Department of Animal Health and Biomedical Sciences, University of Wisconsin, 1655 Linden Drive, Madison, WI 53706, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cryopreservation/HCO3/Cl transporter/metabolism/Na+/H+ antiporter/vitrification
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Open pulled straw vitrification
Two-cell hamster embryos were vitrified using the open-pulled straw (OPS) technique as described (Vajta et al. 1997; Lane et al., 1999b
). French straws (0.5 ml; Minitube, Verona, WI) were pulled to twice the original length over a flame then cut in half and the plugged end removed. Two-cell embryos were incubated for 2 min in 10% ethylene glycol and 10% dimethylsulphoxide (DMSO) and then for between 2030 s in 20% ethylene glycol, 20% DMSO, and 0.67 mol/l sucrose. Ten to 12 embryos were pipetted into a 1 µl drop of the second cryopreservation solution and then loaded into a pulled straw using capillary action. The straw containing the embryos was plunged directly into liquid nitrogen. Embryos were stored in liquid nitrogen for between 1 and 21 days. For warming, embryos were expelled from the straw in H-H10 containing 0.25 mol/l sucrose by the pressure from warming of the medium in the straw. Embryos remained in this medium for 5 min before transfer to H-H10 medium containing 0.125 mol/l sucrose for a additional 5 min. Embryos were washed twice more in medium H-H10 for 5 min each and then either placed in culture or assessed for intracellular pH regulation. This OPS procedure has previously been successful for cryopreserving hamster 2-cell embryos, resulting in morula/blastocyst development rates of around 50% and in normal fetal development after transfer (Lane et al., 1999b
)
Embryo culture
Embryos were cultured in 35 µl drops of medium HECM-10 in groups of 1012 under mineral oil (Sigma Chemical Co.) at 37°C in a humidified atmosphere of 10% CO2, 5% O2 and 85% N2.
Measurement of intracellular pH
Intracellular pH was measured using the pH sensitive probe 2', 7'-Bis (2-carboxyethyl)-5-(and-6)-carboxy fluorescein (BCECF; Molecular Probes, Eugene, OR, USA). Two-cell embryos were loaded with 0.7 µmol/l BCECF using the acetoxymethyl ester (BCECF-AM) for 20 min at 37°C in bfHH3t. Embryos were washed twice in medium without the probe and 812 embryos placed in a temperature-controlled chamber (Biophysica, Baltimore, MD, USA) at 37°C. For assessment of HCO3/Cl transporter activity the chamber was gassed with 10% CO2, 5% O2, and 85% N2. Measurement of pHi was achieved using a Nikon Diaphot inverted microscope connected by a Nikon Dual Optical Path Tube to a Photometrics PXL cooled camera (Huntington Beach, CA, USA) for high resolution recording of epifluorescent images. Analysis of fluorescent images was performed using Metamorph/Metafluor hardware and software (Universal Imaging Corporation, West Chester, PA, USA). Emission wavelength was set to 530 nm and the ratio of fluorescence intensities of excitation wavelengths 500 (pH sensitive) to 450 nm (pH insensitive) was obtained for each embryo. Fluorescent ratios were calibrated in situ using a nigericin/high K+ method at four pH values: 6.7, 7.0, 7.4 and 7.8 for acid-loading and 7.0, 7.3, 7.7 and 8.1 for alkaline loading. The ratio of fluorescence intensity was linearly proportional to pH (r2 = 0.972).
Determination of recovery from acidosis
Intracellular acidosis was induced by a NH4Cl pulse after measurement of baseline pHi. Embryos were incubated for 10 min with 25 mmol/l NH4Cl, which resulted in an immediate alkalization due to rapid equilibration of NH3 across the membrane. A slower movement of NH4+ results in a slow acidification. Subsequent removal of the NH4Cl from the medium causes acidification as the NH3 leaves the cell rapidly, leaving behind the H+ which entered the cell as NH4+ (Boron and DeWeer, 1976; Roos and Boron, 1981
). Recovery was assessed for 20 min following removal of NH4+and calculated by determining the gradient of a tangent to the recovery curve (Baltz et al., 1990
; Lane et al., 1998
).
Determination of recovery from alkalosis
Following measurement of baseline pHi, the chamber was flushed with Cl-free medium for 57 min which, due to the reverse activity of the HCO3/Cl exchanger, causes a small increase in pHi. A further larger intracellular alkaline load was induced by incubation with 25 mmol/l NH4Cl in medium containing Cl. This produces an immediate increase in pHi. Recovery from alkalosis was assessed for a subsequent 20 min. Recovery was calculated by determining the gradient of the tangent to the recovery curve (Baltz et al., 1991; Zhao et al., 1995
; Lane et al., 1999a
).
Assessment of embryo metabolism
Glycolytic activity and oxidative metabolism of 2-cell embryos were determined by incubation with the radioisotopes [5-3H]-glucose and [U-14C]-lactate respectively. Individual embryos were incubated in a 3 µl drop of medium HECM-10 containing 5.0 mmol/l [U-14C]-lactate (0.025 µCi/µl) and 0.5 mmol/l glucose (0.002 µCi/µl) on the lid of a microcentrifuge tube over 1.5 ml of 25 mmol/l NaHCO3 (O'Fallon and Wright, 1986; Rieger et al. 1992
). The tubes were gassed with 10% CO2: 5% O2:85% N2, sealed and incubated at 37°C for 3 h. Sham preparations of medium without embryos were included to control for non-specific breakdown of the label. Total radioactivity was determined by adding 3 µl of medium containing the label to the lid of a vial and inverting the tube to mix with the NaHCO3 trap. After the 3 h incubation period, the tubes were opened and 1 ml of the NaHCO3 trap was placed into a scintillation vial containing 200 µl of 0.1 mol/l NaOH, and the vials stored overnight at 4°C. The following day, 10 ml of scintillation fluid (Ultima Gold; Packard, Meriden, CT, USA) was added to each vial, the vials vortexed and counted for 4 min. Glycolytic activity and oxidative metabolism were calculated using the recovery efficiency of the radioisotope as previously described (Rieger et al., 1992
).
Statistical analysis
Differences between treatments for pHi measurements and oxidative metabolism were assessed using analysis of variance followed by Bonferroni's multiple comparison procedure. Data for glycolytic activity was assessed using a non-parametric test due to the variances being significantly different between the treatments. Differences between treatments were therefore assessed using a KruskallWallis test followed by a Dunn's test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Measurement of Na+/H+ antiporter activity after vitrification
Control 2-cell embryos recovered quickly from an induced acidosis (0.210 ± 0.08 pH units) and after the 20 min recovery period the pHi of the embryos had returned to that at the beginning of the incubation (Figure 1; Table II
). In contrast, the rate of recovery from acidosis by vitrified 2-cell embryos measured immediately following warming was significantly reduced compared to freshly collected embryos (Table II
). Consequently, at the end of the 20 min incubation period, the pHi that the embryos had re-established was significantly lower than the initial resting pHi (Table II
). It also appeared that the rate of recovery had levelled off at this lower pHi (Figure 2
). Of the nine replicates performed at this time point, only embryos from one replicate were able to restore pHi to that observed before acid loading.
|
|
Determination of HCO3/Cl exchanger activity following vitrification
Removing Cl from the medium for freshly collected 2-cell embryos resulted in an increase in pHi of around 0.2 pH units. These control embryos also exhibited high rates of recovery from alkalosis and at the end of the 15 min incubation period, pHi was restored to that observed at the beginning of the experiment. In contrast, when embryos that had been vitrified were examined immediately following warming, the increase in pHi induced by removing Cl from the medium was reduced by half compared to the controls (P < 0.05; Table III). The rate of recovery from alkalosis induced by NH3 was also significantly decreased in vitrified 2-cell embryos examined immediately following warming (Table III
). Consequently, the final pHi at the end of the recovery period was also significantly higher in these embryos (Figure 2
). Embryos that were examined 2 h after warming exhibited an increase in pHi when Cl was removed from the medium. This increase in pHi was similar in magnitude to that in the control embryos. However, the rate of recovery from the induced alkalosis was still significantly slower in these embryos compared to the control embryos (Table III
). Therefore, the final pHi at the end of this recovery period was significantly higher than in the control vitrified embryos. A similar pattern of pHi regulation was observed in embryos that were examined 4 h after warming. The increase in pHi induced by Cl was similar to the control embryos although the rate of recovery was significantly less (Table III
). However, 2-cell embryos 4 h after warming were able to restore pHi to initial values, although the time taken to recover pHi was slower than the control embryos (Figure 2
). By 6 h after warming, the pattern of recovery from alkalosis by 2-cell embryos that had been vitrified was equivalent to that of the control embryos (Table III
; Figure 2
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activities of the Na+/H+ antiporter and the HCO3/Cl exchanger are important for normal embryo development in culture. When either transporter is inhibited, development is reduced. It appears that the reduction in both Na+/H+ antiporter and HCO3/Cl exchanger activities results in an abnormal increase in the baseline pHi. The mechanisms for the decrease in activity of the pHi regulatory transport proteins following cryopreservation and warming are unknown. Both the Na+/H+ antiporter and the HCO3/Cl transporter activities are dependent on their sensitivity in detecting alterations in pHi from the normal set-point (Lane et al., 1998; 1999a). For both transporters, increasing the magnitude of the alterations from the set-point increases the activity of the transporters. In vitrified hamster embryos examined 0 or 2 h after warming, it is clear that this sensitivity to the alterations in cytoplasmic pH by the transporters is impaired. In the case of the Na+/H+ antiporter, a change in the phosphorylation or stimulation of the antiporter is normally manifest as an alkaline shift in the pH dependency curve (Grinstein and Rothstein, 1986
). This was not observed in the present study (data not shown). Rather it appears that there is a desensitization of the activation system for both the Na+/H+ antiporter and HCO3/Cl transporter systems. Because both of these transport systems operate by a transmembrane gradient, for Na+ and Cl respectively, it is possible that, in cryopreserved embryos immediately after warming, these gradients are disturbed, which reduces the ability to transport either H+ or HCO3. Although this study focused on the transporters for the regulation of pHi, it is possible that other transporter systems and regulatory channels will be similarly affected following cryopreservation. Activity of the facilitated glucose transporter GLUT 1, which is expressed in mouse embryos, is also significantly impaired in blastocysts that had been cryopreserved at the 2-cell stage (Uechi et al., 1997
). In addition to membrane pHi regulation, mitochondrial pH and Ca2+ regulatory mechanisms may also be affected.
Intracellular pH is known to be a potent regulator of cell metabolism and energy production (Busa and Nuccitelli, 1984). Therefore, the increase in pHi of around 0.2 pH units that was observed for 4 h following warming may cause aberrations in several cellular functions. Incubating 2-cell embryos for 4 h with the weak alkali TMA increased pHi by around 0.2 pH units, the same as the increase in pHi induced by cryopreservation. Increasing the pHi of these embryos significantly altered embryo metabolism. Glycolytic activity was increased by incubation with 10 mmol/l TMA and oxidative capacity was substantially reduced by both 5 and 10 mmol/l TMA.
The flux-generating step of glycolysis, the enzyme phosphofructokinase (PFK) is very sensitive to changes in pH (Paetkau and Lardy, 1967). Small increases in PFK activity can cause disproportionately large increases in glycolytic pathway activity. In some cells, PFK activity can be altered 10- to 20-fold by a pH change of only around 0.1 units (Danforth, 1965
; Trivedi and Danforth, 1966
). The increase in pHi that was observed in hamster embryos for 4 h after warming would stimulate the enzyme PFK resulting in increased glycolytic activity. Indeed, bovine blastocysts assessed immediately after cryopreservation had an increased rate of glycolysis compared to the same embryos measured before freezing (Gardner et al., 1996
). However, the biggest effect of an increase in pHi on hamster embryo metabolism was a reduction in oxidative metabolism. Oxidative metabolism, specifically metabolism of lactate, appears to be the preferred energy-generating pathway of the cleavage stage hamster embryo (McKiernan et al., 1991
). Interestingly, an increase in glycolysis by cleavage stage hamster embryos resulted in impaired oxidative metabolism (Seshagiri and Bavister, 1991
). A similar effect has been linked to the `2-cell block' in mouse embryos (Gardner and Lane, 1993
). This phenomenon, which was first described in rapidly dividing tumour cells, is known as the Crabtree effect (Crabtree, 1929
; Koobs, 1972
).
In addition to the reduced activity of the pHi transport systems Na+/H+ antiporter and HCO3/Cl exchanger, the intrinsic buffering capacity of hamster 2-cell embryos that had been cryopreserved was reduced immediately following warming. It has recently been reported that the addition of Eagle's non-essential amino acids significantly enhances the ability of mouse cleavage stage embryos to buffer both an acid (Edwards et al., 1998) and alkaline load (L.J.Edwards and D.K.Gardner, personal communication). Therefore, it may be prudent to include these amino acids in the culture medium for embryos following cryopreservation when the embryo has a reduced ability to regulate pHi. While some of these amino acids were present in the culture medium for the hamster embryo in the present study, their concentration is 10 times lower than that reported to help in the buffering of mouse embryos.
In conclusion, this study demonstrated that 2-cell hamster embryos that were vitrified had an elevated pHi immediately after warming. The activities of the major pHi regulatory systems in the hamster embryo, the Na+/H+ antiporter and HCO3/Cl exchanger, were significantly reduced after freezing, and were not restored until 6 h after warming. During this time, the embryos have a significantly reduced ability to regulate pHi, which could have major effects on their subsequent developmental competence.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baltz, J.M., Biggers, J.D. and Lechene, C. (1991) Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange. J. Biol. Chem., 266, 1721217217.
Bavister, B.D., Leibfried, M.L. and Lieberman, G. (1983) Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol. Reprod., 28, 235247.[Abstract]
Begg, D.A. and Rebhun, L.I. (1979) pH regulates the polymerization of actin in the sea urchin egg cortex. J. Cell Biol., 83, 241248.[Abstract]
Boron, W.F. and DeWeer, P. (1976) Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors. J. Gen. Physiol., 67, 91112.[Abstract]
Busa, W.B. and Nuccitelli, R. (1984) Metabolic regulation via intracellular pH. Am. J. Physiol., 246, R409R438.
Crabtree, H.G. (1929) Observations on the carbohydrate metabolism of tumors. Biochem. J., 23, 536545.
Damien, M., Luciano, A.A. and Peluso, J.J. (1990) Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change. Hum. Reprod., 5, 212216.[Abstract]
Danforth, W.H. (1965) Activation of glycolytic pathway in muscle. In Chance B, Estabrook, R.W. and Williamson, J.B. (eds), Control of Energy Metabolism. Academic Press, New York, pp. 287298.
Edwards, L.E., Williams, D.A. and Gardner, D.K. (1998) Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum. Reprod., 13, 34413448.[Abstract]
Gardner, D.K. and Lane, M. (1993) The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: Alleviation of the 2-cell block is associated with the restoration of in vivo metabolic pathway activities. Biol. Reprod., 48 (Suppl 1), 152.
Gardner, D.K., Pawelczynski, M. and Trounson, A.O. (1996) Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation. Mol. Reprod. Dev., 44, 472475.[ISI][Medline]
Grinstein, S. and Rothstein, A. (1986) Mechanisms of regulation of the Na/H exchanger. J. Membr. Biol., 90, 112.[ISI][Medline]
Koobs, D.H. (1972) Phosphate mediation of the Crabtree and Pasteur effects. Science, 178, 127133.[ISI][Medline]
Lane, M. and Bavister, B.D. (1999) Regulation of intracellular pH in bovine oocytes and cleavage stage embryos. Mol. Reprod. Dev., 54, 396401.[ISI][Medline]
Lane, M., Baltz, J.M. and Bavister, B.D. (1998) Regulation of intracellular pH in hamster preimplantation embryos by the Na+/H+ antiporter. Biol. Reprod., 59, 14831490.
Lane, M., Baltz, J.M. and Bavister, B.D. (1999a) Bicarbonate/chloride exchange regulates intracellular pH of embryos but not oocytes of the hamster. Biol. Reprod., 61, 452457.
Lane, M., Bavister, B.D., Lyons, E.A. and Forest, K.T. (1999b) Container-less vitrification of mammalian oocytes and embryos; adaptation of proven technology for protein crystals to live cells. Nature Biotech, 7, 12341236.
Leclerc, C., Becker, D., Buehr, M. and Warner, A. (1994) Low intracellular pH is involved in the early embryonic death of DDK mouse eggs fertilized by alien sperm. Dev. Dyn., 200, 257267.[ISI][Medline]
McKiernan, S.H., Tasca, R.J. and Bavister, B.D. (1991) Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage. Hum. Reprod., 6, 6475.[Abstract]
Menezo, Y. and Veiga, A. (1997) Cryopreservation of blastocysts. In Gomel, V. and Leung, P.C.K., (eds), In vitro Fertilization and Assisted Reproduction. Monduzzi Editore, Bologna, pp. 4953
O'Fallon, J.V. and Wright, R.W. Jr (1986) Quantitative determination of pentose phosphate pathway in preimplantation mouse embryos. Biol. Reprod., 34, 5864.[Abstract]
Paetkau, V. and Lardy, H.A. (1967) Phosphofructokinase. Correlation of physical and enzymatic properties. J. Biol. Chem., 242, 20352042.
Regula, C.S., Pfeiffer, J.R. and Berlin, R.D. (1981) Microtubule assembly and disassembly at alkaline pH. J. Cell Biol., 89, 4553.
Rieger, D., Loskutoff, N.M. and Betteridge, K.J. (1992) Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. J. Reprod. Fertil., 95, 585595.[Abstract]
Roos, A. and Boron, W.F. (1981) Intracellular pH. Physiol. Rev., 61, 296434.
Seshagiri, P.B. and Bavister, B.D (1991) Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight-cell embryos: evidence for the `Crabtree effect'. Mol. Reprod. Dev., 30, 105111[ISI][Medline]
Trivedi, B. and Danforth, W.H. (1966) Effect of pH on the kinetics of frog muscle phosphofructokinase. J. Biol. Chem., 241, 41104112.
Uechi, H., Tsutsumi, O. and Morita, Y. et al. (1997) Cryopreservation of mouse embryos affects later embryonic development possibly through reduced expression of the glucose transporter GLUT1. Mol. Reprod. Dev., 48, 496500.[ISI][Medline]
Vajta, G., Booth, P.J. and Holm, P. et al. (1997) Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo-Lett., 18, 191195.[ISI]
Zhao, Y. and Baltz, J.M. (1996) Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development. Am. J. Physiol., 271, C1512C1520.
Zhao, Y., Chauvet, P.J. and Alper, S.L. et al. (1995) Expression and function of bicarbonate/chloride exchangers in the preimplantation mouse embryo. J. Biol. Chem., 270, 2442824434.
Submitted on May 17, 1999; accepted on November 5, 1999.