Ciapa B and Philippe L (2013), Intracellular and extracellular pH and Ca are b...

ECB-ART-42917

PLoS One 2013 Jun 05;86:e66113. doi: 10.1371/journal.pone.0066113.

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Intracellular and extracellular pH and Ca are bound to control mitosis in the early sea urchin embryo via ERK and MPF activities.

Ciapa B , Philippe L .

Abstract
Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na(+)/H(+) exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life.

PubMed ID: 23785474
PMC ID: PMC3681939
Article link: PLoS One

Genes referenced: impact LOC100887844 LOC115919910 LOC576066 LOC590297 pus1

Article Images: [+] show captions

	Figure 1. ERK and CDK activities control Cai and pHi after fertilization.A. Time courses of Cai changes after fertilization in eggs treated or not (a) 10 mins after sperm addition (time zero) with 2 µM U0126 (b) or 20 µM Roscovitine (c). One typical recording is shown for each condition. Cai transients occur at time of pronuclear migration (black arrows) and at mitosis (grey arrow). Inset images taken 80 mins after fertilization show normal division in control eggs (a), mitotic alterations with U0126 (b) and absence of mitosis with Roscovitine (c). B. Time course of pHi changes after fertilization of control eggs. C. Relative changes in pHi (a) and Cai (b) levels in eggs treated or not (control) with 2 µM U0126 or 20 µM Roscovitine. The mean levels of pHi recorded from 60 and 65 mins following sperm addition are expressed relative to that of unfertilized egg (arbitrarily taken as 1). Mean levels of Cai recorded during 5 mins at time of the mitotic peak (grey arrow and line in Fig. A), i.e. between 35–40 mins in control eggs, 30–35 mins in U0126 and 25–30 mins in Roscovitine treated eggs, are expressed as the percentage of the fertilization Cai peak arbitrarily taken as 100. The total number of eggs monitored is indicated for each condition (brackets). Values (mean +/− sem) are significantly higher than that of control eggs (**, student test, p<0.01) or not significantly different (ns).
	Figure 2. Changes in Cai and pHi during mitosis depend on external Ca++ and Na+.Eggs were transferred in the different media after fertilization (arrow). Two examples representative of n measurements (number in brackets) are shown in each condition. Variations in pHi (a), evolution of Cai (b) and images of eggs taken 70 min after fertilization (insets in a). A. Transfer in 0Na (1), Am (2) or Ac (3). B. Effect of additional absence of external Ca after transfer in 0Na0Ca (1), Am0Ca (2) or Ac0Ca (3). C. Mean levels of pHi (a) and Cai (b) changes recorded from 60–65 mins after sperm addition in Figs. A and B. pHi changes (a) are expressed relative to that of unfertilized egg (arbitrarily taken as 1) while Cai changes (b) are expressed as the percentage of the fertilization Cai peak arbitrarily taken as 100 (zero is the unfertilized level). Values (means +/− sem) obtained in the absence of external Ca are significantly different (student test, p<0.01, two black stars) or not (ns, black letters) from control. Values obtained in the absence of Ca are significantly different (p<0.01, two grey stars) or not (ns, grey letters) from those obtained in the presence of Ca. Number of eggs is indicated for each condition (brackets).

	Figure 4. P-ERK activity controls mitosis and survival of fertilized eggs.A. Images taken 3 hours after fertilization of eggs transferred or not (control) 25 mins after fertilization in Ac containing or not 2 µM U0126. B. The experiment shown in A was performed three times and the data obtained pooled. Eggs were also transferred in 0Na or Am. % of divided eggs (a), embryos (b) or fragmented eggs (c) were determined 3 hours after fertilization (filled histograms) except for divided control eggs that were counted 80 mins after sperm addition (hatched histograms). 42–78 total embryos were counted in each condition. Values (means +/− sem) obtained in the presence of U0126 (grey histogram) are significantly different (student test, p<0.01, two stars) or not (ns) to those obtained without this drug (black histogram).
	Figure 5. Cai and pHi depend on the presence of external HCO3–.A. Observation 80 mins after fertilization. Eggs develop in ASW (Control) or are transferred 20 mins after fertilization into ASW deprived of Ca (0Ca), HCO3 − (0HCO3) or of Na+ and Ca++ (0Na0Ca). B. Time course after fertilization of pHi (a) and Cai (b) in ASW, 0Na and 0Na0Ca in the presence or not of HCO3 −. Examples of results obtained in absence of HCO3-, i.e. in 0HCO3 (1), 0Na0HCO3 (2) and 0Na0Ca0HC03 (3), are depicted in different grey colours and representative of n determinations (grey number in brackets). In each condition, one typical control experiment performed with the same population of eggs in the presence of HCO3 −, i.e. in ASW (1), 0Na (2) and 0Na0Ca (3) is shown in black curve and representative of n determinations (black number in brackets).
	Figure 6. Na/Ca inhibitors reduce inhibition of mitosis induced in the absence of external Na.Eggs were let to develop in ASW (Control) or transferred 20 min after fertilization in 0Na containing or not (0Na) the different inhibitors, BHC, SN6 or KB. Observation 80 min after fertilization (A) and compiled assessment of results (means +/− sem) from 3 experiments that gave similar results (B). Total number of eggs counted in each condition is indicated in brackets in B.

References [+] :

Amirand, Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis. 2001, Pubmed