This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Funahashi, H. Search for Related Content PubMed PubMed Citation Articles by Funahashi, H.

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

The Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan

Correspondence should be addressed to H Funahashi; Email: hirofun{at}cc.okayama-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6–9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50–250 µmol/l. When 50 µmol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5–82.0% vs 90.5–94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 µmol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9–45.8% vs 31.7–34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 µmol/l bME. Although the presence of 50 µmol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6–37.7%), 50 µmol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3–76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
A high incidence of polyspermic penetration is still constituting a major obstacle in the in vitro production of normal porcine embryos (Funahashi 2003), although historical problems, such as unsuccessful male pronuclear formation and low developmental competence, have been overcome by reducing oxidative stress with thiols during in vitro maturation (IVM) and culture (IVC) for early development. Supplementation with antioxidants – such as beta-mercaptoethanol (bME) and cysteamine – during IVM has been found to improve intracellular glutathione content in oocytes and the developmental competence after in vitro fertilization (IVF) in several species (Comizzoli et al. 2000, Mizushima & Fukui 2001, de Matos et al. 2002, Songsasen & Apimeteetumrong 2002, Rodriguez-Gonzalez et al. 2003). In pigs, the addition of cysteine and bME during IVM also enhanced the incidence of male pronuclear formation and the developmental competence of IVF embryos (Funahashi et al. 1997, Abeydeera et al. 1998). However, bME in IVM medium has been known to improve the quality of blastocysts, as determined by the cell number, but not the efficiency of in vitro blastocyst formation in cows (Takahashi et al. 2002), swamp buffalo (Songsasen & Apimeteetumrong 2002) and sheep (de Matos et al. 2002). Furthermore, the incidence of blastocyst formation and the quality of embryos have also been increased by reducing the oxidative stress during IVC of IVM-IVF porcine embryos (Kikuchi et al. 2002). However, little is known about the effect of antioxidants, such as bME, during the IVF period on the penetrability of spermatozoa into porcine oocytes and on early development. Supplementation with thiol components during IVF procedures may be beneficial to the oocyte quality, and consequently to the incidences of normal fertilization and the developmental competence. Since reactive oxygen species like superoxide anion or hydrogen peroxide have been suggested to promote sperm capacitation in humans (de Lamirande & Gagnon 1993, Leclerc et al. 1997), the presence of bME may prevent sperm capacitation, as an oxidative reaction. In fact, the addition of antioxidants during a conventional IVF period has been demonstrated to reduce penetration rates (Blondin et al. 1997) and the subsequent development of bovine embryos to the morula and blastocyst stages (Ali et al. 2003).

Recently, the author and a colleague found that a transient co-culture of porcine oocytes with spermatozoa in the presence of caffeine followed by an additional culture in the absence of sperm cells and caffeine effectively decreased the incidence of polyspermic penetration without any reduction in the penetration rate (Funahashi & Romar 2004). Since this new in vitro fertilization (IVF) system can separate the process of sperm capacitation and zona binding from the following process of sperm penetration under different conditions, this system may be useful in analyzing further details of the effects of bME during the IVF period.

Therefore, the major objectives of the present study were: to determine the effect of bME addition during a transient co-culture period on sperm capacitation and zona binding; and to determine the effects of addition of bME to the following culture medium on sperm penetration in IVF of porcine IVM oocytes, and also early development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Chemicals and culture media
KCl, KH₂PO₄, MgCl₂·6H₂O, CaCl₂·2H₂O, sodium citrate and citric acid were purchased from Ishizu Pharmaceutical Co., Ltd (Osaka, Japan). NaCl and paraffin liquid were obtained from Nacalai Teque Inc. (Kyoto, Japan). Other chemicals were purchased from Sigma.

The medium used for the collection of oocyte–cumulus complexes and washing was modified TL-HEPES-PVA medium composed of 114 mmol NaCl/l, 3.2 mmol KCl/l, 2 mmol NaHCO₃/l, 0.34 mmol KH₂PO₄/l, 10 mmol sodium lactate/l, 0.5 mmol MgCl₂·6H₂O/l, 2 mmol CaCl₂·2H₂O/l, 10 mmol HEPES/l, 0.2 mmol sodium pyruvate/l, 12 mmol sorbitol/l, 0.1% (w/v) polyvinylalcohol, 25 µg gentamicin/ml and 65 µg potassium penicillin G/ml. The basic IVM medium (OMM37) used was BSA-free North Carolina State University 37 (NCSU37) medium (Petters & Wells 1993) supplemented with 0.6 mmol cysteine/l, 5 µg insulin/ml, 50 µmol bME/l and 10% (v/v) porcine follicular fluid (Funahashi et al. 1997). The basic IVF medium was modified Medium199 (m-M199), which is Medium199 with Earle’s salts (Gibco; Invitrogen Corp.) supplemented with 3.05 mmol glucose/l, 2.92 mmol hemi-calcium lactate/l, 0.91 mmol sodium pyruvate/l, 12.00 mmol sorbitol/l, 25 µg gentamicin/ml, 65 µg potassium penicillin G/ml, and 0.4% (w/v) BSA (Sigma; catalog number A4378). The medium used as semen diluent was modified Modena solution prepared with 152.64 mmol glucose/l, 23.46 mmol sodium citrate/l, 11.9 mmol NaHCO₃/l, 6.99 mmol EDTA-2Na/l, 46.66 mmol Tris/l, 15.10 mmol citric acid/l and 25 mg gentamicin sulfate/l. All media without modified TL-HEPES-PVA and modified Modena solution were equilibrated under paraffin liquid at 39 °C in an atmosphere of 5% CO₂ in air over-night prior to incubation with oocytes. Porcine follicular fluid was prepared from antral follicles (3–6 mm in diameter) as described previously (Funahashi et al. 1994a).

Preparation and culture of cumulus–oocyte complexes
Ovaries were collected from slaughtered prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl containing 75 mg potassium penicillin G/l and 50 mg streptomycin sulfate/l. Cumulus–oocyte complexes were aspirated through an 18-gauge needle into a disposable 10 ml syringe from antral follicles (3–6 mm in diameter) on the surface of ovaries, washed three times with modified TL-HEPES-PVA medium, and then collected in a fresh modified TL-HEPES-PVA medium at room temperature (Wongsrikeao et al. 2004). Oocytes were matured in an IVM system that has been reported to produce blastocysts and piglets efficiently following IVF and embryo transfer (Funahashi et al. 1997). Briefly, 50 cumulus–oocyte complexes with uniform ooplasm and a compact cumulus cell mass were washed three times with OMM37 supplemented with 1 mmol dibutyryl cAMP/l, 10 iu equine chorionic gonadotropin (eCG)/ml and 10 iu human chorionic gonadotropin (hCG)/ml, and subsequently cultured in 500 µl of the same medium covered with paraffin oil for 20 h at 39 °C in an atmosphere of 5% CO₂ in air. The complexes were then transferred to 500 µl OMM37 (without dibutyryl cAMP, eCG and hCG) after washing three times with the same medium. The complexes were cultured for an additional 24 h (Funahashi et al. 1994a). After culture, oocytes were stripped of cumulus cells by pipetting with 0.1% (w/v) hyaluronidase and were washed three times with m-M199.

Preparation of fresh boar spermatozoa
Semen-rich fractions (30–50 ml) were collected from a Berkshire boar by the gloved-hand method at a local experimental station and were diluted four times with modified Modena solution. The diluted semen samples were transported to the laboratory within 2 h of collection. After washing once by centrifugation at 750 g for 3 min, spermatozoa were re-suspended at a concentration of 1 x 10⁸ cells/ml in modified Modena solution containing 5 mmol cysteine/l and 20% (v/v) boar seminal plasma (Funahashi & Sato 2005). Diluted sperm suspension was kept overnight at room temperature. Just before use, stored spermatozoa were washed three times by centrifugation at 750 g for 3 min with modified TL-HEPES-PVA solution and then re-suspended at a concentration of 1 x 10⁸ cells/ml in m-M199.

In vitro fertilization
After dilution to 5 x 10⁵ cells/ml with m-M199, 50 µl diluted sperm suspension were inseminated in the same volume of m-M199 containing 10 mmol caffeine-benzoate/l (final concentrations of spermatozoa and caffeine-benzoate were 2.5 x 10⁵ cells/ml and 5 mmol/l respectively). Thirty denuded oocytes were co-cultured with spermatozoa in 100 µl droplets under paraffin oil for 10 min. The oocytes were gently washed once with sperm-free and caffeine-free m-M199, transferred to a 500 µl well of fresh caffeine-free m-M199 and the culture continued for 6 or 9 h at 39 °C in an atmosphere of 5% CO₂ in air.

Parthenogenetic oocyte activation and labeling oocytes with fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA)
Denuded oocytes were washed three times with m-M199 containing 5 mmol caffeine-benzoate/l (m-M199–caffeine) and treated with 100 µmol calcium ionophore/l (Wang et al. 1998b) in the same medium for 5 min for parthenogenetic activation. After the treatment, oocytes were washed three times with m-M199–caffeine, transferred to a 500 µl well of fresh m-M199–caffeine and then the culture continued for 1 h at 39 °C in an atmosphere of 5% CO₂ in air.

After the culture, the oocytes were washed once with TL-HEPES-PVA and fixed with 3% paraformaldehyde in TL-HEPES-PVA for 30 min at room temperature. As described in a previous report (Katayama et al. 2002), the oocytes were processed and stained with 20 µg lectin/ml from Archis hypogaea (peanut)-conjugated FITC (FITC-PNA) and 400 µl propidium iodide/ml for 30 min. After rinsing, the oocytes were mounted in an anti-fade medium. The surface of the oocyte was observed using a confocal laser scanning microscope (MRC1024, Nippon Bio-Rad Laboratories, Tokyo, Japan).

Chlortetracycline (CTC) fluorescence assessment of spermatozoa
The methods used for CTC analysis were essentially those described previously (Funahashi et al. 2000) with minor modification. Briefly, 8 µl of 100 µg Hoechst bis-benzimide 33258/ml was added to 792 µl of sperm suspension. After mixing, each suspension was incubated for 3 min at room temperature in the dark, then layered onto 3 ml of 3% (w/v) polyvinylpyrolidone (PVP-40) in TL-HEPES-PVA and centrifuged at 750 g for 3 min. The pelleted spermatozoa were resuspended in 45 µl TL-HEPES-PVA and 45 µl of this suspension were added to 45 µl CTC solution, containing 750 µmol CTC/l, 5 mmol cysteine-HCl/l, 130 mmol NaCl/l and 20 mmol Tris/l (pH 7.8). Sperm cells were fixed by adding 8 µl of 12.5% (w/v) paraformaldehyde in 0.5 mol Tris–HCl/l (pH 7.4). The CTC solution was prepared daily. Slides were prepared by placing 10 µl of the fixed sperm suspension on a slide and one drop of 0.22 mol 1,4-diazabicyclo[2.2.2]octane/l dissolved in glycerol:TL-HEPES-PVA (9:1) which was then mixed in order to retard the fading of fluorescence. A coverslip was added and sealed with colorless nail varnish. Spermatozoa were assessed under a phase-contrast microscope, equipped with epifluorescent optics, on the same day. Each cell was first observed under u.v. illumination (excitation at 330–380 nm, emission at 420 nm) to determine the live/dead status; the sperm cells showing bright blue staining of the nucleus were considered to be dead and were not counted. More than 100 live sperm were then examined under blue-violet illumination (excitation at 400–440 nm, emission at 470 nm) and classified according to CTC staining patterns. The three fluorescent staining patterns identified were: F, with uniform fluorescence over the whole sperm head; B, with a fluorescence-free band in the post-acrosome region; AR, with almost no fluorescence over the sperm head except for a thin band of fluorescence in the equatorial segment.

Glutathione assay
Five microliters of 1.25 M phosphoric acid was added to a 1.5 ml microfuge tube containing 30–50 oocytes in 5 µl of 0.2 M sodium phosphate supplemented with 10 mM EDTA (pH 7.2, stock buffer). The samples were stored in a freezer (–80 °C) until assayed. The total content of glutathione per oocyte was determined using the 5,5^'-dithiobis(2-nitrobenzoic acid)-glutathione disulfide (DTNB-GSSG) reductase recycling assay (Anderson 1985) with modification (Funahashi et al. 1994b). Briefly, 700 µl of 0.33 mg NADPH/ml in stock buffer, 100 µl of 6 mM DTNB in the stock buffer and 190 µl water were added with mixing into the microfuge tube. To initiate the reaction, 10 µl glutathione reductase were added with mixing. The formation of 5-thio-2-nitrobenzoic acid was followed continuously with a spectrophotometer (Ultraspec-2000, Pharmacia Biotech Ltd, Cambridge, UK) from 30 s to 3 min of reaction with a reading recorded every 15 s. Glutathione standards (0.05–1.0 nmol) and a sample lacking glutathione were also assayed. The total content of glutathione was determined (Calvin et al. 1986).

Experimental design
The experimental design is schematically represented in Fig. 1. In the first experiment, the effect of bME on sperm function was examined using the CTC fluorescence assessment. Washed and re-suspended spermatozoa were diluted at 1 x 10⁶ cells/ml in m-M199–caffeine and various concentrations (0, 2, 10, 50 and 250 µmol/l) of bME and then cultured for 2 h at 39 °C in an atmosphere of 5% CO₂ in air. After the culture, CTC patterns of these sperm cells were compared with those of spermatozoa cultured in bME-free and caffeine-free m-M199. Data were obtained from eight replicated experiments.

View larger version (46K): [in this window] [in a new window]   Figure 1 Schematic representation of experimental design, showing the different treatments.

In the third experiment, the effect of bME during the culture following a transient co-culture on oocyte glutathione content was examined. After a transient co-culture with spermatozoa for 10 min, oocytes were cultured in the absence or presence of 50 µmol bME/l. At 6 h after insemination, 30–50 morphologically normal oocytes were sampled to determine intracellular glutathione content. Data were obtained from 5 or 6 replicated experiments.

In the fourth experiment, the effect of bME on the exocytosis of cortical granules after oocyte activation was examined. In the absence or presence of 50 µmol bME/l, denuded oocytes were treated with 100 µmol calcium ionophore/l in m-M199–caffeine for 5 min and then cultured in m-M199–caffeine for 1 h. Cultured oocytes were fixed, labeled with FITC-PNA and propidium iodide and then the fluorescence was observed in the cortex region of the oocyte under a confocal laser scanning microscope. To compare the degree of cortical reaction, the mean pixel intensity of the cortex region of the oocyte was measured using the accessory software of the Laser Scanning Confocal Imaging System (LaserSharp; Nippon Bio-Rad Laboratories, Tokyo, Japan). Values taken out the background intensity were compared. Data were obtained from four replicated experiments.

In the last experiment, the in vitro development of oocytes fertilized in the absence and presence of bME was examined. After a transient co-culture with spermatozoa at a concentration of 2.5 x 10⁵ cells/ml in the absence or presence of 50 µmol bME/l in m-M199–caffeine for 10 min at 39 °C in an atmosphere of 5% CO₂ in air, the oocyte culture continued in caffeine-free m-M199 containing 50 µmol bME/l. As controls, some oocytes were not exposed to bME during the transient co-culture and the following culture until 6 h after insemination. Those oocytes were then moved again to modified NCSU37 medium supplemented with 0.4% (w/v) BSA (Sigma, A8022), 0.6 mmol cysteine/l, 50 µmol bME/l and 5 µg insulin/ml and then cultured for 7 days at 39 °C in an atmosphere of 5% CO₂ in air. Data were obtained from four replicated experiments.

Statistical analysis
Statistical analyses of results were used for treatment comparisons and were carried out by one- or two-way ANOVA using the JMP 5.0 (SAS Institute, Inc., Cary, NC, USA) program. If the P value was smaller than 0.05 in ANOVA, Tukey–Kramer’s HSD test was carried out using the same program. All percentage data were subjected to arcsine transformation before statistical analysis. In order to show percentage data in tables, those data were transformed back to the original percentages. All data were expressed as means ± S.E.M. P 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Effect of bME on the CTC pattern of spermatozoa (experiment 1)
The presence of bME affected CTC patterns when spermatozoa were cultured in m-M199–caffeine for 2 h (Fig. 2). In bME-free medium, the presence of 5 mmol caffeine-benzoate/l in m-M199 decreased the incidence of intact (F pattern) cells and increased the capacitated (B pattern) and acrosome-reacted (AR pattern) cells. Although 2 µmol bME/l did not affect (P > 0.05) the stimulatory effect of caffeine-benzoate, the presence of bME at more than 10 µmol/l neutralized the stimulatory effect of caffeine-benzoate in a concentration-dependent manner. CTC patterns of spermatozoa cultured in the presence of 50 and 250 µmol bME/l in m-M199–caffeine did not differ from those of spermatozoa cultured in bME-free and caffeine-free m-M199.

View larger version (31K): [in this window] [in a new window]   Figure 2 Effect of bME on sperm characteristics as determined by CTC fluorescence patterns in fresh boar spermatozoa: F, intact (uncapacitated) cells; B, capacitated cells; AR, acrosome-reacted cells. Spermatozoa were cultured in the absence or presence of various concentrations of bME in m-M199 or m-M199–caffeine for 2 h. Different letters above the bars indicate statistically significant differences (P < 0.05).

View larger version (37K): [in this window] [in a new window]   Figure 3 Glutathione contents of porcine oocytes before (0 h) and after a transient co-culture with spermatozoa for 10 min in m-M199–caffeine and the following culture for 6 h in the absence or presence of bME in caffeine-free m-M199. Numbers in parentheses indicate the total number of oocytes assayed. Bars with different letters differ statistically (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 4 Degree of cortical granule exocytosis after calcium ionophore treatment, as evaluated by the distribution of FITC-PNA at the cortex region of oocytes. In the absence (CONT) or presence of 50 µmol bME/l (bME), denuded oocytes were treated with calcium ionophore for 5 min, cultured for 1 h and then processed. After taking out the background intensity, the mean pixel intensities of the cortex region of the oocytes were plotted.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

In the current study, fresh spermatozoa were co-cultured with oocytes for a transient period (10 min) in a fertilization decreased containing caffeine-benzoate just after washing, without preincubation to induce capacitation, and then only oocytes with binding spermatozoa were subjected to the following culture in caffeine-free m-M199 until 9 h after insemination. Supplementation with bME during a transient co-culture period decreased, but not completely, the incidence of sperm penetration, whereas bME in m-M199–caffeine prevented the stimulating effect of caffeine on sperm capacitation and the spontaneous acrosome reaction during culture for 2 h. In a conventional IVF system, it has been reported that the addition of catalase during the co-culture period reduces sperm penetration rates in cows (Blondin et al. 1997). Supplementation with other antioxidants such as superoxide dismutase, catalase and N-acetyl-L-cysteine, during IVF also decreases the subsequent rate of bovine embryo development to the morula and blastocyst stages (Ali et al. 2003). Therefore, these results suggest that, even during a short co-culture period, the presence of bME is effective in preventing to some extent, or delaying, capacitation of boar spermatozoa and consequently appears to reduce the incidence of sperm penetration (at 9 h after insemination).

In contrast, the presence of bME during culture in caffeine-free m-M199 after a transient co-culture significantly decreased the incidence of polyspermy oocytes and the mean number of spermatozoa in a penetrated egg, but did not affect sperm penetration rate. In the present study, to compare the effect of bME treatment on monospermic penetration directly, the incidence of monospermy was shown as a percentage of total mature oocytes examined. Thus, monospermy or polyspermy rates were not affected by the penetration rates. The results suggest that a transient co-culture period in the presence of caffeine-benzoate (10 min) should be enough to induce sperm capacitation and penetration, even when followed by an additional culture in the presence of bME in caffeine-free medium. Therefore, the current new IVF system – composed of a transient co-culture in m-M199–caffeine and the following culture in the presence of bME in caffeine-free m-M199 – is valid for increasing the incidence of normally penetrated eggs without any reduction in penetration rate. However, the presence of bME during the culture after a transient co-culture period reduced the incidence of penetrated oocytes developing to the pronuclear stage (by 9 h after insemination). bME during the culture period following a transient co-culture appears to delay the time of sperm penetration, rather than prevent pronuclear formation since it affects the sperm function associated with capacitation and acrosome reaction.

The current observations showed that oocyte glutathione content decreased during culture until 6 h following insemination. This observation is inconsistent with our previous observations that intracellular glutathione content decreased in oocytes penetrated in vitro (Funahashi et al. 1995), probably due to a sperm enzyme associated with degradation of glutathione (Funahashi et al. 1996). However, the presence of bME during the culture for 6 h following a transient co-culture with spermatozoa minimized the decrease in oocyte glutathione content, as compared with culture in the absence of bME. This evidence indicates that oocytes suffer oxidative stress during IVF, in contrast to conditions in the oviduct where the internal scavenger system reduces stress.

It has been reported that oocytes matured in the recent IVM conditions possess equal ability to release cortical granules on sperm penetration in vitro when compared with in vivo-matured porcine oocytes (Wang et al. 1998a). However, the current data demonstrate that the presence of bME during chemical activation of oocytes significantly improved the degree of cortical granule exocytosis of IVM oocytes 1 h after activation and that supplementation with bME during culture following a transient co-culture, the period of sperm penetration, decreased the incidence of polyspermic penetration. Thus, the rate of cortical exocytosis may be reduced even during IVF processes, by the oxidative stress. Although Boquest et al.(1999) have reported that the addition of glutathione during insemination did not affect the rate of polyspermic fertilization, the polyspermy rate was reduced even in oocytes matured in a medium supplemented with bME (Whitaker 1990, Mizushima & Fukui 2001). In the present study, oocyte glutathione content was higher when oocytes were cultured in a medium containing bME following a transient co-culture. Therefore, supplementation with bME during IVM and sperm penetration not only affects the sperm function associated with fertilization, but also minimizes the decrease in intracellular glutathione content of oocytes during sperm penetration and somewhat improves the degree of cortical granule exocytosis, and consequently appears to reduce the incidence of polyspermic penetration.

The addition of antioxidants, such as cysteine (Ali et al. 2003), ascorbic acid (Comizzoli et al. 2000, Tatemoto et al. 2001), cysteamine (Rodriguez-Gonzalez et al. 2003), l-alpha-aminobutyrate (Whitaker 1990) and bME (Whitaker 1990, Mizushima & Fukui 2001), during IVM improves oocyte glutathione concentrations and early development to the blastocyst stage after IVF in several species. However, these effects appear to be dependent on species and antioxidants (de Matos et al. 2002, Songsasen & Apimeteetumrong 2002). In pigs, supplementation with cysteine and bME during IVM improved oocyte competence to develop to the blastocyst stage after IVF (Funahashi et al. 1997, Abeydeera et al. 1998). Therefore, storage of transcripts encoding for antioxidant enzymes during oocyte maturation could be important in pigs in order to allow the embryo to acquire the aptitude to develop (Guerin et al. 2001). The incidence of blastocyst formation and the quality of embryos have also been enhanced by reducing the oxidative stress during IVC after IVF (Caamano et al. 1996, Kikuchi et al. 2002, Takahashi et al. 2002). In the present study, when bME was supplemented during the culture period after a transient co-culture, the percentage blastocyst formation was not improved. However, the decrease in intracellular glutathione content was minimized, and the degree of cortical reaction at oocyte activation, the incidence of monospermic penetration and the quality of embryos (as determined by the number of cells in a blastocyst) were improved. Furthermore, the presence of bME during a transient co-culture of gametes for only 10 min decreased the incidence of blastocyst formation, probably due to a decreased incidence of sperm penetration, but did not affect the incidence of monospermic penetration and the quality of embryos. It has also been shown that although polyspermic IVM–IVF porcine embryos containing multiple pronuclei can develop to the blastocyst stage at the same percentage as monospermic IVM–IVF embryos, the mean cell number of blastocysts (especially inner cell mass) derived from polyspermic embryos was lower than that of monospermic blastocysts (Han et al. 1999). Therefore, a reduction in oxidative stress in oocytes during sperm penetration in vitro, by adding bME after a transient co-culture, appears to improve the incidence of monospermic penetration by affecting the oocyte function associated with fertilization, and consequently the quality of blastocysts.

In conclusion, supplementation with thiol components during IVF procedures, except for a transient co-culture period permitting sperm capacitation and/or spermzona binding, has several beneficial effects: preventing excessive sperm capacitation; minimizing the decrease in oocyte glutathione content through sperm penetration; promoting cortical granule exocytosis; and increasing the incidence of normal fertilization. These beneficial effects of bME consequently appear to improve the quality of IVF embryos.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The author thanks the Okayama Prefectural Center for Animal Husbandry and Research for supplying fresh boar semen. This work was supported by grants from the Ito Foundation and Grant-in-Aid for Scientific Research (C16580230) of the Japan Society for the Promotion of Science. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 23 February 2005
First decision 19 May 2005
Revised manuscript received 27 August 2005
Accepted 8 September 2005

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

Abeydeera LR, Wang WH, Cantley TC, Prather RS & Day BN 1998 Presence of beta-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology 50 747–756.[CrossRef][Web of Science][Medline]

Aitken J & Fisher H 1994 Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 16 259–267.[CrossRef][Web of Science][Medline]

Ali AA, Bilodeau JF & Sirard MA 2003 Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development. Theriogenology 59 939–949.[CrossRef][Web of Science][Medline]

Anderson ME 1985 Determination of glutathione and glutathione disulfide in biological samples. In Methods in Enzymology, vol 113, Glutamate, Glutamine, Glutathione, and Related Compounds, pp 548–555. Ed. A Meister. Academic Press, San Diego.

Blondin P, Coenen K & Sirard MA 1997 The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. Journal of Andrology 18 454–460.[Abstract/Free Full Text]

Boquest AC, Abeydeera LR, Wang WH & Day BN 1999 Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro. Theriogenology 51 1311–1319.[CrossRef][Web of Science][Medline]

Caamano JN, Ryoo ZY, Thomas JA & Youngs CR 1996 Beta-mercaptoethanol enhances blastocyst formation rate of bovine in vitro-matured/in vitro-fertilized embryos. Biology of Reproduction 55 1179–1184.[Abstract]

Calvin HI, Grosshans K & Blake EJ 1986 Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova: relevance to sperm nucleus transformation in the fertilized egg. Gamete Research 14 265–275.[CrossRef][Web of Science]

Casillas ER & Hoskins DD 1970 Activation of monkey spermatozoa adenyl cyclase by thyroxine and triiodothyronine. Biochemical and Biophysical Research Communications 40 255–262.[CrossRef][Web of Science][Medline]

Comizzoli P, Marquant-Le Guienne B, Heymen Y & Renard JP 2000 Onset of the first S-phase is determined by a paternal effect during the G1-phase in bovine zygotes. Biology of Reproduction 62 1677–1684.[Abstract/Free Full Text]

de Lamirande E & Gagnon C 1993 Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radical Biology and Medicine 14 157–166.[CrossRef][Web of Science][Medline]

de Matos DG, Gasparrini B, Pasqualini SR & Thompson JG 2002 Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology 57 1443–1451.[CrossRef][Web of Science][Medline]

Funahashi H 2003 Polyspermic penetration in porcine IVM-IVF systems. Reproduction, Fertility and Development 15 167–177.[CrossRef][Medline]

Funahashi H & Romar R 2004 Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine. Reproduction 128 789–800.[Abstract/Free Full Text]

Funahashi H & Sato T 2005 Select antioxidants improve the function of extended boar semen stored at 10 °C. Theriogenology 63 1605–1616.[CrossRef][Web of Science][Medline]

Funahashi H, Cantley TC & Day BN 1994a Different hormonal requirement of porcine oocyte-complexes during maturation in vitro. Journal of Reproduction and Fertility 101 159–165.[Abstract/Free Full Text]

Funahashi H, Cantley TC, Stumpf TT, Terlouw SL & Day BN 1994b Use of low salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization. Biology of Reproduction 51 633–639.[Abstract]

Funahashi H, Stumpf TT, Cantley TC, Kim NH & Day BN 1995 Pronuclear formation and intracellular glutathione content of in vitro-matured porcine oocytes following in vitro fertilisation and/or electrical activation. Zygote 3 273–281.[Web of Science][Medline]

Funahashi H, Machaty Z, Prather RS & Day BN 1996 -Glutamyl-transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs. Molecular Reproduction and Development 45 485–490.[CrossRef][Web of Science][Medline]

Funahashi H, Cantley TC & Day BN 1997 Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biology of Reproduction 57 49–53.[Abstract]

Funahashi H, Asano A, Fujiwara T, Nagai T, Niwa K & Fraser LR 2000 Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro. Molecular Reproduction and Development 55 117–124.[CrossRef][Web of Science][Medline]

Guerin P, El Mouatassim S & Menezo Y 2001 Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Human Reproduction Update 7 175–189.[Abstract/Free Full Text]

Han YM, Abeydeera LR, Kim JH, Moon HB, Cabot RA, Day BN & Prather RS 1999 Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro. Biology of Reproduction 60 1110–1113.[Abstract/Free Full Text]

Katayama M, Koshida M & Miyake M 2002 Fate of the acrosome in ooplasm in pigs after IVF and ICSI. Human Reproduction 17 2657–2664.[Abstract/Free Full Text]

Kikuchi K, Onishi A, Kashiwazaki N, Iwamoto M, Noguchi J, Kaneko H, Akita T & Nagai T 2002 Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biology of Reproduction 66 1033–1041.[Abstract/Free Full Text]

Kim JG & Parthasarathy S 1998 Oxidation and the spermatozoa. Seminars in Reproductive Endocrinology 16 235–239.[Web of Science][Medline]

Leclerc P, de Lamirande E & Gagnon C 1997 Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. Free Radical Biology and Medicine 22 643–656.[CrossRef][Web of Science][Medline]

Mizushima S & Fukui Y 2001 Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium. Theriogenology 55 1431–1445.[CrossRef][Web of Science][Medline]

O’Flaherty C, Beconi M & Beorlegui N 1997 Effect of natural anti-oxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa. Andrologia 29 269–275.[Web of Science][Medline]

Petters RM & Wells KD 1993 Culture of pig embryos. Journal of Reproduction and Fertility, Supplement 48 61–73.

Rodriguez-Gonzalez E, Lopez-Bejar M, Mertens MJ & Paramio MT 2003 Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes. Molecular Reproduction and Development 65 446–453.[CrossRef][Web of Science][Medline]

Songsasen N & Apimeteetumrong M 2002 Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes. Animal Reproduction Science 71 193–202.[CrossRef][Web of Science][Medline]

Takahashi M, Nagai T, Okamura N, Takahashi H & Okano A 2002 Promoting effect of beta-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos. Biology of Reproduction 66 562–567.[Abstract/Free Full Text]

Wang WH, Abeydeera LR, Prather RS & Day BN 1998a Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Molecular Reproduction and Development 49 308–316.[CrossRef][Web of Science][Medline]

Wang WH, Machaty Z, Abeydeera LR, Prather RS & Day BN 1998b Parthenogenetic activation of pig oocytes with calcium ionophore and the block to sperm penetration after activation. Biology of Reproduction 58 1357–1366.[Abstract/Free Full Text]

Whitaker MJ 1990 Cell cycle control proteins are second messenger targets at fertilization in sea-urchin eggs. Journal of Reproduction and Fertility, Supplement 42 199–204.

Wongsrikeao P, Karja NWK, Budiyanto A, Mtango NR, Murakami M, Nii M & Otoi T 2004 Meiotic competence and DNA fragmentation of porcine oocytes from ovaries stored in various temperatures. Reproduction, Fertility and Development 16 283–284.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Funahashi, H. Search for Related Content PubMed PubMed Citation Articles by Funahashi, H.