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Regular article |
a School of Bioresources, Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan
ABSTRACT
The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.
apoptosis, cumulus cells, meiosis, ovum, stress
INTRODUCTION
The mammalian oocyte and its surrounding cumulus cells are metabolically coupled through gap junctions, that provide a unique means of entry into the ooplasm for several metabolites [1, 2]. Cumulus cells have a close connection with oocytes during the course of maturation in mammals [3–5]. Gonadotropins, steroids, and other factors from the follicle cells also interact with oocytes to provide essential support for in vivo maturation of oocytes [6]. It is generally accepted that cumulus cells support the maturation of oocytes to the metaphase II stage and greatly enhance cytoplasmic maturation, which is responsible for the capacity to undergo normal fertilization and subsequent embryonic development. Several studies have indicated that cumulus-denuded oocytes (DOs) can undergo meiotic maturation in mice [7], rats [8], sheep [9], and cattle [10, 11] in vitro. Chian et al. [10] and Kim et al. [12] demonstrated that although the presence of cumulus cells coupled to bovine oocytes was not necessary for nuclear maturation, the developmental competence of DOs after in vitro fertilization (IVF) was prominently lower than that of cumulus-oocyte complexes (COCs). These findings indicate that the poor developmental competence of DOs might be caused by the lack of cytoplasmic maturation of oocytes.
During fertilization, glutathione (GSH) content in oocytes participates in sperm decondensation in parallel with oocyte activation [13]. Recently, the addition of cysteamine to maturation medium increased male pronuclear formation after IVF in porcine oocytes [14, 15]. In bovine oocytes, the addition of cysteamine to maturation medium resulted in an increase in GSH content and an improvement in the rate of embryo development to the blastocyst stage, suggesting that the beneficial effects of cysteamine on in vitro maturation and subsequent development after IVF were mediated by GSH [16]. The synthesis of GSH in COCs during in vitro maturation has been reported in mice [17], hamsters [18], pigs [19], and cattle [20]. Cumulus cells contributed to the stimulatory effect on cysteine- and cysteamine-induced GSH synthesis in bovine COCs, indicating that cumulus cells play an important role in oocyte GSH synthesis during in vitro maturation [21].
Glutathione is the major nonproteinous sulfhydryl compound in mammalian cells and is well known to play an important role in protecting the cell from oxidative damage [22]. Depending on the site of formation and on the tension of oxygen, reactive oxygen species (ROS) such as superoxide anions (O2-), hydroxyl radicals (OH-), and hydrogen peroxide (H2O2) can cause lipid peroxidation and enzyme inactivation, resulting in cell damage by promoting hydroxyl radical formation [23]. In vitro culture is maintained at higher concentrations of O2 than the in vivo environment and, therefore, results in increased production of ROS [24]. Recent studies have shown that the redox status of the cell, resulting from an accumulation of ROS and a decrease of antioxidant levels, is involved in inducing apoptotic cell death [25–27], and GSH presumably plays a critical role in regulating apoptosis by influencing the redox status [28]. Oxidative stress induces aneuploidy during mouse oocyte development, indicating that it is implicated in maternal aging-associated infertility [29]. The presence of reactive oxygen scavenger in culture medium during in vitro maturation resulted in the increase in the percentage of embryos developed at the 64-cell stage following IVF in bovine oocytes [30].
It is thus speculated that cumulus cells play an important role in not only acquiring the developmental competence associated with oocyte cytoplasmic maturation but also protecting oocytes against irremediable cell damage encompassed by oxidative stress during oocyte maturation. However, it remains unknown whether cumulus cells have a functional role in protecting oocytes from oxidative stress. The present study was conducted to examine the protective effect of cumulus cells on cell damage caused by the hypoxanthine-xanthine oxidase (XOD) system during in vitro maturation of porcine oocytes. After treatment of COCs and DOs with or without XOD for 44 h, meiotic maturation, DNA cleavage and damage, caspase-3 activity, and oocyte GSH content were determined in each experimental group. Cleavage and damage of DNA were determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively.
MATERIALS AND METHODS
All chemicals used in the present study were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.
Collection of Oocytes
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, Ltd., Tokyo, Japan) at 30°C. Within 2 h postslaughter, the follicular contents were recovered by excising the visible small antral follicles (about 2–5 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 COCs with uniform ooplasm and a compact cumulus cell mass were prepared in Hepes-buffered Tyrodes medium containing 0.01% (w:v) polyvinyl alcohol (H-TL-PVA [31]). The DOs were obtained by mechanically removing cumulus cells from COCs with a narrow-bore pipette. Before culture, COCs and DOs were washed three times with H-TL-PVA.
Maturation Culture of Oocytes
The basic medium for maturation culture of oocytes was BSA-free NCSU37 (NCSU37 [32]) supplemented with 0.6 mM cysteine, 2% (v:v) minimual essential medium (MEM) amino acids solution (Gibco BRL, Grand Island, NY), 1% (v:v) MEM nonessential amino acids solution (Gibco), 0.04 U/ml ovine FSH, 0.02 U/ml ovine LH, 10% (v:v) porcine follicular fluid, and 1 mM hypoxanthine. After washing in the basic medium, each group of 20 COCs and 20 DOs was individually transferred into 100-µl droplets of the basic medium that was previously equilibrated in a CO2 incubator. After 20 h of maturation culture, the oocytes were washed and transferred to 100-µl droplets of the basic medium without hormonal supplementation for an additional 24 h of culture. Some oocytes were cultured for the entire period in the medium added with 1 mU/ml XOD to expose to ROS during in vitro maturation. All media containing oocytes were covered with mineral oil and cultured at 39°C in an atmosphere of 5% CO2 in air. After a total 44 h of maturation culture, COCs were treated to strip their cumulus cells by pipetting through a narrow-bore pipette in H-TL-PVA containing 0.1% (w:v) hyaluronidase.
Assessment of Nuclear Status
After maturation culture with or without XOD, 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 their nuclear status using a phase-contrast microscope at 400x magnification.
Analysis of DNA Cleavage and Damage
The COCs and DOs cultured in medium with or without XOD as well as oocytes freshly isolated from their follicles (freshly isolated oocytes) were transferred into 0.1% (w:v) protease solution in H-TL-PVA at room temperature to remove the zona pellucida, and washed quickly in PBS (Gibco) containing 3 mg/ml BSA (PBS-BSA). A portion of zona-free oocytes was then immediately fixed for 1 h in 4% paraformaldehyde prepared in PBS containing 0.1 mg/ml PVA. After fixation, the oocytes were washed three times in PBS-BSA and incubated for 10 min on ice in PBS containing 0.5% Triton X-100. The oocytes were washed three times in PBS-BSA and incubated with fluorescein-conjugated dUTP and TdT (TUNEL reagents; In situ apoptosis detection kit; Takara Shuzo Co., Kusatsu, Japan) at 37°C for 1 h in the dark. The oocytes were then washed three times in PBS-BSA and mounted between a coverslip and a nonfluorescence glass slide supported by four columns of Vaseline and paraffin (9:1). The slides were sealed with clear nail polish and examined using a fluorescent microscope.
Another portion of zona-free oocytes was employed for comet assay reported by Singh et al. [33] to detect DNA damage in single cells. Ten to fifteen oocytes were mixed with 10-µl drops of 2% low-melting agarose (SeaPlaque GTG agarose; FMC BioProducts, Rockland, ME) at 40°C on the glass slide, and the cell suspension was immediately covered with a coverslip. The slides were then kept at 4°C for 10 min to allow solidification of the agarose. After gently removing the coverslip, the slides stuck with the oocytes-embedded agarose were immersed in a lysing solution (1% N-lauroyl-sarcosine, 2.5 M NaCl, 10 mM EDTA, 10 mM Tris, pH 10, and 1% Triton X-100) for 1 h to lyse the cells and permit DNA unfolding. The slides were then placed on a horizontal gel electrophoresis unit and equilibrated for 20 min in TBE electrophoresis buffer. Electrophoresis was conducted for 20 min at 50 V. After electrophoresis, the slides were stained with 10 µg/ml 4',6-diamidino-2-phenylindole (DAPI) for 10 min, washed with distilled water, and then covered with a coverslip. The slides were sealed with clear nail polish and examined using a fluorescent microscope. The length of migrated DNA of 20–25 oocytes in each experimental group was individually measured using a micrometer.
Assay of Caspase-3 Activity
After the incubation period with or without XOD, oocytes were washed three times in H-TL-PVA, and groups of 40 oocytes were put into 0.6-ml microfuge tubes containing 20 µl STKM buffer (0.25 M sucrose, 50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2 and 0.25% Triton X-100), and lysed for 15 min on ice. Cell lysates were stocked at -80°C until assay. Caspase-3 activity was colorimetrically measured using Ac-Asp-Glu-Val-Asp 4-methyl-coumaryl-7-amide (Ac-DEVD-MCA) (Peptide Institute Inc., Osaka, Japan) as a substrate. The assay was performed by incubating 20 µl of cell lysates with 178 µl of reaction buffer (100 mM Hepes, pH 7.5, 20% [v:v] glycerol, 5 mM dithiothreitol, 0.5 mM EDTA) and 2 µl of 10 mM substrate at 37°C for 2 h. Release of 7-amino-4-methyl-coumarin (AMC) from Ac-DEVD-MCA by the enzyme reaction was spectrophotometrically monitored at 370 nm with a UV spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) and its quantity determined based on its standard curve.
Assay of GSH Content
The COCs and DOs treated with or without XOD, in addition to freshly isolated oocytes, were assayed for GSH content. Oocytes were washed three times in the stock buffer (0.2 M sodium phosphate buffer containing 10 mM EDTA, pH 7.2), and groups of 30 oocytes in 5 µl of stock buffer were transferred to 1.5-ml microfuge tubes, and 5 µl of 1.25 M H3PO4 was added. Samples were stored at -80°C until assay. The intracellular concentration of GSH in oocytes was determined using the 5,5'-dithio-bis(2-nitrobenzoic acid)-glutathione disulfide (DTNB-GSSG) reductase recycling assay as described by Funahashi et al. [31], which detects both GSH and GSSG in the oocyte. Briefly, 700 µl of 0.33 mg/ml NADPH in the stock buffer, 100 µl of 6 mM DTNB in the stock buffer, and 190 µl of distilled water were added and mixed in a microfuge tube. Ten microliters of 250 units/ml glutathione reductase (Boehringer Mannheim, Mannheim, Germany) was added to initiate the reaction. The absorbance was monitored continuously at 412 nm using a spectrophotometer (JASCO Co., Tokyo, Japan) for 3 min and its quantity determined based on its standard curve.
Statistical Analysis
Statistical analyses of data from four replicate trials were carried out by ANOVA and Fishers protected least significant difference test using the STATVIEW program (Abacus Concepts, Inc., Berkeley, CA). All percentage values were subjected to arcsine transformation before statistical analysis. All values are expressed as mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.
RESULTS
Effects of Cumulus Cells on the Meiotic Progression of Porcine Oocytes Treated with XOD
There was no significant difference in the percentage of germinal vesicle breakdown (GVBD) between COCs treated with or without XOD (P > 0.05) (Table 1). However, the maturation rate of COCs at the metaphase II stage was significantly decreased to 69% by treatment with XOD, compared with that of untreated oocytes (86%) (P < 0.05). This inhibitory effect of XOD on the meiotic maturation of porcine oocytes was more markedly observed in the absence of cumulus cells during culture. The maturation rate of DOs treated without XOD was suppressed to 66%, and DOs treated with XOD showed significantly lower percentages of GVBD and maturation (38% and 28%, respectively) (P < 0.05). In particular, in DOs with the meiotic resumption blocked by XOD treatment, the incidence of unique degenerated oocytes possessing fragmented chromatin bodies or obscurely decondensed chromatin within their GVs was drastically increased (Fig. 1, A and B). When either COCs or DOs were cultured for 44 h in medium without XOD, these degenerated oocytes were never observed (Fig. 1C).
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Effects of Cumulus Cells on the DNA Cleavage and Damage of Porcine Oocytes Treated with XOD
To confirm the occurrence of DNA cleavage in degenerated oocytes after treatment with XOD for 44 h, the following set of oocytes was subjected to the TUNEL assay in which free 3'-ends of the oocyte DNAs were labeled with fluorescein-conjugated dUTP molecules. Fluorescence microscopy revealed no incorporation of the labeled nucleotide on the metaphase plate in oocytes matured in the absence of XOD, and some of these oocytes showed a positive TUNEL reaction only in the polar body (Fig. 2A). In contrast, a large number of oocytes, blocked at the GV stage and induced to degenerate by exposure to ROS, displayed clear and extensive DNA labeling as revealed by clumps of brightly fluorescent chromatin in their GVs (Fig. 2B).
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To confirm further the effect of cumulus cells on the DNA damage caused by oxidative stress, the extent of DNA migration of a single oocyte was measured by the comet assay following treatment of COCs and DOs with or without XOD for 44 h. As shown in Figure 3A, the length of DNA migration of COCs was not significantly increased after culture in the absence or presence of XOD (67.4 ± 7.8 or 81.0 ± 8.9 µm, respectively), compared with that of freshly isolated oocytes (59.4 ± 4.5 µm) (P > 0.05). In these matured oocytes, an increase in the length of migration was found only in DNA derived from the first polar body (Fig. 4A). Exposure of DOs to ROS caused irremediable damage to oocyte DNA, and the length of DNA migration in XOD-treated DOs was remarkably increased to 226.5 ± 11.8 µm compared with that of untreated DOs (90.4 ± 9.7 µm) (P < 0.05). A photomicrograph of a typical DO exposed to ROS is presented in Figure 4B.
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Effects of Cumulus Cells on the Caspase-3 Activity of Porcine Oocytes Treated with XOD
To confirm the role of cumulus cells in preventing activation of apoptotic factors in oocytes exposed to ROS, we measured caspase-3 activity in each experimental group of oocytes using its specific substrate, Ac-DEVD-MCA. The activation of caspase family proteinases, particularly caspase-3, has been reported to be an event conserved in essentially all apoptotic cell death paradigms [34]. Caspase-3 activity of COCs matured in the absence or presence of XOD was 58.3 ± 10.7 pmol/oocyte or 67.7 ± 3.7 pmol/oocyte, respectively, and these values were higher than that of freshly isolated oocytes (37.0 ± 4.3 pmol/oocyte) (P < 0.05) (Fig. 3B). However, caspase-3 activity of DOs treated with XOD was remarkably higher (123.3 ± 3.9 pmol/oocyte) than that of DOs treated without XOD (72.2 ± 4.2 pmol/oocyte) (P < 0.05).
Effects of Cumulus Cells on the GSH Content of Porcine Oocytes Treated with XOD
As shown in Figure 3C, GSH content was significantly increased in COCs after maturation culture despite the treatment with or without XOD (7.3 ± 0.2 pmol/oocyte or 7.2 ± 0.1 pmol/oocyte, respectively), compared with that of freshly isolated oocytes (5.6 ± 0.1 pmol/oocyte) (P < 0.05). In contrast, GSH content in DOs was significantly reduced to 3.9 ± 0.3 pmol/oocyte even in the absence of XOD (P < 0.05), and the addition of XOD to culture medium did not cause further reduction of GSH content in DOs (3.8 ± 0.2 pmol/oocyte).
DISCUSSION
It has been reported that cumulus cells are involved in the cytoplasmic maturation of oocytes followed by the acquisition of developmental competence [2, 10, 35–38]. However, few studies have focused on the functional roles of cumulus cells in protecting oocytes from cell damage. The present findings indicated that cumulus cells, during in vitro maturation, efficiently prevented porcine oocytes from cell damage caused by oxidative stress. Moreover, ROS can induce apoptotic cell death in oocytes cultured without cumulus cells, judging from the findings that DNA cleavage by TUNEL, the increased length of DNA migration by comet assay, and the activation of caspase-3 were prominently detected in DOs treated with XOD. To our knowledge, this study is the first that directly showed that cumulus cells were involved in protection of porcine oocytes against cell damage caused by ROS during in vitro maturation.
In the present study, porcine oocytes cultured without cumulus cells were highly susceptible to oxidative stress, and the incidence of degeneration, the length of DNA migration, and caspase-3 activity were significantly increased in DOs exposed to ROS compared with those of DOs cultured without XOD. In COCs exposed to ROS, however, only the maturation rate was reduced and other detrimental effects of ROS were not recognized. These different responses to oxidative stress between COCs and DOs may be associated with the different abilities to synthesize GSH in these oocytes throughout in vitro culture. GSH is the major nonproteinous sulfhydryl compound present in mammalian cells and it plays an important role in protecting cells against oxidative stress [22]. After 44 h of maturation culture, GSH content was significantly increased in COCs, whereas DOs possessed markedly lower levels of GSH content than that of freshly isolated oocytes. de Matos et al. [21] reported that cumulus cells play an important role on the GSH synthesis enhanced by addition of cysteine and cysteamine during bovine oocyte maturation in vitro.
The aberrant patterns of microfilament organization in porcine oocytes during in vitro maturation were distinct from those in oocytes matured in vivo, suggesting that this inadequate microfilament organization impairs the function of cytoplasmic organelles controlling pronuclear formation and polar body formation in the oocyte [39]. The generation of ROS during in vitro culture is thought to cause reduced sperm motility [40, 41], lipid peroxidation [42], decreased capacity of sperm-oocyte fusion [30, 43] and retarded development of embryos [44–46]. As reported by Liu et al. [47], the occurrence of apoptosis was observed in mouse embryos exposed to the SH-specific oxidant diamide, indicating that alteration of the thiol-redox status by oxidative stress in embryos may result in cell cycle arrest and/or apoptotic cell death. In the present study, ROS generated by the hypoxanthine-XOD system during maturation periods provoked apoptotic cell damage to only those oocytes cultured without cumulus cells, suggesting that cumulus cells effectively protect oocytes against apoptosis caused by ROS through the enhancement of the GSH content in oocytes.
In addition to GSH synthesis, other mechanisms in cumulus cells may be involved in protecting oocytes against apoptotic cell death by ROS. Recently, Faure et al. [48] reported that when mitochondria were added in excess to cytosol, cytostatic factor (CSF) extracts prepared from the metaphase II-arrested Xenopus oocytes effectively blocked the induction of apoptosis, but those prepared from parthenogenetically activated eggs had no inhibitory effect on apoptosis. They suggested that a resistant factor to apoptotic signaling was included in CSF extracts of the metaphase II-arrested oocytes. It has been reported that the sustained high levels of maturation-promoting factor (MPF) activity, responsible for the meiotic arrest at the metaphase II stage, are maintained by CSF activity [49–51]. The MPF activity of COCs is significantly higher than that of DOs during porcine oocyte maturation [52]. The present findings revealed that caspase-3 activity of DOs cultured with XOD was significantly increased during in vitro culture and there was no significant difference in the caspase-3 activity between COCs and DOs cultured without XOD. Taken together, this suggests that the CSF activity of porcine oocytes cultured with cumulus cells may be higher than that cultured without cumulus cells. This means that COCs have less sensitivity to apoptotic signals triggered by oxidative stress during in vitro maturation. Furthermore, it was reported that estradiol functioned as a reactive oxygen scavenger during pregnancy-mediated luteal rescue folliculogenesis [27], and cumulus cells stimulated by FSH were implicated in steroidogenesis during culture of porcine oocytes [53].
In mice, apoptosis was observed in metaphase II-arrested oocytes from aged mice [54, 55], and the occurrence of apoptotic oocytes was induced by treatment with anticancer drugs [56]. The primary features of cells undergoing apoptosis are described as ‘nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments,’ subsequently referred to as apoptotic bodies. In the present study, however, fragmentation was not observed in porcine oocytes undergoing apoptosis by oxidative stress (data not shown). Additionally, when porcine oocytes were cultured for 44 h in medium containing the apoptosis-inducing agent, etoposide, fragmentation could not be detected irrespective of the high incidence of the cell damage resulting from apoptotic changes (unpublished data). Although the reason for this difference between the two species is unknown, the chromatin fragmentation within GVs was apparently observed in porcine apoptotic arrested oocytes that underwent degeneration at the GV stage. Furthermore, we detected brighter TUNEL fluorescence and an increased length of DNA migration of the first polar body in morphologically normal oocytes arrested at the metaphase II stage. The positive reaction of TUNEL in the first polar body was also found in mouse oocytes [54]. We previously observed that the polar bodies always underwent degeneration during early stages of embryonic development [57]. Thus, it is clear that the polar bodies extruded in the perivitelline space are immediately induced to die via apoptosis.
It could be concluded that porcine oocytes cultured in vitro underwent apoptotic cell death by oxidative stress as follows: the meiotic arrest, DNA cleavage and damage, and the activation of caspase-3 were observed in the oocytes exposed to ROS generated by the hypoxanthine-XOD system, which resulted in apoptotic cell death. However, cumulus cells played a critical role in protecting oocytes against apoptosis by means of the enhancement of the GSH content in oocytes. Further studies are required to investigate the functional mechanisms of cumulus cells, implicated in protecting against apoptosis and supporting the developmental competence in oocytes during maturation.
ACKNOWLEDGMENTS
We are grateful to the staff of the Meat Inspection Office in the city of Fukuyama, Japan, for supplying the porcine ovaries.
FOOTNOTES
First decision: 22 March 2000.
1 This study was supported by a grant from the Ministry of Education, Science and Culture of Japan (no. 11760196). 
2 Correspondence. FAX: 81 8247 4 0191; hidettmt{at}bio.hiroshima-pu.ac.jp
Accepted: April 21, 2000.
Received: February 29, 2000.
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