Intracellular pH Regulation by Sodium-Hydrogen Exchanger Isoforms in Preimplantation Mouse Embryos
Creators: name: Siyanov, Violetta
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2015
Subjects: NHE
Slc9a
pHi regulation
Embryo
Amiloride
Cariporide
S3226

description: Intracellular pH (pHi) impacts many cellular mechanisms including cellular metabolism, gene expression, cell volume regulation, cell survival and proliferation. Most cells use two general pHi regulatory mechanisms: HCO3-/Cl- antiporters (AE, Slc4a family) to reduce internal alkaline load, and Na+/H+ exchangers (NHE, Slc9a family) that protect cells from acidosis. Previous studies with preimplantation (PI) embryos have shown robust activity of HCO3-/Cl- exchanger in all stages of development. It was also determined that inhibition of this exchange with the stilbene AE inhibitor 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) was detrimental to embryo development from the 2‐cell stage to blastocyst when cultured at high external pH. In this study I investigated which of the five known plasma membrane NHE isoforms was present and active within mouse PI embryos and their role as pHi regulators throughout preimplantation embryo development. In mouse oocytes and preimplantation embryos, mRNAs were detected encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in fully-grown oocytes through one-cell stage embryos and then generally lower levels after the two-cell stage. No transcripts for NHE2 (SLC9A2) or NHE5 (SLC9A5) were detected. Measurements of intracellular pH during recovery from acidosis, induced by transient ammonium pulse, suggested that recovery occurred and was mediated by NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). This recovery was inhibited by 1 mM amiloride, a general NHE inhibitor. The observed residual recovery was attributed to passive passage of protons across the membrane, rather than the activity of NHE4 (an amiloride-resistant isoform), since no further decrease in recovery rates from acidosis was observed upon amiloride increase to 5 mM. Furthermore, recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over that achieved with cariporide alone, indicating that NHE3 did not play a functional role in pHi regulation at any stage assessed. Another regulator of intracellular pH against acidosis, previously reported to be active in oocytes and 1-cell embryos, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. This indicated that NHE1 is likely the only significant regulator of pHi in preimplantation mouse embryos, at least after the 1-cell stage. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development, as assessed by development to the blastocyst stage and cell lineage allocation. However, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not further decrease embryo development to the blastocyst stage more extensively under conditions of chronic acidosis than at normal pH. This suggests that mouse PI embryos have a restricted ability to counteract chronic acidosis by means of pHi regulatory mechanisms, despite clearly being able to recover from acute acidosis via NHE1 activity.