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BOR - Papers in Press, published online ahead of print September 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032813
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BIOLOGY OF REPRODUCTION 72, 127–134 (2005)
DOI: 10.1095/biolreprod.104.032813
© 2005 by the Society for the Study of Reproduction, Inc.

Anti-Hyaluronidase Oligosaccharide Derived from Chondroitin Sulfate A Effectively Reduces Polyspermy During In Vitro Fertilization of Porcine Oocytes1

Hideki Tatemoto2,3, Norio Muto4, Sun-Deok Yim4, and Tadashi Nakada3

Department of Bioproduction,3 Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan School of Bioresources,4 Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 µg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1–100 µg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 µg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10–100 µg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25–30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 µg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 µg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

early development, in vitro fertilization, ovum, polyspermy, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recent progress of in vitro maturation (IVM) and in vitro fertilization (IVF) technologies has increased the availability of mammalian embryos for the study of early zygotic development. Successful methods for in vitro production of IVM-IVF-derived porcine embryos to the blastocyst stage have been developed [1–8]. However, in pigs, the abnormally high incidence of polyspermy after IVM and IVF is a major problem and often exceeds 50% [9– 11]; hence, various improvements are applied to the method of porcine IVF to decrease the polyspermy rate under conditions maintaining a higher oocyte penetration rate. The addition of oviductal epithelial cells [12, 13] to the fertilization medium or oviductal fluid [14] to the prefertilization medium has been used to reduce the incidence of polyspermy in porcine oocytes, suggesting that a factor, or factors, secreted from the oviduct may be associated with the functional block of polyspermy during IVF. In particular, the addition of porcine oviduct-specific glycoprotein (OGP) to IVF medium at concentrations of 10–50 µg/ml significantly reduced not only the polyspermy rate with no effect on the sperm penetration but also the number of sperm bound to the zona pellucida (ZP) [15, 16].

In general, sperm binding to the ZP has been described to occur in two phases. During the primary binding phase, one or more carbohydrate-binding proteins on the sperm plasma membrane interact with zona glycoproteins to mediate sperm attachment [17]. In mouse oocytes, oligosaccharides with galactose in either {alpha}- or ß-linkage are demonstrated to be effective inhibitors of the primary binding of sperm to ZP3 glycoprotein [18, 19]. In the secondary binding phase, which takes place following the acrosome reaction, PH-20 on the posterior head plasma membrane and inner acrosomal membrane interacts with the ZP. PH-20 is a glycosyl phosphatidylinositol-anchored membrane protein [20], and its homolog has been identified in guinea pigs [21, 22], mice [23, 24], monkeys [25–27], and humans [28]. It is interesting that PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for hyaluronic acid-induced cell signaling, and a receptor for the ZP glycoproteins surrounding the oocyte [29]. According to the findings reported by Primakoff et al. [30], the hyaluronidase-active site is presumed to exist in the PH-20 N-terminal region (41 kDa on SDS-PAGE). In addition, the sequence of amino acid residues 17–307 in PH-20 is shown to be homologous with that of bee venom hyaluronidase [31]. The specific antibody to PH-20 strongly inhibits not only the secondary binding of sperm to guinea pig egg ZP2 glycoprotein [22, 32, 33] but also the hyaluronidase activity of sperm PH-20 protein and the sperm penetration into oocytes [23, 26]. However, to our knowledge, there is no report describing the effects of oligosaccharides modifying the primary binding of sperm to ZP and the sperm hyaluronidase activity derived from PH-20 on the interactions between sperm and oocytes during IVF of porcine oocytes.

As reported by Toida et al. [34], O-sulfonated chondroitin sulfate can block the activity of hyaluronidase (50% inhibitory concentration; IC50 = 1.35 µg/ml). Because chondroitin sulfate is ordinarily synthesized while cumulus cells are expanded during oocyte maturation in pigs [35], it can be speculated that chondroitin sulfate A-derived oligosaccharide (ChSAO) added to IVF medium may serve a critical role in decreasing the incidence of polyspermic fertilization in porcine oocytes as a consequence of regulating the hyaluronidase activity and/or the sperm-ZP adhesion. Therefore, in the present study, the following experiments were undertaken: 1) to determine the inhibitory effect of ChSAO on the activity of hyaluronidase extracted from boar sperm; 2) to evaluate the effects of ChSAO added to IVF medium on fertilization responses, including penetration rate and incidence of polyspermy in porcine oocytes matured in vitro; 3) to determine whether treatment with ChSAO during IVF can enhance development to the blastocyst stage; and 4) to examine the effects of ChSAO on sperm binding to the ZP of oocytes, the modification of ZP glycoproteins after oocyte activation, and the increase in the incidence of acrosome reaction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals used in the study were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

Culture Media

The culture medium used for oocyte maturation was BSA-free North Carolina State University (NCSU) 37 medium [36] supplemented with 0.57 mM cysteine, 250 µM ascorbic acid 2-O-{alpha}-glucoside (Hayashibara Biochem. Lab., Okayama, Japan), 0.04 U/ml ovine FSH, 0.02 U/ml ovine LH, and 10% (v/v) porcine follicular fluid. Porcine follicular fluid was aspirated from follicles 2–6 mm in diameter, centrifuged at 10 000 x g for 15 min at 4°C to remove cellular debris and stored at –30°C until use. The basic medium used for IVF was essentially the same as the modified Tris-buffered medium (mTBM) used by Abeydeera and Day [5]. This mTBM was designated as IVF medium by supplementation with 2 mM caffeine sodium benzoate and 0.1% (w/v) BSA and as sperm preincubation medium by supplementation with 4 mM caffeine sodium benzoate and 0.4% (w/v) BSA. Embryos were cultured in NCSU37, which was designated as in vitro culture (IVC) medium supplemented with 0.4% (w/v) BSA.

Preparation of ChSAO

Chondroitin sulfate A (from bovine trachea; 2 g) was dissolved in 10 ml of 20 mM acetate buffer (pH 5.0) containing 30 mM NaCl at 4°C overnight and thoroughly digested by the addition of 20 mg hyaluronidase at 37°C overnight in a shaking bath. The enzymatic hydrolysate was heated at 100°C for 3 min and centrifuged at 10 000 x g for 20 min. The supernatant was applied to a Sephadex G-75 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and eluted with Milli-Q water. Absorbance was monitored at 230 nm. Fractions containing oligosaccharides with molecular weights less than 10 kDa were pooled, then lyophilized. The powder was dissolved in 5 ml of Milli-Q water and applied to a Sephadex G-25 column (Amersham Pharmacia Biotech). Fractions containing oligosaccharides with molecular weights of around 1 kDa, composed mainly of tetrasaccharide, were pooled and then lyophilized. This preparation was designated as ChSAO and used in this study.

Extraction of Sperm Hyaluronidase

Hyaluronidase was extracted from boar sperm according to the method of Li et al. [37]. For sperm preparation, frozen-ejaculated spermatozoa of Landrace were thawed (39°C) and washed twice by centrifugation at 400 x g for 4 min in Dulbecco PBS (Invitrogen, Carisbad, CA) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA) at pH 7.2. The sperm pellet was resuspended at 4 x 108 sperm/ml in the sperm preincubation medium and then incubated for 90 min at 39°C to induce capacitation. After preincubation, sperm suspensions were washed by centrifugation at 400 x g for 10 min in Hepes-buffered saline (HBS; 5 mM Hepes, 150 mM NaCl, pH 7.0). The sperm pellet was resuspended in 3 ml of HBS, layered onto 3 ml of 40% Percoll (Amersham Pharmacia Biotech), then centrifuged at 300 x g for 15 min to remove BSA. After washing three times by resuspension in 10 ml of HBS and centrifugation, the sperm pellet was resuspended at 2 x 108 sperm/ml in an appropriate volume of cold HBS containing protease inhibitors (20 mM EDTA, 1 mM p-hydroxymercurobenzenzoate, 5 mM N-ethymaleimide, and 1 mM beszamidine). After addition of Triton X-100 to a final concentration of 1% (v/v), the sperm suspension was vortexed for 5 min and then centrifuged at 10 000 x g for 15 min at 4°C. The supernatant was stored at –80°C until assayed for hyaluronidase activity.

Microplate Assay of Hyaluronidase Activity

Hyaluronidase activity was determined as described earlier [37], with the following modifications. Hyaluronic acid sodium salt (derived from microorganism; Nacalai Tesque, Kyoto, Japan) was dissolved in 50 mM Tris-HCl (pH 7.0) at a concentration of 8 mg/ml at 4°C overnight. Agarose was dissolved in 50 mM Tris-HCl (pH 7.0) at a concentration of 0.9% (w/ v) by heating and maintained at 55°C. One volume of the hyaluronic acid solution preheated at 55°C was mixed with nine volumes of the agarose solution, and then the warm hyaluronic acid-agarose mixture (100 µl) was dispensed into each well of the microplate on a temperature-controlled hot plate (37°C). The microplate was allowed to stand at room temperature to let the gel set.

To examine the effect of ChSAO on hyaluronidase activity, 25 µl of sperm-extract solution, 25 µl of 50 mM Tris-HCl (pH 7.0), and 50 µl of ChSAO solution at the final concentration range of 0–100 µg/ml were mixed and preincubated for 1 h at room temperature before addition to the microplate well. Each well was filled with 100 µl of the sperm-extract-ChSAO mixture and incubated at 37°C for 15 h. After the solution was discarded and each well was washed three times with 150 µl of 50 mM Tris-HCl (pH 7.0), 100 µl of 10% (w/v) cetylpyridinium chloride in distilled water was added to each well and incubated at room temperature for 1 h. The degree of turbidity of each well was determined at 415 nm with an automated microplate reader (Model 550; Bio-Rad Laboratories, Richmond, CA). The units of hyaluronidase activity were determined based on the standard curve of hyaluronidase activity of bovine testicular hyaluronidase with a specific activity of 500 U/mg (H-3506; Sigma).

IVM, IVF, and IVC

Ovaries were collected from maturing gilts at a local slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl containing 100 mg/ L kanamycin sulfate (Meiji Seika, Tokyo, Japan) at 30°C. Within 2 h postslaughter, the follicular contents were recovered by excising the visible small antral follicles (about 2–6 mm in diameter) on the ovarian surface using a razor, and by scraping the inner surface of the follicle walls with a disposable surgical blade. Only cumulus-oocyte complexes (COCs) with uniform ooplasm and a compact cumulus cell mass were collected and washed three times with Hepes-buffered Tyrode medium containing 0.01% (w/v) PVA (H-TL-PVA). After washing in IVM medium, groups of 20 COCs were transferred into 100-µl droplets of IVM medium that had been previously covered with mineral oil and equilibrated in a 5% CO2 incubator. After 20 h of maturation culture, the oocytes were washed and transferred to 100-µl droplets of IVM medium without hormonal supplementation for an additional 24 h of culture.

After a total 44 h of maturation culture, some portions of the COCs were freed from their cumulus cells by treatment with H-TL-PVA containing 0.1% (w/v) hyaluronidase, followed by repeated passage through a narrow-bore pipette. Cumulus-intact and cumulus-free oocytes were washed three times with IVF medium, and 25–30 oocytes were transferred to 50-µl droplets of IVF medium containing 0, 0.1, 1, 10, or 100 µg/ml ChSAO, which had been covered with warm mineral oil. The droplets containing oocytes were kept in an incubator for 1 h until spermatozoa were added for fertilization. After thawing and washing frozen-ejaculated spermatozoa as described earlier, the sperm pellets were resuspended at 4 x 108 cells/ml in sperm preincubation medium and then incubated for 90 min at 39°C. After sperm preincubation, 50 µl of diluted sperm suspension in IVF medium containing 0, 0.1, 1, 10, or 100 µg/ml ChSAO was added to a droplet containing oocytes at a final sperm concentration of 1 x 106 cells/ml. Oocytes were coincubated with spermatozoa for 7 h at 39°C in an atmosphere of 5% (v/v) CO2 in air.

After insemination, oocytes were removed from fertilization drops, washed three times, and cultured in 50-µl drops of IVC medium at 39°C in an atmosphere of 5% CO2 in air. At 48 and 168 h after IVF, the cleavage rate and blastocyst formation, respectively, were evaluated under a stereomicroscope. The percentages of cleavage and development to the blastocyst stage were determined from the number of oocytes that were placed into the maturation media.

Assessment of Fertilization Parameters

After 10 h of in vitro insemination, groups of 30–40 oocytes were mounted, fixed in acetic acid-ethanol (1:3, v/v) for 72 h, stained with 1% (w/v) lacmoid in 45% (v/v) acetic acid, and examined for fertilization parameters under a phase-contrast microscope at 400x magnification. Oocytes were designated as penetrated when one or more sperm heads and/ or male pronuclei and corresponding sperm tails were present. The rates of polyspermy, male pronuclei formation, and mean number of sperm per oocyte were determined from the oocytes penetrated.

Sperm-ZP Binding Assay

After IVM culture, cumulus-free oocytes were transferred to IVF medium and coincubated with capacitated spermatozoa for 2 h in the presence of increasing concentrations of ChSAO (0–100 µg/ml) as described earlier. After the coincubation, the oocytes and bound sperm were gently pipetted 10 times with a wide-bore pipette to remove loosely bound sperm in H-TL-PVA, and fixed at room temperature for 40 min by 2% formaldehyde. The oocytes were then placed into 50-µl drops of H-TL-PVA containing 10 µg/ml of bis-benzimide Hoechst 33342 and incubated for 10 min. The oocytes were washed in H-TL-PVA, mounted, and the sperm tightly bound to ZP were counted under the fluorescent microscope (Olympus, Tokyo, Japan).

Oocyte Activation and Assessment of ZP Modification

To control the precise timing of activation, oocytes for assessment of ZP modification were artificially activated by electrostimulation as described earlier [38, 39]. Current pulses for stimulation of oocytes were provided using a cell-fusion apparatus (SSH-1; Shimadzu, Kyoto, Japan). After IVM culture, cumulus-free oocytes were preincubated for 1 h in IVF medium added with or without 100 µg/ml ChSAO. Then the oocytes were transferred to electroporation medium (0.3 M mannitol, 0.1 mg/ml PVA, 100 µM CaCl2·H2O, 100 µM MgCl2·6H2O) and stimulated twice by direct-current pulses of 60 V/mm for 30 µsec with a parallel stainless steel wire chamber spaced 1 mm apart on a glass slide at 2-min intervals. Electrostimulated oocytes were further cultured in IVF medium with or without 100 µg/ml ChSAO. It had been previously confirmed that more than 85% of oocytes underwent entry into interphase with pronucleus formation by this procedure [39].

The modification of a single porcine ZP after oocyte activation was analyzed by enhanced chemiluminescent detection of a biotinylated ZP. ZPs were biotinylated according to the method of Moos et al. [40]. Intact ZPs of a single oocyte were isolated as previously described by Kurasawa et al. [41] and biotinylated with water-soluble succinimidyl-6-(biotinamido) hexanoate (NHS-lethal concentration-Biotin; Pierce, IL). NHS-lethal concentration-Biotin was dissolved in 0.1 M NaHCO3 (pH 8.3) at a concentration of 3 µM, and three volumes of this solution were mixed with one volume of a buffer (pH 7.4) containing 150 mM NaCl, 20 mM Hepes, and 3 mg/ml polyvinylpyrrolidone, and used as a biotinylating solution. The isolated ZPs were incubated in the biotinylating solution for 4 h at room temperature and washed five times with H-TL-PVA. Biotinylated ZPs were subjected to SDS-PAGE on a 7.5% polyacrylamide gel and transferred to nitrocellulose membranes (Hybond ECL Western; Amersham Pharmacia Biotech). The membranes were blocked for 1 h at room temperature with 3% (v/v) teleostean skin gelatin in Tris-buffered saline (pH 7.4) containing 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20 (TBS-T-gelatin), then incubated for 50 min at room temperature with 0.1% (v/v) streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) in TBS-T-gelatin, and washed with TBS-T. The bound peroxidase was detected with an Amersham ECL detection kit and an ECL-mini camera (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The amount of each band was quantified by using an Image PC (Scion Co., Frederick, MD). The data were expressed in terms of the fold strength of biotinylated-ZP1 band intensity present in the metaphase II-arrested oocytes (nonelectrostimulated oocytes).

Chlortetracycline Fluorescence Assay

To determine sperm capacitation and acrosome reaction, the fluorescence assay was performed according to the method described previously [42]. The buffer containing 750 µM chlortetracycline (CTC), 20 mM Tris, 130 mM NaCl, and 5 mM cysteine was kept at 4°C and shielded from light. After 90 min of preincubation, sperm were incubated for 2 h in IVF medium in the presence or absence of 100 µg/ml ChSAO, as described earlier. The sperm suspension (20 µl) was mixed with an equal volume of the CTC solution. After 10 sec, 5 µl of 1% glutaraldehyde in 1 M Tris buffer was added. One drop (10 µl) of the sperm suspension was put on a glass slide and covered with a coverslip. Slides were examined under the fluorescence microscope. At least 250 spermatozoa were counted on each slide. Spermatozoa were classified as uncapacitated (F pattern), having uniform fluorescence over the entire head; capacitated (B pattern), having a fluorescence-free dark band in the postacrosomal region; or acrosome-reacted (AR), having barely detectable fluorescence over the whole surface of the head.

Statistical Analysis

Values are presented as the mean of four independent experimental replicates. Variation between experiments is illustrated using SEM. For evaluation of the differences between groups, data on the percentage of fertilization parameters, embryo development, and CTC fluorescence patterns were checked for homogeneity, pooled, and then subjected to contingency-table analysis followed by the Tukey test for nonparametric multiple comparisons [43]. Statistical analyses of data on hyaluronidase activity, mean number of spermatozoa per penetrated oocyte, the number of sperm binding to ZPs, and amount of ZP1 glycoproteins were carried out by the Shapiro-Wilk normality test and analysis of variance followed by the Tukey-Kramer test. All statistical analyses were carried out with the Statistical Analysis System R software package (Cary, NC). A probability of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of ChSAO on Hyaluronidase Activity

The data in Figure 1 show the effect of ChSAO on the hyaluronidase activity of extracts of preincubated boar spermatozoa. A significant decrease in hyaluronidase activity (118 ± 16 U/ml) was shown at a concentration of ChSAO of 0.1 µg/ml compared with that in the absence of ChSAO (279 ± 18 U/ml) (P < 0.05). The hyaluronidase activities were progressively inhibited with increasing ChSAO concentrations and completely blocked at concentrations of 10 µg/ml or higher.



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  		FIG. 1. Inhibitory effect of ChSAO on the hyaluronidase activity of preincubated boar sperm extract. Values are expressed as the mean ± SEM. Values with different superscripts are significantly different (P < 0.05)

Effect of ChSAO on IVF and Subsequent Embryo Development

After IVM culture of COCs, cumulus-intact or cumulus-free oocytes were inseminated in the presence of ChSAO at various concentrations (0–100 µg/ml) to examine the effects of ChSAO on IVF parameters. In cumulus-intact oocytes (Table 1), the penetration rate decreased with increasing concentrations of ChSAO in the IVF medium, and treatments with 50 and 100 µg/ml ChSAO significantly decreased the incidence of sperm penetration into the oocytes (45% and 39%, respectively) compared with that of oocytes treated without ChSAO (63%) (P < 0.05). Moreover, the incidence of polyspermic fertilization was significantly reduced to 28% in the presence of 100 µg/ml ChSAO compared with that in untreated oocytes (52%) (P < 0.05). The degree of cumulus dispersion by sperm was observed without any differences in comparison with the control. In contrast, the inhibitory effect of ChSAO on sperm penetration in cumulus-free oocytes was not found at the concentrations tested, and a high incidence of penetration (74%) was sustained even in the presence of 100 µg/ml ChSAO (Table 2). Interestingly, increased ChSAO concentrations significantly decreased polyspermy (25–30%) compared with the control (53%), resulting in a reduction of the mean number of sperm per oocyte, from 1.7 sperm to 1.3–1.4 sperm (P < 0.05). In both cumulus-intact and cumulus-free oocytes, there was no effect of ChSAO on the formation of male pronuclei. Consequently, the addition of 100 µg/ml ChSAO to the IVF medium in cumulus-free oocytes was selected for the subsequent experiment to evaluate the early embryo development to the blastocyst stage.


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  		TABLE 1. Effect of ChSAO on fertilization parameters of cumulus-intact porcine oocytes matured and fertilized in vitro


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  		TABLE 2. Effect of ChSAO on fertilization parameters of cumulus-free porcine oocytes matured and fertilized in vitro

As shown in Figure 2, the cleavage rate of the oocytes fertilized in the presence of 100 µg/ml ChSAO (64% ± 4%) did not differ from that in the absence of ChSAO (67% ± 4%). In the oocytes treated without ChSAO during IVF, 22% ± 3% of oocytes developed to the blastocyst stage, but a significantly higher proportion of oocytes (33% ± 4%) could develop to the blastocyst stage following in vitro insemination in IVF medium supplemented with 100 µg/ ml ChSAO (P < 0.05). Blastocysts derived from oocytes treated with or without 100 µg/ml ChSAO during IVF contained the same nucleus number (48.6 ± 1.3 or 48.9 ± 1.5, respectively) when fixed and stained with 10 µg/ml of bis-benzimide Hoechst 33342.



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  		FIG. 2. Effects of ChSAO on cleavage and blastocyst formation rates of porcine oocytes fertilized in vitro. In vitro-matured oocytes were preincubated and fertilized in the presence of 0 or 100 µg/ml ChSAO. Values are expressed as the mean ± SEM. The numbers of oocytes examined are indicated in parentheses. Within the same category, values with different superscripts are significantly different (P < 0.05)

Effect of ChSAO on Sperm Binding to ZP

To determine whether the decrease of polyspermy in cumulus-free oocytes resulted from the inhibitory effect of ChSAO on the sperm-zona adhesion, the number of sperm bound to ZP was observed in the presence of various concentrations of ChSAO (0–100 µg/ml). The number of sperm bound on the oocytes concentration-dependently decreased with ChSAO, and the number of sperm bound was significantly decreased to 35.8 ± 2.3 sperm/oocyte by the addition of 1 µg/ml ChSAO compared with that in the absence of ChSAO (57.1 ± 5.2 sperm/oocyte; Fig. 3) (P < 0.05). The inhibitory effect of ChSAO on the binding of sperm to ZP became more obvious in the oocytes treated with 100 µg/ml ChSAO (28.5 ± 1.9 sperm/oocyte).



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  		FIG. 3. Effect of ChSAO on sperm binding to the ZP of cumulus-free oocytes. Oocytes were matured in vitro, then the cumulus cells were removed and fertilized for 2 h in the presence of various concentrations of ChSAO (0–100 µg/ml). Values are expressed as the mean ± SEM. The numbers of oocytes examined are indicated in parentheses. Values with different superscripts are significantly different (P < 0.05)

Effect of ChSAO on ZP Modification Accompanied with Oocyte Activation

The effect of ChSAO on ZP modification was examined to determine if ChSAO is associated with the block of polyspermy by changing the properties of ZP glycoproteins following the exocytosis of cortical granules. When biotinylated ZPs of a single oocyte arrested at the metaphase II stage were subjected to SDS-PAGE, three bands, with an average molecular weight of 92, 69, and 55 kDa were observed by the ECL detection system, which were designated as ZP1, ZP2, and ZP3, respectively (Fig. 4). Electrostimulation of matured oocytes especially revealed a reduction in the amount of the biotinylated-ZP1 band, although no striking difference in the amount of the ZP1 bands between the oocytes treated with and without 100 µg/ml ChSAO was detected throughout the time course after electrostimulation (Fig. 5). The amounts of biotinylated-ZP1 glycoprotein addressed from oocytes treated with or without ChSAO reached near minimum levels after 4 h postelectrostimulation (14.5 ± 2.9 or 16.5 ± 4.8, respectively).



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  		FIG. 4. Representative photograph of zona glycoproteins analyzed by biotinylation and ECL detection in a single oocyte after electrostimulation. Metaphase II-arrested oocytes were artificially activated by electrostimulation and the intensity of biotinylated-ZP1 band was analyzed by ECL as described under Materials and Methods. Each lane represents the profile from a single isolated ZP



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  		FIG. 5. Time-course change in amount of biotinylated-ZP1 glycoprotein in porcine oocytes after electrostimulation in the absence or presence of 100 µg/ml ChSAO. Values are expressed as the mean ± SEM. The numbers of oocytes examined are indicated in parentheses. The maximum value for ZP1 of a matured oocyte (0 h) is taken to be 100. Within the same treatment groups, values with different superscripts are significantly different (P < 0.05)

Effect of ChSAO on Sperm Capacitation and Acrosome Reaction

To determine whether the decrease in the number of sperm bound to ZP was responsible for the increase in the incidence of acrosome reaction, the sperm capacitation and acrosome reaction during incubation in IVF medium with or without 100 µg/ml ChSAO were studied using the CTC test (Table 3). There was no difference in the proportions of B pattern (capacitated) or AR pattern (acrosome reacted) between spermatozoa treated with or without ChSAO. Despite the addition of ChSAO to IVF medium, the capacitation pattern was found in 19–22% of spermatozoa, and treatment with ChSAO did not prominently induce sperm to undergo the acrosome reaction.


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  		TABLE 3. Effect of ChSAO added to the IVF medium on CTC fluores cence patterns of spermatozoa after 2 h of incubation


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In porcine oocytes fertilized in vitro, remarkably low normal fertilization rates, resulting from a high rate of polyspermy, have retarded advances in porcine IVF. This polyspermy, often reaching levels greater than 50%, remain to be solved [9–11, 44]. Various types of equipment have been applied to the method of porcine IVF to improve the incidence of normal fertilization [12, 15, 16, 45]. The present study was conducted to examine the effects of antihyaluronidase oligosaccharide, ChSAO, on IVF parameters of cumulus-intact and cumulus-free oocytes in pigs.

When cumulus-intact oocytes were inseminated in IVF medium containing ChSAO, the penetration and the polyspermic fertilization rates were significantly lower compared with those of oocytes without ChSAO. Although the degradation of hyaluronic acid by hyaluronidase extracted from boar sperm was perfectly blocked by ChSAO at concentrations of 10 µg/ml or higher (Fig. 1), the cumulus dispersion and the sperm penetration were not completely obstructed even in the presence of a sufficient amount (100 µg/ml) of ChSAO (Table 1). Cherr et al. [25] demonstrated that sperm PH-20 is the only hyaluronidase in cynomolgus monkey sperm and that the enzyme is active at physiological pH before the acrosome reaction. When anti-mouse PH-20 IgG was added to IVF medium in cumulus-intact mouse oocytes, the sperm remained outside or only partially entered the cumulus, and no sperm reached the ZP, as a result of inhibition of PH-20 hyaluronidase activity [23]. As reported by Li et al. [37], four polyphenols (tannic acid, kaempferol, quercetin, and apigenin) inhibited the activity of hyaluronidase extracted from cynomolgus monkey sperm in a concentration-dependent manner at a range of 50–200 µM, and these polyphenols had inhibitory effects on monkey sperm penetration into cumulus-intact hamster oocytes. Moreover, apigenin at a concentration of 250 µM could inhibit monkey sperm hyaluronidase activity and reduce the penetration of monkey sperm into monkey cumulus oophorus [46]. It has been also shown that the hyaluronidase activity of mouse sperm PH-20 and cynomolgus monkey sperm PH-20, as well as the recombinant form of cynomolgus monkey PH-20, are inhibited by apigenin [23, 46, 47]. However, we found that the inhibitory effect of apigenin on the activity of hyaluronidase extracted from boar sperm was much weaker than that of ChSAO, and apigenin could not completely block the enzyme activity even at a concentration of 250 µM (unpublished data). It is interesting that mouse sperm lacking PH-20 mRNA show delayed cumulus dispersion and a significantly lower rate of fertilization than wild-type sperm, but fertilization still occurs despite the absence of sperm PH-20 because of the presence of other hyaluronidase(s) presumably within the acrosome of mouse sperm [24]. Therefore, the present findings suggest that the water-soluble ChSAO can readily block the hyaluronidase activity of soluble form extracted from boar sperm but may have difficulty in inhibiting the enzyme activity of the membrane-bound form in the sperm during the fertilization processes. According to Cherr et al. [25] and Li et al. [48], there are two different forms of PH-20 exhibiting hyaluronidase activity in macaque sperm, the 54-kDa soluble form and the 64-kDa membrane-bound form of the proteins.

In cumulus-free oocytes, ChSAO at concentrations of 10 µg/ml or higher decreased the incidence of polyspermy while maintaining high penetration rates (Table 2). This strong prevention of polyspermy in cumulus-free oocytes treated with 100 µg/ml ChSAO led to a significantly higher proportion of in vitro development to the blastocyst stage compared with that of oocytes treated without ChSAO (Fig. 2). The pathological condition of polyspermy is a very early cause of death for the zygote [49]. Data in the present study also indicate that the addition of ChSAO at concentrations of 10 µg/ml or higher to IVF medium strongly decreased the number of sperm bound to the ZP (Fig. 3). In agreement with these results, it has been reported that the addition of OGP to IVF medium can reduce the incidence of polyspermy and increase the number of embryos at the blastocyst stage in porcine oocytes as a consequence of the reduction in the number of sperm bound to the ZP of the oocytes [15]. In mice, small serine/threonine-linked (O-linked) oligosaccharides, isolated from purified mouse-ZP3 following extensive digestion by alkaline reduction, inhibited primary binding of sperm to eggs [50–52]. Although the structures of mouse ZP3 O-linked oligosaccharides have not been determined, the ability of oligosaccharides to inhibit primary binding of sperm to egg is dependent on several parameters, including the size and branching pattern of the oligosaccharide and the nature of the sugar residue at the nonreducing end [18]. In contrast with mouse ZP3, asparagine-linked (N-linked) carbohydrate chains in porcine ZP3{alpha} play a major role in mediating the primary binding of boar sperm to the ZP [53, 54]. In the present study, it becomes clear that ChSAO causes an effective decrease in the incidence of polyspermy in cumulus-free oocytes through a reduction in the number of bound sperm. Although it is not yet clarified that this phenomenon elicited by treatment with ChSAO results from blocking either the primary binding related to carbohydrate-mediated events or the hyaluronidase activity associated with PH-20 protein, the addition of ChSAO to the IVF medium of cumulus-free porcine oocytes seems to be an efficient method for producing a large number of normal fertilized oocytes.

In mouse oocytes, the modification of ZP occurs immediately after fertilization, resulting in the inhibition of further sperm penetration [55]. We previously revealed that complete ZP modification (i.e., minimum levels of biotinylated-ZP1 glycoprotein) of porcine oocytes was seen after 3.5 h of electrostimulation, and this substantial decrease in the amount of the ZP1 glycoprotein during oocyte activation took place in accordance with induction of zona hardening and pronase resistance [38, 56]. The time required for porcine ZP modification is greatly delayed as compared with that observed in mouse oocytes [40], and this delayed ZP modification in porcine oocytes may account for the notable increase in the frequency of polyspermy during IVF. According to very recent findings reported by Asano and Niwa [57], the polyspermic penetration after insemination with sperm at a concentration of 1 x 106 cells/ml was inhibited in porcine oocytes, which were pretreated with an appropriate concentration (6.25 µM) of calcium ionophore A23187 to induce the moderate exocytosis of cortical granules in advance. Kim et al. [14] found that preculture of porcine oocytes in a medium containing 30% oviductal fluid reduced the incidence of polyspermy as a result of an increase in the complete exocytosis of cortical granules and the resistance of ZP to dissolution by pronase at the time of sperm penetration. However, in the present study, the time-course change in the amount of biotinylated-ZP1 glycoprotein after electrostimulation in oocytes treated with ChSAO did not differ from that of oocytes treated without ChSAO (Figs. 4 and 5). The lack of a correlation between the resistance of ZP to pronase dissolution and the reduction of polyspermy in porcine oocytes treated with OGP has also been suggested by Kouba et al. [15]. Moreover, because acrosome-intact, and not acrosome-reacted sperm, can bind primarily to the zona in mice [58] and pigs [59], it may be considered that the disordered acrosome reaction caused by treatment with ChSAO might generate the decrease in the number of sperm binding to ZP. However, a large increase in the percentage of acrosome-reacted sperm in the presence of ChSAO was not detected by the CTC fluorescence assay reported here (Table 3). This result is consistent with previous findings that solubilized mZP3 induced mouse sperm to undergo acrosome reaction, whereas O-linked oligosaccharide released from mZP3 inhibited binding of sperm to oocytes but did not induce acrosome reaction [50, 60]. Therefore, these findings confirm that the effect of ChSAO on the reduction in polyspermy and the number of sperm bound to ZP is never associated with the time-course change in ZP modification immediately after oocyte activation and the incidence of the acrosome-reacted sperm.

In summary, the present study demonstrated that i) ChSAO strictly inhibited the hyaluronidase activity extracted from boar sperm; ii) treatment with ChSAO during IVF interfered with sperm penetration into cumulus-intact oocytes, but not cumulus-free oocytes; and iii) ChSAO effectively reduced the incidence of polyspermy in cumulus-free oocytes under conditions maintaining high sperm penetrability, resulting from a reduction in the number of sperm bound to ZP without influences on ZP modification or acrosome reaction. It is concluded that ChSAO used in the present study appears to be an efficient probe for promoting the normal fertilization process in porcine oocytes during IVF. Further studies are required to investigate the detailed mechanism by which ChSAO interacts with the sperm PH-20 protein and/or sperm-zona adhesion molecules and to elucidate whether the inhibitory effect of ChSAO on sperm hyaluronidase activity is directly involved in sperm-ZP interactions.


   ACKNOWLEDGMENTS
 
We are grateful to the staff of the Meat Inspection Office of the city of Oosato, Japan, for supplying the porcine ovaries.


   FOOTNOTES
 
1 Supported by a Grant-in-Aid for Young Scientists (14760181 to H.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Back

2 Correspondence. FAX: 81 98 895 8757; hidettmt{at}agr.uryukyu.ac.jp Back

Received: 4 June 2004.

First decision: 28 June 2004.

Accepted: 26 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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