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a Laboratory for Reproductive Medicine, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
b Department of Ob/Gyn, Women & Infants Hospital and Brown University, Providence, Rhode Island 02905
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal cells balance the redox system and defense systems to protect against reactive oxygen species (ROS). If such defenses fail or excess ROS formation prevails, oxidative stress and cell death ensue. The cell death may occur via necrosis or apoptosis. Necrosis typically arises from acute pathological stimuli, while apoptosis typically is associated with mild toxic stimulation over prolonged periods of time. Apoptosis, or programmed cell death, also plays an important role in development and differentiation [15]. Evidence suggests that some oocytes undergo apoptosis or degenerate during development and aging [4, 16]. After fertilization, oocytes separate from their cumulus cells; thus the zygotes provide a unique system for study of the cellular basis of apoptosis during early development, because no cell-to-cell interactions exist, in contrast to the situation in most apoptotic systems [17]. The apoptotic and survival factors in mouse blastocysts recently have been described [18]. However, so far, the apoptotic process in mammalian zygotes remains unclear. Diamide, a specific thiol oxidant [19, 20], was shown to induce apoptotic changes resembling the natural process. Disruption of mitochondrial transmembrane potential occurred early [1]. We hypothesize that thiol-redox status may determine the commitment of zygotes either to cell death or to development. To clarify the effects of thiol oxidation on early embryos, we used an in vitro model of mouse zygotes treated with diamide. Apoptotic cell death in blastocysts also was studied as a comparison. The involvement of specific thiol oxidation-induced embryonic death also was investigated with the specific anti-thiol oxidant, dithiothreitol (DTT).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Equine CG used for superovulation was purchased from Calbiochem (La Jolla, CA).
Animals, Embryo Recovery, and Culture
Female B6C3F1 or CF1 mice (6 wk old) were purchased from Charles River (Boston, MA) and subjected to a 14L:10D light cycle for at least 2 wk before use. Animals were housed according to procedures approved by the Marine Biological Laboratory and Women and Infants Hospital Animal Care Committees. B6C3F1 or CF1 males of proven fertility were used for mating. Female mice were superovulated by i.p. injection of 7.5 IU eCG followed 46–48 h later by injection of 7.5 IU hCG; they were then mated individually with males. Next morning, females with mating vaginal plugs were selected and killed by cervical dislocation at 22–23 h after hCG injection. Zygotes (Day 1) enclosed in cumulus masses were released from the ampullae into modified Hepes-buffered KSOM with 14 mM Hepes and 4 mM sodium bicarbonate, containing 0.03% hyaluronidase, and cumulus cells were then removed by gentle pipetting. Cumulus-free zygotes were washed in Hepes-buffered KSOM three times and then in preequilibrated modified KSOM three times, pooled, and randomly distributed to treatment groups. The modified KSOM used for in vitro culture was supplemented with nonessential amino acids and 2.5 mM Hepes ([21, 22]; personal communication with John D. Biggers, Harvard Medical School, Boston, MA). Embryos were cultured in 50-µl droplets of KSOM under mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air. The in vitro-developed blastocysts at Day 4 in KSOM were able to develop to viable offspring after embryo transfer (15 of 34). In vivo blastocysts of B6C3F1 mice were flushed from uterine horns at 92–96 h after hCG injection and mating.
Pharmacological Treatments and Assessment of Embryo Development
Diamide (azodicarboxylic acid bis[dimethylamide]) and DTT (DL-dithiothreitol) 25 mM stocks were prepared in sterile, distilled water, aliquoted, and then stored frozen. The chemicals, diluted to the desired concentrations in the KSOM, were equilibrated in the incubator for 2 h prior to treatments. To ensure that the observed effects of DTT were not mediated by interaction with diamide itself, we performed additional experiments in which we avoided the equilibration step and instead treated embryos simultaneously with both diamide and DTT. Because we obtained identical results, we performed most of the experiments with the equilibration step. In diamide dose-response experiments, zygotes were exposed to 0, 12.5, 25, 50, 100, and 200 µM diamide for 3 h, washed extensively, and then cultured in KSOM for 3 (at Day 4 for B6C3F1) or 3.5 (at Day 4.5 for CF1) days. They were assessed for cleavage at Day 2 and then for development to blastocysts. Diamide treatment conditions were selected on the basis of previous similar dosages with hamster oocytes [6, 23]. Based on dose-response experiments, appropriate concentrations of diamide and DTT were chosen for the antioxidant inhibition experiments, in which the percentage of cleavage and blastocyst development, the total number of nuclei, and propidium iodide (PI)-positive and Hoechst-stained fragmented nuclei in blastocysts were evaluated. Blastocysts that developed in vivo at Day 4 from B6C3F1 mice were treated with diamide and/or DTT and then checked at 24 h for the number of nuclei and the PI-positive and Hoechst-stained fragmented nuclei.
Apoptotic Assessment, Mitochondrial Distribution, and Fluorescence Microscopy
For cell death assessment, embryos were stained with the cell-impermeant dye PI (20 µg/ml; Molecular Probes, Eugene, OR) and the cell-permeant dye Hoechst 33342 (20 µg/ml) for 15 min, washed, and then observed under an inverted microscope (Zeiss Axiovert 100TV, Oberkochen, Germany) equipped with fluorescence optics. The nucleus or pronucleus was visualized with Hoechst dye through UV illumination. The PI stain was observed with a rhodamine filter. Staining with PI and/or Hoechst has been used routinely for quantitative analysis of apoptotic cells. Apoptotic cells show condensed or fragmented nuclei that can be distinguished easily from normal cells [24–27]. Apoptosis is a relatively slow cellular process, and therefore zygotic pronuclei demonstrating PI-positive stain less than 24 h after treatment were considered to have undergone necrosis in the present experiment. Nuclei with PI-positive staining or fragmented chromatin revealed by Hoechst at later than 24 h were counted as apoptotic. The concept of apoptosis was originally developed from the observation of shrinkage necrosis reported by Kerr in 1972 (see review by Harmon and Allan [28]). Despite the many new techniques that have been developed as markers of apoptosis (DNA ladders, flow cytometry, in situ nick translation analysis, etc.), morphological changes still provide the most reliable criteria for recognizing the process [28, 29]. Therefore, in addition to fluorescence analysis, apoptosis also was examined by differential interference contrast (DIC) optics for observation of changes in cellular morphology, including plasma membrane blebbing, cell shrinkage, and condensation of nuclei, all of which are indicative of apoptosis [29, 30].
The distribution of mitochondria within cytoplasm after 3 h of treatment was determined by rhodamine 123 (Rh123) stain [31]. Zygotes were incubated with 10 µg/ml Rh123 (Molecular Probes) for 15 min, washed, and then observed with Zeiss fluorescence microscopy as indicated above.
Statistical Analysis
One-way ANOVA was utilized for comparisons of treatment means. Significant difference was defined as P < 0.01 and no difference defined as P > 0.05. The chi-square test was used for confirmation of the analysis of differences in the case of percentage comparison. The entire statistical analysis was carried out using the Data Analysis program in Microsoft (Redmond, WA) Excel 97.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the dose-response experiment, with each treatment group including at least 45 embryos in three replicates, 12.5 µM diamide treatment for 3 h did not have significant effects on the percentage of cleavage or blastocyst development of B6C3F1 mouse zygotes when compared to values in untreated controls (P > 0.05). However, diamide at concentrations equal to or higher than 25 µM completely inhibited cleavage and arrested further development of zygotes. At the end of treatment, pronuclear morphology under DIC optics differed markedly among the groups (Fig. 1). Zygotes from control and DTT-added groups showed clear boundaries around their pronuclei, while those treated with diamide alone displayed obscure or absent boundaries around their pronuclei depending on the dosage employed. The 50 µM diamide treatment resulted in markedly obscured pronuclei (Fig. 1B). Hoechst staining further confirmed that the pronuclear boundary was obscured in diamide-treated zygotes (Fig. 1E). Moreover, mitochondrial distribution was altered in diamide-treated zygotes. Perinuclear mitochondria were sparse. Instead, mitochondria were dispersed toward the central cytoplasm (Fig. 1H). In contrast, control and DTT-treated zygotes showed typical, perinuclear mitochondria distribution, as observed in hamster embryos [31].
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At 24 h after treatment, when zygotes arrested at the one-cell stage were stained with PI and Hoechst, they showed variable permeability to PI, depending on treatment. None of the zygotes treated with 25 µM or 50 µM diamide displayed PI-positive pronuclei, although one polar body typically appeared PI positive that could serve as a positive internal control for the staining procedure (Fig. 2, A and B). Pronuclei of a few zygotes (30%) treated with 100 µM diamide exhibited PI staining. However, all zygotes treated with 200 µM diamide showed PI-positive staining in their pronuclei (Fig. 2C and Table 1). As 50 µM was the intermediate dosage for induction of embryonic cell cycle arrest and death, but not necrosis, zygotes treated with this dosage of diamide were further checked at 48 h after treatment and were chosen for the subsequent anti-oxidation experiment with 50 µM DTT. Figure 3 shows that 50 µM diamide induced zygotic apoptosis, characterized by blebbing of plasma membranes, cell shrinkage, and condensation of pronuclei. At 72 h after treatment, one-cell zygotes showed PI-positive stain in the pronuclei, whereas the Hoechst stain was bright throughout the whole cytoplasm, regardless of the diamide dosage.
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In the anti-oxidation experiment, 50 µM diamide also inhibited zygotic division and development in CF1 mice (Fig. 4A). No difference (P > 0.05) existed in the percentage of cleavage and blastocyst development among control (92.3% and 71.6%, n = 42), 50 µM DTT (94.9% and 72.1%, n = 32), and 50 µM diamide plus 50 µM DTT (96.2% and 74.2%, n = 20) groups. Moreover, cleavage and development rates were significantly higher (P < 0.01) than those in the 25 µM diamide-treated group (15.4% and 11.5%, n = 23). However, when zygotes were treated with 50 µM diamide for 3 h, followed by 50 µM DTT for 3 h, all (n = 21) arrested at the one-cell stage and showed morphological changes similar to those observed after 50 µM diamide treatment alone. Similarly, 50 µM diamide (n = 41) completely inhibited zygotic division and development in B6C3F1 mice, as seen in the previous dose-response experiment. However, 50 µM diamide plus 50 µM DTT treatment (n = 39) resulted in cleavage (94.1%) and blastocyst development rates (90.4%) that were similar (P > 0.05) to those for control (n = 40) and treatment with 50 µM DTT alone (n = 38) (Fig. 4B). It is noteworthy that the B6C3F1 and CF1 mouse strains responded similarly to diamide, diamide/DTT cotreatment, and DTT treatment alone.
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Further analysis with PI and Hoechst stain and fluorescence microscopy showed no significant difference (P > 0.05) in the total cell number, or the apoptotic cell number, in blastocysts derived from either CF1 or B6C3F1 mouse zygotes (Fig. 5). The total cell numbers of blastocysts derived from CF1 zygotes in the control, the 50 µM diamide plus 50 µM DTT, and the 50 µM DTT group were 48.5 ± 13.7 (n = 22), 54.5 ± 15.5 (n = 14), and 49.6 ± 13.3 (n = 23), respectively (Fig. 5A). Similarly, the total cell number of blastocysts derived from B6C3F1 zygotes also did not differ among control (56.6 ± 7.7, n = 13), 50 µM diamide plus 50 µM DTT (53.3 ± 7.6, n = 14), and 50 µM DTT (59.5 ± 5.6, n = 19) groups (Fig. 5B). The Hoechst staining of total nuclei and fragmented nuclei, as well as the PI-positive staining of condensed nuclei and sometimes fragmented nuclei, is shown in Figure 6.
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An additional experiment, represented in Table 1, showed that appropriately matching the concentration of DTT with diamide determined whether DTT could block the effects of oxidative damage caused by diamide. The 50+50, 100+100, or 200+200 µM diamide plus DTT combinations led to similar rates of zygotic division and blastocyst development (P > 0.05). The developed blastocysts looked similar in morphology and the average cell numbers were comparable, at least in the 200+200 combination group (50.4 ± 9.6, n = 10), to those of the control group. By contrast, mismatches in concentrations failed to prevent cell death by DTT. DTT at 100 µM failed to protect against necrosis triggered by 200 µM diamide. No zygotes that had been treated with 200 µM diamide plus 100 µM DTT cleaved or developed further.
For in vivo blastocysts, 50 µM diamide treatment for 3 h significantly reduced cell number and increased apoptosis, as indicated by Hoechst-stained fragmented nuclei (Fig. 7). The average cell number in blastocysts after 24 h in the diamide-treated group (72.4 ± 24.5) was significantly lower (P < 0.01) than that in the control group, the diamide plus DTT group, or the DTT group (108.7 ± 12.1, 100.0 ± 13.2, or 111.5 ± 17.0, respectively). In contrast, the number of fragmented nuclei stained by Hoechst in the diamide-treated group (an average of 3.2 per blastocyst) was significantly higher (P < 0.01) than in the other three groups (0.4–0.9). Again, DTT appeared to prevent the harmful effects of diamide in blastocysts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Depending on the dosage of the oxidant, zygotes displayed either characteristics of apoptosis (50 µM), a mixture of apoptotic and necrotic death (100 µM), or necrotic cell death (200 µM). Similarly, Jurkat cells undergo apoptosis or necrosis in response to diamide oxidation in a concentration-dependent manner [2]. Apoptosis is a relatively slow cellular process. In comparison to 100 and 200 µM diamide, which induced PI-positive staining in pronuclei in less than 24 h and therefore necrosis, 50 µM diamide acted more slowly, consistent with initiating apoptosis rather than necrosis. Forty-eight hours after treatment, zygotes began to exhibit classical characteristics of apoptosis, including membrane blebbing, cell shrinkage, and nuclear condensation [28, 29]. In the end, all apoptotic zygotes appeared to undergo necrosis, possibly because no neighboring cells existed to communicate with and eliminate apoptotic cells. We hypothesize, therefore, that zygotes exposed to thiol oxidant or other oxidants initially undergo cell cycle arrest and then initiate the apoptotic pathway, but cannot complete the final events of nuclear fragmentation under moderate conditions. These zygotes presumably end up with leaky membranes and become PI positive. In somatic cells, this process has been called secondary necrosis [26, 32]. We have not examined the exact stage of cell cycle arrest (G1, G2, or S) after diamide treatment. As zygotes treated with diamide for 3 h did not cleave and PI-positive stain was not observed within 24 h, which is long enough for normal cell cycle progression, we assumed that the cell cycle was arrested. After 48 or 72 h, progressive PI-positive pronuclei appeared, implying that cell death occurred later. In fact, cell cycle perturbations may be closely related to apoptosis [33]. At higher doses of the oxidant, zygotes do start the apoptotic process, as evidenced by the appearance of cell shrinkage. This process might be earlier than cell cycle arrest but cannot be completed before necrosis takes place, which may be due to extensive lipid peroxidation [34], leading to destruction of the cell membrane and thus increased permeability to PI stain. Our results suggest that thiol oxidation plays an important role in zygotic cell death and that the intensity of oxidative stress may determine which death pathway is triggered.
One striking feature of apoptosis in zygotes is the absence of nuclear fragmentation that typifies apoptosis in somatic cells. In zygotes, pronuclei appeared condensed but stayed round. Perhaps this feature highlights a difference between zygotic pronuclei and somatic nuclei. Diamide did induce nuclear fragmentation in blastocysts, which might imply that the signaling from cell-to-cell contact is necessary for nuclear changes. Moreover, at the blastocyst stage, the effects of diamide-induced oxidative stress were less extensive than at the zygotic stage. GSH content, a target of oxidation, decreases significantly from unfertilized oocytes and zygotes to blastocysts [7], a finding that stresses the importance of GSH for development at earlier stage. These findings suggest that embryos at earlier stages may be more sensitive to oxidation damage.
Apparently the capacity of embryos to counteract oxidative damage induced by diamide is quite limited. The embryonic cells arrest and then proceed to apoptosis. DTT protected against diamide-induced oxidative damage when added before or concurrently with the embryos' exposure to diamide. Thus, DTT is an efficient and powerful protective agent against oxidative damage in either zygotes or blastocysts. Similarly, DTT was shown to counteract thiol oxidation in mature and aging oocytes [4, 5]. GSH is a major antioxidant in cells, and its presence in mitochondria is well documented [13]. Possible roles of cellular thiols other than GSH might exist in apoptotic T cells [2]. GSH has protective functions against apoptosis, possibly because it may be a downstream regulator of Bcl-2, a key survivor factor [3, 35]. Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols [9, 36]. Cytochrome c also may be involved in the antioxidant defense system of mitochondria and in apoptosis [37]. These findings also indicate that the apoptotic process could be prevented. Thus, DTT could be considered a pharmacological anti-apoptotic agent, in contrast to such naturally occurring antioxidants as Bcl-2. As zygotes treated with diamide and then incubated with DTT did not survive, the thiol oxidation-induced zygotic cell death apparently was irreversible.
It has been suggested that DNA degradation and nuclear fragmentation are the hallmark of apoptosis. This may not always hold, especially for early embryos, for several reasons. First, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) staining usually is apparent only at a late stage of apoptosis, much later than PI staining. Secondly, cells without nuclei can undergo apoptosis by apoptotic stimuli through pathways that are suppressed by Bcl-2 expression [38, 39]. Thirdly, in cells without adjacent cell signals, like zygotes, nuclei most likely keep their condensed form without fragmentation. Thus, morphologic change still is an effective and simple way to evaluate apoptosis in zygotes. Apoptosis in blastocysts is conveniently determined by PI and Hoechst staining, which do not require fixation and which allow instant observations, in contrast to TUNEL staining. For control blastocysts, PI positivity averaged 2.0 ± 1.8 and Hoechst 1.2 ± 1.1 (n = 25), whereas TUNEL positivity averaged 2.1 ± 1.6 (n = 33) (unpublished results). The percentage detected by PI and Hoechst staining is slightly higher than that obtained with TUNEL staining [18, 30], again suggesting that TUNEL-positive nuclei appear only late in apoptosis.
A possible target for diamide's effects is the mitochondria. Cells exposed to oxidants were shown to have disturbed mitochondrial function [1, 12], together with calcium-dependent events [40, 41]. It has been demonstrated that these events lead to cell death [42]. A redistribution of mitochondria from the perinuclear to a more intermediate region was observed in the zygotic cytoplasm after diamide treatment, which can be indicative of alterations of mitochondrial function. Mitochondrial GSH oxidation was found to correlate with age-associated, oxidation-induced damage to mitochondrial DNA, and therefore it was suggested that GSH plays an important role in the protection against free radical damage that occurs during aging [13].
Age-associated decline in fertility is common in female humans and in other long-lived mammalian species. Oxidative stress can induce aneuploidy during meiotic division in vitro of mouse oocytes; moreover, antioxidant therapy counteracts the disturbing effects of diamide and maternal aging on meiotic division and chromosomal segregation [4]. GSH oxidation by diamide also could induce disruption of the meiotic spindle, chromosome aggregation, and thus abnormal fertilization in oocytes [6]. This may be related to the later embryonic aneuploidy. Diamide also has been shown to disrupt spindle microtubules in somatic cells [43]. Although the precise mechanism involved in triggering apoptosis by diamide is not clear, the induced apoptosis might be mediated by both the modulation of cellular redox status and the activation of the intracellular signaling pathways.
Mitochondria, and specifically mtDNA, have been shown to be important targets of age-associated free radical attack [44]. The frequency of mtDNA deletions in oocytes from older women was higher than in oocytes from younger women [45]. Mitochondrial DNA mutations or deletions are closely associated with the aging process [46]. It has been shown that antioxidants protect against GSH oxidation and mtDNA damage [13]. On the other hand, it appeared that pronuclear boundaries in diamide-treated zygotes were disrupted. Moreover, the nucleolus disappeared after diamide treatment (Fig. 1, B and E). It has been suggested that a conserved mechanism of cellular aging might be related to nucleolar structure damage [47, 48]. The present thiol oxidation and antioxidation experiments in one-cell zygotes suggest that both mitochondria and nuclei may be affected in thiol-oxidative stress. As oxidation and mitochondrial alterations are closely associated with aging and thiol oxidation is involved in the process, exploring thiol oxidation in development by using in vitro zygotes might be a good model for study of aging-associated infertility. Further investigations in these molecular processes will help to clarify the roles of redox status during early development and aging.
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FOOTNOTES |
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2 Correspondence: David Keefe, Women & Infants Hospital and Brown University, Dept. of Ob-Gyn, 101 Dudley Street, Providence, RI 02905. FAX: 401 453 7500; dkeefe{at}smtp.wihri.org
Accepted: May 25, 1999.
Received: January 25, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1997; 56:1088–1096.[Abstract]This article has been cited by other articles:
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