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ABSTRACT: The coupled action of the Na(+) /H(+) exchanger NHE1 and the HCO3 (-) /Cl(-) exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation. In contrast, NHE1 activity stimulated by decreased cell volume was low in GV oocytes but became active during meiotic maturation as the oocyte detached from the zona pellucida. It then decreased again in mature eggs similar to activity stimulated by acidosis. The subcellular localization of NHE1 was investigated with YFP-tagged NHE1. Exogenous NHE1 expressed in GV oocytes localized to the plasma membrane and resulted in increased Na(+) /H(+) exchanger activity, but only when co-expressed with Calcineurin Homologous Protein 1 (CHP1). When oocytes expressing functional NHE1 were matured to eggs, however, membrane localization of NHE1 and Na(+) /H(+) exchanger activity were lost. It was unknown why NHE1 and AE2 activities are suppressed during meiotic maturation. Maintenance of cell volume in preimplantation embryos requires glycine accumulation via the GLYT1 transporter, a process unique to eggs and early embryos that is initiated during meiotic maturation. When NHE1 and AE2 activities were maintained in GV oocytes by exogenous expression, glycine accumulation was inhibited. We propose that NHE1-mediated acute cell volume regulation is inactivated during meiotic maturation to allow preferential accumulation of glycine in eggs. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Physiology 04/2013; · 3.87 Impact Factor
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ABSTRACT: The earliest stages of preimplantation embryos are particularly sensitive to increased osmolarity, even within the physiological range. This sensitivity contributed to persistent developmental arrest, even when embryos were cultured in vitro in older, conditioned culture media, and seems to arise when embryos at the 1- and 2-cell stages accumulate inorganic ions used for cell volume homeostasis at too high a level, through activation of coupled Na(+) /H(+) and HCO 3-/Cl(-) exchange. Such accumulation of inorganic ions can be disruptive since, above a certain level, the increased ionic strength disrupts cellular biochemistry and macromolecular functions and alters membrane potential. To counter this, embryos have evolved mechanisms of cell volume regulation that are unique to early preimplantation embryogenesis. The primary role of these is glycine accumulation via the GLYT1 transporter, with a secondary contribution by betaine accumulation via the SIT1 transporter. Independent cell-volume regulation first arises in the oocyte only after ovulation is triggered, when the strong oocyte-zona pellucida adhesion present in germinal vesicle stage oocytes in the ovarian follicle is released and GLYT1 becomes activated to begin accumulating glycine. Open questions still remain regarding how these processes are regulated. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.
Molecular Reproduction and Development 09/2012; · 2.53 Impact Factor
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ABSTRACT: Preimplantation mouse embryos are particularly sensitive to increased osmolarity within their normal physiological range. The detrimental effects can be alleviated by organic osmolytes such as glycine transported into early embryos, an effect thought to be due to the organic osmolyte replacing a portion of intracellular inorganic ions accumulated during acute cell volume regulation. However, no mechanism of cell volume regulation dependent on inorganic ions has been identified in preimplantation embryos. We found that decreased cell volume rapidly activated Na(+) /H(+) exchange in preimplantation mouse embryos. This activity was likely mediated by the NHE1 (Slc9a1) isoform, since it was blocked by the highly selective NHE1 inhibitor, cariporide, which also inhibited the ability of the 1-cell embryo to maintain cell volume. How NHE1 is activated by decreased cell volume is not generally well understood. Full activation of NHE1 by decreased cell volume in 2-cell mouse embryos required the activity of the tyrosine kinase Janus kinase 2 (Jak2), since NHE1 activation was inhibited by the general tyrosine kinase inhibitor genistein, several selective inhibitors of Jak2, and dominant negative Jak2 expressed in 2-cell embryos. Decreased cell volume furthermore resulted in increased tyrosine phosphorylation of proteins in 2-cell embryos detected both by anti-phosphotyrosine antibody and an antibody directed against active phospho-Jak2. Thus, Jak2 apparently serves as a cell volume sensor in embryos. Evidence from pharmacological inhibitors further indicated that NHE1 activation by decreased cell volume was dependent on calmodulin activity, likely downstream of Jak2, and required active phospholipase C. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
Journal of Cellular Physiology 06/2012; · 3.87 Impact Factor
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ABSTRACT: Betaine (N,N,N-trimethylglycine) added to culture media is known to substantially improve the development of preimplantation mouse embryos in vitro, and to be imported into 1-cell embryos by a transporter that also accepts proline. Here, we found that the betaine/proline transporter is active in preimplantation mouse embryos only for a short period of development, between the 1- and 2-cell stages. Betaine/proline transport was activated after fertilization, beginning approximately 4 hours post-egg activation and reaching a maximum by approximately 10 hours. One- and 2-cell embryos contained endogenous betaine, indicating that a likely function for the transporter in vivo is the accumulation or retention of intracellular betaine. The appearance of transport activity after egg activation was independent of protein synthesis, but was reversibly blocked by disruption of the Golgi with brefeldin A. We assessed two candidates for the betaine/proline transporter: SIT1 (IMINO; encoded by Slc6a20a) and PROT (Slc6a7). mRNA from both genes was present in eggs and 1-cell embryos. However, when exogenously expressed in Xenopus oocytes, mouse PROT did not transport betaine and had an inhibition profile different from that of the embryonic transporter. By contrast, exogenously expressed mouse SIT1 transported both betaine and proline and closely resembled the embryonic transporter. A morpholino oligonucleotide designed to block translation of SIT1, when present from the germinal vesicle stage, blocked the appearance of betaine transport activity in parthenogenotes. Thus, SIT1 is likely to be a developmentally restricted betaine transporter in mouse preimplantation embryos that is activated by fertilization.
Development 01/2009; 135(24):4123-30. · 6.60 Impact Factor
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ABSTRACT: Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO(3)(-)/Cl(-) exchange--a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO(3)(-)/Cl(-) exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO(3)(-)/Cl(-) exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established.
Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO(3)(-)/Cl(-) exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO(3)(-)/Cl(-) exchange in mature MII eggs. A GFP-tagged form of the HCO(3)(-)/Cl(-) exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation.
Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO(3)(-)/Cl(-) exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.
PLoS ONE 01/2009; 4(10):e7417. · 4.09 Impact Factor
