Osmoregulation and cell volume regulation in the preimplantation embryo.

The early preimplantation mammalian embryo possesses mechanisms that regulate intracellular osmolarity and cell volume. While transport of osmotically active inorganic ions might play a role in this process in embryos, the major mechanisms that have been identified and studied are those that employ organic osmolytes. Organic osmolytes provide a substantial portion of intracellular osmotic support in embryos and are required for their development under in vivo conditions. The main osmolytes that have been identified in cleavage stage embryos are accumulated via two transport systems of the neurotransmitter transporter family active in early preimplantation embryos--the glycine transport system (GLY) and the beta-amino acid transport system (system beta). While system beta has been established to have a similar role in many other cells, this is a novel function for the GLY transport system. The intracellular concentration of organic osmolytes such as glycine in early preimplantation embryos is regulated by tonicity, allowing the embryo to regulate its volume against shrinkage and to control its internal osmolarity. In addition, the cells of the embryo can regulate against an increase in volume via controlled release of osmolytes from the cytoplasm. This is mediated by a swelling-activated anion channel that is also highly permeable to a range of organic osmolytes, and which closely resembles similar channels found in many other cell types (VSOAC channels). Together, these mechanisms appear to regulate cell volume in the egg through the early cleavage stages of embryogenesis, after which there are indications that the mechanisms of osmoregulation change.

Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo.

Anion channels activated by increased cell volume are a nearly ubiquitous mechanism of cell volume regulation, including in early preimplantation mouse embryos. Here, we show that the swelling-activated anion current (I(Cl,swell)) in early mouse embryos is cell-cycle dependent, and also that this dependence is developmentally regulated. I(Cl,swell) is present both in first meiotic prophase (germinal vesicle stage) mouse oocytes and in unfertilized mature oocytes in second meiotic metaphase, and it persists after fertilization though the 1-cell and 2-cell stages. I(Cl,swell) was found to remain unchanged during metaphase at the end of the 1-cell stage. However, I(Cl,swell) decreased during prophase and became nearly undetectable upon entry into metaphase at the end of the 2-cell stage. Entry into prophase/metaphase was required for the decrease in I(Cl,swell) at the end of the 2-cell stage, since it persisted indefinitely in 2-cell embryos arrested in late G(2). There is considerable evidence that the channel underlying I(Cl,swell) is not only permeable to inorganic anions, but to organic osmolytes as well. We found a similar pattern of cell cycle and developmental dependence in the 1-cell and 2-cell stages for the swelling-induced increase in permeability to the organic osmolyte glycine. Thus, entry into metaphase deactivates I(Cl,swell) in embryos, but only after developmental progression through the 2-cell stage.

Differences in intracellular pH regulation by Na(+)/H(+) antiporter among two-cell mouse embryos derived from females of different strains.

Regulation of intracellular pH (pH(i)) by two-cell-stage embryos derived from female mice of three different strains (CF-1, Balb/c, and BDF) was investigated. Embryos recovered at a slow rate from intracellular acidosis produced by a pulse of NH(4)Cl; the rate did not differ significantly among strains. Recovery was reversibly inhibited by amiloride or the absence of Na(+), implicating Na(+)/H(+) antiporter activity. The threshold pH(i) (setpoint) below which Na(+)/H(+) antiporter activity was elicited was approximately 7.15 for each strain. No recovery from induced acidosis occurred in the absence of external Na(+) in any strain, and thus embryos could be maintained in acidosis for an extended period. Upon reintroduction of Na(+), embryos derived from either CF-1 or BDF females recovered at a slow rate comparable to that measured in embryos not maintained for a period in Na(+)-free medium, but embryos derived from Balb/c females consistently recovered at a highly accelerated rate. This accelerated recovery appeared to be due, in part, to an activation of the Na(+)/H(+) antiporter in Balb/c-derived embryos, which did not occur in CF-1- or BDF-derived embryos. Thus, embryos derived from different strains of female mice differ in their control of mechanisms for pH(i) regulation.

Intracellular pH regulation in human preimplantation embryos.

We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8-cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+)- and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range.

Glycine transport by single human and mouse embryos.

Mouse zygotes and early cleavage-stage embryos have previously been shown to utilize glycine as an organic osmolyte, accumulating it to oppose any decrease in cell volume. Such glycine uptake in early cleavage-stage mouse embryos is via the glycine-specific Gly transporter. Mouse embryos also possess swelling-activated channels which function to release osmotically active glycine and other osmolytes when cell volume becomes too large. In this study it was found that human cleavage-stage embryos also transported glycine via a similarly saturable, sarcosine-inhibitable transporter, implying that the Gly transporter also mediates glycine transport in human embryos. Mouse zygotes have previously been shown to accumulate more intracellular glycine when cultured at increased osmolarities for 24 h. It was found in the current study that this ability was lost as preimplantation mouse embryo development proceeded, and that early cleavage-stage human embryos may also be capable of such osmosensitive accumulation of glycine. Finally, using spare human eggs which had failed to fertilize or cleave, the presence of swelling-activated currents resembling those in mouse zygotes was demonstrated. These data indicate that osmoregulation in early human embryos occurs via similar mechanisms as in the mouse.

Bicarbonate/chloride exchange regulates intracellular pH of embryos but not oocytes of the hamster.

The ability to regulate intracellular pH (pH(i)) is essential for normal cell development and differentiation. This study was an investigation of the regulatory system used by the hamster oocyte and preimplantation embryo to regulate pH(i) in the alkaline range. Recovery from alkalosis by late 1-cell and 2-cell embryos was rapid, and physiological pH(i) levels could be restored within 10 min. Recovery from an induced alkaline load was dependent on the chloride concentration in the external medium and sensitive to a stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonic acid that inhibits bicarbonate and chloride exchange. Therefore the recovery from alkalosis by hamster embryos appears to be via activity of the HCO(3)(-)/Cl(-) exchanger that was activated above a pH(i) set point of 7.24. In contrast, hamster oocytes and early 1-cell embryos (collected 3-4 h post-egg activation) could not recover from an intracellular alkalosis, and pH(i) remained elevated. Therefore, the hamster oocyte and the early 1-cell embryo still undergoing pronuclear formation lack an active HCO(3)(-)/Cl(-) exchanger for the restoration of pH(i). Inability to restore pH(i) from an alkali challenge resulted in a reduced ability of embryos to develop to the morula/blastocyst stages in culture, indicating that HCO(3)(-)/Cl(-) exchange is involved in physiological regulation of pH(i).

Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development.

We report here that at least one major pHi-regulatory mechanism, the HCO3-/Cl- exchanger, is quiescent in unfertilized mouse eggs but becomes fully activated during early development following fertilization. Zygotes (8-12 h postfertilization) exhibited a marked intracellular alkalinization upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. In addition, efflux of Cl- from eggs upon external Cl- removal was much slower than that from zygotes, indicating additional pathways for Cl- to cross the plasma membrane in zygotes. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. Following in vitro fertilization (IVF), increased HCO3-/Cl- exchanger activity was first detectable about 4 h postfertilization and reached the maximal level after about 8 h. The upregulation of HCO3-/Cl- exchanger activity after fertilization appeared to occur by activation of existing, inactive exchangers rather than by synthesis or transport of new exchangers, as the increase in activity following IVF was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. This activation may depend on the Ca2+ transients which follow fertilization, as suppression of these transients, using the Ca2+ chelator BAPTA, reduced subsequent upregulation of HCO3-/Cl- exchanger activity by about 50%. Activation of pHi-regulatory systems may be a widespread feature of the earliest period of embryonic development, not restricted to species such as marine invertebrates as previously believed.

Estimates of mouse oviductal fluid tonicity based on osmotic responses of embryos.

Zygotes and early cleavage-stage embryos are very sensitive to increased osmolality in vitro, although the tonicity of their in vivo environment, oviductal fluid, is unknown. A preference for low osmolality in vitro might imply similar conditions in vivo or be specific to culture. Previous electron probe x-ray microanalysis measurements of total ion content predicted oviductal fluid osmolalities of 310-360 mOs/kg, higher than osmolalities tolerated by mouse zygotes in vitro. However, such indirect estimates may not reflect the tonicity experienced by embryos. We have now used embryos themselves as osmosensors to determine the tonicity of mouse oviductal fluid. In one method, we measured the mean volume of zygotes in undiluted oviductal fluid and compared this to the mean volumes measured for zygotes in media spanning a range of osmolalities. The osmolality corresponding to the measured mean volume in oviductal fluid was taken to be isotonic. In another, independent method, the sizes of zygotes and two-cell embryos were measured as a function of time beginning immediately after removal from oviducts. The osmolality in which the embryos neither swelled nor shrank was taken to be isotonic. Both methods yielded approximately the same range for the tonicity of oviductal fluid: around 290-300 mOs/kg.

Volume-regulated anion and organic osmolyte channels in mouse zygotes.

Whole-cell currents in mouse zygotes were measured using the patch-clamp technique in whole-cell mode. Upon exposure to hypotonic medium, patch-clamped zygotes increased in volume and developed a large swelling-activated current. The swelling-activated current was blocked by Cl- channel blockers, and the magnitude of the current and reversal potential were dependent on the Cl- gradient. Thus, the swelling-activated current had the properties of a current mediated by anion channels. However, in addition to being permeable to Cl- and I- (with I- having the greater permeability), there was also a significant swelling-activated conductance to aspartate and taurine, indicating that the swelling-activated channels in zygotes conduct not only inorganic anions but organic osmolytes as well. This swelling-activated anion and organic osmolyte pathway likely underlies the ability of zygotes to recover from an increase in volume, and it may function to regulate intracellular amino acid concentrations.

Na+/H+ antiporter activity in hamster embryos is activated during fertilization.

This study characterized the activation of the regulatory activity of the Na+/H+ antiporter during fertilization of hamster embryos. Hamster oocytes appeared to lack any mechanism for the regulation of intracellular pH in the acid range. Similarly, no Na+/H+ antiporter activity could be detected in embryos that were collected from the reproductive tract between 1 and 5 h post-egg activation (PEA). Activity of the Na+/H+ antiporter was first detected in embryos collected at 5.5 h PEA and gradually increased to reach maximal activity in embryos collected at 7 h PEA. Parthenogenetically activated one-cell and two-cell embryos demonstrate Na+/H+ antiporter activity, indicating that antiporter activity is maternally derived and initiated by activation of the egg. The inability of cycloheximide, colchicine, or cytochalasin D to affect initiation of antiporter activity indicates that antiporter appearance is not dependent on the synthesis of new protein or recruitment of existing protein to the cell membrane. In contrast, incubation of one-cell embryos with sphingosine did inhibit the appearance of Na+/H+ antiporter activity, showing that inhibition of normal protein kinase C activity is detrimental to antiporter function. Furthermore, incubation of oocytes with a phorbol ester which stimulates protein kinase C activity induced Na+/H+ antiporter activity in oocytes in which the activity was previously absent. Incubation with an intracellular calcium chelator also reduced the appearance of antiporter activity. Taken together, these data indicate that the appearance of Na+/H+ antiporter activity following egg activation may be due, at least in part, to regulation by protein kinase C and intracellular calcium levels.

Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter.

This study was an investigation of the mechanisms for the regulation of intracellular pH (pHi) by hamster preimplantation embryos. The resting pH values of hamster embryos were similar at the 1-cell (7. 19 +/- 0.34), 2-cell (7.21 +/- 0.21), and 8-cell (7.22 +/- 0.41) stages. Cleavage-stage hamster embryos alleviated intracellular acidosis by activity of the Na+/H+ antiporter. The rate of recovery from acidosis was similar for embryos at 1-cell, 2-cell, and 8-cell stages. When Na+/H+ antiporter activity was inhibited by either incubation in Na+-free medium or the presence of an inhibitor, pHi was unable to recover to initial levels. Instead, pHi remained acidic. The Na+/H+ antiporter was also found to contribute to baseline pH regulation, as incubation in Na+-free medium resulted in an immediate intracellular acidification. The set point for Na+/H+ antiporter was pH 7.14. There was no evidence at any developmental stage for activity of either Na+-dependent HCO3-/Cl- exchanger or H+-ATPase in the regulation of pHi. Inhibition of the Na+/H+ antiporter by an amiloride derivative significantly reduced the ability of 2-cell embryos to develop in culture when challenged with acidosis, indicating that the Na+/H+ antiporter is an essential regulator of pHi.

Fluorophore toxicity in mouse eggs and zygotes.

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading--which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM)--and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl-). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.

Osmolarity-dependent glycine accumulation indicates a role for glycine as an organic osmolyte in early preimplantation mouse embryos.

Mouse zygotes and early cleavage-stage embryos are sensitive to increased osmolarity. However, development can occur at higher osmolarities if any of a number of organic compounds are present. One of the most effective of these is glycine. We have found that the amount of glycine accumulated by embryos during in vitro culture from the zygote to two-cell stage depends on the osmolarity of the medium, with significantly more glycine accumulated at 310 or 340 mOsM than at 250 mOsM. The accumulated glycine is largely retained in a freely diffusible form, as it can be released via a swelling-activated pathway in two-cell embryos. Increased glycine accumulation does not seem to depend on an increase in its rate of transport. The transport rate is not higher in two-cell embryos that have been cultured from zygotes in hypertonic vs. normal medium, and hypertonicity only slightly stimulates transport in zygotes. Our results indicate that glycine functions as an organic osmolyte in early mouse embryos.

Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement.

Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]i in swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaea neurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 microM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]e or very low [Ca]e level (0.9 mM or 0.13 microM, respectively). In neither case did the [Ca]i increase to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]i clamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]i did not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]i clamped at 0.1 microM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.

Culture with Matrigel inhibits development of mouse zygotes.

PURPOSE: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. METHODS: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. RESULTS: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. CONCLUSIONS: Matrigel may be a deleterious addition to embryo culture or coculture systems.

Routes of Cl- transport across the trophectoderm of the mouse blastocyst.

The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.

Cell volume regulation by the mouse zygote: mechanism of recovery from a volume increase.

Mouse zygotes regulate their volumes after cell swelling. This regulatory volume decrease (RVD) is rapid and complete. RVD in zygotes was inhibited by K+ or Cl- channel blockers, indicating the participation of such channels in volume recovery. The channels are separate entities, as indicated by the ability of the cation ionophore gramicidin to restore RVD when K+ channels are blocked but not when Cl- channels are blocked. Intracellular Ca2+ concentration increased with cell swelling. Nevertheless, RVD occurred normally in zygotes loaded with the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which prevented Ca2+ from increasing above its normal resting concentration. Thus an increase in intracellular Ca2+ is not necessary for zygote RVD; consistent with this, inhibitors of Ca(2+)-activated K+ channels had little or no effect on RVD. RVD in zygotes was also completely inhibited by millimolar amounts of extracellular ATP. ATP has been shown to inhibit current passed by the volume-sensitive organic osmolyte-Cl- channel in other cells, and thus zygotes may have such a channel participating in RVD.

Organic osmolytes and embryos: substrates of the Gly and beta transport systems protect mouse zygotes against the effects of raised osmolarity.

Mouse embryo development is identically inhibited by raised osmolarity, whether produced by added NaCl or raffinose, demonstrating that high osmolarity is itself detrimental to embryos. In the face of increased osmolarity, cells in the brain and kidney, and likely many other cells, accumulate nonperturbing organic osmolytes in their cytoplasm. In the presence of any of a number of organic compounds that were proven or probable substrates of either the Gly or the beta transport systems, mouse embryo development in vitro was protected from raised osmolarity. Zygotes developed past the "2-cell block," and with most Gly or beta substrates, to the blastocyst stage. The most effective osmoprotectants were glycine, glutamine, betaine, proline, beta-alanine, and hypotaurine; several others were partially effective. A model Gly substrate, glycine, was effective at a much lower concentration (EC50 = 50 microM) than was a model beta substrate, beta-alanine (EC50 = 1.3 mM). The protective effect of these two compounds was additive, indicating a common mode of action. The various effective compounds tested do not all share metabolic pathways or other such properties in common. Thus, it is likely that cleavage-stage mouse embryos utilize them, in large part, as organic osmolytes.

Intracellular ion concentrations and their maintenance by Na+/K(+)-ATPase in preimplantation mouse embryos.

We have measured the amounts of Na+, K+ and C- in preimplantation mouse embryos (1-cell, 2-cell and morula) using electron probe X-ray microanalysis. The levels of these ions do not vary much over this period, and are approximately the same as those found in other mammalian cells, contrary to previous reports. We have confirmed that preimplantation embryos exhibit Na+/K(+)-ATPase activity at all stages examined, and have shown that the ATPase maintains high K+/Na+ ratios (12-16) in all these embryonic stages, comparable to those seen in other healthy cells; this is in contrast to the low ratios reported in earlier work. Inhibition of the Na+/K(+)-ATPase results in the slow exchange of intracellular K+ for extracellular Na+ (half-time approximately 5 h), indicating that Na+/K(+)-ATPase activity maintains steep Na+ and K+ gradients in preimplantation mouse embryos as it does in most other cells.

Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development.

HCO3-/Cl- exchanger activity, which regulates intracellular pH (pHi) in the alkaline range, has been shown to be present throughout preimplantation mouse embryo development and to be necessary for embryo viability. We have characterized HCO3-/Cl- exchange activity and its regulation of pHi throughout preimplantation development (1-cell, 2-cell, morula, and blastocyst stages). Embryos at each stage can recover from alkalosis. Recovery was dependent on external [Cl-], activated by increased pHi, and inhibited by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and external HCO3-. Dependence of exchanger activity on external [Cl-] and pHi remained unchanged during preimplantation development. However, the concentration at which DIDS inhibits the maximal response by 50% increased significantly (by approximately 5-fold) after the one-cell stage. In addition, HCO3-/Cl- exchange activity decreased over the course of development, with significantly lower activity at the morula and blastocyst stages relative to the one- and two-cell stages, coinciding with the movement of embryo from the high pH environment of the oviduct to the lower pH environment of the uterus.