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Gamete Biology |
Instituto de Biología y Medicina Experimental,3
Departamento de Química Biológica,4
Grupo de Investigaci;aaon en Biología Evolutiva,5 Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gamete biology, meiosis, oocyte development, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Catecholestrogens, which are endogenous estradiol metabolites, have been proposed to have a local regulatory role in the ovary. They have been shown to influence cell differentiation and proliferation of granulosa, theca, and luteal cells of different species [2–4]. However, to our knowledge, no reports on the actions of catecholestrogens in the bovine ovary have appeared. The two hydroxylases (CYP1A1 and CYP1B1, respectively) that convert estradiol to 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), as well as the aryl hydrocarbon receptor (AHR) that regulates these enzymes at the transcriptional level, have been shown to be present in the ovary [2, 5]. The AHR receptor has no known endogenous ligands, but it can be activated by environmental contaminants [6]. Another enzyme, the catechol-O-methyl transferase that converts 2-OHE2 into 2-methoxyestradiol (2-MOE2), is also expressed in the ovarian follicle [2]. Finally, substantial amounts of catecholestrogens have been quantitated by gas chromatography-mass spectroscopy in human and mare follicular fluid [7, 8].
Both 2-OHE2 and 4-OHE2 have been shown either to mimic or to modulate estradiol action [9]. These effects, however, are not mediated by interaction with the classical estrogen receptors, and the mechanism of action of catecholestrogens remains unclear.
On the other hand, 2-MOE2 is known to inhibit tumor cell growth and angiogenesis. Two possible mechanisms have been proposed for this growth-inhibitory activity: inhibition of superoxide dismutase with a consequent increase in intracellular free radicals that leads to apoptosis [10] and inhibition of tubulin polymerization by interacting at the colchicine site [11, 12].
The goal of the present study was to determine the possible effects and mechanism of action of catecholestrogens during oocyte maturation and early embryo development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte Collection and In Vitro Maturation
Bovine ovaries were obtained at a local slaughterhouse from beef cows and heifers and were transported to the laboratory in physiological saline at 30–35°C. Cumulus-oocyte complexes (COCs) were collected by aspiration from follicles (diameter, 2–8 mm) using a 18-gauge needle attached to a 5-ml syringe. Only oocytes having homogeneous cytoplasm and unexpanded cumulus cells were selected. Groups of 40–50 COCs were washed three times and placed in 500 µl of Tissue Culture Medium 199 (TCM199) with Earle salts (Gibco BRL, Grand Island, NY) and bicarbonate (2.2 g/L, pH 7.4) in multidish, four-well plates (Nunc, Roskilde, Denmark). The medium was supplemented with 5% fetal bovine serum (FBS; Gibco BRL), 20 ng/ml of ovine FSH (oFSH; NIDDK-oFSH-20; NIH), and 50 µg/ml of gentamicin sulfate and was overlaid with 150 µl of mineral oil. The COCs were then matured with or without catecholestrogens (10 ng/ml to 1 µg/ml; E2470, E2500, and E2490; Steraloids, Inc., Newport, RI) for 24 h at 38.5°C under 5% CO2 in air in a humidified atmosphere. Catecholestrogens stocks (mg/ml) were prepared in ethanol. When roscovitine was used to inhibit meiosis progression at the germinal vesicle stage, oocytes were incubated in TCM199 with Earle salts and bicarbonate (2.2 g/L, pH 7.4) supplemented with 25 µM roscovitine for 24 h, washed, and then subjected to in vitro maturation (IVM) as indicated above.
In Vitro Fertilization
Frozen bull semen was thawed and washed twice by centrifugation (260 x g for 5 min) with Brackett-Oliphant (BO) medium [13] containing 5 mM theophylline without BSA. After removing the supernatant, the sperm pellet was diluted in BO medium containing 5 mM theophylline, 10 µg/ml of heparin, and 5 mg/ml of BSA at a final concentration of 1 x 107 spermatozoa/ml [14]. After 24 h of IVM, oocytes reached the metaphase II stage. Then, expanded COCs were washed twice in BO medium and placed in 100-µl drops of sperm suspension (20–25 oocytes/drop) under mineral oil in a 35-mm culture dish (Nunc). Incubations were carried out for 7 h at 38.5°C in 5% CO2 in air in a humidified atmosphere.
In Vitro Culture
After fertilization, presumptive zygotes were transferred to Hepes-buffered TCM199 supplemented with 5% FBS and stripped of remaining cumulus cells by repeated pipetting through a small-bore glass pipette. Then, groups of 40–50 presumptive zygotes were placed in 500 µl of synthetic oviduct fluid [15] supplemented with Eagle nonessential amino acids and essential amino acids [16] and 8 mg/ml of BSA-fatty acid-free (SOFaa-BSA) medium in multidish, four-well plates (Nunc). The medium was overlaid with 150 µl of mineral oil, and embryos were cultured for 8 days at 38.5°C in 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere. Cleavage was assessed 48 h postinsemination (Day 2), and blastocyst development was recorded at Day 8 postinsemination.
Immunocytochemistry
Immunocytochemistry was carried out with a slight modification of the method described by Navara et al. [17].
After maturation with or without catecholestrogens, expanded COCs were stripped of cumulus cells by using 0.2% hyaluronidase and pipetting oocytes through a small-bore glass pipette. Zona pellucidae were removed by a brief treatment with 0.2% pronase. After a 20-min recovery at 38.5°C, zona-free oocytes were fixed and permeabilized in a microtubule-stabilizing buffer (100 mM PIPES, 5 mM MgCl2·6H2O, 2.5 mM EGTA, 2% formaldehyde, 0.5% Triton X-100, and 1 µM paclitaxel [pH 7.0]) for 40 min at 38.5°C. Nonspecific binding of the primary and secondary antibodies were blocked by a 40-min incubation in blocking solution (2% BSA, 2% nonfat dry milk, 2% normal goat serum, 100 mM glycine, and 0.01% Triton X-100 in PBS). Microtubule localization was performed using a mouse monoclonal antibody to 
-tubulin (B-5-1-2). The primary antibody was detected using an fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody. Each antibody was applied for 40 min at 38.5°C and rinsed between antibody applications with blocking solution. The DNA was fluorescently detected with 4 µg/ml of ethidium bromide (EtBr)
Slides were examined using a Zeiss LSM 510 laser-scanning confocal microscopy.
Cytogenetic Analysis
Embryos from the 2- to 10-cell stage (Day 2) developing from oocytes exposed to 2-MOE2 during IVM (2-MOE2 group) or not exposed (control group) were prepared and examined to characterize their chromosome composition. Colchicine (1 µg/ml) was added to the culture medium (SOFaa-BSA medium), and embryos were incubated for 6 h at 38.5°C in 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere. Each embryo was incubated in 1% sodium citrate for 10 min at room temperature, placed on a glass slide, fixed, and spread with a drop of methanol-acetic acid (1:1). After drying, slides were stained in a 3% Giemsa solution (Merck, Whitehouse Station, NJ) for 10 min. Metaphases were examined at 400x and 1000x magnification and photographed in a Leica DM LB photomicroscope with AGFAPAN film APX 25. Nuclear stages were classified as interphase, metaphase, and nonanalyzable metaphase (gross overspreading or clumped chromosomes). Each analyzable metaphase was classified according to the chromosome number as diploid (2n = 60), haploid (n = 30), or aneuploid. Each embryo was classified according to the ploidy of its blastomeres as diploid, haploid, mixoploid (embryos with diploid and aneuploid blastomeres), and aneuploid (all analyzable metaphases were aneuploid).
Comet Assay
Detection of DNA damage in individual embryos was carried out with a slight modification of the method described by Takahashi et al. [18]. Embryos were washed twice in PBS plus polyvinylpyrrolidone (4 mg/ml), mixed with 1% low-melting-point agarose in PBS at 39°C, and placed on a slide initially coated with 1% high-melting-point agarose. After solidification, they were incubated in lysing buffer for 3 h at 4°C (10 mM Tris, 1% N-lauroylsarcosine, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100, and 10 µg/ml of proteinase K). Then, slides were removed from lysing solution and placed on an electrophoresis unit filled with fresh buffer (300 mM NaOH and 1 mM Na2EDTA) and allowed to equilibrate for 20 min. Electrophoresis was conducted for 20 min at 25 V. After electrophoresis, slides were neutralized with 0.4 M Tris-HCl (pH 7.5). The DNA was stained with a 20-µl drop of EtBr (4 µg/ml). The DNA damage was quantified by measuring the length of the streak of DNA comet tail using a Zeiss LSM 510 laser-scanning confocal microscope.
Statistical Analysis
Data are presented as the mean percentage of at least three independent experimental replicates. Variation between experiments is illustrated using the SEM. For evaluation of the differences between groups, data of the three replicates were checked for homogeneity, pooled, and then subjected to chi-square analysis. Differences with P < 0.05 were regarded as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neither cleavage rates nor blastocyst production were significantly affected by the addition of estradiol (1 µg/ml) to the maturation medium of bovine oocytes (Fig. 1). In contrast, addition of catecholestrogens to the maturation medium at a concentration of 1 µg/ml did not affect the cleavage rate but was associated with developmental arrest at the morula stage (Fig. 2). Consequently, a marked decrease in the blastocyst production was observed on Day 8 in 2-MOE2- and 2-OHE2-treated groups and, although less pronounced, in the 4-OHE2-treated group (Fig. 1). Because of the higher potency of 2-MOE2 in the inhibition of early embryonic development, this steroid was used in all subsequent experiments.
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When no oil was used for IVM, the effect of 2-MOE2 was significant at doses as low as 100 ng/ml (300 nM) (Fig. 3). The effects of 2-MOE2 were also evident when serum was omitted from maturation medium and were not reversed by addition of estradiol (data not shown).
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Effects of 2-MOE2 Addition During Embryo Culture
Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. No significant effects in the morula-to-blastocyst transition were observed when 2-MOE2 was added on Day 6 (Fig. 4).
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Effect of Antioxidants
Huang et al. [10] have shown that the inhibitory actions of 2-MOE2 on human leukemia cells were reversed by the addition of certain antioxidants (ambroxol, N-acetylcysteine), thus suggesting that the effects of this steroid resulted from an inhibition of superoxide dismutase. Therefore, we tried to determine whether antioxidants or agents capable of counteracting the activity of free radicals could also reverse the inhibition of embryo development produced by 2-MOE2 when added during IVM.
As shown in Table 1, the tested agents, cysteamine (an antioxidant routinely added to IVM media in some experimental protocols [19]) and ambroxol, were unable to alter significantly the inhibition elicited by 2-MOE2. In addition, no significant effects could be observed with other antioxidants tested, such as N-acetylcysteine, dimethyl sulfoxide, or 2-mercaptoethanol (data not shown).
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Comparative Effects with Colchicine
In view of the known interaction between 2-MOE2 and tubulin at the colchicine site [11], we decided to compare the effects of 2-MOE2 with those of colchicine. Colchicine, when added during IVM, produced a similar inhibition of blastocyst production without altering the cleavage rate. However, in contrast with 2-MOE2, colchicine was able to block embryo development completely when added at any time during embryo culture (Table 2).
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Effect of 2-MOE2 on Meiotic Spindle Assembly
When analyzed by immunocytochemistry using tubulin-specific antibodies, oocytes exposed to 2-MOE2 showed marked alterations in the meiotic spindle assembly when compared with control oocytes. Most of them showed multipolar spindles with large arrays of microtubules, and some presented multiple spindles. In contrast, in colchicine-treated oocytes, the morphology of the spindle was different, and no microtubule arrays could be observed (Fig. 5).
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Cytogenetic Analysis
A total of 189 embryos in the 2- to 10-cell stage (Day 2) were examined. Although 124 embryos showed 289 analyzable metaphases, 65 showed only interphasic nuclei or nonanalyzable metaphases (Fig. 6). As shown in Table 3, a significant increase was observed in the proportion of aneuploid metaphases in embryos exposed to 2-MOE2 during oocyte IVM. Moreover, only 8% of metaphases of these embryos were diploid. When we analyzed ploidy in the embryo as a whole (according to the ploidy of its blastomeres), we found that 82% of 2-MOE2-exposed embryos were completely aneuploid, only 3% were diploid and 10% mixoploid (Table 4). On the contrary, only 20% of control embryos were aneuploid, but 40% were diploid and 36% mixoploid. With regard to the proportion of aneuploid embryos in control oocytes, the process of in vitro production influences chromosome complement, and the estimates from previous studies have been variable (14–80% of chromosomally abnormal embryos), possibly because of the different culture systems used [20]. Another significant difference observed in the present study was that 2-MOE2-exposed embryos showed a higher proportion of nonanalyzable metaphases (23%, n = 92) compared with control embryos (1%, n = 97, P < 0.01).
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Effects on Apoptosis
To characterize the nature of the developmental arrest of embryos exposed to 2-MOE2 during IVM, DNA damage was analyzed by a comet assay at Day 6 postinsemination. The extent of damage was quantified by measuring the length of the DNA comet tail detected following electrophoresis. An increase of the DNA comet tail was observed in embryos from 2-MOE2-treated oocytes: 138 ± 18 µm (n = 58) vs. 37 ± 9 µm (n = 61) in the control group (Fig. 7).
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Effect of Delayed Maturation
We then tested whether assembly of the meiotic spindle was required for the inhibitory effect of 2-MOE2 during IVM. Oocytes were exposed to 2-MOE2 in the presence of roscovitine, a specific cyclin-dependent kinase 2 inhibitor that produces a reversible arrest of meiotic maturation. After a 24-h exposure, media were changed and oocytes allowed to progress into meiotic maturation in the absence of 2-MOE2. Under these conditions, 2-MOE2 failed to produce any significant changes in either cleavage rates or blastocyst formation (Table 5).
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On the other hand, when oocytes were exposed to 2-MOE2 during the 24- to 48-h maturation period, the cleavage rate was not affected. The blastocyst rate, however, was significantly lower (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo, bovine oocytes acquire full developmental competence within the ovulatory follicle before ovulation. This first requires the completion of nuclear maturation (i.e., the progression of meiosis from prophase I to metaphase II, when meiotic arrest is imposed until fertilization). This next requires the completion of a separate set of molecular changes, such as protein synthesis and relocation of organelles and mRNA (referred as cytoplasmic maturation). Nuclear and cytoplasmic maturation programs can proceed independently, but developmental competence is only achieved when both processes proceed while closely integrated. Deficiencies during the maturation process may lead to impairment of development that can be expressed at early stages of development, such as during fertilization or cleavage, as well as at late stages, such as during pregnancy or at birth [21].
Using an in vitro embryo production system, we demonstrated that during maturation, exogenous estradiol has no effect, whereas its metabolite 2-MOE2 impairs the acquisition of full developmental competence. Oocytes exposed to 2-MOE2 were able to be fertilized, to cleave, and to develop until the morula stage, but they were unable to reach the blastocyst stage. Furthermore, this effect was stage-specific, because the development of preimplantation embryos exposed to 2-MOE2 after cleavage was less affected.
Although estradiol is routinely added to IVM media in some experimental protocols [22, 23], the effects of this steroid on oocyte maturation and/or developmental competence remain controversial. In addition, the capability of bovine granulosa cells to synthesize estradiol in culture [24] may explain the lack of effect of the exogenous steroid. On the other hand, although catecholestrogens have been shown to interact with estrogen receptors in some experimental models [25, 26], the inhibitory effects reported herein do not seem to be related to an interference with estrogen-mediated events, because they were not reversed by the addition of an excess of exogenous estradiol.
The effects of 2-MOE2 were also observed in the absence of serum. This also argues against a possible mediation by binding to serum proteins such as sex hormone-binding globulin, as previously proposed by Rosner et al. [27].
Huang et al. [10] suggested that 2-MOE2 inhibits tumor cell growth and angiogenesis through the inhibition of superoxide dismutase, with a consequent increase in intracellular free radicals that leads to apoptosis. This hypothesis has been questioned [28]. Data presented in the present study indicated that the effect of 2-MOE2 could not be reversed by any of the antioxidant or free radical scavengers tested, thus suggesting that the inhibitory effects do not involve changes in superoxide dismutase activity.
The ability of 2-MOE2 to interact with tubulin at the colchicine site has been well documented [11], and the consequent alteration of microtubule polymerization has been postulated to be the reason for the inhibition of cell proliferation in many cell types produced by this steroid [12]. The similitude between the effects of 2-MOE2 and those of colchicine on embryo development as well as the marked alterations observed in the meiotic spindle in the presence of this catecholestrogen strongly suggest that its deleterious effects are mediated by its interaction with tubulin. Interestingly, despite these alterations in the meiotic spindle, the embryos were capable of progressing up to the morula stage. Because mistakes in the segregation of chromosomes have disastrous consequences, leading to the production of abnormal gametes, a stringent control mechanism would have been expected. In most cell types, the spindle-assembly checkpoint is the mechanism responsible for proofreading the spindle structure for mistakes, ensuring that after mitosis, each daughter cell obtains a complete set of chromosomes (for review, see [29]). Nevertheless, despite this control mechanism seeming to be highly conserved from yeast to mammals and being well documented during male meiosis [30], our observations with bovine oocytes support the general idea that with regard to this surveillance mechanism, the female gamete is an exception [31].
In lower vertebrates, such as Xenopus sp., sea urchin, and zebrafish, these checkpoint mechanisms do not operate during early cleavage [32] until the midblastula transition stage, when zygotic transcription is initiated. In humans, considering the high incidence of chromosomal abnormalities in preimplantation embryos, it was postulated [33] that cell-cycle checkpoints would be absent or restricted during the early stages of development, becoming functional after embryonic genome activation [34, 35]. Hence, because exposure of oocytes to 2-MOE2 during maturation would induce missegregation of chromosomes, the lack of a functional spindle-assembly checkpoint at this stage is consistent with the development of aneuploid embryos observed after in vitro fertilization. Although mixoploid embryos can proceed to the blastocyst stage and even produce viable pregnancies, haploid or complete aneuploid embryos are always arrested at early stages of development [20, 35, 36]. Thus, because 82% of the embryos exposed to 2-MOE2 during IVM were aneuploid, 5% haploid, 10% mixoploid, and 3% diploid, we conclude that the developmental arrest of these embryos is caused by their chromosomal abnormalities. In the bovine, activation of the embryonic genome takes place around the 8-cell stage [37]; thus, our data agree with the relationship observed between embryonic genome activation and developmental arrest of aneuploid embryos.
Our results strongly suggest that the aneuploidy and developmental arrest of embryos exposed to 2-MOE2 during IVM is a consequence of an alteration in oocyte nuclear maturation. Consistent with this idea, when meiosis progression was blocked during 2-MOE2 exposure and these oocytes were then subjected to subsequent standard maturation, fertilization, and culture conditions, development to the blastocyst stage was not different from that in the control group.
Effects of 2-MOE2 During Culture
The effects of 2-MOE2 exposure during the preimplantation period seem to be more intriguing, because colchicine and 2-MOE2 did not have the same effect when added to the culture medium at the morula stage. Colchicine produced a complete block in the passage from morula to blastocyst, whereas 2-MOE2 did not. These differences may arise from the different effects of both compounds on tubulin dynamics. One colchicine molecule binds to one tubulin molecule and prevents its polymerization. In contrast, 2-MOE2 binds to tubulin at the colchicine site and seems to be incorporated into the polymer, increasing its resistance to depolymerization [11]. Because the metaphase spindle assembly is suspended in a state of dynamic equilibrium between polymerization and depolymerization (with a high turnover), colchicine causes the whole spindle to disassemble rapidly, whereas 2-MOE2 drastically affects its architecture, stabilizing arrays of microtubules that lack any spatial organization. Although neither of these two effects seems to be compatible with normal embryonic development, the observation that 2-MOE2 does not inhibit the passage from morula to blastocyst suggests that the alterations produced by this compound may be compatible with progression of the cell cycle despite aberrations in spindle architecture. In this case, blastocyst formation would not be a satisfactory end point to assess embryo quality, because further development is very likely to be seriously compromised by aneuploidy.
Many chemicals affect chromosome segregation in oocytes [38–40]; however, the fact that 2-MOE2 is an endogenous estradiol metabolite makes its biological significance unclear. This catecholestrogen has been found in the follicular fluid of some species at concentrations comparable to those affecting tubulin polymerization [7, 8]. Furthermore, an increase in the production of 2-OHE2 has been reported in large, preovulatory follicles of porcine ovaries [41].
Our current hypothesis is that rather than a physiological process, alterations in oocyte maturation elicited by high concentrations of 2-MOE2 may play a role in development of the infertility syndromes associated with some environmental contaminants known to activate the AHR receptor. These agents are potent inducers of the enzymes responsible for the conversion of estradiol into catecholestrogens in granulosa cells [42]. For example, 2-OHE2 can be converted into 2-MOE2 by the ovarian catechol O-methyl transferase [43]. In this context, it is interesting to note that cigarette smoke has been found to be responsible for both activating oocyte AHR and producing nondisjunction in meiosis [44, 45].
The suggestion that age-related aneuploidies in humans may be caused by an altered oocyte maturation because of the aberrant hormone levels often found in women as they approach menopause [46] is also remarkable. Because human embryos have a abnormal chromosome number more often than those of any other species [47], and because increasing evidence indicates that the aneuploidy found in human fetuses originates mostly during female meiosis [31], it is tempting to speculate that 2-MOE2 could be involved. Hence, although high concentrations of 2-MOE2 during oocyte maturation cause the arrest of embryo development, lower concentrations may elicit subtler spindle alterations and chromosomal errors that could lead to miscarriage or other nonlethal genetic problems.
Taken together, our results show that in addition to their previously demonstrated intraovarian actions, abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from alterations in the meiotic spindle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Lino Baranao, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. FAX: 54 11 4786 2564; lbaranao{at}dna.uba.ar
Received: 18 February 2003.
First decision: 4 March 2003.
Accepted: 16 July 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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