Calculation of Na<Superscript>+</Superscript>/Cl<Superscript>−</Superscript> Selectivity in Tight Junctions between Epithelial Cells by Methods of Nonlocal Electrostatics with Pole Models of Dielectric Function without an Overscreening Effect

A mathematical model of Na⁺/Cl^? selectivity in tight junctions (TJs) between epithelial cells was developed. It was demonstrated that Na⁺/Cl^? selectivity in TJs depends on the total charge of amino-acid residues of claudin macromolecules within TJs, as well as on the ion-distribution coefficients between TJs and free solution. It was demonstrated that the obtained formulas predict a change of Na⁺/Cl^? selectivity in TJs for Cl^?/Na⁺ selectivity if the sign of the total charge of amino-acid residues in TJ changes. The calculated Na⁺/Cl^? selectivity value for MDCK cells coincides with the experimental data of (Colegio et al., 2002). To calculate a change in ion-solvation energy during their transition to TJs, formulas for nonlocal electrostatics with one-, two-, and three-pole models of dielectric function without taking into account the overscreening effect were used.     

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 10⁶ oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC‐dextran flux) in Caco‐2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E‐cadherin. Claudin 3, zonula occludens‐1 (ZO1) and α‐catenin were also significantly decreased, whereas claudins 1 and 2 and β‐catenin were not altered. Substantial downregulation of occludin, claudin 4, and E‐cadherin was also observed in response to CP infection ex vivo in mouse enteroid‐derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP‐infected mouse ileum and jejunum but were unaltered in Caco‐2 cells. Further, bafilomycin‐A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco‐2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection‐induced diarrhoea.     

Roles of ERα during mouse trophectoderm lineage differentiation: revealed by antagonist and agonist of ERα

During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8‐cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose‐dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.     

Asynchronous CDX2 expression and polarization of porcine trophoblast cells reflects a species‐specific trophoderm lineage determination progress model

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula‐stage embryos. In TE cells, polarization can regulate Hippo and Rho‐associated kinase (Rho‐ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula‐stage embryos but only in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species‐specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four‐cell stage porcine embryos resulted in increased CDX2‐positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, but the polarization progress is not conserved between the pig and the mouse, and results in a species‐specific trophoblast determination progress model.     

Nepro is localized in the nucleolus and essential for preimplantation development in mice

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2‐cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria‐associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus‐associated protein, and its loss leads to the apoptosis before blastocyst formation in mice.     

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

mRNA expression levels of tight junction protein genes in mouse brain capillary endothelial cells highly purified by magnetic cell sorting

Tight junctions (TJs) are an important component of the blood-brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of brain capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 10⁶ cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the brain capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-brain barrier in cooperation with claudin-5.     

Effect of Homologous Peptides of Differentiation Factors HLDF and PEDF on Preimplantation Development of Mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351–356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84–89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2–4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Modulation of embryonic NA+-K+-ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K⁺) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose-dependent negative effect of increasing K⁺ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two-cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-AT-Pase), we used two other conditions known to inhibit the Na⁺-K⁺-ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K⁺ concentrations (1.4 mM) or in medium supplemented with ouabain (50 μM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K⁺ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K⁺ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na⁺-K⁺-ATPase during the two-cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.     

Endothelia of term human placentae display diminished expression of tight junction proteins during preeclampsia

This study explores the molecular composition of the tight junction (TJ) in human term placenta from normal women and from patients with preeclampsia, a hypertensive disorder of pregnancy. Maternal endothelial dysfunction is a critical characteristic of preeclampsia; hence, we have analyzed its impact on placental vessels. The study concentrates on the TJ because this structure regulates the sealing of the paracellular route. We have found that, in placental endothelial vessels, TJ components include the peripheral protein ZO–1 and the integral proteins occludin and claudins 1, 3, and 5. During preeclampsia, the amounts of occludin and ZO–1 exhibit no significant variation, whereas those of claudins 1, 3, and 5 diminish, suggesting the presence of leakier TJs in the endothelia of the preeclamptic placenta, possibly in response to the decreased perfusion of this organ during preeclampsia. We have unexpectedly found that, in normal placentae, the multinucleated syncytiotrophoblast layer displays claudin 4 at the basal surface of the plasma membrane, and claudin 16 along the apical and basolateral surfaces. The presence of membrane-lined channels that cross the syncytiotrophoblast constituting a paracellular pathway has been determined by transmission electron microscopy and by the co-immunolocalization of claudin 16 with the plasma membrane proteins Na⁺K⁺-ATPase and GP135. Since claudin 16 functions as a paracellular channel for Mg²⁺, its diffuse pattern in preeclamptic placentae suggests the altered paracellular transport of Mg²⁺ between the maternal blood and the placental tissue.This work was supported by grants 45691-Q from the Mexican Council for Science and Technology (CONACYT) and 2005/1/I/012 from the Research Promotion Fund of the Mexican Institute of Social Security (IMSS/FOFOI).     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Role of claudin interactions in airway tight junctional permeability

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.     

Role of occludin, a tight junction protein, in blastocoel formation, and in the paracellular permeability and differentiation of trophectoderm in preimplantation mouse embryos

Tight junctions (TJ) are critical for blastocoel formation in mammalian embryos. The present study aimed to examine the role of tight junctions in the differentiation of the trophectoderm (TE), and in the pluripotency of blastomeres, as well as in the formation and integrity of the blastocoel. We examined the effect of occludin antibody on blastocoel formation, blastocyst permeability, and expression of H19 and Oct-4, markers of TE differentiation and blastomere pluripotency, respectively. Eight-cell mouse embryos and morulae were cultured in the presence or absence of occludin antibody for 31 h. Occludin antibody inhibited blastocoel formation and increased permeability of the TE of nascent and expanding blastocysts to FITC-dextran (4 kDa), a permeability tracer. At the same time Oct-4 expression increased while expression of H19 became barely detectable. These observations indicate that occludin is involved in establishing the blastocoel, as well as in maintaining its impermeability, and that the development of tight junction is critical for TE formation in mouse embryos.     

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid‐gastrulation

Transgenic mice with a defined cell‐ or tissues‐specific expression of Cre‐recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre‐expression from Cdx1 regulatory elements. The expression of Cre‐recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre‐mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development. genesis 47:204–209, 2009. © 2009 Wiley‐Liss, Inc.     

Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.