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L M Wiley

Publications and source records attributed to L M Wiley.

At least 19 recordsLinked to original sources
PubMed1992

Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development.

Epidermal growth factor receptors (EGF-Rs) are expressed at increasing levels on mouse preimplantation embryos. Immunofluorescence assays were used to show that unfertilized eggs and 2-cell embryos have a very low level of reactivity to antimouse EGF-R antibodies, but by the 4-cell stage and later the reactivity increases. The synthesis of EGF-R protein was verified at the blastocyst stage by immunoprecipitation of a 170-kDa metabolically labeled protein. The EGF-R protein is expressed on cell plasma membrane surfaces, but after compaction at the 8-cell stage concentrates on the apical cell surfaces. We also find a low level of expression of EGF-R protein on inner cell mass cells; thus, all cell lineages express receptors from the beginning of gestation. The receptor protein synthesized by the 8-cell embryo and later is probably translated from embryonic transcription, since reverse transcription-polymerase chain reaction indicates increasing levels of mRNA starting after the 4-cell stage. However, we also detected maternal mRNA in zygotes and 2-cell embryos. The pervasive nature of EGF-R expression throughout development suggests important roles for these receptors which could include autocrine and paracrine stimulation.

Animals↗
PubMed1992

Effects of maternal exposure to trichloroethylene (TCE) on cell proliferation in the mouse preimplantation embryo.

Pregnant mice were treated with trichloroethylene (TCE) at doses ranging from 0.01 micrograms/kg body weight to 483 mg/kg body weight when embryos were traversing the pronuclear stages of development. No treatment-related effect was seen on total number of embryos recovered from oviducts at the 2 to 4 cell stage of development. The 4-cell embryos were tested for cellular proliferative disadvantage by the embryo chimera assay in which embryos from treated mothers (experimental embryos) are paired with embryos from nontreated mothers (control embryos) to form aggregation chimeras. No significant cell proliferation decreases were observed for any of the experimental embryos across all doses examined.

Administration, Oral↗
PubMed1991

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

Animals↗
PubMed1991

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos.

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.

Animals↗
PubMed1991

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay.

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.

Animals↗
PubMed1990

Cell polarity and development of the first epithelium.

In the 4 1/2 to 5 days between fertilization and implantation, the mouse conceptus must gain the abilities to implant and produce an embryo. Each of these is the sole developmental responsibility of one of two cell types forming the blastocyst, trophectoderm and inner cell mass (ICM), respectively. Trophectoderm is a polarized transporting epithelium while the ICM is an aggregate of non-epithelial pluripotent stem cells. These two cell types originate from the division of polar blastomeres when their cleavage furrows parallel their apical surfaces. Blastomeres polarize in response to asymmetric cell--cell contact, and understanding the mechanism of this induction is regarded as the key to understanding the origin of trophectoderm and ICM. Here we propose a model based on transcellular ion current loops for the induction of cell polarity during the development of the first epithelium, trophectoderm.

Animals↗
PubMed1989

Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons.

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport--phlorizin, which inhibits brush border (apical) Na+/glucose symporters, and ouabain, which inhibits Na+/K+-ATPase, thereby modifying the transmembrane Na+ gradient--were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain (L.M. Wiley, 1984, Dev. Biol. 105, 330-342) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10(-5) M; phlorizin, 10(-6) M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10(-4) M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.

Animals↗
PubMed1989

A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice.

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

Animals↗
PubMed1988

Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres.

Cell surface and cytoplasmic polarity is exhibited by the blastomeres of mouse preimplantation embryos following compaction at the 8-cell stage of cleavage. It has been hypothesized that cytoplasmic polarity is initiated by plasma membrane functions of polar blastomeres that are absent from apolar blastomeres. To test this hypothesis the plasma membranes of "test" polar and apolar 8-cell- and 16-cell-stage blastomeres were inserted into the plasma membrane of "carrier" 4-cell-stage blastomeres by polyethylene glycol-mediated fusion of carrier-test blastomere pairs. After a 4-hr culture period each heterokaryon was scored for the distribution of two marker organelles--lipid droplets and nuclei--with respect to their proximity to the plasma membrane insert from the test blastomere. Plasma membrane inserts from polar test blastomeres were identified by labeling their apical domains with fluorescently tagged (succinylated) concanavalin A. The incidence of polar heterokaryons (those exhibiting a discrete fluorescently labeled area of plasma membrane corresponding to the apical domain inherited from the test blastomere) was 55/85 (69%) and 48/79 (61%) for 8-cell-stage and 16-cell-stage test blastomeres, respectively. In all polar heterokaryons, both nuclei were subjacent to the fluorescent label (apical domain of a polar plasma membrane insert), while the majority of lipid droplets resided in the hemisphere opposite the fluorescent label. In all 61 apolar heterokaryons examined (those lacking a discrete fluorescently labeled plasma membrane area) both nuclei were centrally located and lipid droplets were randomly distributed. These observations are consistent with the hypothesis that cytoplasmic polarity can be initiated by properties that distinguish the plasma membranes of polar blastomeres from those of apolar blastomeres.

Animals↗
PubMed1988

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Animals↗
PubMed1988

Trophectoderm: the first epithelium to develop in the mammalian embryo.

The first epithelium to appear during mammalian embryogenesis is the trophectoderm, a polarized transporting single cell layer that comprises the wall of the blastocyst. The trophectoderm develops concurrently with blastocoele fluid production as the morula develops into a blastocyst. The process whereby the morula becomes a fluid-filled cyst is called 'cavitation', which can be regarded as the first functional expression of the trophectoderm phenotype. The outer layer of cells of the morula comprise the nascent trophectoderm and are already morphologically polarized prior to the onset of cavitation. A major working hypothesis in the field of mammalian embryogenesis is that such polarization is a prerequisite for the initiation of cavitation. To test this hypothesis we examined morulae for their ability to cavitate during treatments that modify morphological polarity in nascent trophectoderm cells. These treatments included ouabain, different concentrations of extracellular K and Na, cytochalasins and colcemid. Ouabain and extracellular K and Na affect the activity of Na/K-ATPase, which has been implicated in the maintenance of morphological polarity of nascent trophectoderm cells. Cytochalasins and colcemid also modify apical-basal polarity of nascent trophectoderm cells and impair cavitation. The endpoints that were monitored included incidence of cavitation, rate of cavitation and the status of morphological polarity of nascent trophectoderm cells. Collectively, all of these treatments indicate that there is a functional association between an asymmetric distribution of organelles along the apical-basal axis of nascent trophectoderm cells and the ability of the embryo to produce nascent blastocoele fluid efficiently. In addition, the preimplantation embryo appears to possess two mechanisms for accumulating blastocoele fluid.

Animals↗
PubMed1987

Reproductive effects of chlorpromazine exposure to female mice: cell proliferation disadvantage revealed by the Chimera Embryo Assay.

Administration of chlorpromazine-HCl at 5 to 15 mg/kg bodyweight to pregnant CD-1 mice at 24 h after human chorionic gonadotropin (hCG) (20-23 h after mating) inhibited blastocyst formation and reduced the cell number of embryos recovered at 95 h after hCG. When embryos are recovered at the two- to four-cell stage (48-50 h after hCG) and cultured for an additional 47 h (to 95 h after hCG) or 72 h (to 120 h after hCG), blastocyst formation and embryo cell number were similarly reduced. When the dose range was reduced to 0.5 to 2 mg/kg bodyweight, no significant effect of the drug was observed on blastocyst formation or on embryo cell number. However, when aggregation chimeras were formed between embryos recovered from drug-exposed females and from untreated females, a decrease in cell proliferation rate of the embryo from the drug-exposed female was observed at a dose of 2 mg/kg bodyweight. This result indicates that exposing pregnant mice to chlorpromazine-HCl at doses as low as 2 mg/kg bodyweight can induce a potential for decreased cleavage rate in their pre-implantation embryos that can be revealed by challenging those embryos by direct contact with embryos from nonexposed females. Finally, when four-cell stage embryos recovered from untreated females cultured in the presence of chlorpromazine (0.1-25 mM), blastocyst formation and embryo cell number were significantly reduced in a dose-dependent manner. This last result suggests that in vivo the drug may act directly on the embryo from the pronuclear stage to the early morula stage of development.

Animals↗
PubMed1987

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

Animals↗
PubMed1986

Immunofluorescent localization of immunoglobulins on the cell surface of mouse oocytes and preimplantation embryos.

Living mouse oocytes and preimplantation embryos were assayed by indirect immunofluorescence for their ability to adsorb heterologous serum proteins from culture media to their cell surfaces. Bovine and human immunoglobulins of the IgG class were adsorbed by the oocytes and all stages of preimplantation embryos, while IgG of mouse or goat origin was not. In contrast, none of the serum albumins was adsorbed to the cell surfaces of mouse oocytes and preimplantation embryos. From the differential binding of IgG of some, but not all, of the species that were tested, we concluded that these cell surface IgG "receptors" on mouse oocytes and preimplantation embryos are likely to be heterophilic in nature. Similar observations were made irrespective of the strain of mice used to provide the oocytes and embryos. These observations raise the question of whether human oocytes and preimplantation embryos should also be assayed for their ability to adsorb animal serum proteins that are possible candidates as a substitute protein supplement for human serum in culture medium used in human in vitro fertilization/embryo transfer programs.

Animals↗
PubMed1986

Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro.

Mouse preimplantation embryos were used to test the effects of three culture parameters on development of 2-cell stage embryos to the late morula stage (40 to 43 hours of culture, 65 to 80 hours after fertilization): concentration of potassium (K)--0.6 mM, 1.4 mM, and 6.0 mM; type of protein supplement, bovine serum albumin (BSA), and bovine serum immunoglobulins (BSIG); and number of embryos (2 or 20) cultured per 10- to 12-microliter drop of T6 culture medium under paraffin oil. Cell number per embryo of cultured late morulae was compared with that of same-age embryos developing in vivo. The developmental rate to late morula stage with 20 embryos per drop of medium was media containing 0.6- and 1.4-mM K, 0.15% BSA, 0.15% BSIG greater than medium containing 1.4-mM K, 0.3% BSA greater than all media containing 6.0-mM K (P less than 0.01 comparing media containing 0.6- or 1.4-mM K with media containing 6.0-mM K). For a given medium, developmental rate was significantly slower for 2 embryos per drop than for 20 embryos per drop of medium with those media containing either 0.3% BSA (for all concentrations of K tested) or 6.0-mM K (for 0.3% BSA and for 0.15% BSA + 0.15% BSIG). Finally, cell number per embryo was: in vivo-developed embryos greater than medium containing 1.4-mM K, 0.15% BSA, 0.15% BSIG much greater than 6.0- or 1.4-mM K, 0.3% BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents.

Eight- to sixteen-cell stage mouse morulae were dissociated with Ca2+-free medium into blastomeres that were labeled with fluoresceinated-succinylated Con A (FS-Con A) to mark their apical-basal axes. The vibrating probe was then used to map their extracellular current patterns. The average current density around normal blastomeres approached the resolution of the probe system (0.2 microA/cm2) and was undetectable in the majority of blastomeres. Since the current density at the measuring point outside the cell is known to increase with cell size in other systems, enlarged blastomeres were created by fusing together blastomeres of 4-cell stage embryos in 45% polyethylene glycol. Enlarged blastomeres were then aggregated with normal blastomeres using phytohemagglutinin and cultured to the 8- to 16-cell stage to allow them to become polarized. Such aggregates were then dissociated with Ca2+-free medium to recover polarized, enlarged blastomeres. The enlarged blastomeres were 30-65 microns in diameter and 70% of them generated a detectable current; currents were detected around 83% of those blastomeres larger than 40 micron in diameter. The current pattern in these most reliable cases was predominantly inward apical (11/16 or 69%) and outward basal (15/16 or 94%), with lateral currents about three-fold smaller in amplitude than these apical-basal currents. Lateral currents were undetectable in 53% of the cases. Preliminary data suggest that the inward current is carried in part by Na+ influx and is independent of the Na+,K+-ATPase over the short term. Transcellular ion currents were detectable as long as 4 hr after dissociation, and the apical-basal current pattern was usually stable during that time. In contrast, the fluorescent cap of FS-Con A faded within 7-30 min at 35 degrees C but remained stable in 0.1% azide or 1.5 micrograms/ml cytochalasin D. The electrical polarity therefore persisted after the apical cap of Con A fluorescence was no longer visible. We propose that these transcellular ion currents may be involved in the establishment of blastomere polarity and describe a mechanism of action in an "ion current polarization" hypothesis.

Animals↗