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Departments of Obstetrics and Gynecology3
Physiology,4 Yamagata University School of Medicine,Yamagata 990-9585, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, calcium, developmental biology, fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we demonstrated in the mouse oocyte that in vivo postovulatory aging (insemination at 20 h after hCG injection) significantly alters the pattern of Ca2+ oscillations in a fertilized oocyte [11]. Compared with freshly ovulated oocytes (14 h after hCG injection), aged oocytes showed individual Ca2+ oscillations that were high in frequency and low in amplitude [11, 12]. These changes in Ca2+ oscillations may arise from alterations in Ca2+ handling in aged oocytes, particularly those in the endoplasmic reticulum (ER), because the rate of [Ca2+]i reuptake by the ER was slow in aged oocytes [11, 13]. Such incomplete restoration of [Ca2+]i can result from depletion of intracellular ATP ([ATP]i), because Ca2+ reuptake is dependent on the availability of [ATP]i near the ER Ca2+ ATPases.
Consequently, our hypothesis is that the poor developmental competency in a fertilized, aged oocyte may arise from inadequate intracellular handling of Ca2+ due to partial depletion of ATP in the aged oocyte. Increases in ATP consumption should occur at fertilization. Synchronizing with such increases in ATP utilization, mitochondria must up-regulate oxidative ATP production quickly and precisely so that the level of [ATP]i is sustained and subsequent energy-consuming processes in oocyte development can proceed. We undertook the present study to demonstrate dynamic changes in [ATP]i at fertilization. We also examined changes in the regulation of [ATP]i caused by postovulatory aging in mouse oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human tubal fluid (HTF) medium (101.6 mM NaCl, 4.69 mM KCl, 0.20 mM MgSO4, 0.37 mM KH2PO4, 21.4 mM Na-lactate, 2.78 mM glucose, 25.0 mM NaHCO3, 0.33 mM Na-pyruvate, 2.04 mM CaCl2, and 100 IU/ml penicillin) was used for oocyte incubation and measurements of intracellular concentration of free Mg2+ ([Mg2+]i) and [Ca2+]i. Hepes-HTF medium (21.0 mM Na-Hepes and 4.0 mM NaHCO3 were substituted for 25.0 mM NaHCO3 of the HTF medium) was used for oocyte handling in air. All the media except for those used in the carbonyl cyanide m-chlorophenylhydrazone (CCCP) experiment (Figs. 1–3) contained 0.5% BSA (Sigma, St. Louis, MO). The HTF medium was used after equilibration to 5% CO2 in air at 37°C (pH 7.4). Reagents for culture media preparation were of tissue-grade quality.
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Gamete Preparations
This study was carried out with permission from the Committee of Animal Experimentation, Yamagata University School of Medicine.
Oocytes
B6C3F1 hybrid female mice aged 4 to 6 wk were superovulated by i.p. injection of 10 IU eCG (Teikokuzouki, Tokyo, Japan), followed 48 h later by 10 IU hCG (Mochida, Tokyo, Japan). Using a fine needle, unfertilized oocytes were released from the oviduct at 12.5 or 18.5 h post-hCG into droplets of Hepes-HTF medium containing 300 IU/ml hyaluronidase (Sigma). Then, cumulus cells were removed by several aspirations with a narrow-bore pipette. Cumulus-free oocytes were washed five times in the Hepes-HTF medium and incubated in the HTF medium at 37°C in 5% CO2 in air. Finally, zonae pellucidae were mechanically removed after treatment with 25 IU 
-chymotrypsin (Sigma), and denuded oocytes were used for all experiments. In the present study, oocytes released from the oviduct at 12.5 and 18.5 h post-hCG were designated as "fresh" and the "aged" oocytes, respectively. Measurements of [Mg2+]i and [Ca2+]i were conducted 1.5 h following the release of oocytes from the oviducts.
Sperm ICR male mice (8 to 12 wk of age) were killed by cervical dislocation, and the cauda epididymides and spermiducts were resected. These were cut into small pieces in HTF medium and kept at 37°C for 90 min in 5% CO2 in air to allow capacitation. After capacitation the supernatant containing spermatozoa was collected (<1 x 104 spermatozoa/ml) and used for insemination. Insemination was conducted while stopping the perfusion in the measuring cuvette. To avoid polyspermy, excess spermatozoa were washed out from the measuring cuvette immediately after the first sperm attachment with the oocyte was confirmed visually with the aid of a microscope. In a preliminary experiment, we confirmed that the incidence of polyspermy under these conditions was negligible. Fertilization occurred in 16.9% (66/390) of the fresh oocytes and in 20.9% (90/438) of the aged oocytes (not significant [NS], chi-square test), while polyspermy was found in only 1.5% (1/66) and 2.2% (2/90) of fresh and aged oocytes (NS, chi-square test), respectively.
Simultaneous Measurements of [Ca2+]i and [Mg2+]i
As reported previously [11], [Ca2+]i was measured using a Ca2+-sensitive fluorescent dye, Fura-PE3. Fura-PE3 is the same as Fura-2 in its spectral properties but is retained within the cell for a longer period of time, thus being suitable for the present prolonged measurements. Using the same optical system, we also measured [Mg2+]i using a Mg2+-sensitive fluorescent dye, Magnesium Green (MgG), to indirectly assess [ATP]i (see Discussion). Excitation light wavelengths were 340/380 nm, and 480 nm for Fura-PE3 and MgG, respectively. Fluorescence measurements were conducted at >400 nm and >510 nm for Fura-PE3 and MgG, respectively. Measurements of [Ca2+]i and [Mg2+]i were conducted almost simultaneously (the delay between these two measurements was <0.1 sec). These paired measurements were conducted every 5 sec. The method for calculating the absolute [Ca2+]i value was described previously [12]. Gradual decreases in the MgG fluorescence due to photobleaching were compensated for by a linear extrapolation. Because it was not possible to calibrate MgG fluorescence for [ATP]i, MgG fluorescence was represented as the percentage of baseline (before experimental interventions) fluorescence intensity.
Zona pellucida-free oocytes were incubated for 40 min at 37°C in HTF medium containing 5 µM MgG acetoxymethyl ester (Molecular Probes, Eugene, OR) and 5 µM Fura-PE3 acetoxymethyl ester (Wako, Osaka, Japan). Then, the oocytes were transferred to the HTF medium in a plastic culture dish and incubated for a further 20 min to allow for equilibration of the dyes. Finally, these oocytes were transferred to the HTF medium in a temperature-controlled measuring cuvette placed on the stage of an inverted microscope (Diaphot-TMD; Nikon, Tokyo, Japan).
To test the feasibility of using MgG as a tool for [ATP]i measurement, we used an uncoupler of mitochondrial oxidative phosphorylation, CCCP, to induce rapid ATP depletion. In this experiment, oocytes were perfused with the HTF medium at 1 ml/min. With this perfusion rate, exchange of medium in the measuring cuvette was completed within 5 min. Then, we conducted the simultaneous measurements of [Mg2+]i and [Ca2+]i in the fresh and the aged oocytes to elucidate the aging-related changes in ATP regulation.
In Vitro Determination of Intracellular ATP Content
For a quantitative assessment of intracellular ATP content, the luciferin-luciferase assay of ATP was performed in lysed oocytes using a commercial kit (LL-100-1; TOYO INK, Tokyo, Japan). Unfertilized and fertilized (5 h after in vitro fertilization) mouse oocytes were prepared according to the manufacturer's instruction, and luciferin luminescence was measured using a luminometer (Luminescencer-JNR AB-2100; ATTO, Tokyo, Japan). ATP content was represented per oocyte.
Statistics
Values are expressed as the mean ± SEM. A Student t-test and two-way analysis of variance were used to compare the means when statistical significance was reached at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Administration of 2 µM CCCP elicited rapid increases in MgG fluorescence and [Ca2+]i in the unfertilized fresh oocytes, and this retuned nearly to baseline after CCCP was removed in all fresh oocytes (n = 10; Fig. 1A). MgG fluorescence increased by 23% ± 2% at the end of the CCCP challenge. These increases in MgG fluorescence appeared to reflect increases in [Mg2+]i released as a result of rapid ATP hydrolysis and inhibition of ATP synthesis [14]. In vitro measurements of ATP content by luciferin-luciferase in CCCP-treated fresh oocytes were consistent with this continuous assessment of [ATP]i (Fig. 2).
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In aged oocytes, MgG fluorescence and [Ca2+]i also abruptly increased with CCCP administration, but the behavior of MgG fluorescence differed considerably (Fig. 1, B and C). In fact, 56% (9/16) of the aged oocytes died (judged from [Ca2+]i tracing and morphological changes) during the CCCP challenge (Fig. 1C), and 3 out of 7 aged oocytes that survived the CCCP challenge showed sustained elevation of MgG fluorescence after washout of CCCP (Fig. 1B). Also, disruption of [Ca2+]i regulation was noted in 12 of the 16 aged oocytes (Fig. 1, B and C).
A possibility that changes in [Ca2+]i might have affected MgG fluorescence was examined by suppressing [Ca2+]i changes. Preincubation with a Ca2+ chelater, 1,2-bis(o-Aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM; Sigma) abolished the increase in [Ca2+]i upon administration of CCCP, while significant increase in MgG fluorescence (111% ± 2%, n = 10) was still present (Fig. 3).
Oxidized flavoproteins (FAD++) contain endogenous (mitochondrial) fluorophore excitation/emission spectra that overlap those of MgG. Autofluorescence of FAD++ changes according to the mitochondrial metabolic state [15]. To examine whether FAD++ fluorescence interferes with the measurement of MgG fluorescence, an oocyte was treated with 100 µM digitonin while measuring MgG fluorescence. Immediately after permealization of the oolemma by digitonin, MgG fluorescence leveled off to almost zero, indicating that the origin of the fluorescence was in the cytosol (data not shown). In addition, the magnitude of autofluorescence in oocytes was only 0.4% of MgG fluorescence. Thus, in our MgG fluorescence measurement, autofluorescence arising from mitochondrial FAD++ could be ignored.
Intracellular ATP Content in Unfertilized and Fertilized Oocytes
As shown in Figure 4, ATP content of unfertilized oocytes was comparable in both fresh (0.16 ± 0.01 pmol/ oocyte) and aged (0.14 ± 0.01 pmol/oocyte) oocytes. The ATP contents determined 5 h after in vitro fertilization were significantly increased in both fresh (0.22 ± 0.01 pmol/ oocyte) and aged (0.18 ± 0.01 pmol/oocyte) oocytes. The fertilized fresh oocytes showed a higher intracellular ATP content than the fertilized aged oocytes (P < 0.05).
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Calcium Oscillations and [ATP]i in Fresh and Aged Oocytes
Figure 5 shows representative data demonstrating [Ca2+]i and changes in MgG fluorescence before and after fertilization in fresh (Fig. 5A) and aged (Fig. 5B) oocytes. The frequency of Ca2+ oscillations was significantly increased in the aged oocytes compared with that of fresh oocytes, whereas the amplitude of Ca2+ oscillations was significantly decreased in aged oocytes compared with that of fresh oocytes (Table 1).
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Synchronizing with the first transient elevation of [Ca2+]i at fertilization, MgG fluorescence abruptly increased in both fresh and aged oocytes (indicated by arrow A in Fig. 5). In fresh oocytes, this MgG fluorescence transient was followed by a significant decrease below baseline (indicated by arrow B). Such a secondary drop in MgG fluorescence was independent of the sperm dose, because the similar change in MgG fluorescence was demonstrated in unfertilized oocytes treated with strontium or thimerosal (Fig. 6).
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Magnesium green fluorescence behaved quite differently in aged oocytes. Figure 7 summarizes the differences in the MgG fluorescence pattern between fresh and aged oocytes at fertilization. The intensity of MgG fluorescence before insemination was comparable in fresh (4907 ± 267 arbitrary units [au]; n = 27) and aged oocytes (5930 ± 594 au; n = 15; NS), indicating consistent loading of the fluorescent dye. The first transient elevation in MgG fluorescence was significantly larger in aged (11.0% ± 0.6% of the baseline MgG fluorescence intensity) than in fresh (7.5% ± 0.6%) oocytes. Then, MgG fluorescence decreased to below baseline in fresh oocytes; the fluorescence levels were –10.4% ± 0.7% and –13.0% ± 1.3% at 0 and 60 min after the first transient elevation in MgG fluorescence, respectively. In contrast, in aged oocytes, MgG fluorescence returned only to baseline (–2.9% ± 0.8%, –2.3% ± 0.9% at 0 and 60 min, respectively).
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In fresh oocytes, each Ca2+ spike was associated with a drop in MgG fluorescence (Fig. 5A), whereas such small fluctuations appeared to be dampened in aged oocytes (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dynamic measurement of [ATP]i in single living cells is quite difficult to perform at the present time. Recently, Dumollard et al. [15, 16] successfully applied the conventional luciferin-luciferase assay of ATP in single living mouse oocytes. Although this technique is relatively accurate at least in aerobic cells, it is technically difficult to conduct with a fluorescent measurement of [Ca2+]i using, for example, Fura-2, due to the difference in the measuring mode (luminescence vs. fluorescence). In the present study, we indirectly assessed [ATP]i using a Mg2+-sensitive fluorescent dye, MgG. The rationale for using Mg2+ for the assessment of [ATP]i is as follows: 1) the intracellular Mg2+ pool is predominantly present as MgATP2–, 2) the affinity of ATP4– for Mg2+ is about 10-fold greater than that of ADP at physiological pH and, therefore, 3) Mg2+ increases when ATP hydrolysis exceeds the rephosphorylation of ADP to ATP and vice versa [14]. Another important point is that excitation/emission wavelengths for MgG (480 nm/>510 nm) do not overlap those of Fura-PE3 (340 nm and 380 nm/>400 nm). Thus, the simultaneous measurement of [Mg2+]i and [Ca2+]i was possible. This technique has been used to continuously monitor ATP response to metabolic inhibitions in single cardiomyocytes [14, 17] and adrenal chromaffin cells [18]. This technique was particularly useful in the present study because we were able to precisely determine, from the [Ca2+]i tracing, the time point at which fertilization took place and relate it to ATP regulation.
After the double loading of Fura-PE3 and MgG in single oocytes, we could simultaneously assess changes in [Ca2+]i and [ATP]i with a satisfactory temporal resolution in unfertilized (Fig. 1) and fertilized (Fig. 5) mouse oocytes. As expected, rapid depletion of ATP by an uncoupler of oxidative phosphorylation (2 µM CCCP) quickly elevated MgG fluorescence. The MgG signal may not be entirely free from some calcium signal because MgG is also sensitive to a high concentration of Ca2+ (Kd = 
6 µM). However, the bleed-through appeared low enough not to hamper the interpretation of the signals because 1) elevation of MgG fluorescence was demonstrated when the increase in [Ca2+]i was suppressed by 10 µM BAPTA (Fig. 3) and 2) the MgG signals moved in the opposite direction in Figure 5A than would have been predicted for a signal entirely due to calcium bleed-through. In addition, the magnitude of oxidized flavoprotein autofluorescence that overlaps MgG fluorescence was substantially low.
As shown in Figures 5 and 7, fertilization in fresh oocytes induced immediate increases in MgG fluorescence that synchronized with the first transient elevation in [Ca2+]i. Thus, the rate of ATP consumption exceeded that of the production at this time point. After the transient elevation in MgG fluorescence lasting for 4.7 ± 0.2 min, MgG fluorescence significantly decreased to below baseline in fresh oocytes (Figs. 5 and 7). Decreases in MgG fluorescence imply that ATP production relatively exceeded consumption. Thus, these changes in MgG fluorescence reflect significant alterations in the balance of ATP production and consumption at fertilization.
Although quantitative data are lacking in mammalian oocytes, fertilization should drastically increase the energy requirement of the oocyte. Consequently, mitochondrial ATP production must be stimulated to meet the increased metabolic demands at fertilization. In fresh oocytes, fertilization appeared to shift the set point of intracellular ATP regulation at a level higher than before fertilization. Among many factors that are up-regulated at fertilization, we speculate that elevated ATP requirement for sustaining Ca2+ oscillations [15] may be of particular importance because a similar shift in ATP production/consumption could be demonstrated by artificially induced Ca2+ spikes in the absence of the sperm (Fig. 6).
These findings are consistent with more direct measurement of intracellular ATP using the luciferin-luciferase technique in single mouse oocytes. Dumollard et al. [15] reported significant increases in luciferin luminescence after insemination of sperm in 9 of 25 mouse oocytes, while in 16 of 25 oocytes, luciferase luminescence was unchanged. Those researchers suggested that the signal that mediates metabolic readjustment at fertilization is sperm-induced changes in [Ca2+]i. Although the transient elevation in [Ca2+]i at fertilization is likely to stimulate ATP production (probably via Ca2+-sensitive dehydrogenases in mitochondria), ATPs are also consumed in the ER Ca2+ ATPases to restore [Ca2+]i to baseline. Thus, the new equilibrium point of [ATP]i following the transient increase in [Ca2+]i at fertilization may be determined by a subtle balance of stimulated ATP consumption and production, and therefore may be significantly affected by the integrity of the oocyte tested.
These findings are also consistent with the separate in vitro measurement of ATP content conducted before and 5 h after fertilization (Fig. 4). Several studies have shown using the conventional luciferin-luciferase in vitro assay that intracellular ATP content did not change appreciably in unfertilized and fertilized oocytes of mouse [19], sheep [20], and human [21]. The new equilibrium level of [ATP]i after fertilization may change with oocyte development. Within the time frame of the present experiment, fertilization appeared to induce more than enough increases in ATP production.
In contrast to fresh oocytes, MgG fluorescence in in vivo-aged oocytes behaved quite differently (Figs. 5 and 7). The increase in MgG fluorescence at fertilization was longer (5.7 ± 0.6 min; P < 0.05 vs. fresh oocytes) and larger, indicating that readjustment of ATP production occurred slowly. After the first [Ca2+]i transient fluctuation, MgG fluorescence returned to near baseline (Fig. 7), indicating that readjustment of ATP production was not enough compared with that of fresh oocyte. These findings are also consistent with the separate in vitro assay of intracellular ATP content (Fig. 4). From these results, we conclude that in vivo-aged mouse oocytes failed to readjust ATP regulation at fertilization.
Intracellular ATP has diverse effects on cellular functions. Particularly, in embryogenesis, a close relationship between intracellular ATP content in a pre-embryo and the developmental potency of the early cleavage stage of embryos was demonstrated [21, 22]. Furthermore, the intracellular ATP content of the cleavage-stage human embryo (8-cell to 10-cell) might be related to developing speed because the embryo that showed a slow speed of development tended to have a low intracellular ATP content [23].
Aging of oocytes induces significant biochemical and morphological alterations [24, 25]. Among these, we focused associated changes in intracellular ATP content because Ca2+ oscillations critically depend on ATP availability near the ER. From this standpoint, relative depletion of ATP in aged fertilized mouse oocytes demonstrated here should have special relevance. Because the pattern of Ca2+ oscillations appears to have a significant effect on oocyte development [10], the failure in readjustment of ATP regulation at fertilization in aged oocytes might be responsible, via Ca2+ oscillations, for the poor developmental potency of aged oocytes [12, 25–27].
Aging-related mitochondrial dysfunction has been suggested in the oocyte [27]. As a cause for compromised mitochondrial functions in the aged oocyte, oxygen radical damages to mitochondria appear relevant [28], because the postovulatory aged oocyte may be exposed to oxidative stresses for a prolonged time in the oviduct [29, 30]. In fact, we have demonstrated that exposing fresh mouse oocytes to a low concentration of H2O2 resulted in the alteration of Ca2+ oscillations, and reductions in the fertilization rate and blastocyst formation that were similar to those in aged oocytes [12]. Furthermore, Tarin et al. [31] demonstrated that deleterious effects of oocyte aging on fertilization and oocyte development were partially reversed by addition of a reducing agent, dithiothreitol, in culture medium. Reactive oxygen species exert detrimental effects in mitochondrial functions through oxidative damages to mitochondrial DNA, proteins, and lipids [32–34]. Thus, it is presumable that an increased level of oxidative stress in aged oocytes affects regulation of ATP production in mitochondria, resulting in the incomplete readjustment of ATP regulation at fertilization. Such incompleteness in the regulation of ATP in aged oocytes may directly or indirectly (via changes in Ca2+ oscillations) affect embryo development. If so, mitochondria might be one of the targets for therapeutics toward rejuvenation of aged oocytes.
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FOOTNOTES |
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2 Correspondence: Eiji Takahashi, Ph.D., Department of Physiology, Yamagata University School of Medicine, 2-2-2 Iida-nishi Yamagata 990-9585, Japan. FAX: 81 23 628 5215; eiji{at}med.id.yamagata-u.ac.jp
Received: 17 August 2004.
First decision: 13 September 2004.
Accepted: 12 January 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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