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a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706
b Loeb Research Institute and Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, Ontario, Canada K1Y 4E9
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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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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The basic medium used in this study was a modified version of hamster embryo culture medium-3 [13] supplemented with 0.5 mM taurine and lactate concentration reduced to 1 mM L-lactate (H3t) [3]. The pH of medium H3t in an atmosphere of 10% CO2, 5% O2, and 85% N2 is 7.3. For some experiments requiring a chloride-free medium, sodium chloride was replaced with sodium gluconate (cfH3t). For embryo collection and manipulation, a Hepes-buffered modification of H3t was used, in which the bicarbonate was replaced with Hepes to maintain pH at 7.35 (bfHH3t). When either 25 mM NH4Cl or NH4SO4 was added to the medium, the pH was adjusted to 7.35. All chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
Medium for calibration of pH consisted of 100 mM KCl, 25 mM NaCl, 21 mM Hepes, and 75 mM sucrose. The medium was adjusted to either pH 7.1, 7.4, 7.7, or 8.1. Immediately prior to use, nigericin (10 µg/ml; Molecular Probes, Eugene, OR) and valinomycin (5 µg/ml) were added to the calibration media from 1000-strength stock solutions dissolved in dimethyl sulfoxide (DMSO).
Medium for prolonged embryo culture was hamster embryo culture medium-10 (HECM-10) [14]. HECM-10 was prepared from stocks on the day before culture and stored at 4°C.
The weak alkali trimethylamine was added to culture medium from a concentrated stock solution (100-strength) on the morning of the experiment. DIDS (4,4'-diisothiocyanatostilbene-2,2'-di-sulfonic acid) was used to inhibit HCO3-/Cl- exchange. DIDS was prepared as a concentrated stock (1000-strength) in DMSO and was added to the medium immediately prior to the experiment. A concentration of 100 µM was chosen as this concentration has previously been shown to maximally inhibit HCO3-/Cl- exchanger in embryos [11].
Animals
Oocytes and embryos were collected from 3- to 4-mo-old golden hamster females. Multiple ovulations were induced by an i.p. injection of eCG (Pregnyl; Diosynth, Chicago, IL) on the day of postestrus. Oocytes were collected from unmated females immediately after ovulation. One-cell- and two-cell-stage embryos were collected from females mated to males on Day 4 of the estrous cycle. One-cell embryos were collected at either 3–4 h post-egg activation (PEA) by sperm (timings described by Bavister et al. [15]) or 7–8 h PEA. Embryos collected at 3–4 h PEA regularly have only 1 pronucleus, while 1-cell embryos collected at 7–8 h PEA have 2 pronuclei and 2 polar bodies. Two-cell embryos were collected the following day at 32 h PEA. Oocytes and embryos were flushed from the oviduct with medium bfHH3t at 37°C. Oocytes and 3–4-h PEA 1-cell embryos were denuded of surrounding cumulus cells by incubation with 0.5 mg/ml hyaluronidase (Sigma) for 1 min.
Measurement of pHi
The pHi was assessed using fluorescence measurements with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein (BCECF; Molecular Probes). Oocytes and embryos were loaded with 0.7 µM BCECF using the acetoxymethyl ester (BCECF-AM) for 20 min at 37°C in bfHH3t. This concentration of BCECF and loading time were found to be optimal for subsequent embryonic development (data not shown). Between 8 and 12 oocytes or embryos were washed in medium without the probe, and embryos were placed in a temperature-controlled chamber (Biophysica, Baltimore, MD) set at 37°C. Solutions were changed by flushing 20 ml of medium through the chamber using a syringe pump. Measurement of pHi was achieved using a Nikon Diaphot (Garden City, NY) inverted microscope connected by a Nikon Dual Optical Path Tube to a Photometrics PXL cooled camera (Huntington Beach, CA) for high-resolution recording of epifluorescent BCECF images. Image analysis of fluorescent images was performed using Metamorph/Metafluor hardware and software (Universal Imaging Corporation, West Chester, PA). Emission wavelength was set to 530 nm, and the ratio of fluorescence intensities of excitation wavelengths 500 (pH sensitive) to 440 nm (pH insensitive) was obtained for each embryo. The ratio of fluorescence intensities is linearly proportional to pH, and fluorescent ratios were calibrated in situ using a nigericin/high K+ method at four pH levels: 6.9, 7.2, 7.5, and 7.8 [16]. Additional controls were conducted with the pH indicator BCECF to ensure that the pH of the medium in the measurement chamber remained constant for up to 2 h.
Chloride Removal Assay for HCO3-/Cl- Exchanger Activity
The activity of the HCO3-/Cl- exchanger depends on the coupled transport of external chloride into the cell with the export of bicarbonate. Therefore, in cells with an active HCO3-/Cl- exchanger, removal of external chloride from the medium will cause the exchanger to run backward, resulting in bicarbonate entering the cell and increasing pHi. Therefore a further assay for the presence of the HCO3-/Cl- transporter is used to measure any change in pHi following incubation in Cl--free medium [12]. This assay was used to initially assess the presence of the HCO3-/Cl- exchanger in hamster oocytes and embryos.
Induction of Intracellular Alkalosis and Calculation of Recovery Rates
Embryos in the chamber were allowed to sit in medium H3t for 5 min before baseline pHi was determined. After measurement of baseline pHi, the chamber was flushed with a Cl--free medium (cfH3t), and all embryos remained in this Cl--free medium for 5–7 min. An intracellular alkaline load was subsequently induced by incubation with H3t supplemented with 25 mM NH4Cl or cfH3t supplemented with NH4SO4 (for chloride-free recovery). This produces an immediate increase in pHi due to the rapid diffusion of NH3 across the membrane. The rate of recovery from alkalosis was calculated by calculating the tangent to the recovery curve (dpH/dt) [1, 10, 11].
Determination of Set Point of Activity of the HCO3-/Cl- Exchanger
The set point for activation of the HCO3-/Cl- exchanger was calculated by determining the recovery rates at specific levels of pHi in the presence or absence of external chloride. When the data obtained in the presence of Cl- with those in the absence of Cl- are plotted, the intersection of the two graphs indicates the set point of activation of the HCO3-/Cl- exchanger.
Embryo Culture
Two-cell embryos were collected as described and cultured in 35-µl drops of medium HECM-10 in groups of 10–15 in 35-mm Petri dishes (Falcon, CTC Biomedical Inc., Carrollton, TX) under mineral oil (Sigma). Embryos were cultured in a water-jacketed incubator (Forma Scientific, Marietta, OH) at 37°C in an atmosphere of 10% CO2, 5% O2, and 85% N2. Development to the morula/blastocyst stages was assessed after 48 h of culture.
Statistical Analysis
Differences in increases in pH by the chloride removal assay were assessed by Student's t-test. Differences in the rates of recovery from alkalosis in the various media formulations were determined by ANOVA followed by Bonferroni's Procedure for Multiple Comparisons. Embryo development in culture was assessed using linear-logistic regression in which the error distribution was assumed to be binomial. The null hypothesis of no treatment effect against a treatment effect was tested using the log-likelihood ratio statistic. A value of p < 0.05 was considered significantly different.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initially the Cl- removal assay was used to detect the presence of HCO3-/Cl- exchanger activity. Removing chloride from medium (H3t) resulted in an immediate alkalization of the blastomeres of 2-cell embryos (Fig. 1a). The mean increase in pHi induced by incubation in Cl--free medium (cfH3t) for 2-cell embryos was 0.201 ± 0.027 pH units (Table 1). Addition of 100 µM DIDS to cfH3t, which inhibits HCO3-/Cl- transport, reduced the increase in pHi to 0.054 ± 0.009 (Fig. 1a). In contrast, in hamster oocytes, removal of Cl- from the medium (cfH3t) caused only a small increase in pHi of 0.037 ± 0.006 pH units (Fig. 1b; Table 1). Addition of DIDS to cfH3t did not alter the magnitude of this small increase in pHi (0.042 ± 0.009 pH units; Fig. 1b). Similar to observations for oocytes, removing Cl- from the external culture medium (incubation with cfH3t) resulted in only a small change in pHi in 1-cell embryos collected between 3 and 4 h PEA (0.033 ± 0.007 pH units). Addition of DIDS to medium cfH3t also did not affect this small increase in pHi (Table 1). In contrast, 1-cell embryos collected at 7–8 h PEA exhibited a large increase in pHi when Cl- was removed from the culture medium (0.179 ± 0.041 pH units). Furthermore, addition of DIDS to medium cfH3t significantly reduced the amplitude of this increase in pHi (Table 1).
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Recovery of pHi from Alkaline Loading Induced by NH3
For 2-cell embryos incubated in the control medium (H3t), recovery from an intracellular alkalosis induced by incubation with NH4+ was fast, and pHi returned to around 7.2 usually within 10 min (Fig. 2; Table 2). In contrast, recovery from alkalosis by 2-cell embryos in the absence of Cl- in the medium (cfH3t) leveled off after around 2 min, and recovery was always incomplete. Intracellular pH recovered only to 7.49 ± 0.06 in the absence of Cl- (Fig. 2; Table 2). Recovery was not affected by the presence or absence of Na+ (data not shown). Recovery from alkalosis by the 2-cell hamster embryo is Cl- sensitive and therefore mediated by the HCO3-/Cl- exchanger.
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Incubation of hamster oocytes with NH4+ resulted in an initial decrease in pHi that then leveled off, and pHi was not restored to the normal physiological range but remained at 7.47 ± 0.09 (Fig. 3, Table 2). This pattern of recovery for oocytes and the rates of recovery were equivalent in either the absence or presence of Cl- (Fig. 3) or the presence of DIDS (Table 2).
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The patterns of recovery from alkalosis by 1-cell embryos varied depending on the time at which the embryos were collected after egg activation by the sperm. For 1-cell embryos collected between 3 and 4 h PEA, the patterns of recovery were similar to those observed for oocytes. Recovery from alkalosis in control medium by 3–4 PEA 1-cell embryos leveled off after the initial nonspecific recovery, likely due to slow diffusion of NH4+ [10]. Rates of recovery from alkalosis were not altered by the absence of Cl- (cfH3t) or by the presence of DIDS (Table 2). In contrast, 1-cell embryos that were collected 7–8 h PEA exhibited recovery patterns similar to those observed for 2-cell embryos. The rate of recovery and ability to recover pHi to normal ranges by 7–8 h PEA 1-cell embryos were both Cl- dependent and DIDS sensitive, indicating that the recovery was mediated by the HCO3-/Cl- exchanger (Table 2).
Cl- Dependency of HCO3-/Cl- Exchanger
The dependency on external Cl- concentration for the recovery from alkaline loading by 2-cell embryos was assessed. The rate of recovery from alkalosis was very low in embryos when there was no external chloride (Fig. 4). Increasing the chloride concentration to 40 mM significantly increased the rate of recovery and also decreased the final pHi that was restored after the 15-min incubation period (Fig. 4). Increasing the external chloride to 80 mM further increased the recovery rate, and highest rates of recovery were observed when the Cl- concentration was increased to 113 mM (Fig. 4). Increasing the external chloride concentration also increased the ability of the embryos to restore pHi to initial levels after the 15-min incubation period (Fig. 4).
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Determination of the Set Point of the HCO3-/Cl- Exchanger
In the presence of 113 mM Cl-, activity of the HCO3-/Cl- exchanger increased monotonically as the deviation of pHi from the initial baseline levels increased (Fig. 5). In contrast, recovery rates in the absence of Cl- or the presence of DIDS were relatively constant across a range of pHi. The set point of HCO3-/Cl- exchange was calculated by determining the intercept of the lines indicating recovery in the presence and absence of Cl-. The set point of activation in 2-cell embryos was determined to be 7.24 (Fig. 5). A similar analysis of the pHi recovery rates determined in oocytes revealed that the rates observed in the presence or absence of chloride did not converge, providing further evidence for the absence of HCO3-/Cl- exchanger activity in hamster oocytes.
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Role of HCO3-/Cl- Exchanger in Preimplantation Development in Culture
Initially the effect of incubating 2-cell hamster embryos with the weak alkali trimethylamine on pHi was assessed. Incubation with 5 mM or 10 mM trimethylamine significantly increased pHi from 7.18 ± 0.02 in control embryos incubated in HECM-10 to 7.30 ± 0.04 (p < 0.05) and 7.37 ± 0.05 (p < 0.01; n = at least 30 embryos per treatment), respectively. Two-cell embryos were subsequently cultured for 48 h in either HECM-10 or HECM-10 supplemented with 100 µM DIDS, HECM-10 supplemented with either 5 or 10 mM trimethylamine, or HECM-10 supplemented with either 5 or 10 mM trimethylamine and 100 µM DIDS. The addition of DIDS to the culture medium did not affect morula/blastocyst development, although there was a small decrease in blastocyst development (Fig. 6). Addition of either 5 or 10 mM trimethylamine, which increased pHi by 0.12 and 0.19 pH units, respectively, resulted in a decrease in morula/blastocyst development and also significantly reduced blastocyst development (Fig. 6). Development to the morula stage was further reduced in the presence of the weak alkali trimethylamine and DIDS. Furthermore, blastocyst development was prevented in the presence of both trimethylamine (5 mM or 10 mM) and DIDS (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the 2-cell embryo, hamster oocytes and early 1-cell embryos collected 3–4 h PEA (when embryos are still undergoing pronuclei formation) do not appear to have any active HCO3-/Cl- transport mechanism for the regulation of pHi. When an intracellular alkalosis was invoked, hamster oocytes and early 1-cell embryos were unable to recover pHi to initial resting levels, and pHi remained significantly elevated. It therefore appears that the activity of the HCO3-/Cl- exchanger in hamster embryos is activated several hours after fertilization. Prior to 4 h PEA, hamster embryos and oocytes lack any detectable mechanism for the alleviation of an alkaline load.
This pattern of activation of HCO3-/Cl- exchanger activity throughout hamster oocyte and embryo development is very similar to that reported for HCO3-/Cl- exchange in the early mouse embryo. Mouse oocytes also lack functional HCO3-/Cl- exchange [12]. Therefore, mouse oocytes also cannot restore pHi, which remains elevated after induction of intracellular alkalosis [12]. This lack of activity of the HCO3-/Cl- exchanger in mouse oocytes continued for 3–4 h after activation of the egg by the spermatozoa. After this time, exchanger activity appeared gradually between 4 and 8 h postinsemination [12]. In the mouse, the mRNA transcripts for the exchanger were detectable in oocytes and embryos, and the initiation of functional activity was due to the activation of existing protein in the oocyte [12]. In hamsters, active HCO3-/Cl- exchange could also be detected in 1-cell embryos collected 7–8 h PEA by the spermatozoa (present study). It is possible that induction of HCO3-/Cl- exchanger activity in hamster embryos is also due to an activation of existing proteins that is associated with fertilization.
Similar to the HCO3-/Cl- exchanger, activity of the Na+/H+ antiporter, which regulates pHi in the acid to neutral range, is absent in hamster oocytes and early 1-cell embryos still completing pronuclei formation [6]. Activity of the Na+/H+ antiporter could not be detected in hamster embryos until around 6 h PEA. Presence of active Na+/H+ exchange was associated with egg activation and was induced by a process dependent on protein kinase C [6]. Therefore, for the hamster embryo, both transport systems for the regulation of pHi (Na+/H+ exchange and HCO3-/Cl- exchange) are activated in the several hours following egg activation. This timing of exchanger appearance coincides with the calcium oscillations that continue for 5–6 h after egg activation.
Under normal culture conditions, the role of the HCO3-/Cl- exchange in the regulation of pHi may be low as evidenced by the set point of around 7.2. This set point for activation is similar to that reported for the developing mouse embryo [11, 12] and for other cell types [9, 25]. Therefore, when hamster embryos were cultured in a controlled environment where the external pH was maintained close to 7.2, addition of the inhibitor DIDS resulted in only a small decrease in morula/blastocyst development. However, when embryos were challenged with a small increase in pHi (around 0.2 units) in the presence of DIDS, the ability to develop to the morula stage was severely compromised, and none of the embryos were able to develop to the blastocyst stage. On the basis of the bicarbonate concentration in oviduct fluid and CO2 level [26, 27] measured within the oviduct, it is presumed that the pH of oviduct fluid is alkaline (around pH 7.6). HCO3-/Cl- exchanger activity would be very high in the oviduct and would therefore have a very important role in the regulation of pHi in vivo.
Regulation of pHi in the alkaline range in hamster 1-cell (collected after 7 h PEA) and 2-cell embryos is provided by the HCO3-/Cl- exchanger. This exchanger is activated when the pHi increases above the set point of activation of 7.24 to restore pHi to physiological levels. Interestingly, hamster oocytes and early embryos collected in the hours immediately following sperm penetration and egg activation do not have active HCO3-/Cl- exchangers to regulate pHi. Thus it appears that the ability to regulate pHi in the hamster embryos is activated during fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Michelle Lane, 799 East Hampden Ave., Suite 300, Englewood, CO 80110. FAX: 303 788 4438; mlane{at}colocrm.com
Accepted: March 18, 1999.
Received: December 17, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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