl chains (see below), lipids were extracted from sperm stored in the presence of antioxidants.

All experiments involving the use of mice adhered to the Canadian Council on Animal Care guidelines and were reviewed and approved by the Animal Care Committee of the Ottawa Health Research Institute.

Sperm Motility and Viability

Motility of washed capacitated and PGC capacitated sperm was estimated by placing a 10-µl drop of the sperm suspension in KRB-BSA (containing ~5 million sperm/ml) onto a glass slide, which was then viewed under an inverted Nikon TMS microscope (Nikon Canada, Inc., Mississauga, ON) at 100x magnification. To assess sperm viability, the sperm samples were treated with 0.5 µg/ml of propidium iodide at 37°C under 5% CO2 for 5 min. A drop of the sperm samples was placed onto a glass slide, topped with a coverslip, and viewed under a Zeiss IM35 Microscope (Zeiss Canada, Toronto, ON) using a rhodamine filter. Sperm that excluded the fluorescent dye, propidium iodide, were scored as viable.

In Vitro Sperm-ZP Binding

Washed capacitated and PGC capacitated sperm were evaluated for their ability to bind to cumulus-free, ZP-intact mature eggs (prepared as described by Hogan et al. [34]) using the in vitro sperm-ZP binding assay [30]. Briefly, 60 000 motile capacitated sperm (washed or PGC) were incubated (37°C, 5% CO2, 30 min) with 20–25 eggs in a 60-µl droplet of KRB-BSA. Because washed capacitated sperm consistently had lower motility (70%–80%) than PGC capacitated sperm (90%–100%), the total number of gametes (motile + immotile) added to the egg drop of the washed sample always was higher than that of the PGC sample (~75 000– 86 000 sperm for the washed sample vs. 60 000–67 000 sperm for the PGC sample). Following washing of the loosely bound sperm by pipetting the sperm-egg complexes through a drawn Pasteur pipette (bore diameter, ~250 µm), the number of sperm bound per egg was counted under an inverted microscope at 200x magnification. Student t-test was used to analyze significant differences in the numbers of sperm bound per egg between the washed capacitated and PGC capacitated sperm samples in each experiment as well as the average data from the mean values of these numbers obtained from three experimental days.

In Vitro Fertilization

Approximately 500 000 motile sperm of the washed and PGC sperm samples, each precapacitated in KSOM supplemented with 0.3% BSA (KSOM-BSA) for 1 h, were incubated (37°C, 5% CO2) in 500 µl of KSOM-BSA with 20–25 cumulus-free, ZP-intact mature eggs (prepared as described previously [34]). After 8 h of gamete coincubation, eggs were assessed for their fertilization status under a Nikon Diaphot inverted phase-contrast microscope at 400x magnification. Fertilized eggs were those containing two pronuclei. Data were expressed as percentages of total eggs fertilized in the coincubation drop. To assess the level of parthenogenesis, eggs were incubated under the same conditions but without sperm addition. This level was consistently 0%. The Student t-test was used to analyze significant difference in the average data for the percentage of eggs fertilized by washed capacitated sperm versus PGC capacitated sperm on three experimental days.

Analysis of Noncapacitated, Capacitated, and Acrosome-Reacted Sperm by Chlortetracycline Staining

Chlortetracycline (CTC) staining of mouse sperm was done according to the method described by Lee and Storey [35] with minor modifications. The CTC working solution (20 mM Tris, 130 mM NaCl, 5 mM cysteine, 750 µM CTC, pH 7.8) was prepared fresh on each experimental day and stored light-protected at 4°C until use. Equal volumes of the PGC capacitated or washed capacitated sperm sample (~100 µl, 5 million sperm/ml) and prewarmed (37°C) CTC solution were mixed in a light-protected Eppendorf tube. To this mixture, 12.5% glutaraldehyde in 1 M Tris buffer (pH 7.8) was added to give a final concentration of 0.1%. An aliquot (~8 µl) of this sperm suspension was then placed onto a glass slide and topped with a coverslip. The slides were examined within 2 h under a Zeiss i.m. 35 Microscope with a BP 400-440 excitation filter and an LP 470K barrier filter. At least 200 sperm on each slide were assessed for the noncapacitated, capacitated, intermediate (between capacitated and acrosome-reacted), and acrosome-reacted patterns as described previously [35]. Experiments were repeated three times on different days. Significant differences of each CTC staining pattern between the PGC capacitated sperm sample and the washed capacitated sperm sample were analyzed by ANOVA.

Immunoblotting of Sperm Tyrosine Phosphorylated Proteins

One million sperm from the PGC capacitated or washed capacitated sperm sample were pelleted by centrifugation (5000 x g, 1 min, room temperature) in a Heraeus Biofuge 15 microcentrifuge (Heraeus Instruments, Stratos, Germany), washed in 1 ml of PBS, resuspended in 30 µl of less of SDS-PAGE sample buffer [36] without 2-mercaptoethanol, and boiled for 5 min. After centrifuging at 5000 x g for 3 min, the collected supernatant was treated with 2.5% 2-mercaptoethanol, boiled for 5 min, and then subjected to SDS-PAGE [36], followed by immunoblotting [37] with 0.5 µg/ml of mouse monoclonal antiphosphotyrosine immunoglobulin G (4G10; Upstate Biotechnology, Lake Placid, NY). Antigen-antibody recognition was then probed using secondary horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody (Bio-Rad, Hercules, CA) at 1:5000 dilution and an enhanced chemiluminescence Western blot detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). Tris-buffered saline (20 mM Tris-HCl, 137 mM NaCl, pH 7.4) containing 5% nonfat powdered milk was used to dilute antibodies as well as to block the nitrocellulose membrane for nonspecific binding with the antibodies.

Analysis of Sperm DNA

Sperm DNA was quantified by a modification of the method outlined by Labarca and Paigen [38] as previously described [12], which was based on the binding of Hoechst 33258 to DNA of live cells treated with a high salt concentration (2 M NaCl) to allow separation of DNA strands and, thus, intercalation of the Hoechst dye. Specifically for sperm cells, 100 mM dithiothreitol (DTT) was added to the sperm suspension to decondense the highly compact sperm chromatin by disrupting protamine disulfide bonds as well as to minimize the adherence of sperm to the surface of the assay tubes. Calf thymus DNA (Sigma) was used as a standard. All sperm DNA samples were assayed in duplicate. The sensitivity of the assay was 1 µg of DNA [12].

Extraction of Sperm Lipids

Lipids were extracted from all sperm samples following a modification of the method outlined by Bligh and Dyer [39] as described by Kates [40]. The details of this procedure have been described previously for sperm [12, 13].

High-Performance Thin-Layer Chromatography of Sperm Lipids

High-performance thin-layer chromatography (HPTLC) HPK silica gel 60-Å plates (particle size, 5 µm; 10 x 10 cm; thickness, 200 µm; Whatman, Kent, U.K.) were prewashed by ascending thin-layer chromatography (TLC) in chloroform/methanol (1:1, v/v) and activated at 100°C for 1 h. Total sperm lipids (~50 µg) in chloroform were applied as a thin band (width, 0.5 cm) onto the plate along with authentic lipid standards (phosphatidylcholine [PC; porcine liver], phosphatidylethanoloamine [PE; porcine liver], sphingomyelin [SM; bovine brain], cardiolipin [bovine heart], cholesterol [bovine brain], SGG [ram testis], galactosylglycerolipid [GG; ram testis], diacylglycerol [DAG; 16:0/16:0], and triacylglycerol [TAG; 16:0/18:0/16:0]). All phospholipid standards and cholesterol were purchased from Doosan Serdary Research Laboratories (Englewood Cliffs, NJ), and the mole amounts of phospholipid standards were quantified using the phosphorus assay described below. The DAG (16:0/16:0) and TAG (16:0/18:0/16:0) were obtained from Sigma. The SGG was prepared in our laboratory as described previously [41], and GG was then generated by acid desulfation of SGG [42]. For separation of sperm phospholipids, SGG, and cholesterol, the HPTLC plate was developed by ascending chromatography (35 min, 25°C) in chloroform/methanol/H2O (65:32:4, v/v/v) [32, 33]. For separation of neutral lipids (cholesterol, diglyceride [DG], and triglyceride [TG]), a developing solvent consisting of petroleum ether/ ethyl ether/acetic acid (80:20:1, v/v/v) was used [40]. The plate was then air-dried and stained (30 min, 25°C) with 0.03% Coomassie brilliant blue G-250 in 30% methanol plus 100 mM NaCl with gentle agitation. All lipids were stained blue. The plate was destained (5 min, 25°C) with 30% methanol in 100 mM NaCl.

Densitometric analysis using ScionImage software for Windows (Scion Corporation, Frederick, MD) was performed on the Coomassie brilliant blue G250-stained HPTLC plate to determine the mole amounts of DG and TG in the isolated sperm lipids as well as the distribution of each phospholipid subclass [43]. Because these lipid classes and phospholipid subclasses of the same mole amounts were not stained equally with Coomassie brilliant blue G-250, a standard curve was constructed for each lipid by plotting the analyzed optical density values of the lipid bands versus the lipid amounts (in moles). For DG and TG, DAG (16:0/16:0) and TAG (16:0/18:0/16:0) were used to construct the standard curves, and the mole amounts were calculated from the weighed lipids using their known molecular weights. The mole amounts of all phospholipid standards were determined from the phosphate assay (see below).

Quantitation of Cholesterol, Phospholipids, and SGG

Cholesterol in extracted sperm lipids was quantified by the modified fluorometric method described by Gamble et al. [44], which used cholesterol oxidase, peroxidase, and p-hydroxyphenylacetic acid. Cholesterol was oxidized first by cholesterol oxidase to 4-cholestene-3-one and H2O2. The H2O2 and p-hydroxyphenylacetic acid then served as substrates of peroxidase to generate a stable fluorescence product. In the present study, the assay volume was reduced so that the assay could be performed in a microtiter plate. Briefly, sperm lipids in chloroform were dried under N2 and then resuspended in methanol. An aliquot (10 µl) of this solution containing lipids extracted from 0.2 to 0.5 million sperm was transferred into the well of a black 96-well plate (Corning, Inc., Corning, NY), and 100 µl of the reaction mixture were added to each well. The reaction mixture was made by combining 4 ml of 0.1 M potassium phosphate buffer (pH 7.4) with 1 ml of cholesterol oxidase (1 U/ml; Sigma), 1 ml of horseradish peroxidase (10 U/ml; Sigma), 0.5 ml of 0.5% Triton X-100, 0.5 ml of sodium cholate (8.6 mg/ml), and 1.5 ml of p-hydroxyphenylacetic acid (6 mg/ml); all reagents were made up in 0.1 M potassium phosphate buffer (pH 7.4). After incubation of the plate for 30 min at 37°C followed by 15 min at room temperature, the fluorescence intensity of the reaction product was measured at the excitation and emission wavelengths of 325 and 415 nm, respectively, using a SpectraMAX GeminiXS (Molecular Devices, Sunnyvale, CA). All sperm lipid samples were assayed in triplicate. The sensitivity of the assay was 0.05 µg.

Phospholipids were quantified according to the method described by Duck-Chong et al. [45] as modified by Tanphaichitr et al. [12]. The assay was based on phospholipid digestion by magnesium nitrate at high temperature to inorganic phosphate followed by its color reaction with molybdate and malachite green. An aliquot of sperm lipids extracted from approximately 0.5 million sperm was used for each assay tube. The amount of inorganic phosphorus was quantified from a standard curve of KH2PO4 (0–0.1 µg of phosphorus) that was treated in the same way as the samples. All sperm lipid samples were assayed in triplicate. The sensitivity of the assay was 0.02 µg.

The only sulfolipid in mouse sperm [9], SGG was quantified according to the method described by Kean [46] as modified by Weerachatyanukul et al. [33]. This quantification was based on the formation of a color complex of SGG with cationic Azure A dye (Sigma). An aliquot of sperm lipid in chloroform, extracted from approximately 20 million sperm, was used for each assay point. A standard curve was constructed using SGG purified from ram testis [41]. All sperm lipid samples were assayed in duplicate. The sensitivity of the assay was 3 µg.

Analyses of Fatty Acid Methyl Esters and Dimethyl Acetals Generated from PC Phosphatidylethanolamine and Fatty Acid Methyl Esters Generated from SM

Lipids extracted from washed sperm, washed capacitated, and PGC capacitated sperm were subjected to preparative TLC (using a K6 silica gel 600-Å plate; particle size, 10 µm; thickness, 250 µm; 20 x 20 cm; Whatman) following the protocols for plate treatment and solvent development described above for HPTLC. However, the extracted lipid was applied to the TLC plate as a thin, 15-cm band. Standard PC, PE, and SM also were applied as a thin, short band. The plate was developed for 90 min at 25°C in a solvent system consisting of chloroform/methanol/acetic acid/water (50:37.5:3.5:2, v/v/v/v) [47], which gave the best separation of all phospholipid subclasses from each other and from SGG (compared to the two other solvent systems described above for HPTLC). Individual phospholipid classes were located by staining the plate in Rhodamine 6G (Sigma) solution (0.005% in water). The PC and PE bands were scraped gently from the plate. The scraped silica powder was directly treated (95°C, 4 h in a closed tube) with 0.6 N methanolic HCl [40]. This treatment hydrolyzed the ester bonds of glycerophospholipids, generating fatty acid methyl esters (FAMEs). Mammalian sperm also contain an appreciable amount of plasmalogens [48]. The sn-1 chain of plasmalogens is an alkyl-1-enyl ether linkage, whereas the sn-2 chain possesses a fatty acyl chain. The acid methanolysis procedure releases a FAME from the sn-2 chain and a fatty aldehyde dimethylacetal (DMA) from the sn-1 chain from a plasmalogen [40]. However, this 0.6 N methanolic HCl treatment is not sufficient to hydrolyze the N-acyl fatty acid chain in SM; more drastic conditions (2 N methanolic-HCl at 95°C) must be used [40]. After the methanolysis reaction, the tube was placed at –20°C for 30 min, followed by addition of a one-tenth volume of cold water. The mixture was then extracted twice with a volume of cold petroleum ether (4°C) equal to that of the methanol, and the tube was centrifuged (300 x g, 1 min, 4°C) to separate the methanol/water and petroleum ether phases. The latter phase, containing FAMEs and DMAs, was dried under a stream of N2. These dried FAME and DMA samples were reconstituted in toluene for analyses by GC/MS.

The C16:0 DMA and C18:0 DMA standards were prepared from palmitaldehyde and stearic aldehyde (both from K & K Laboratories, Inc., Carlsbad, CA) as described above for the methanolysis of phospholipids. The dried DMAs were then weighed, reconstituted in toluene, and subjected to GC/MS.

Gas Chromatography/Mass Spectrometry

A Varian 3400 gas chromatograph equipped with an autosampler (CTC Model A-200S) was operated using the Magnum software system 2.40 (Varian, San Jose, CA). The GC column was a DB-5 narrow-bore capillary column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.1 µm). The GC injection port, transfer line, and detector manifold temperatures were 270, 280, and 300°C, respectively. The linear velocity of the helium carrier gas was 28.57 cm/sec. The column temperature initially was held at 80°C for 1 min and then increased at a rate of 10°C/min to 300°C, where it was held for 20 min. Ionization was in the electron-impact mode with an emission current of 10 µA, and the electron multiplier voltage was 1650 V. The mass range scanned was 40–650 atomic mass units with the automatic gain control setting to obtain the total ion spectrum. The scan rate was 1 sec–1. The sperm FAME plus DMA samples (1 µl) prepared in toluene were applied to the GC by automated injection. Linear (R2 = 0.99) calibration curves were established for the FAMEs and DMAs by analysis of solutions of authentic standards. Blanks were analyzed along with samples to detect any cross-contamination between analyses. A FAME mixture "grain" of standards was obtained from Supelco (Bellefonte, PA). The cis-4,7,10,13,16,19-DHA (C22:6n-3) methyl ester and cis-7,10,13,16,19-docosapentaenoic acid (C22:5n-6) methyl ester were purchased from Sigma. Methylarachidonate was from Doosan Serdary Research Laboratories. The C16:0 DMA and C18:0 DMA were prepared as described above and analyzed by GC/MS as described for the sperm lipid samples. Molecular ions (M/e) of the DMA generated from palmitaldehyde were 255 (weak peak) and 75 (strong peak), which is typical of C16:0 DMA, whereas corresponding values of the DMA generated from stearic aldehyde were 283 and 75, which is typical of C18:0 DMA. Fatty acid methyl ester peaks were identified by both their GC retention times (as compared to those of standards) and by their mass spectra. They were then quantified by comparison of their GC peak areas with that of an internal standard (methylheptadecanoate).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The PGC capacitated mouse sperm sample possesses a higher population of sperm with morphology typical of mature gametes (complete chromatin condensation, complete formation of mitochondria, and absence of coating membrane vesicles) compared with the washed capacitated sperm (see Introduction), although the majority of sperm from both preparations was viable, as shown by their ability to exclude propidium iodide (95%–100% for the PGC capacitated sperm sample and 90%–92% for the washed capacitated sperm sample). However, the ability of PGC capacitated and washed capacitated mouse sperm to bind to the egg ZP and to fertilize eggs has not, to our knowledge, been compared previously. Figure 1A shows that a greater number of PGC capacitated sperm bound to the egg ZP compared with washed capacitated sperm in all three experiments performed (25 ± 2 vs. 15 ± 1, P < 0.001). Corroborating this result was the higher ability of PGC capacitated sperm to fertilize eggs in vitro (74 ± 6 vs. 57 ± 8 for washed capacitated sperm, P < 0.05) (Fig. 1B). Although the percentage of the motile population in the PGC capacitated sperm sample was consistently higher than that of washed capacitated sperm (≥90% vs. ~70%–80%), the same number of motile sperm of both sperm types was used for egg coincubation in both the gamete-binding and the in vitro fertilization assays. Therefore, the higher fertilizing ability of PGC capacitated sperm, as observed by the increase in the number of sperm bound per egg and in the in vitro fertilization rates, would be attributed to possibilities other than the percentage sperm motility. First, the hyperactivated motility patterns showing an elevated beat frequency of the sperm tail and a larger amplitude of the sperm movement were more obvious in the PGC capacitated sperm sample than in the capacitated washed sperm preparation when assessed under a light microscope. Therefore, PGC capacitated sperm may have a stronger thrust to penetrate the ZP. Second, the PGC sample may have a higher population of capacitated gametes. CTC has been used widely to assess microscopically the percentages of noncapacitated (F pattern), capacitated (B pattern), intermediate (between capacitated and acrosome-reacted, S pattern), and acrosome-reacted (AR pattern) gametes in a sperm sample. Figure 2A shows that the capacitated sperm population in the PGC sample was slightly (15%) but significantly (P < 0.005) higher than that in the washed sample. The intermediate plus acrosome-reacted sperm population in the PGC sample was proportionally lower in the washed sample (P < 0.005). However, the percentage of the noncapacitated sperm population (F pattern) was the same in both samples. Nonetheless, the slight increase in the capacitated sperm population in the PGC sample could not account entirely for their significant increases in ZP-binding ability and in vitro fertilization compared with the washed sample (Fig. 1), especially because a higher number of sperm (motile + immotile) from the washed sample was used consistently for gamete coincubation as a result of the lower percentage of its motile population (see Materials and Methods). This suggested that the capacitated sperm population, as detected by CTC staining (B pattern), of the two sperm preparations differed in their biochemical and functional properties.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. Sperm fertilizing ability of PGC capacitated sperm and washed capacitated sperm. A) Sperm-ZP binding ability. Data from each experiment were expressed as the mean + SD of sperm bound per egg from the three replicates of gamete coincubates on each experimental day. B) In vitro fertilizing ability. In both A and B, data are expressed as the mean + SD of the mean values from the three experiments. n, Total number of eggs assessed in each sample. *Significant difference between data from washed capacitated sperm samples and data from PGC capacitated sperm (P < 0.001 for sperm-ZP binding results and P < 0.05 for in vitro fertilization results)



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. A) Distribution of chlortetracycline staining patterns in PGC capacitated sperm and washed capacitated sperm. Four staining patterns (F, Noncapacitated; B, capacitated; S, intermediate; AR, acrosome reacted) [35] were observed in both sperm types. B) Sperm tyrosine phosphorylation patterns of PGC capacitated sperm and washed capacitated sperm. Lane 1: washed capacitated sperm; Lane 2: PGC capacitated sperm. Molecular masses of protein standards (kDa) are shown on the right. Proteins extracted from an equal number of PGC capacitated or washed capacitated sperm (0.5 million) were used for gel loading. The result shown is representative of three replicate experiments, and the value of each sperm type is expressed as mean ± SD of the three sperm counts. *Significant difference between data from washed capacitated sperm samples and data from PGC capacitated sperm (P < 0.005)

Sperm protein tyrosine phosphorylation has been shown to be highly correlated with capacitation [49, 50]. Figure 2B shows that both the PGC capacitated sperm sample and the washed capacitated sperm sample possessed a similar pattern of protein tyrosine phosphorylation, as revealed by immunoblotting using 4G-10 antiphosphotyrosine antibody. The immunoreactive bands included sperm proteins of molecular weights ranging from approximately 45 to 250 kDa. The immunoreactive intensity of only two phosphorylated proteins (i.e., those with molecular masses of 64 and 60 kDa) was higher in the PGC capacitated sperm sample. These results suggested that the capacitation stage of PGC sperm may be slightly more advanced than that of washed sperm, although it may not account completely for the observed increases in ZP binding and in vitro fertilization competence of PGC capacitated sperm. Alternatively, this enhancement of fertilizing ability may result from changes at the plasma membrane levels in PGC capacitated sperm, one of which may be exposure and/or enrichment of ZP-binding ligands on the sperm surface.

One of the ZP-binding ligands that exists in an appreciable amount is the male germ cell-specific SGG (constituting 10 mol% of total sperm lipids) [28, 29, 51]. To determine whether the level and/or the proportion of SGG was increased in PGC capacitated sperm compared with washed sperm, we quantified SGG and other major lipids (cholesterol, phospholipids, DG, and TG) in both sperm types. Because mouse sperm tend to adhere to the tube wall surface within 10 min in suspension, we used the amount of DNA instead of the sperm number as a denominator of the sperm lipid content. The DNA assay involved treating sperm with DTT, which prevented sperm adherence to the tube wall, thereby allowing the assay to be carried out at a later time [12]. Both PGC capacitated and washed capacitated sperm had the same amount of DNA (4.1 ± 0.2 vs. 4.0 ± 0.4 pg/sperm) (Table 1). The levels of SGG in PGC capacitated and washed capacitated sperm were not essentially different (0.120 ± 0.006 vs. 0.139 ± 0.027 nmol/µg DNA). In contrast, the levels of both cholesterol and phospholipids in PGC capacitated sperm were markedly lower than in washed capacitated sperm (only 36% and 42%, respectively, as compared with the levels in washed capacitated sperm). However, the levels of the minor sperm lipids, DG and TG, in PGC capacitated sperm and washed capacitated sperm were very similar (Table 1). Considering cholesterol plus phospholipids plus SGG plus DG plus TG as 100% of sperm lipids, the percentage of SGG in washed capacitated sperm was 8.3%, compared to 14.5% in PGC capacitated sperm. The molar ratios of SGG to cholesterol and to phospholipids also were markedly higher in PGC capacitated sperm (1.00 and 0.29 vs. 0.41 and 0.14 in washed capacitated sperm) (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Levels and molar ratios of various lipid classes in washed capacitated sperm and PGC capacitated sperm.a

It is possible that the marked decrease of phospholipids in PGC capacitated sperm compared to washed capacitated sperm was confined to specific phospholipid subclasses. However, HPTLC results (Fig. 3) argue against this hypothesis. Densitometric analyses of lipids from two HPTLC runs revealed that the average contents (as mol% of total phospholipids, see Materials and Methods) of the three main phospholipids, PC, PE, and SM, in washed capacitated sperm were 58%, 26%, and 11%, respectively. In PGC capacitated sperm, these phospholipids subclasses had similar contents, with PC being the highest (47%), followed by PE (25%) and SM (14%). Cardiolipin existed as a minor phospholipid in both washed capacitated and PGC capacitated sperm samples (5% and 14%, respectively). However, a minor lipid band, migrating slightly faster than SM, was present in washed capacitated but not in PGC capacitated sperm. This lipid did not stain purple with the orcinol dye (data not shown), indicating that it was not a glycolipid. Another Coomassie blue-stained lipid with a higher mobility than SGG also was observed in both PGC capacitated and washed capacitated sperm. The identity of both lipids (each denoted by a question mark symbol in Fig. 3) remains to be determined.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 3. HPTLC of lipids extracted from PGC capacitated sperm and washed (W) capacitated sperm. Lipids extracted from approximately 5 million capacitated sperm were used for HPTLC. The HPTLC plate was developed in chloroform/methanol/H2O (65:32:4, v/v/v). Staining of lipids was performed using Coomassie brilliant blue G-250. Band positions of lipid standards are labeled on the side of the HPTLC illustration. Two bands, labeled with a question mark, with one present only in the washed capacitated sperm samples, are unidentified lipids. The chromatogram shown is representative of two HTPLC replicates

The PGC sperm immediately acquired hyperactivated motility patterns on suspension in medium. This suggested that their plasma membrane may have a higher fluidity than that of washed sperm. In addition, a greater ability of PGC capacitated sperm to fertilize eggs as compared with that of washed capacitated sperm (Fig. 1B) may result not only from their higher level of ZP binding (Fig. 1A) but also from their enhanced capability to undergo fusion events pertinent to fertilization (acrosome reaction and sperm-egg plasma membrane fusion). Because lipids play an important role in regulating membrane fluidity, we characterized further the lipid profile of PGC capacitated sperm in comparison with that of washed capacitated sperm. The phospholipid molecular species in PGC sperm may be different from those of washed sperm. In particular, a higher proportion of phospholipid species containing HUFA likely would contribute to high membrane fluidity [27], which is required for hyperactivated motility and membrane fusion events. Phospholipids, especially PC and PE, of mouse testicular germ cells and epididymal sperm have been shown to possess HUFAs [16, 51]. In contrast, our initial analysis of FAMEs generated from SM of washed capacitated mouse sperm revealed the presence of palmitic acid (C16: 0) as the major fatty acid (70%), followed by stearic acid (C18:0; 20%) and oleic acid (C18:1n-9; 10%), and the absence of HUFA. These results are in agreement with those of previous FAME analyses of SM isolated from mouse testis [52]. Therefore, we concentrated our efforts on characterizing FAMEs from PC and PE, the two main phospholipid subclasses of both washed capacitated and PGC capacitated sperm. Figures 4 and 5 show that acidic methanolysis of PC and PE, respectively, from both washed capacitated and PGC capacitated sperm generated FAMEs as their major products and DMAs as their minor products. This indicated that the hydrocarbon chains of PC and PE of both sperm samples were linked mainly to the glycerol backbone through ester linkages, whereas minor amounts of PC and PE existed as plasmalogens. The PE of both washed capacitated and PGC capacitated sperm possessed higher proportions of DMAs than the corresponding PC molecules (Figs. 4 and 5).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. Composition of FAMEs and DMAs in PC of washed capacitated sperm and PGC capacitated sperm. The distribution profile (in mol%) is representative of two replicate analyses



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 5. Composition of FAMEs and DMAs in PE of washed capacitated sperm and PGC capacitated sperm. The distribution profile (in mol%) is representative of two replicate analyses

Palmitic acid (C16:0) was one of the representative saturated fatty acids present in both PC and PE from washed capacitated and PGC capacitated sperm. In PC, it constituted 23% and 38% of total FAMEs plus DMAs in washed capacitated sperm and PGC capacitated sperm, respectively (Fig. 4). The corresponding percentages of palmitic acid in PE were 41% and 53%, respectively (Fig. 5). Stearic acid (C18:0) was the other major saturated fatty acid present in both PC and PE of washed capacitated sperm (41% and 35%, respectively) (Figs. 4 and 5). However, the distribution of C18:0 fatty acid in both phospholipid subclasses decreased dramatically in PGC capacitated sperm (to 2% and 18% of total FAMES + DMAs in PC and PE, respectively) (Figs. 4 and 5). Our acidic methanolysis products also contained appreciable amounts of C16:0 DMA in PE from both washed capacitated and PGC capacitated sperm (18% and 22% of total FAMEs + DMAs, respectively) (Fig. 5). This indicated that this hydrocarbon chain was present at the sn-1 position of PE plasmalogen. In contrast, the proportion of C16:0 DMA in PC was much lower in washed capacitated sperm (5%), although its percentage increased to 16% in PGC capacitated sperm (Fig. 4).

In PC and PE of both washed capacitated and PGC capacitated sperm, small amounts (<5% of total FAMEs + DMAs) of the mono-unsaturated fatty acid, oleic acid (C18: 1n-9) (Figs. 4 and 5), were found. Also, HUFAs in PE were absent in washed capacitated sperm and were minimally present (<5% as C20:4n-6) in PGC capacitated sperm (Fig. 5). In contrast, the proportion of HUFAs in PC was 27% in washed capacitated sperm and 42% in PGC capacitated sperm (Fig. 4). In washed capacitated sperm, HUFAs of PC consisted of DHA (C22:6n-3; 12%), arachidonic acid (C20: 4n-6; 10%), and docosapentaenoic acid (C22:5n-6; 5%). On the other hand, the main HUFA species in PC of PGC capacitated sperm was C22:6n-3 (41%), whereas C20:4n-6 and C22:5n-6 in PC appeared in trace amounts (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the percentages of the motile population in the PGC capacitated and washed capacitated mouse sperm samples were high (≥70%), the present study showed, to our knowledge for the first time, that PGC capacitated mouse sperm had higher fertilizing ability than washed capacitated mouse sperm (Fig. 1). The results corresponded with the greater degree of hyperactivated movement of PGC capacitated sperm observed visually under a light microscope. However, the slight increase of the CTC-stained B pattern (designated as "capacitated acrosome intact") [35] in PGC capacitated sperm (15% higher than in washed capacitated sperm) could not explain their higher degrees of ZP-binding ability and in vitro fertilization (1.7- and 1.4-fold, respectively, the values of washed capacitated sperm), especially because the total number of sperm incubated with eggs was always higher for the washed sperm sample. The lack of tight correlation between the distribution of PGC capacitated sperm in the CTC-stained B pattern and the fertilizing ability of these sperm was not surprising, because CTC is believed to bind calcium in hydrophobic environments [35], and therefore sperm with the CTC-stained ß pattern may not represent the whole population of capacitated sperm having fertilizing ability.

Sperm tyrosine phosphorylation has been shown to correlate with capacitation [49, 50, 53–55]. The mechanism of this sperm protein modification is through the cAMP and protein kinase A pathway, which is initiated by cholesterol efflux and the increase in intracellular bicarbonate levels [49, 50, 54, 55]. The PGC capacitated sperm showed a slight increase in sperm protein tyrosine phosphorylation (Fig. 2B). The cholesterol:phospholipid ratio in PGC capacitated sperm also was lower than that in washed capacitated sperm (Table 1), suggesting that cholesterol efflux may occur at a greater extent in PGC sperm. This may lead to the slightly higher level of tyrosine phosphorylation in these sperm, and it may account, in part, for their improved ability to bind to the ZP and to fertilize the egg. However, other mechanisms also may contribute to the observed higher fertilizing ability of PGC capacitated sperm.

In the present report, we demonstrated that the lipid profiles of PGC capacitated and washed capacitated sperm differed, mainly in three aspects. First, the levels of cholesterol and phospholipids, the two main sperm lipid classes, were markedly lower in PGC capacitated sperm (Table 1). These results corroborated those previously described in mouse and human sperm [12, 13] and the ultrastructural findings that PGC sperm were devoid of coating membrane vesicles [5, 9]. Presumably, these vesicles were enriched in cholesterol and phospholipids, and their absence in PGC sperm likely would allow exposure of ZP ligands on the sperm head surface. One of these ZP-binding ligands was SGG [51], the levels of which were constant in both PGC capacitated and washed capacitated sperm (Table 1). However, because of a significant decrease of cholesterol and phospholipids in PGC capacitated sperm, SGG became enriched in these sperm. The proportion of SGG in PGC capacitated sperm was 1.75-fold the corresponding value in washed capacitated sperm (14.5% vs. 8.3%) (Table 1). This would impart a greater opportunity to PGC capacitated sperm to bind to the ZP (as shown in Fig. 1A), especially when no more coating membrane vesicles were present to hinder SGG and other ZP adhesion molecules on the sperm surface to interact with the ZP.

The higher percentage of SGG as well as its increased ratio to cholesterol and phospholipids (Table 1) in PGC capacitated sperm suggested that SGG contributed more to the membrane architecture in these sperm compared to washed capacitated sperm. Recently, we showed that the majority of SGG exists in lipid rafts of capacitated sperm [51]. Lipid rafts are liquid-ordered, cholesterol-containing microdomains, which are considered to be platforms of cell adhesion and signaling molecules [56, 57]. In somatic cells, glycosphingolipids are present in lipid rafts, contributing to the raft formation via hydrophobic interaction and hydrogen bonding with each other as well as with cholesterol [58, 59]. However, glycosphingolipids exist at a very low level in mammalian sperm [19]. SGG, the major glycolipid of mammalian sperm (present at ~10 mol% of total mammalian sperm lipids [29, 51]) [table], may interact with cholesterol in a manner analogous to that of glycosphingolipids in somatic cells, thus contributing to the formation of lipid rafts. In fact, our model membrane studies have verified the interaction between SGG and cholesterol [51; unpublished results]. In addition, SGG can interact with each other via both hydrogen bonding and the Van der Waal force [31]. The higher percentage of SGG in PGC capacitated sperm therefore would result in relative enrichment of lipid raft microdomains in these sperm. Our unpublished results reveal that isolated mammalian sperm lipid rafts bind to ZP with the same specificity employed by intact sperm. Therefore, in addition to the direct ZP-binding ability of SGG, its enrichment in PGC capacitated sperm may account for the increased ZP-binding ability of these sperm via enhancement of sperm raft formation.

The third lipid parameter that was distinctively different between PGC capacitated sperm and washed capacitated sperm was the presence of a higher percentage of HUFAs in PC of the former sperm type. Specifically, DHA (C22: 6n-3) was the only prominent HUFA of PC in PGC capacitated sperm, constituting approximately 40% of total FAMEs plus DMAs (Fig. 4). In contrast, HUFAs of PC in washed capacitated sperm comprised arachidonic acid (C20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA, the percentage sum of which was only 25% of total FAMEs plus DMAs (Fig. 4). Because of its highest number of double bonds, DHA would enhance fluidity of biomembranes to the greatest extent [27]. This enhanced fluidity would be beneficial for fusion events during fertilization (acrosome reaction and sperm-egg plasma membrane fusion). Because HUFA-containing phospholipids do not interact well with cholesterol and SGG (unpublished results), they likely exist in the nonraft fluid-phase domains [59]. Despite the enrichment of DHA in PGC capacitated sperm, this sperm type appeared to have a lower rate of spontaneous acrosome reaction compared with washed capacitated sperm (Fig. 2A). This may result from the raft microdomains possibly existing as islands surrounded by the fluid phase. As discussed above, compared with washed capacitated sperm, PGC capacitated sperm membranes may contain a higher proportion of lipid raft microdomains, and this would reduce the continuity of the fluid phase areas in these sperm. On the other hand, the higher spontaneous acrosome reaction rate of washed capacitated sperm may result from their possession of arachidonic acid (20:4n-6) containing PC. Arachidonic acid, which can be released from its parental phospholipids by the action of phospholipase A2 [60], is known to have an acrosome reaction-inducing effect [61, 62]. However, HUFA-containing phospholipids may be beneficial for the ZP-induced acrosome reaction. The current concept holds that the interaction between multivalent binding ligands and their receptors in lipid rafts would lead to raft clustering/coalescence, resulting in formation of macrorafts and simultaneous activation of downstream signaling events [63, 64]. Because ZP sulfoglycoproteins contain a number of glycan chains, which are known as the sperm-binding entities [65, 66], macrorafts on sperm also may be generated as a result of sperm-ZP binding. Because HUFA-containing phospholipids contribute significantly to biomembrane fluidity [15, 27], they likely would facilitate raft clustering and, thus, macroraft formation and subsequent downstream signaling events, the final outcome of which would be the acrosome reaction in sperm. Therefore, the PGC capacitated sperm, having a higher proportion of HUFA-containing PC, would have a higher potential in macroraft formation and a greater tendency to undergo the ZP-induced acrosome reaction compared to washed capacitated sperm, and this would be reflected in their higher in vitro fertilization ability, as shown in the present report (Fig. 1B).

Our findings that both SGG- and DHA-containing PC are enriched in PGC capacitated sperm suggested that the sperm plasma membrane had to undergo remodeling events in both lipid raft (liquid-ordered) and fluid-phase microdomains to gain their high fertilizing ability. Our present results argue for the following two points: First, Percoll gradient centrifugation should be used routinely for preparing mouse sperm for fertilization-related studies. Second, sperm lipid contents, especially SGG and DHA, should be considered to be biomarkers for sperm fertilizing ability in addition to the B pattern of CTC-stained sperm and sperm tyrosine phosphorylation. At present, it is unclear whether a population of sperm already exists that uniquely possess higher amounts of SGG, DHA-containing PC molecular species, and other molecules that provide them with higher fertilizing ability and whether Percoll gradient centrifugation simply selects this sperm population. Alternatively, the process of Percoll gradient centrifugation may generate these high-quality sperm, or the combination of both possibilities may be responsible for the generation of PGC capacitated sperm with high fertilizing ability. The PGC capacitated sperm may be analogous to oviductal sperm in the in vivo situation. Sperm that have reached the oviduct likely have higher fertilizing ability than the remaining sperm that are left behind in the uterus. The higher fertilizing ability of oviductal sperm may reflect their inherently better quality as well as modification of their cell membranes during their swimming through the glycan/glycoprotein matrices in the female reproductive tract [67, 68]. Currently, we are using the experimental approaches described in the present report to characterize lipid profiles of oviductal sperm.


   ACKNOWLEDGMENTS
 
The authors thank Ms. Terri van Gulik for assistance in manuscript preparation.


   FOOTNOTES
 
1 Supported by NSERC (grant RGPIN183958 to N.T.). A.F. and M.B.K. were awardees of Ontario Graduate Scholarships (OGS), and H.X. is an awardee of a Strategic Training Initiative in Research in the Reproductive Health Sciences (STIRRHS) bursary. Back

2 Correspondence: Nongnuj Tanphaichitr, Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada. FAX: 613 761 5365; ntanphaichitr{at}ohri.ca Back

Received: 7 September 2004.

First decision: 30 September 2004.

Accepted: 15 October 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hammadeh ME, Zavos PM, Rosenbaum P, Schmidt W, Comparison between the quality and function of sperm after semen processing with two different methods. Asian J Androl 2001 3:125-130[Medline]
  2. Kelly MP, Corson SL, Gocial B, Batzer FR, Gutmann JN, Discontinuous Percoll gradient preparation for donor insemination: determinants for success. Hum Reprod 1997 12:2682-2686[Abstract/Free Full Text]
  3. Gravance CG, Champion ZJ, Sax-Gravance SK, Casey PJ, Percentage of normal sperm heads is significantly increased by Percoll separation of semen. Int J Androl 1998 21:116-119[CrossRef][Medline]
  4. Prakash P, Leykin L, Chen Z, Toth T, Sayegh R, Schiff I, Isaacson K, Preparation be differential gradient centrifugation is better than swim-up in selecting sperm with normal morphology (strict criteria). Fertil Steril 1998 69:722-726[CrossRef][Medline]
  5. Tanphaichitr N, Millette CF, Agulnick A, Fitzgerald LM, Egg-penetration ability and structural properties of human sperm prepared by Percoll-gradient centrifugation. Gamete Res 1988 20:67-81[CrossRef][Medline]
  6. Mai A, Weerachatyanukul W, Tomietto M, Wayner DD, Wells G, Balhorn R, Leader A, Cyr JL, Tanphaichitr N, Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome-reacted human sperm. Mol Reprod Dev 2002 63:471-479[CrossRef][Medline]
  7. Miyake M, Coney P, Iritani A, Kling OR, Motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of Percoll. Gamete Res 1989 24:49-57[CrossRef][Medline]
  8. Chen MJ, Bongso A, Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum Reprod 1999 14:759-764[Abstract/Free Full Text]
  9. Tanphaichitr N, Smith J, Kates M, Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse sperm. Biochem Cell Biol 1990 68:528-535[Medline]
  10. Phelps MJ, Liu J, Benson JD, Willoughby CE, Gilmore JA, Critser JK, Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation. Biol Reprod 1999 61:1031-1041[Abstract/Free Full Text]
  11. Flesch FM, Gadella BM, Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta 2000 1469:197-235[Medline]
  12. Tanphaichitr N, Zheng Y, Kates M, Abdullah N, Chan A, Cholesterol and phospholipid levels of washed and Percoll-gradient centrifuged mouse sperm: presence of lipids possessing inhibitory effects on sperm motility. Mol Reprod Dev 1996 43:187-195[CrossRef][Medline]
  13. Sugkraroek P, Kates M, Leader A, Tanphaichitr N, Levels of cholesterol and phospholipids in freshly ejaculated sperm and Percoll gradient-pelleted sperm from fertile and unexplained infertile men. Fertil Steril 1991 55:820-827[Medline]
  14. Ollero M, Powers RD, Alvarez JG, Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage. Mol Reprod Dev 2000 55:326-334[CrossRef][Medline]
  15. Zalata AA, Christophe AB, Depuydt CE, Schoonjans F, Comhaire FH, The fatty acid composition of phospholipids of spermatozoa from infertile patients. Mol Hum Reprod 1998 4:111-118[Abstract/Free Full Text]
  16. Grogan WM, Franham WF, Szopiak BA, Long chain polyenoic acid levels in viably sorted, highly enriched mouse testis cells. Lipids 1981 16:401-410[CrossRef][Medline]
  17. Beckman JK, Gray ME, Coniglio JG, The lipid composition of isolated rat spermatids and spermatocytes. Biochim Biophys Acta 1978 530:367-374[Medline]
  18. Brouwers JF, Gadella BM, Van Golde LM, Tielens AG, Quantitative analysis of phosphatidylcholine molecular species using HPLC and light scattering detection. J Lipid Res 1998 39:344-353[Abstract/Free Full Text]
  19. Nikolopoulou M, Soucek D, Vary J, Changes in the lipid content of boar sperm plasma membranes during epididymal maturation. Biochim Biophys Acta 1985 815:486-498[Medline]
  20. Parks JE, Lynch DV, Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 1992 29:255-266[CrossRef][Medline]
  21. Connor WE, Weleber RG, DeFrancesco C, Lin DS, Wolf DP, Sperm abnormalities in retinitis pigmentosa. Invest Ophthalmol Vis Sci 1997 38:2619-2628[Abstract/Free Full Text]
  22. Poulos A, Very long chain fatty acids in higher animals—a review. Lipids 1995 30:1-14[Medline]
  23. Coniglio JG, Testicular lipids. Prog Lipid Res 1994 33:387-401[CrossRef][Medline]
  24. Aveldano MI, Long and very long polyunsaturated fatty acids of retina and spermatozoa: the whole complement of polyenoic fatty acid series. In: Bazan NG (ed.), Neurobiology of Essential Fatty Acids. New York: Plenum Press; 1992:231–242
  25. Lenzi A, Gandini L, Maresca V, Rago R, Sgro P, Dondero F, Picardo M, Fatty acid composition of spermatozoa and immature germ cells. Mol Hum Reprod 2000 6:226-231[Abstract/Free Full Text]
  26. Conquer JA, Martin JB, Tummon I, Watson L, Tekpetey F, Fatty acid analysis of blood serum, seminal plasma and spermatozoa of normozoospermic versus asthenozoospermic males. Lipids 1999 34:793-799[CrossRef][Medline]
  27. Farkas T, Kitajka K, Fodor E, Csengeri I, Lahdes E, Yeo YK, Krasznai Z, Halver JE, Docosahexaenoic acid-containing phospholipid molecular species in brains of vertebrates. Proc Natl Acad Sci U S A 2000 97:6362-6366[Abstract/Free Full Text]
  28. Murray RK, Narasimhan R, Glycoglycerolipids of animal tissues. In: Kates M (ed.), Glycolipids, Phosphoglycolipids, and Sulfoglycolipids. New York: Plenum Press; 1990:321–361
  29. Ishizuka I, Chemistry and functional distribution of sulfoglycolipids. Prog Lipid Res 1997 36:245-319[CrossRef][Medline]
  30. Tanphaichitr N, Smith J, Mongkolsirikieart S, Gradil C, Lingwood C, Role of a gamete specific sulfoglycolipid-immobilizing protein on mouse sperm-egg binding. Dev Biol 1993 156:164-175[CrossRef][Medline]
  31. Attar M, Kates M, Bou Khalil M, Carrier D, Wong PTT, Tanphaichitr N, A Fourier-transform infrared study of the interaction between germ cell-specific sulfogalactosylglycerolipid and dimyristoylglycerophosphocholine. Chem Phys Lipids 2000 106:101-114[CrossRef][Medline]
  32. White D, Weerachatyanukul W, Gadella B, Kamolvarin N, Attar M, Tanphaichitr N, Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding. Biol Reprod 2000 63:147-155[Abstract/Free Full Text]
  33. Weerachatyanukul W, Rattanachaiyanont M, Carmona E, Furimsky A, Mai A, Shoushtarian A, Sirichotiyakul S, Ballakier H, Leader A, Tanphaichitr N, Sulfogalactosylglycerolipid is involved in human gamete interaction. Mol Reprod Dev 2001 60:569-578[CrossRef][Medline]
  34. Hogan B, Costantini F, Lacy E, Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1994:127–216
  35. Lee MA, Storey BT, Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence. Biol Reprod 1985 33:235-246[Abstract]
  36. Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  37. Towbin H, Gordon J, Immunoblotting and dot immunobinding-current status and outlook. J Immunol Methods 1984 72:313-340[CrossRef][Medline]
  38. Labarca C, Paigen K, A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980 102:344-352[CrossRef][Medline]
  39. Bligh EG, Dyer WJ, A rapid method of total lipid extraction and purification. Canadian J Biochem Physiol 1959 31:911-917
  40. Kates M, Technique of lipidology: isolation, analysis and identification of lipids. In: Burdon RH (ed.), Laboratory Techniques in Biochemistry and Molecular Biology. New York: Elsevier; 1986:100– 278
  41. Tupper S, Wong PTT, Kates M, Tanphaichitr N, Interaction of divalent cations with germ cell specific sulfogalactosylglycerolipid and the effects on lipid chain dynamics. Biochemistry 1994 33:13250-13258[CrossRef][Medline]
  42. Alvarez JG, Storey BT, Hemling ML, Grob RL, High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa. J Lipid Res 1990 31:1073-1081[Abstract]
  43. Ohta K, Sato C, Matsuda T, Toriyama M, Lennarz W, Kitajima K, Isolation and characterization of low density detergent-insoluble membrane (LD-DIM) fraction from sea urchin sperm. Biochem Biophys Res Commun 1999 258:616-623[CrossRef][Medline]
  44. Gamble W, Vaughan M, Kruth HS, Avigan J, Procedure for determination of free and total cholesterol in micro or nanogram amounts suitable for studies with cultured cells. J Lipid Res 1978 19:1068-1070[Abstract]
  45. Duck-Chong CG, A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate. Lipids 1979 14:492-497[CrossRef]
  46. Kean EL, Rapid, sensitive spectrophotometric method for quantitative determination of sulfatides. J Lipid Res 1968 9:319-327[Abstract]
  47. Holub BJ, Watson SP, Methods for measuring agonist-induced phospholipid metabolism in intact human platelets. In: Watson SP (ed.), Platelets: A Practical Approach. New York: Oxford University Press; 1996:236–257
  48. Alvarez JG, Lopez I, Touchstone JC, Storey BT, Thin layer chromatography of phospholipid composition in mouse and rabbit spermatozoa. J Liquid Chromatogr 1987 10:3557-3573
  49. Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS, Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 1995 121:1129-1137[Abstract]
  50. Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf GS, Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995 121:1139-1150[Abstract]
  51. Tanphaichitr N, Bou Khalil M, Weerachatyanukul W, Kates M, Xu H, Carmona E, Attar M, Carrier D, Physiological and biophysical properties of male germ cell sulfogalactosylglycerolipid. In: De Vriese S (ed.), Lipid Metabolism and Male Fertility. Champaign, IL: AOCS Press; 2003:125–148
  52. Grogan WM, Distribution of long chain polyenoic acids among phospholipids of mouse testis. Lipids 1981 16:940-942[CrossRef][Medline]
  53. Asquith KL, Baleato RM, McLaughlin EA, Nixon B, Aitken RJ, Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition. J Cell Sci 2004 117:3645-3657[Abstract/Free Full Text]
  54. Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG, Kopf GS, Cholesterol efflux-mediated signal transduction in mammalian sperm: ß-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J Biol Chem 1999 274:3235-3242[Abstract/Free Full Text]
  55. Visconti PE, Ning X, Fornes MW, Alvarez JG, Stein P, Connors SA, Kopf GS, Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. Dev Biol 1999 214:429-443[CrossRef][Medline]
  56. Anderson RG, Jacobson K, A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 2002 296:1821-1825[Abstra