Amino Acid transport mechanisms in mouse oocytes during growth and meiotic maturation.

Ottawa Hospital Research Institute, Ottawa, Canada.
Biology of Reproduction (Impact Factor: 4.03). 08/2009; 81(6):1041-54. DOI: 10.1095/biolreprod.109.079046
Source: PubMed

ABSTRACT Amino acids are transported into cells by a number of different transport systems, each with their own specific range of substrates. The amino acid transport systems active in preimplantation embryos and the amino acids required by embryos for optimal development have been extensively investigated. Much less is known about amino acid transport systems active in growing and meiotically maturing oocytes or about developmental changes in their activity. As a first step in determining the array of amino acid transporters active in oocytes, the transport characteristics of nine amino acids were measured in small, medium, and large growing oocytes; in fully grown germinal vesicle (GV)-stage oocytes; in metaphase I oocytes; and in metaphase II eggs. Whether each of 11 classically defined amino acid transport systems was likely active in oocytes at each stage was determined using assays based on measuring the transport of radiolabeled amino acids into oocytes and the effect of a limited set of potential competitive inhibitors. Six amino acid transport systems were found to be active during oocyte growth or maturation. L, b(0,+), and ASC/asc were active throughout oocyte growth and maturation, increasing during growth. In contrast, GLY, beta, and x(c)(-) had little or no activity during growth but became activated during meiotic maturation. Surprisingly, the presence of follicular cells surrounding medium growing oocytes or cumulus cells surrounding GV oocytes did not confer amino acid transport by additional transport systems not present in the oocyte. In some cases, however, follicular cells coupled to the oocyte enhanced uptake of amino acids by the same systems present in the oocyte.

  • [Show abstract] [Hide abstract]
    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; · 4.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.
    Reproduction Fertility and Development 01/2010; 22(6):1032-40. · 2.58 Impact Factor


Available from