Sun L and Machaca K (2004), Ca(2+)(cyt) negatively regulates the initiation...

XB-ART-3737

J Cell Biol 2004 Apr 01;1651:63-75. doi: 10.1083/jcb.200309138.

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Ca(2+)(cyt) negatively regulates the initiation of oocyte maturation.

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Ca(2+) is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca(2+) in mitosis. In contrast, there has been a long-standing debate as to whether Ca(2+) signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca(2+) (Ca(2+)(cyt)) plays a dual role during Xenopus oocyte maturation. Ca(2+) signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca(2+)(cyt) signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca(2+)-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca(2+)(cyt) acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca(2+)(cyt) delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca(2+) modulates both the cell cycle machinery and nuclear maturation during meiosis.

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Species referenced: Xenopus laevis
Genes referenced: ccnb1 cdk1 mapk1 mos tmem11 tub

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	Figure 1. Role of extracellular and store Ca2+ load in GVBD. (A) Oocytes were incubated in l-15 containing the indicated Ca2+ concentrations, stimulated with progesterone, and GVBD scored every half hour. (B) Cells were incubated in media with different calcium concentrations: low Ca2+ (L-Ca) 50 μM Ca2+; normal Ca2+ (N-Ca) 0.6 mM Ca2+; and high Ca2+ (H-Ca) 5 mM Ca2+, and treated with either 2 μM thapsigargin for 3 h (Thap) or 10 μM ionomycin for 5 min (Ion) before progesterone addition. Control (Con) refers to cells incubated N-Ca and stimulated with progesterone. For the treatments described no GVBD was observed in the absence of progesterone, indicating that the different manipulation are not sufficient to induce maturation. (C) The rate of oocyte maturation, quantified as the time at which 50% of the oocytes have undergone GVBD (GVBD50). The GVBD50 for each indicated treatment were normalized to GVBD50 in the N-Ca control. (D) Maximal percentage of cells that undergo GVBD for the different treatments. (C and D) Asterisks indicate significantly different means (P ≤ 0.0146, n = 6). (E and F) Prolonged Ca2+ store depletion does not inhibit GVBD. Oocytes were incubated with or without 2 μM thapsigargin for the times indicated (from 3 to 48 h) before progesterone addition. The rate of maturation (GVBD50) and maximal GVBD are shown. For all experiments GVBD was directly confirmed by fixing and bisecting oocytes. Error bars are SEM.
	Figure 2. Buffering Ca2+cyt with BAPTA accelerates meiosis entry. (A) GVBD time course of BAPTA-injected cells. Oocytes were incubated in L-Ca medium and treated in one of the following ways: injected with 500 μM BAPTA (BAPTA); incubated in 2 μM thapsigargin for 3 h (Thaps); or treated with both BAPTA and thapsigargin (Thaps-BAPTA) in the order and for the times indicated. The control group (Con) refers to 5 μg/ml of progesterone alone. (B and C) The rate of maturation (B, GVBD50), and maximal GVBD (C) are shown for the different treatments. Letter designations refer to significantly different means (P < 0.018, n = 5).
	Figure 3. Effects of high Ca2+cyt on meiosis entry. (A and B) GVBD time course of oocytes incubated in media containing increasing Ca2+ concentrations: 50 μM, 0.6 mM, 1.5 mM, 3 mM, and 5 mM for L-Ca, N-Ca, H1.5-Ca, H3-Ca, and H5-Ca, respectively, with (B) or without (A) thapsigargin (T) treatment (2 μM, 3 h). Oocytes with high Ca2+ (3 and 5 mM) had a diffuse white spot on the animal pole, but when fixed and cut the nucleus was present although flattened and close to the membrane. In addition, oocyte degeneration was observed in these treatments, which partly contributes to lower GVBD levels. No degeneration was seen in 1.5 mM Ca2+ (H1.5-Ca). (C and D) Relative GVBD50 and maximal GVBD for the different treatments. For GVBD50 (C) the letters above the bars indicate significantly different means (P < 0.05, n = 3). Oocytes in H3-Ca2+ and H5-Ca2+ are excluded from this analysis because they rarely reach GVBD50. For maximal GVBD (D) the asterisk indicates significantly different means (P < 0.0155, n = 3).
	Figure 4. ICa,Cl as marker for Ca2+cyt levels. ICa,Cl was recorded from control cells (untreated), cells treated with thapsigargin (2 μM, 3 h), or injected with 500 μM BAPTA to estimate store Ca2+ load and the levels of Ca2+ influx. ICa,Cl provide endogenous reporters of Ca2+ release from stores (ICl1) and Ca2+ influx from the extracellular space (IClT) as described in the text. (A) Voltage protocol and representative current traces of the Ica,Cl. ICl1 is a sustained current recorded upon depolarization to +40 mV (trace t), whereas IClT is a transient current detected only when the +40 mV pulse is preceded by a hyperpolarization step to −140 mV (traces x–z). Note that at high 5 mM Ca2+ levels, Ca2+ influx at −140 mV activates an inward Cl− current (trace z). The current traces shown are from the control oocyte in B. The time at which each trace was obtained is indicated in B. (B–G) Oocytes were incubated in Ca2+-free Ringer (F-Ca) and treated with ionomycin to release store Ca2+. The levels of ICl1 induced in response to ionomycin provide a measure of store Ca2+ load. ICl1 (squares) is plotted as the maximal current at the end of the +40 mV pulse as indicated by the arrow in A (left). After store depletion oocytes were sequentially exposed to solutions containing the indicated Ca2+ concentration: L (50 μM Ca2+), N (0.6 mM Ca2+), H1.5 (1.5 mM Ca2+), H3 (3 mM Ca2+), and H5 (5 mM Ca2+). Store depletion activates Ca2+ influx through the SOCE pathway, which activates IClT. IClT (circles) is plotted as the maximal current during the second +40 mV pulse as indicated by the arrow in A (right). B, D, and F show the time course of ICl1 and IClT in control, thapsigargin, and BAPTA-treated cells, respectively. The time of solution changes and ionomycin (Ion.) addition are indicated above each panel. C, E, and G show statistical analysis of ICl1 and IClT. ICl1 levels were significantly different in the three treatments (P ≤ 0.0041, n = 5–7). No ICl1 was detected in the thapsigargin treatment indicating complete Ca2+ store depletion. For IClT in each panel the asterisks indicate significantly different means: (C) Con, P ≤ 0.015, n = 5; (E) Thaps, P ≤ 0.00012, n = 7; (G) BAPTA, P = 0.00022, n = 6.
	Figure 5. MPF and MAPK kinetics. (A) GVBD time course for oocytes incubated in N-Ca, L-Ca, or treated with 2 μM of thapsigargin for 3 h or injected with 500 μM BAPTA. (B and C) Phospho-MAPK (P-MAPK; B) and MPF activity (C) in the different treatments. At each time point as indicated, lysates were assayed for P-MAPK levels using an anti–P-MAPK specific antibody, and for MPF levels measured as histone H1 kinase activity. MAPK and MPF assays were performed on the same lysate. For P-MAPK Western blots were also probed for α-tubulin (Tub) to provide a loading control. The time course is divided into two phases: (1) after progesterone addition and (2) after GVBD. The 50 and 37 molecular weight markers are shown on the left of each gel. These data are representative of three similar experiments.
	Figure 6. Spindle structure. Oocytes were stained for tubulin and DNA with Sytox orange to visualize spindle structure. (A) The treatment designation for N-Ca, L-Ca, Thaps, and BAPTA is as indicated in Fig. 5. P refers to cells in late prophase with clustering of the microtubules and chromosome condensation. PMI, prometaphase I; MI, metaphase I; A, Anaphase I; PMII, prometaphase II; MII, metaphase II; EP, early prophase with chromosome condensation but microtubules still spread over a large area; Ab, abnormal spindle structure; PM-L, prometaphase-like spindle with short or slightly disorganized structure; DS, double spindle with two overlapping spindles. (B and C) Polar body extrusion. (B) Representative images showing an oocyte with a polar body (PB) matured in N-Ca medium 3 h after GVBD (top); and an oocyte matured in L-Ca medium 3 h after GVBD, with no polar body (bottom). (C) Normalized polar body emission levels from 2–4 h after GVBD in the different treatments: N-Ca (0.6 mM); L-Ca (50 μM); thapsigargin (Thaps, 2 μM for 3 h); injected with 500 μM BAPTA (BAPTA); incubated in N-Ca until GVBD and then switched to L-Ca medium (N-L); or incubated in L-Ca until GVBD and then switched to N-Ca (L-N). Asterisks above the bar indicate significantly different means from N-Ca and N-L (P < 0.002; n = 3). Bars, 10 μm.
	Figure 7. Mapping the site of action of Ca2+cyt on meiosis entry. (A) GVBD time course for oocytes incubated in N-Ca solution (N), or L-Ca, and treated with thapsigargin (T). Oocyte maturation was induced with progesterone (N and T), PKI (PKI), Mos RNA 10 ng (Mos), cyclin B1 RNA 10 ng (Cy), or Δ87cyclin B1 protein ∼200 pg (CyP). (B) Relative time to GVBD50 normalized to GVBD50 in the N-Ca for each activator. P values are shown above the graph, n = 4–7. (C) Biochemical analysis of the cell cycle machinery. Maturation was induced as described in A and lysates were collected from immature oocytes (ooc), at hourly intervals after progesterone addition as indicated, at first GVBD (GVBD) and at GVBD50 (G50). For the GVBD50 time point we collected lysates form oocytes that have undergone GVBD (white spot, w) and those that have not (no white spot, nw). (D) Working model for the role of Ca2+cyt in oocyte maturation/meiosis (see text for details). Dashed arrows indicated yet unknown steps in the cascade. The change in arrow shape and the bold font indicate transitions between the different stages of meiosis. M I, meiosis I; PB, polar body emission; M II, meiosis II.

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