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Biology of Reproduction 67, 1419-1429 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Developmental Changes in the Management of Acid Loads During Preimplantation Mouse Development1

E.A. Hardinga, C.A. Gibba, M.H. Johnsona,b, D.I. Cooka,c, and M.L. Day2,a

a Department of Physiology, University of Sydney, New South Wales 2006, Australia b Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, United Kingdom c Medical Foundation of the University of Sydney, New South Wales 2006, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular pH recovery in Quackenbush Swiss mouse preimplantation embryos following acid loading was investigated under conditions of H+-monocarboxylate cotransporter inactivity. Isoform-sensitive inhibitors of Na+-H+ exchange (NHE) were used to block the Na+-dependent component of the response. A biphasic dose-response curve for HOE-694 and N-methylisopropylamiloride (MIA) suggested that two isoforms (putatively NHE1 and NHE3) are active in the oocyte, 1-cell, and 2-cell stages. By the blastocyst stage, loss of one of the MIA-sensitive NHE activities (putatively NHE3) was observed in isolated inner cell masses, and an MIA-resistant component of the recovery was identified. The MIA-resistant component was inhibited by 2 mM amiloride and enhanced by external K+ and by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, suggesting NHE4 activity. However, unlike NHE4 in other tissues, the MIA-resistant component did not transport Li+ in exchange for H+, and reverse transcription-polymerase chain reaction detected NHE4 mRNA in the oocyte but not in later stages. Trophoblast, whether in intact or collapsed blastocysts, did not show measurable NHE activity or MIA-sensitive activity during recovery from acid load. Both trophoblast and pluriblast manifested an H+ conductance in response to acid load. This H+ conductance was first detected at the 8-cell stage and was blocked by zinc in the isolated inner cell mass but not in trophoblast. No other effective inhibitors of its activity were found.

conceptus, developmental biology, embryo, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The experimental study of mammalian preimplantation development and the efficient application of the knowledge gained to clinical and veterinary practice depend on the successful in vitro creation and culture of a conceptus. However, further optimization of culture media is still necessary [1]. Abundant evidence suggests that even quite short periods of in vitro culture can induce changes in the conceptus. These include changes in metabolism [2–6], genomic imprinting [7–9], gene expression patterns [10–12], and developmental potential, which in extremis may prejudice fetal, perinatal, or postnatal survival [13]. Even in the best culture media, the conceptus probably is simply adapting to the adverse conditions to an extent sufficient to permit survival [14]. The realization of this situation not only places a question mark against the validity of our knowledge about preimplantation development but also may help to explain the variable and relatively poor outcomes of clinical and veterinary in vitro fertilization (IVF) programs [15].

This situation has arisen in part because most media have been developed empirically and without regard to the cellular physiology of the gametes, zygote, and conceptus [16, 17], about which we still know remarkably little [18]. The ionic milieu of a cell is a powerful determinant of its function, influencing the configuration, distribution, and properties of organic molecules both directly and indirectly. The ability of cells of the conceptus to control their ionic composition in response to external ionic stress is, therefore, likely to be an important determinant of developmental progress, but we have only limited knowledge about the ion transporters and channels at these early stages of development. For example, H+ ions can exert particularly powerful influences on cell function [19, 20], and so understanding the nature and properties of the proton transport and buffering systems available to the conceptus is of particular significance. Intracellular pH may also play a role in developmental signaling. For example, pHi has been described as an important trigger for the later developmental events of neural induction [21] and posterior axial development [22]. An acute rise in pHi also characterizes fertilization in sea urchin [23, 24], Xenopus sp. [25], and surf clam [26] and is important for subsequent development. In the mammal, no evidence supports the occurrence of an acute rise in pHi [27–31], but a slower rise does occur [32], which may simply reflect the much longer cell cycles observed in mammals [33]. Abnormalities of intracellular pH (and/or its recovery from acid or alkali load) have been described in association with adverse culture media [34], exposure to cryoprotectants and cryopreservation [35, 36], early lethal genetic mutations [37], and oocyte aging [38]. Changes to the composition of culture medium that improve intracellular buffering also improve embryogenic development [39], and changes in the pH of the subcellular compartments mark transitions through the first developmental cell cycle [40].

The importance of pHi for successful early development was first demonstrated empirically in Bavister's studies on the pH dependence of monospermic fertilization in the hamster [41], which led directly to the first (now historic) paper describing successful IVF in humans [42]. At that time, nothing was known about proton transport in oocytes and blastomeres, but a subsequent claim that the preimplantation mouse conceptus lacked a Na+-H+ exchanger (NHE) and relied on a passive proton conductance to relieve acid loading [43–45] seemed to explain why fertilization was so sensitive to external variation in pH. Later work, however, showed that the experimental conditions under which those initial studies were conducted had, unfortunately, resulted in the activity of an undetected H+-monocarboxylate cotransporter (MCT), which masked the transport activities of an NHE and gave the appearance of a proton conductance [46], a problem that may also have been compounded by differences in the activity of NHE among conceptuses from different mouse strains [47]. A direct study regarding the effects of varying external pH (pHo) on internal pH (pHi) in mouse and human conceptuses has suggested that both rapid and longer-term adaptive responses to both acid and alkali loads are possible and that the nature of these responses varies with the developmental stage [48, 49], confirming the presence of a more complex pHi regulatory system than originally proposed.

Clearly, this regulatory system has a number of components, of which two have so far been developmentally characterized in the early conceptus. First, a Cl--HCO3- exchanger is present in conceptuses from several species, shows developmental variation, and provides a response to alkaline load [49–54]. Second, response to an acid load is provided, in part, by members of the MCT family, the activities of which show developmental variation [32]. Acid load also induces a response by NHE transporters in conceptuses of several species [46, 55–57]. Although two of the seven NHE family members have been detected at the mRNA and protein levels [58], to our knowledge no detailed developmental study of their function has been reported. Here, we provide a functional description of the activity of the NHE family in the early mouse conceptus. We also report the detection of a third H+-transport system responsive to acid load. This third system is Na+ independent, appears to be an H+ conductance, and is first evident at the 8-cell stage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection and Culture of Oocytes and Conceptuses

Quackenbush strain (QS) female mice (3–5 wk old; Laboratory Animal Services, University of Sydney, NSW, Australia) on a fixed 12L:12D photoperiod were superovulated by i.p. injections of 10 IU of eCG (Intervet, Bendigo, VIC, Australia) followed approximately 48 h later by 10 IU of hCG (Intervet). Some of the female mice were then paired overnight with male QS mice (>12 wk old), and mating was ascertained by the presence of a vaginal plug. Studies were performed in accordance with the National Health and Medical Research Council of Australia Guidelines on Ethics in Animal Experimentation and were approved by the University of Sydney, Animal Ethics Committee.

The terminology used throughout is that of Johnson and Selwood [59], in which the product of fertilization is called the conceptus (not the embryo) and the 3.5-day inner cell mass (ICM) consists of pluriblast cells that are surrounded by trophoblast cells. Unfertilized oocytes, 1-cell fertilized, 2-cell stage, 8-cell stage, and blastocysts were obtained from female mice killed by cervical dislocation at 12.5–13, 18–24, 39–44, 69–72, and 93–95 h post-hCG injection, respectively. Unfertilized oocytes and 1-cell fertilized oocytes were teased or flushed from the oviducts into M2 medium [60] containing 4 mg/ml of bovine serum albumin (M2 plus BSA; Sigma-Aldrich, Castle Hill, NSW, Australia). Cumulus cells were removed with hyaluronidase (0.2 mg/ml, type II; Sigma-Aldrich), and oocytes were stored in M2 plus BSA until used. Zygotes were cultured in CZB medium [61] containing 4 mg/ml of BSA under liquid paraffin oil (BDH Merck, Kilsyth, VIC, Australia) in 5% CO2 at 37°C. The 2-cell and 8-cell conceptuses were flushed from the oviducts and blastocysts from the uteri with M2 plus BSA and cultured in M16 plus BSA until use. In experiments involving blastocyst collapse, the 2',7'-bis-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF-AM)-loaded blastocysts were placed in Na+-free solution and collapsed mechanically by gently passing the blastocyst up and down through a pipette tip with an internal diameter slightly less than that of the blastocyst itself [62]. This is sufficient to disrupt physically the tight junctional seal at one or two points in the mural trophoblast and allows equilibration. This collapse mechanism has the advantage compared to use of cytochalasin D for collapse [63] in that most of the intertrophoblast contacts are not disturbed and the disruption is reversible (the seal eventually reforming after a few hours) after the experimental measurements have been completed.

Immunosurgical Isolation of ICMs

Immunosurgery [64] was performed on fully expanded, zona-intact blastocysts by incubation in M2 plus BSA containing heat-inactivated rabbit anti-mouse antiserum (kindly provided by Professor Peter Kaye, University of Queensland, St Lucia, QLD, Australia) at 37°C for 10 min. Blastocysts were then washed four times in M2 plus BSA and incubated for a further 20 min in M2 plus BSA containing guinea pig complement (ICN Biomedicals Australasia, Seven Hills, NSW, Australia) at 37°C. The blastocysts were then washed four times in M2 plus BSA, and the pluriblast cells of the ICM were separated from the dead trophoblast by aspiration through a narrow-bore pipette. The ICM was then washed twice in M16 plus BSA and incubated in M16 plus BSA under liquid paraffin oil in 5% CO2 at 37°C for 30 min before the commencement of loading with BCECF-AM to ensure absence of contaminating fluid-accumulating cells.

Dye Loading and Cytosolic pH Measurements

Oocytes and conceptuses were loaded with the fluorescent pH indicator BCECF-AM as described previously [32, 46]. Briefly, they were rinsed through protein-free M2 and transferred to a drop of the control perfusion solution containing BCECF-AM (0.07 µM for oocytes, 1-cell to morula stages, and isolated ICMs; 0.44 µM for intact blastocysts) under paraffin oil and incubated at 37°C for 15–20 min. After loading, samples were placed on a Cell Tak-coated coverslip (Integrated Sciences, Crows Nest, NSW, Australia), which was then attached using high-vacuum silicone grease to the base of a Perspex perfusion chamber (Department of Physiology, University of Sydney). The chamber was perfused immediately with the control solution at a rate of 1.1 ml/min at 37°C for 10–15 min to equilibrate the samples before commencement of the experiment [65]. Experiments were performed using a 20x Zeiss Neofluar objective (Camperdown, NSW, Australia), which allowed up to six oocytes or cleavage stages or three blastocysts to be observed simultaneously.

Measurements were performed on only one blastomere of each conceptus, because preliminary studies showed no significant differences between blastomeres [46]. Within intact blastocysts, it was possible to identify and record from trophoblast cells only. Cytosolic pH (pHi) was measured using ratiometric imaging, and in vivo calibration was performed daily using K+-rich solutions as previously described [46]. The initial rates were calculated over the linear range of the change in pHi. The general design of experiments that used drugs or variable conditions was to expose embryogenic cells to two sequential acid loads, with the first being a control exposure and the second a test exposure. The recoveries in each were then compared. A second set of control embryogenic cells was then exposed to two sequential pulses, both using control solutions. Data from both sets of pulsed experiments were then compared in an unpaired Student t-test for a specific effect of the test intervention independent of any effect of double pulsing.

Statistics

The data from each conceptus in each single recording (up to six cleavage stages or three blastocysts) were averaged to provide the result for that experiment. All results are presented as the mean ± SEM, with the number of replicate experiments (n) in parentheses. Statistical significance was assessed using paired or unpaired Student t-tests as appropriate.

Solutions

The M2 medium [60] contained (in mM): NaCl (94.66), KCl (4.78), KH2PO4 (1.19), MgSO4 (1.19), CaCl2 (1.71), H-Hepes (20.85), glucose (5.56), Na-lactate (23.85), and Na-pyruvate (0.33). The pH was adjusted to 7.40 at 37°C with NaOH and was checked daily. The CZB medium [61] contained (in mM): NaCl (81.62), KCl (4.83), KH2PO4 (1.18), MgSO4 (1.18), CaCl2 (1.71), L-glutamine (1), Na-lactate (31.3), Na-pyruvate (0.27), H-EDTA (0.11), and NaHCO3 (25). The pH was adjusted to pH 7.40 at 37°C with NaOH or HCl. The M16 medium [60] contained (in mM): NaCl (94.66), KCl (4.83), KH2PO4 (1.18), MgSO4 (1.19), CaCl2 (1.71), NaHCO3 (25.01), glucose (5.56), Na-lactate (23.85), and Na-pyruvate (0.33). The pH was adjusted to pH 7.40 at 37°C with NaOH or HCl. The solutions contained no phenol red or antibiotics. M2 was used as the control perfusion solution during microfluorimetric experiments. The KCl-rich pH calibration solution contained (in mM): KCl (140), MgSO4 (1), CaCl2 (1), Hepes (15), glucose (10), nigericin (0.005), and valinomycin (0.001). All control and test experiments involving ZnCl2 were conducted in phosphate-free medium to prevent precipitation.

Chemicals

Nigericin, p-chloromercuriphenylsulfonate (PCMPS), bafilomycin, L-lactate, pyruvate, sodium metavanadate, and N-ethylmaleimide were purchased from Calbiochem (Croydon, VIC, Australia). The 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) was purchased from Sigma-Aldrich, and the BCECF-AM was purchased from Molecular Probes (Eugene, OR). The SCH28080 was a gift from Schering-Plough Research Institute (Kenilworth, NJ). Culture solutions were made from cell culture-grade reagents obtained from Sigma-Aldrich. The BCECF-AM was stored as a stock solution of 13 mM in water-free dimethyl sulfoxide at -20°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NHE During Preimplantation Development

The mechanisms involved in regulation of intracellular pH during preimplantation QS mouse development were investigated by examining the responses of the embryogenic cells to an acid load produced by the transient addition of NH4Cl to the medium. The addition of NH4Cl to the medium causes NH3 to enter the cells and capture H+ ions, thereby raising the cytosolic pH (pHi). The subsequent removal of NH4Cl leads to the rapid diffusion of NH3 out of the cell and the dissociation of the NH4+ within the cells into H+ and NH3, which in turn causes the pHi to fall sharply (acid load). Previous use of this approach in 2-cell blastomeres showed that the recovery from the cytosolic acidification results from both Na+-dependent NHE [46] and Na+-independent MCT [32, 46].

Seven NHE isoforms have been identified in mice and show differential sensitivity to inhibitors. The NHE isoforms responsible for the Na+-dependent component of recovery were, therefore, investigated using a wide range of concentrations of the isoform selective inhibitor HOE-694 [66]. The concentration-response relationship for HOE-694 is biphasic, with a plateau of inhibition at approximately 1 µM (caused by action on NHE1) and at 50 µM (caused by action on NHE2 and/or NHE3), suggesting that at least two isoforms are active at the 2-cell stage (Fig. 1). Effects of higher concentrations of HOE-694 could not be studied, because they resulted in cell lysis. The NHE activity was confirmed and extended by examining the concentration-dependent effects of the amiloride-analogue N-methylisopropylamiloride (MIA) on the Na+-dependent pHi recovery at different embryogenic stages. In all these experiments, the activity of the MCT was inhibited by either addition of PCMPS (300 µM) or the removal of lactate and pyruvate from the extracellular solution [32]. These interventions prevent the transporter from interfering with measurement of the NHE activity. Addition of 1 µM MIA (to measure putative NHE1 activity) resulted in the Na+-dependent pHi recovery decreasing to 59.6 ± 15.8% of the control level in oocytes, to 59.0 ± 18.3% of the control level in 1-cell fertilized oocytes, and to 17.0 ± 2.6% of the control level at the 2-cell stage (Fig. 2, A–C). Only in the presence of 50–100 µM MIA was complete inhibition of the Na+-dependent recovery achieved at these stages, with the Na+-dependent pHi recovery being reduced to -7.5 ± 11.7% of the control level in the unfertilized oocyte, to -6.7 ± 8.9% of the control level in the 1-cell fertilized oocyte, and to 2.3 ± 1.4% of the control level in the 2-cell stage. The Na+-dependent pHi recovery at later stages was also completely inhibited in the presence of 100 µM MIA, being -29.8 ± 21.1% (n = 6) of the control level in the noncompact 8-cell stage, -4.9 ± 18.4% (n = 4) in the compact 8-cell stage, and 8.4 ± 10.6% (n = 4) in morulae. However, by the blastocyst stage, recovery was found to be more complex.



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  		FIG. 1. Concentration-response curve for the effect of HOE-694 on the Na+-dependent pHi recovery at the 2-cell stage following an NH4Cl pulse. Each point represents the mean ± SEM of 3–18 experiments



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  		FIG. 2. Na+-dependent pHi recovery following an NH4Cl pulse in various MIA concentrations (expressed as % recovery of that seen in nontreated controls) in A) unfertilized oocytes (n = 5–12), B) 1-cell fertilized oocytes (n = 7–15), C) 2-cell stages (n = 4–13), and D) isolated ICMs (pluriblast, n = 3–9) in medium containing 300 µM PCMPS (A–C) or in M2 minus lactate and pyruvate (D). Each point represents the mean ± SEM. Also summarized (E) is the appearance of the MIA-insensitive component of pHi recovery during development for unfertilized oocytes (UF; {diamondsuit}; n = 5), 1-cell fertilized oocytes ({circ}; n = 8), 2-cell stages (; n = 4), noncompact ({square}; n = 7) and compact ({block}; n = 4) 8-cell stages, morulae ({blacktriangleup}; n = 4), and ICMs ({triangleup}; n = 9) in the absence of lactate and pyruvate and in the presence of 100 µM MIA

NHE Activity in Tissues of the Blastocyst

In the pluriblast of isolated ICMs, the Na+-dependent pHi recovery was reduced significantly to 43.4 ± 14.3% of control values with 1 µM MIA (putatively NHE1), but no additional inhibition was observed when the MIA concentration was increased to 50 µM (42.3 ± 28.5%) or to 100 µM (33.1 ± 24.3%) (Fig. 2D), suggesting that NHE2 and/or NHE3 activity was no longer present. These data also suggest that an MIA-insensitive isoform of NHE has appeared in pluriblast during cellular differentiation. This is illustrated comparatively in Figure 2E, which shows the rate of Na+-dependent pHi recovery during each stage of development in the presence of 100 µM MIA. The failure of 100 µM MIA to inhibit the Na+-dependent pHi recovery in the ICM suggests that the NHE4 isoform, which is highly insensitive to amiloride analogues [67–69], may be active de novo in this tissue. Therefore, this possibility was investigated directly.

Apart from the insensitivity of NHE4 to MIA and HOE-694, other features that can be used to distinguish NHE4 from the other isoforms of NHE are the ability of NHE4 to exchange K+ for H+ and the activation of NHE4 by the stilbene DIDS [67]. We thus performed experiments to determine whether the MIA-insensitive NHE activity in isolated ICMs showed these characteristics. When the perfusion solution contained 147 mM K+, the rate of pHi recovery was increased significantly (P = 0.01) (Fig. 3A). This stimulation of the rate of pHi recovery was dependent on the concentration of extracellular K+, being significantly greater at 147 mM K+ than at 48 mM K+ (P = 0.02) (Fig. 3B). The rate of pHi recovery in the presence of 48 mM K+ was only reduced significantly by addition of very high levels of amiloride (2 mM, P < 0.01), which is consistent with it being mediated by an amiloride-insensitive NHE isoform. Furthermore, the rate of pHi recovery following the addition of 48 mM K+ to the perfusion solution was increased by addition of 100 µM DIDS (P = 0.01) (Fig. 3C). These data are consistent with the appearance of NHE4 activity in the ICM. However, unlike K+, extracellular Li+ did not sustain recovery of pHi (Fig. 3A), a finding that is inconsistent with the report that NHE4 in transfected cells catalyzes the exchange of Li+ for H+ [67]. Also, whereas the expression of NHE4 mRNA was detected by reverse transcription-polymerase chain reaction in unfertilized oocytes, it was not detected at any other embryogenic stages (data not shown).



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  		FIG. 3. Characterization of NHE4 activity in the ICM. A) Effect of extracellular cations on the rate of pHi recovery following an NH4Cl pulse. ICMs were incubated in 25 mM NH4Cl for 7 min, and then the extracellular Na+ was replaced by either 147 mM N-methyl-D-glucamine (NMDG+), 147 mM Li+, or 147 mM K+. This resulted in rapid acidification of the cytoplasm, followed by recovery of pHi to the resting level. The initial rate of this pHi recovery was measured over the linear part of the recovery. B) Effect of extracellular K+ concentration on the rate of pHi recovery. C) Effect of addition of extracellular amiloride (2 mM) or DIDS (100 µM) on the rate of pHi recovery. All perfusion solutions contained no lactate, pyruvate, or phosphate but contained 1 mM ZnCl2 to inhibit H+ transport. Data represent the mean ± SEM, with the number of experiments in parentheses

When the pHi recovery following an acid load was investigated in trophoblast cells of expanded blastocysts, it was found that despite inactivation of the MCT, pHi returned to its resting level during the Na+-free period (Fig. 4A). This could have been caused by the presence of Na+ in the blastocoelic fluid. However, we observed the same result in the trophoblast of blastocysts that were collapsed in, and then maintained in, Na+-free medium (Fig. 4B). Moreover, this Na+-independent recovery in collapsed blastocysts was insensitive to 100 µM MIA (n = 3, P = 0.28). We thus used a different strategy in an attempt to quantify NHE activity in the trophoblast. We first measured, under lactate- and pyruvate-free conditions (Fig. 4C), the time courses of the pHi recovery following an acid load both in the presence of Na+ (n = 5) and in its absence (n = 5). We then used standard methods [46, 70] to differentiate these time courses to obtain estimates of the rate of change in pHi (change in pHi; over change in time; dpHi/dt). Finally, we plotted the average estimates of dpHi/dt in the presence or absence of Na+ as a function of pHi (Fig. 4D). These data show that removal of extracellular Na+ had no measurable effect on the rate of recovery of the pHi of the trophoblast following an acid load.



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  		FIG. 4. Representative experimental traces showing the response of pHi in the presence of 300 µM PCMPS to an NH4Cl pulse (25 mM) followed by an Na+-free period in trophoblast of A) expanded blastocysts and B) blastocysts that had been collapsed in Na+-free solution. Also shown (C) is the time course of the pHi recovery in trophoblast of expanded blastocysts in the presence () or absence ({circ}) of Na+ in medium lacking lactate and pyruvate and D) estimates of dpHi/dt in the presence (solid line) or absence (dashed line) of Na+ as a function of pHi calculated from the data in C

H+ Conductance in the Mouse Preimplantation Conceptus

Isolated ICMs (Fig. 5B), unlike the 2-cell stage (Fig. 5A), also showed some recovery from an acid load when lactate, pyruvate, and Na+ were absent from the bathing solution. This Na+-independent H+-transport system, unlike the MCT in 2-cell stage embryos (Figs. 5C and 6A), was insensitive to 300 µM PCMPS (P = 0.58, n = 5) (Figs. 5, C and D, and 6A) and to 1 mM CHC (P = 0.22, n = 6, data not shown). Thus, the cells of the ICM contain an acid extrusion system other than NHE or MCT. This H+ extrusion system was absent at the 4-cell stage and was first observed at the 8-cell stage both before and after compaction (Fig. 6B). The appearance of this H+ transporter depended on activation of the zygotic genome, because 2-cell stages cultured from the late 1-cell stage in 11 µg/ml of {alpha}-amanitin [71] for approximately 65 h (i.e., to 96 h post-hCG) showed no such recovery from acid loading (P = 0.001, n = 6, compared to cultured controls).



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  		FIG. 5. Time course of pHi recovery in 2-cell stages (A and C) and isolated ICMs (B and D) following an NH4Cl pulse (25 mM) in the absence of Na+, lactate, and pyruvate (A and B) and in the absence of Na+ and the presence of 300 µM PCMPS (C and D)



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  		FIG. 6. A) Effect of 300 µM PCMPS on the rate of pHi recovery following an NH4Cl pulse (25 mM) in the absence of Na+, lactate, and pyruvate. Data represent the mean ± SEM of five experiments for each embryogenic stage. B) Appearance of An H+ conductance during embryogenic development and comparison for different stages of the rates of recovery of pHi following an NH4Cl pulse in M2 minus Na+, lactate, and pyruvate. Data represent the mean ± SEM for unfertilized oocytes (UF; {diamondsuit}; n = 10); 1-cell fertilized oocytes ({circ}; n = 17); 2-cell stages (; n = 9); 4-cell stages ({diamond}; n = 8); early noncompact (*; n = 8), noncompact ({square}; n = 12), and compact ({block}; n = 4) 8-cell stages; ICMs ({triangleup}; n = 7); trophoblasts ({blacktriangleup}; n = 9); and collapsed blastocysts ({blacktriangledown}; n = 12)

To further investigate the mechanism(s) responsible for this recovery from an acid load, isolated pluriblast was exposed to various ion-transport inhibitors in medium free of Na+, lactate, and pyruvate (Table 1). These data suggest that an H2DIDS-sensitive anion transporter, a V-type ATPase, and a P-type ATPase were not involved in the recovery. Similarly, 1 mM ouabain, removal of K+ from the bath solution, or increasing K+ in the bath solution to 56 mM had no significant effects (P = 0.61, n = 5; P = 0.29, n = 6; and P = 0.80, n = 4, respectively) compared to the controls, thereby excluding a role for Na+-K+-ATPase or K+-H+-ATPase activity.


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  		TABLE 1. Effect of ion-transport inhibitors on the rate of pHi recovery after acid loading of isolated ICMs.a

Addition of the H+ conductance inhibitor, ZnCl2 (in phosphate-free conditions) at 500 µM reduced significantly the rate of pHi recovery following acid loading (P = 0.02, n = 7). Furthermore, in isolated ICMs, the recovery of pHi following alkalinization caused by removal of lactate and pyruvate was reduced significantly by 500 µM ZnCl2 (P = 0.04, n = 6). These data suggest that the pluriblast possesses An H+ conductance. This H+ conductance does not, however, appear to influence resting pHi in pluriblast, because 500 µM ZnCl2 did not affect resting pHi in either M2 or M2 plus lactate plus pyruvate (P = 0.4, n = 6 and P = 0.5, n = 9, respectively). Compared with controls, 500 µM ZnCl2 had no significant effect on the rate of recovery from acid loading in the trophoblast of expanded or collapsed blastocysts (P = 0.62, n = 7 and P = 0.29, n = 7, respectively) suggesting either a different sensitivity of the H+ conductance to ZnCl2 in this tissue or involvement of an additional H+-transport system that is yet to be characterized.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that the 2-cell stage QS mouse conceptus handles acid load through the expression of both NHE and MCT activity [46]. In a subsequent publication, we examined the H+-carboxylate cotransporter more fully and showed that it has pharmacological characteristics consistent with it being caused by MCT1, an isoform that is expressed in preimplantation mouse conceptuses [32]. We also showed that the activity of this transport system increases until the blastocyst stage [32]. In the present study, we have examined further the NHE isoform activity in response to acid load during preimplantation QS mouse development.

Seven isoforms of NHE have been cloned thus far from mammalian tissues [68, 69, 72, 73]. Two isoforms appear to be restricted to intracellular locations and actions. NHE6 is targeted to endoplasmic and secretory organelles [74], and NHE7 is reportedly targeted to the trans-Golgi network [75]. Of the five NHEs expressed on the cell surface, one, NHE5, is reportedly brain specific [76]. Our understanding of the physiological roles of the remaining four isoforms of surface NHEs is poor. NHE1 and NHE4 are localized in the basolateral membranes of epithelia and NHE2 and NHE3 in the apical membranes, but despite their different locations, all four clearly are capable of relieving an intracellular acid load [72, 73, 77]. Furthermore, NHE1, NHE2, and NHE4 are stimulated by increased extracellular osmolality [78, 79] and have been reported to play a role in the restoration of cell volume following cell shrinkage such as that produced by exposure to hypertonic solutions [72, 73]. In addition, the apically located isoforms, NHE2 and NHE3, have been shown to play important roles in electrolyte transport across gastrointestinal and renal epithelia [80, 81], where they act in concert with anion exchangers to produce net movement of Na+ and Cl- across the apical membrane [73]. The observation that NHE4 can exchange NH4+ for Na+ has lead to the proposal that it may mediate removal of NH4+ from the cells of the renal thick ascending limb [69].

A previous molecular study [58] investigated the presence of the surface membrane isoforms (NHE1–4) during mouse preimplantation development. The mRNA and proteins corresponding only to isoforms NHE1 and NHE3 were detected. The mRNA for NHE1 was reported as being present from oocyte to blastocyst and that for NHE3 as being present in oocyte only, but proteins for both were detected immunocytochemically in the blastocyst. Our approach to the surface isoforms has been functional and indicated that at least three of the isoforms are active during preimplantation development. An isoform sensitive both to 1 µM HOE-694 and to 1 µM MIA is present in the oocyte and early cleavage and is most likely due to NHE1 activity [66, 82], which is consistent with the results of Barr et al. [58]. All remaining activity is inhibited over these same stages of development by 50–100 µM MIA, which indicates an activity due to NHE2 or NHE3, but not NHE4. Given the results of previous molecular studies [58], NHE3 is the most likely additional isoform active at these stages. In addition, 100 µM MIA inhibits all NHE activity during the remainder of cleavage to the morula stage, suggesting that NHE4 is not active throughout this period of development.

By the blastocyst stage, however, the situation becomes more complex. First, in isolated ICMs, 1 µM MIA reduced Na+-dependent pHi recovery by approximately 50%, but no further reduction occurred on exposure to 50–100 µM MIA, suggesting that NHE1 activity remained present but that NHE3 activity had been lost. NHE1, but not NHE3, has been detected immunocytochemically between pluriblast cells [58]. The residual component of the Na+-dependent pHi recovery in the ICM was only sensitive to amiloride at very high doses, which is consistent with it being attributable to NHE4 [66]. As previously reported for NHE4 in other tissues [67, 83], this MIA-insensitive component of Na+-dependent pHi recovery in the conceptus is capable of exchanging K+ for H+ and can be stimulated by the stilbene DIDS. Our one anomalous finding in the conceptus was that this transport system fails to exchange Li+ for H+. Whether this difference is caused by a tissue or species difference or is an indication that we have identified a novel transporter is unclear. We were able to demonstrate the presence of NHE4 mRNA in the unfertilized oocytes but not at later stages. A similar lack of correspondence between message and protein was also described for NHE3 in the conceptus [58] and for NHE4 in the heart, liver, and spleen [77]. Should the MIA-insensitive Na+-dependent H+ transport be attributable to NHE4, it would require the NHE4 protein to have a long half-life and to be activated posttranslationally during emergence of the pluriblast. Several key events in early mouse development are known to be regulated posttranslationally [84–89].

We were surprised to be unable to detect Na+-dependent H+ transport attributable to NHE in trophoblast, either through use of 100 µM MIA or by direct comparison of pHi recovery rates in the presence and absence of extracellular sodium. The latter experiments were performed on both intact blastocysts and blastocysts that had been collapsed in, and then maintained in, Na+-free medium, thereby eliminating the possibility that residual extracellular Na+ might have explained the lack of Na+ dependence. Previously, immunocytochemical data have localized putative NHE1 to the basolateral surface of mural trophoblast and NHE3 to the apical trophoblast [58]. However, immunoreactivity does not necessarily imply activity or give information regarding the level of activity. Indeed, evidence suggests that NHE protein isoforms can undergo posttranslation modification and/or be expressed from different splice variants [77]. To our knowledge, the only previous claim to have detected possible NHE activity in trophoblast [90] was not decisive and did not exclude other Na+-transporting mechanisms as being responsible for the observed phenomena. We should stress that our results do not rule out the possibility of some NHE activity in, for example, Na+ transport into trophoblast cells as part of a blastocoelic fluid secretory mechanism [58, 90]. However, if NHEs are active in the trophoblast, they seem to play a relatively small part in the pHi regulatory response to acid load under the conditions used in our experiments. The Na+-independent, proton-transporting systems seem to play the major role. Our previous results showed that the MCT does play such a role [32]. However, an additional transporter must be active in the experiments reported here, because we had taken care to inactivate the MCT in our current protocol. We did find direct evidence for a third proton transporter.

An H+ conductance is clearly present in both trophoblast and ICM. This conductance first appeared in the 8-cell stage, and its expression was dependent on activation of the embryogenic genome. The H+ conductance in the ICM (but not the trophoblast) was, similar to that in other tissues [91, 92], sensitive to Zn2+; therefore, whether the same H+ conductance mechanism is operating in both tissues of the blastocyst is unclear. A Zn2+-insensitive H+ conductance was previously reported to be present in the mouse 2-cell stage [45], but we found no evidence for this in our studies. The molecular basis of Zn2+-sensitive H+ conductances remains unknown [93]. They are found in highly metabolically active cell types, such as neutrophils [94], eosinophils [95], osteoclasts [96], and microglia [97], in which metabolically generated H+ needs to be lost rapidly [91, 92]. Thus, it is of interest that Zn2+ and other divalent cations, which inhibit the H+ conductance in the ICM, have been reported to be toxic to preimplantation mouse conceptuses [98].

In conclusion, our results suggest that the period of preimplantation development in QS mice is characterized by dynamic developmental changes in the systems available for handling acid load. Three systems have now been described and partially characterized. First, an MCT results mainly, if not exclusively, from MCT1 activity, which progressively increases as development progresses [32]. Second, at least three isoforms of NHE are expressed and show changes in relative expression profile with both developmental stage and emergent tissue lineages. Some of these changes appear to be regulated posttranslationally. The NHE isoforms appear to play less of a role in the response to acid load as development progresses. Third, a proton conductance develops from the 8-cell stage under control of the embryogenic genome and is particularly prominent by the blastocyst stage. These conclusions must inevitably be constrained by two important qualifications already raised in the Introduction. First, given previous evidence that some elements of pHi handling may vary with mouse strain [47], determining which elements of the above scheme are not universal clearly is important. Such an analysis might incidentally provide useful insights into why some strains develop better than others in vitro. Second, all studies were necessarily performed in vitro. Given that in vitro conditions impact on various aspects of early development, the precise proton-transport system detected here might be induced by the exposure to conditions, and varying the culture medium, for example, might vary the nature of the transport systems detected. This problem is fundamental to all in vitro studies. However, the published studies do show what mechanisms are possible. Moreover, a complexity of mechanisms for responding to acid load is not unreasonable, given that in vivo, the oocyte and conceptus must pass through changing microenvironments during passage from follicle to oviduct and then uterus [18], so changing profiles of proton-transport mechanisms may operate there, too. During these transitions, it is not just pH variations that may be encountered. It is important to remember that pHi recovery mechanisms also mediate responses to other environmental changes, such as osmolality and metabolite levels, and are involved in fluid movements across membranes. For example, of the NHE isoforms present, NHE1 and NHE4 may also play a role in cell-volume regulation. Given that we observed no evidence for NHE3 activity in the trophoblast, it seems unlikely that this isoform plays a major role in the transepithelial fluid and electrolyte transport that establishes and maintains the blastocoel. Finally, given the known sensitivity of mouse embryogenic cells to ammonium [99], it is tempting to suggest that NHE4 may, in addition, play a role in removal of ammonium from the cytosol.


   ACKNOWLEDGMENTS
 
We thank Peter Kaye (University of Queensland) for the gift of the rabbit anti-mouse antiserum.


   FOOTNOTES
 
1 Supported by the NHMRC Australia, Medical Foundation of the University of Sydney, The Royal Society, U.K., and The Wellcome Trust, U.K. Back

2 Correspondence: M.L. Day, Department of Physiology (F13), University of Sydney, NSW 2006, Australia. FAX: 61 2 9351 2058; margotd{at}physiol.usyd.edu.au Back

Received: 19 March 2002.

First decision: 11 April 2002.

Accepted: 5 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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G. FitzHarris, V. Siyanov, and J. M. Baltz
Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms
Development, December 1, 2007; 134(23): 4283 - 4295.
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