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<!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.0 20120330//EN" "JATS-archivearticle1.dtd">
<article xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" article-type="research-article"><?properties open_access?><front><journal-meta><journal-id journal-id-type="nlm-ta">J Cell Biol</journal-id><journal-id journal-id-type="iso-abbrev">J. Cell Biol</journal-id><journal-title-group><journal-title>The Journal of Cell Biology</journal-title></journal-title-group><issn pub-type="ppub">0021-9525</issn><issn pub-type="epub">1540-8140</issn><publisher><publisher-name>The Rockefeller University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">11781335</article-id><article-id pub-id-type="pmc">PMC1307503</article-id><article-id pub-id-type="publisher-id">0110059</article-id><article-id pub-id-type="doi">10.1083/jcb.200110059</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Induction of maturation-promoting factor during <italic>Xenopus</italic> oocyte maturation uncouples Ca<sup>2+</sup> store depletion from store-operated Ca<sup>2+</sup> entry</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Machaca</surname><given-names>Khaled</given-names></name></contrib><contrib contrib-type="author"><name><surname>Haun</surname><given-names>Shirley</given-names></name></contrib></contrib-group><author-notes><fn><p>Address correspondence to Khaled Machaca, Ph.D., Department of Physiology, Slot 505, 4301 West Markham St., University of Arkansas Medical Science, Little Rock, AR 72205. Tel.: (501) 603-1596. Fax: (501) 686-8167. E-mail: <email>machacakhaleda@uams.edu</email>
</p></fn></author-notes><pub-date pub-type="ppub"><day>7</day><month>1</month><year>2002</year></pub-date><volume>156</volume><issue>1</issue><fpage>75</fpage><lpage>86</lpage><history><date date-type="received"><day>11</day><month>10</month><year>2001</year></date><date date-type="rev-recd"><day>15</day><month>11</month><year>2001</year></date><date date-type="accepted"><day>19</day><month>11</month><year>2001</year></date></history><permissions><copyright-statement>Copyright © 2002, The Rockefeller University Press</copyright-statement><license license-type="openaccess"><license-p>This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see <ext-link ext-link-type="uri" xlink:href="http://www.rupress.org/terms">http://www.rupress.org/terms</ext-link>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/4.0/">http://creativecommons.org/licenses/by-nc-sa/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Department of Physiology and Biophysics, University of Arkansas Medical Science, Little Rock, AR 72205</p><p>During oocyte maturation, eggs acquire the ability to generate specialized Ca<sup>2+</sup> signals in response to sperm entry. Such Ca<sup>2+</sup> signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during <italic>Xenopus</italic> oocyte maturation of store-operated Ca<sup>2+</sup> entry (SOCE), an important Ca<sup>2+</sup> influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during <italic>Xenopus</italic> oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos–mitogen-activated protein kinase (MAPK)–maturation-promoting factor (MPF) kinase cascade, which drives <italic>Xenopus</italic> oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca<sup>2+</sup> influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.</p></abstract><kwd-group><kwd>calcium signaling; store-operated Ca<sup>2+</sup> entry; oocyte maturation; <italic>Xenopus laevis</italic>; maturation-promoting factor</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>Sexual reproduction requires germ cell fusion, and correct execution of the developmental program. In many species, including mammals and amphibians, fully-grown oocytes in the ovary are not able to be fertilized or support embryonic development, unless they undergo oocyte maturation. Oocyte maturation corresponds to a complex differentiation program that transforms the oocyte into an egg that is fertilization competent, and capable of supporting early embryonic development (<xref rid="bib48" ref-type="bibr">Yamashita et al., 2000</xref>).</p><p>The amphibian oocyte represents one of the best-characterized models for oocyte maturation, at a biochemical as well as morphological level (<xref rid="bib1" ref-type="bibr">Bement and Capco, 1990</xref>). In <italic>Xenopus laevis</italic>, fully grown oocytes (stage VI) in the ovary are arrested at prophase of meiosis I. During oocyte maturation, cells enter meiosis, undergo germinal vesicle (nuclear envelope) breakdown (GVBD),<xref ref-type="fn" rid="fn1">*</xref> complete meiosis I with the extrusion of a polar body, and arrest at metaphase of the second meiotic division (MII) until fertilization (<xref rid="bib25" ref-type="bibr">Masui and Clarke, 1979</xref>). We will refer to mature cells arrested at MII as eggs, to differentiate them from immature oocytes arrested at prophase I. <italic>Xenopus</italic> oocyte maturation is initiated in response to the hormone progesterone, which induces a signal transduction cascade culminating in the activation of maturation-promoting factor (MPF) (for recent review see <xref rid="bib30" ref-type="bibr">Nebreda and Ferby, 2000</xref>). MPF is a complex, composed of a serine/threonine kinase (p34<sup>cdc2</sup>) subunit and a regulatory cyclin B subunit. MPF activation is necessary and sufficient for GVBD and entry into meiosis (<xref rid="bib43" ref-type="bibr">Smith, 1989</xref>). Progesterone-induced maturation requires translation of Mos kinase, which induces the MAP kinase cascade, resulting in MPF activation (<xref rid="bib39" ref-type="bibr">Sagata, 1997</xref>). In addition to the Mos–mitogen-activated protein kinase (MAPK) cascade, other signaling cascades are activated in parallel, resulting in rapid MPF induction. The polo-like kinase cascade is thought to activate Cdc25C phosphatase, which dephosphorylates and activates MPF (<xref rid="bib35" ref-type="bibr">Qian et al., 1999</xref>, <xref rid="bib36" ref-type="bibr">2001</xref>).</p><p>An important component of oocyte maturation is differentiation of the Ca<sup>2+</sup> signaling machinery, which is essential for egg activation after fertilization. An increase in cytoplasmic Ca<sup>2+</sup> at fertilization is the universal signal to initiate development in all species investigated (<xref rid="bib12" ref-type="bibr">Homa et al., 1993</xref>; <xref rid="bib45" ref-type="bibr">Stricker, 2000</xref>). Ca<sup>2+</sup> signals generated at fertilization in <italic>Xenopus</italic> have specific spatial and temporal features that are presumably important for successful egg activation. For example, Ca<sup>2+</sup> signals are crucial for the block to polyspermy (<xref rid="bib4" ref-type="bibr">Cross, 1981</xref>; <xref rid="bib16" ref-type="bibr">Kline and Nuccitelli, 1985</xref>), through the induction of membrane depolarization (<xref rid="bib23" ref-type="bibr">Machaca et al., 2001</xref>) and the activation of cortical granule fusion (<xref rid="bib47" ref-type="bibr">Wolf, 1974</xref>; <xref rid="bib11" ref-type="bibr">Grey et al., 1976</xref>). Increased Ca<sup>2+</sup> is also responsible for releasing the MII block by inactivating cytostatic factor (CSF) (<xref rid="bib26" ref-type="bibr">Meyerhof and Masui, 1979</xref>). Therefore, during oocyte maturation, Ca<sup>2+</sup> signaling pathways undergo their own differentiation followed by functionally significant consequences. In this study, we investigate the regulation of store-operated Ca<sup>2+</sup> entry (SOCE) during <italic>Xenopus</italic> oocyte maturation. SOCE is a ubiquitous Ca<sup>2+</sup> entry pathway in nonexcitable cells, activated in response to depletion of intracellular Ca<sup>2+</sup> stores (<xref rid="bib32" ref-type="bibr">Parekh and Penner, 1997</xref>). SOCE is important for a variety of physiological functions, including regulation of exocytosis (<xref rid="bib18" ref-type="bibr">Koizumi and Inoue, 1998</xref>; <xref rid="bib8" ref-type="bibr">Fomina and Nowycky, 1999</xref>), modulation of Ca<sup>2+</sup> waves (<xref rid="bib10" ref-type="bibr">Girard and Clapham, 1993</xref>; <xref rid="bib6" ref-type="bibr">Dolmetsch and Lewis, 1994</xref>), and activation of T lymphocytes (<xref rid="bib7" ref-type="bibr">Fanger et al., 1995</xref>; <xref rid="bib41" ref-type="bibr">Serafini et al., 1995</xref>). The mechanism by which Ca<sup>2+</sup> store depletion activates SOCE is controversial. Three primary models have been proposed with experimental evidence for each: direct physical coupling between SOCE channels and a Ca<sup>2+</sup> sensor (<xref rid="bib2" ref-type="bibr">Berridge, 1995</xref>; <xref rid="bib15" ref-type="bibr">Kiselyov et al., 1998</xref>); activation of SOCE channels by a diffusible messenger (<xref rid="bib37" ref-type="bibr">Randriamampita and Tsien, 1993</xref>; <xref rid="bib5" ref-type="bibr">Csutora et al., 1999</xref>); and exocytotic insertion of SOCE channels in the plasma membrane (<xref rid="bib44" ref-type="bibr">Somasundaram et al., 1995</xref>; <xref rid="bib49" ref-type="bibr">Yao et al., 1999</xref>).</p><p>We have previously demonstrated that SOCE inactivates during <italic>Xenopus</italic> oocyte maturation (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>), and were interested in elucidating the mechanisms mediating this inactivation. In this study, we show that the induction of MPF is necessary for SOCE inactivation. In contrast, the activation of kinase cascades upstream of MPF does not affect SOCE. However, MPF does not appear to inhibit Ca<sup>2+</sup> influx through SOCE channels, rather it blocks the coupling mechanism between store depletion and SOCE activation.</p></sec><sec><title>Results</title><sec><title>Levels of SOCE during progesterone-induced oocyte maturation</title><p>Depletion of intracellular Ca<sup>2+</sup> stores activates SOCE in stage VI <italic>Xenopus</italic> oocytes (<xref rid="fig1" ref-type="fig">Fig. 1</xref> B). The SOCE current (I<sub>SOCE</sub>) was measured as previously described (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>), using a ramp voltage stimulation from −140mV to +60mV (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A). Current at −140mV (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A, arrow) is plotted over time to visualize the activation of I<sub>SOCE</sub> after Ca<sup>2+</sup> store depletion (<xref rid="fig1" ref-type="fig">Fig. 1</xref> B). Depletion of intracellular Ca<sup>2+</sup> stores in eggs induced to mature with progesterone, did not activate SOCE (<xref rid="fig1" ref-type="fig">Fig. 1</xref> C). A representative current–voltage (I–V) relationship from an oocyte reveals the typical inward rectifying I–V relationship that is a signature of I<sub>SOCE</sub> (<xref rid="fig1" ref-type="fig">Fig. 1</xref> D). In contrast, the I–V relationship in an egg is flat, indicating that store depletion does not activate SOCE in mature eggs (<xref rid="fig1" ref-type="fig">Fig. 1</xref> D).</p><fig id="fig1" position="float"><label>Figure 1.</label><caption><p>
<bold>I<sub>SOCE</sub> during <italic>Xenopus</italic> oocyte maturation.</bold> (A) Voltage protocol used to measure I<sub>SOCE</sub> development and I–V relationship. The membrane potential was stepped to −140 mV (100 ms), and then ramped from −140 to +60 mV (2 s) from a −40 mV holding potential. The protocol was applied once every 30 s. (B and C) I<sub>SOCE</sub> development after Ca<sup>2+</sup> store depletion with ionomycin (10 μM) in an oocyte (B) and an egg (C). Cells were injected with 7 nmol BAPTA to allow for I<sub>SOCE</sub> recording (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>). The current at −140 mV (A, arrow) was plotted over time. La<sup>3+</sup> (100 μM) was added at the end of the experiment to block SOCE and obtain a measure of absolute I<sub>SOCE</sub>. (D) Representative I–V relationships obtained from the ramp voltage stimulation shown in A from an oocyte and an egg. I–V relationships were obtained by subtracting the current after La<sup>3+</sup> addition from the current before addition. (E) Time course of progesterone-mediated GVBD. Progesterone was added to a population of at least 50 oocytes, and the occurrence of GVBD over time was recorded by following white spot appearance on the animal pole. The data shown represent the average ± SE from seven donor females. The time of GVBD<sub>i</sub> ± SD is also shown. GVBD<sub>i</sub> represents the time at which GVBD was first observed in the population. (F and G) I<sub>SOCE</sub> levels from individual oocytes at different times during oocyte maturation. Each square represents the current from a single cell. I<sub>SOCE</sub> data from oocytes are shown before progesterone addition. The data was normalized to average current in oocytes. I<sub>SOCE</sub> data from 80 cells at different times during maturation are shown in F. (G) I<sub>SOCE</sub> levels from cells incubated in progesterone without undergoing GVBD (<italic>n</italic> = 63). The average time ± SD of GVBD<sub>i</sub> is shown for reference.</p></caption><graphic xlink:href="0110059f1"/></fig><p>The time course of oocyte maturation varies between different females. When comparing data from different donors it is helpful to normalize to the time at which 50% of the oocyte population undergo GVBD (GVBD<sub>50</sub>) (<xref rid="fig1" ref-type="fig">Fig. 1</xref> E). GVBD is scored based on the appearance of a white spot on the animal pole due to pigment dispersal (<xref rid="bib43" ref-type="bibr">Smith, 1989</xref>). In these experiments (<xref rid="fig1" ref-type="fig">Fig. 1</xref>, E–G), GVBD<sub>50</sub> was reached 5.6 ± 0.4 h after progesterone addition (<italic>n</italic> = 7). The initial appearance of GVBD in the population (GVBD<sub>i</sub>) was reached 3.8 ± 0.2 h after progesterone addition (<italic>n</italic> = 7) (<xref rid="fig1" ref-type="fig">Fig. 1</xref> E). We measured I<sub>SOCE</sub> at different time points during the maturation process (<xref rid="fig1" ref-type="fig">Fig. 1, F and G</xref>). Before GVBD, there was no significant change in I<sub>SOCE</sub> levels in maturing cells as compared with untreated oocytes (<xref rid="fig1" ref-type="fig">Fig. 1</xref> F). However, I<sub>SOCE</sub> abruptly inactivated as soon as GVBD was observed, and remained inactivated for the remainder of the maturation process (<xref rid="fig1" ref-type="fig">Fig. 1</xref> F). Therefore, once maturing oocytes reach the GVBD stage, and for the rest of the maturation period, SOCE can no longer be activated by store depletion. We have previously shown that this acute inactivation of SOCE does not correlate with the gradual decrease in membrane surface area observed during oocyte maturation. Nor is it due to cortical granule fusion upon Ca<sup>2+</sup> store depletion, which releases proteases at the cell surface (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>).</p><p>We also examined I<sub>SOCE</sub> levels in oocytes incubated in progesterone for prolonged periods of time, without undergoing GVBD (GVBD negative) (<xref rid="fig1" ref-type="fig">Fig. 1</xref> G). In the majority of such cells (34/44), I<sub>SOCE</sub> was detected at levels similar to those in control oocytes (<xref rid="fig1" ref-type="fig">Fig. 1</xref> G), indicating a correlation between I<sub>SOCE</sub> inactivation and GVBD. However, there was a trend for decreased levels of I<sub>SOCE</sub> at time points longer than 0.8 GVBD<sub>50</sub> (<xref rid="fig1" ref-type="fig">Fig. 1</xref> G). GVBD is followed by white spot appearance, indicating completion of nuclear envelope breakdown. Nonetheless, GVBD initiates before white spot appearance as the nuclear envelope breaks down gradually, in a polarized fashion beginning at the vegetal face of the nucleus (<xref rid="bib3" ref-type="bibr">Colman et al., 1985</xref>). Therefore, cells in which I<sub>SOCE</sub> activity was decreased could have initiated GVBD, consistent with a temporal correlation between GVBD and I<sub>SOCE</sub> inactivation.</p><p>I<sub>SOCE</sub> inactivation could be a direct downstream consequence of GVBD. To test this possibility, we removed the nucleus from oocytes (enucleated), and matured them in progesterone. We recorded I<sub>SOCE</sub> from enucleated cells when 100% of the control oocyte group underwent GVBD. Under these conditions in both control and enucleated cells, I<sub>SOCE</sub> was completely inactivated (unpublished data). Furthermore, enucleated cells incubated for the same time in the absence of progesterone had I<sub>SOCE</sub> levels similar to those of untreated oocytes, indicating that removal of the nucleus did not lead to I<sub>SOCE</sub> inactivation. Thus, physical breakdown of the nuclear envelope is not a prerequisite for I<sub>SOCE</sub> inactivation.</p></sec><sec><title>Time course of Mos, MAPK, and MPF activation during oocyte maturation</title><p>As discussed above, oocyte maturation is driven by kinase cascades that lead to MPF activation and entry into meiosis. We next examined whether these kinases are involved in I<sub>SOCE</sub> inactivation. We focused our attention on the Mos–MAPK signal transduction cascade, which is the primary pathway for MPF activation in <italic>Xenopus</italic> oocytes (<xref rid="bib31" ref-type="bibr">Palmer and Nebreda, 2000</xref>). <xref rid="fig2" ref-type="fig">Fig. 2</xref> summarizes the time course of Mos, MAPK, and MPF activation during progesterone-induced oocyte maturation. In this experiment, GVBD<sub>i</sub> occurred at 5.5 h, and GVBD<sub>50</sub> was reached ∼7 h after progesterone addition (<xref rid="fig2" ref-type="fig">Fig. 2</xref> A). MPF activity increased at GVBD<sub>i</sub> and then decreased slightly 30 min after GVBD, indicative of the transition between meiosis I and II. This was followed by a plateau phase of high MPF activity for the reminder of maturation (<xref rid="fig2" ref-type="fig">Fig. 2</xref> B). MAPK activation could be followed with a specific antibody that recognized the phosphorylated (activated) form of the kinase (<xref rid="fig2" ref-type="fig">Fig. 2</xref> C). Phospho-MAPK (P-MAPK) was first detected 3 h after progesterone addition, and increased slowly with a jump at GVBD (<xref rid="fig2" ref-type="fig">Fig. 2</xref> C). Mos kinase levels rose to a low level plateau 2 h after progesterone addition (<xref rid="fig2" ref-type="fig">Fig. 2</xref> D), before any increase in P-MAPK was observed. This is consistent with Mos's role as the upstream kinase that activates the MAPK cascade (<xref rid="bib39" ref-type="bibr">Sagata, 1997</xref>). Mos protein levels remained at the low plateau level until 30 min after GVBD, when a significant increase was observed (<xref rid="fig2" ref-type="fig">Fig. 2</xref> D). This is presumably due to the activation of MPF, which stabilizes the Mos protein (<xref rid="bib39" ref-type="bibr">Sagata, 1997</xref>). As shown in <xref rid="fig1" ref-type="fig">Fig. 1</xref>, SOCE inactivated at the GVBD stage of meiosis, at the same time that MPF and P-MAPK levels increased (<xref rid="fig2" ref-type="fig">Fig. 2, B and C</xref>). It is therefore possible that either MPF, MAPK, or both are involved in SOCE inactivation.</p><fig id="fig2" position="float"><label>Figure 2.</label><caption><p>
<bold>Time course of MPF, MAPK, and Mos activation during progesterone-induced maturation.</bold> (A) Time course of GVBD. GVBD<sub>i</sub> occurred at 5.5 h, and GVBD<sub>50</sub> ∼7.25 h after progesterone addition. At each time point, five cells were pooled to quantify kinase activity. (B–D) MPF, P-MAPK, and Mos levels during oocyte maturation. MPF activity was measured as the histone H1 kinase activity as described in Materials and methods. The levels of P-MAPK and Mos were determined by Western blot analysis using a P-MAPK–specific and anti-Mos antibody, respectively, as described in Materials and methods. In the case of MAPK, the same blot was reprobed with an antibody that detects total MAPK (T-MAPK) levels, showing that MAPK was present in the lysates but was not phosphorylated. To be able to compare P-MAPK and Mos levels between different experiments, we always ran a positive control lysate from eggs on every gel, and normalized P-MAPK and Mos levels to that sample. Therefore, Western data for P-MAPK and Mos throughout this manuscript are normalized to the same egg lysate (see Materials and methods for more details). The time scale is divided into two phases: after progesterone addition; and after GVBD. The 35-kD molecular mass marker is shown on the right of the gels. (E) MPF, P-MAPK, and Mos levels in cells treated with progesterone, but that did not undergo GVBD. GVBD<sub>i</sub> is indicated for reference. This time course of kinase activation is representative of five similar experiments.</p></caption><graphic xlink:href="0110059f2"/></fig><p>We have also determined the time course of activation of these kinases in cells treated with progesterone but that have not undergone GVBD (<xref rid="fig2" ref-type="fig">Fig. 2</xref> E). In this population, MPF activity started to increase past the GVBD<sub>i</sub> time point, eventually reaching levels similar to those observed in GVBD positive cells (<xref rid="fig2" ref-type="fig">Fig. 2</xref> E). P-MAPK levels also increased gradually after GVBD<sub>i</sub>, whereas Mos protein remained at the preGVBD plateau level (<xref rid="fig2" ref-type="fig">Fig. 2</xref> E). Increased MPF and MAPK activity in GVBD negative cells, is consistent with one or both kinases being involved in SOCE inactivation. This is because SOCE levels are lower in oocytes at this stage of maturation (<xref rid="fig1" ref-type="fig">Fig. 1</xref> G).</p></sec><sec><title>SOCE inactivates independently of the MAPK cascade</title><p>The time course of I<sub>SOCE</sub> inhibition and kinase activation during progesterone-induced maturation (<xref rid="fig1" ref-type="fig">Figs. 1</xref> and <xref rid="fig2" ref-type="fig">2</xref>), suggest that the MAPK cascade and/or MPF could be involved in SOCE inactivation. To directly test this possibility we manipulated the levels of MAPK and MPF, and determined the effect on SOCE. This was accomplished by injecting cyclin B1 protein in the presence or absence of a mitogen-activated protein kinase kinase (MEK) inhibitor (<xref rid="fig3" ref-type="fig">Fig. 3</xref>). MEK is the upstream kinase that phosphorylates and activates MAPK (<xref rid="bib31" ref-type="bibr">Palmer and Nebreda, 2000</xref>). Cyclin B1 binds and activates p34<sup>cdc2</sup>, the catalytic subunit of MPF, thus rapidly increasing MPF activity. Cyclin B1 injections have been shown to induce GVBD in the absence of progesterone (<xref rid="bib9" ref-type="bibr">Freeman et al., 1991</xref>).</p><fig id="fig3" position="float"><label>Figure 3.</label><caption><p>
<bold>Correlation between I</bold>
<sub>SOCE</sub>
<bold>and MPF and P-MAPK levels after Δ87cyclin B1 protein injection.</bold> Oocytes were either directly injected with Δ87cyclin B1 protein (A and B) or preincubated in the MEK inhibitor PD98059 (50–100 μM) for 0.5–2 h before Δ87cyclin B1 injection. I<sub>SOCE</sub> levels were recorded at various times after Δ87cyclin B1 injection. After I<sub>SOCE</sub> recording, individual cells were lysed and assayed for MPF and P-MAPK levels. This allowed us to obtain I<sub>SOCE</sub> (squares), MPF (circles), and P-MAPK (triangles) levels from the same oocyte. The data for individual cells are connected by drop lines to help in matching I<sub>SOCE</sub> and kinase activity from the same cell. For Δ87cyclin B1 and PD98059-Δ87cyclin B1 injections, current and kinase data from 40 and 33 cells, respectively, are shown. I<sub>SOCE</sub> levels were normalized to the levels in untreated oocytes, and MPF and MAPK levels were normalized to the levels found in fully mature eggs, as described in <xref rid="fig2" ref-type="fig">Fig. 2</xref>. The time scale was normalized to the time at which GVBD initially appeared in the population (GVBD<sub>i</sub>) after Δ87cyclin B1 injection. The standard deviation of GVBD<sub>i</sub> is shown in A and B (<italic>n</italic> = 7). PD98059-treated cells are plotted on a time scale normalized to GVBD<sub>i</sub> in Δ87cyclin B1–injected cells (A and B). Individual cell data were fitted with a Boltzman function to obtain a general trend of I<sub>SOCE</sub> inactivation and the activation of the different kinases.</p></caption><graphic xlink:href="0110059f3"/></fig><p>In these experiments we wanted to correlate the levels of kinase activity with I<sub>SOCE</sub>. However, the asynchronous nature of <italic>Xenopus</italic> oocyte maturation introduced significant variability, making such correlations difficult to draw. To circumvent this problem, we performed these studies on single oocytes, i.e., we recorded I<sub>SOCE</sub> levels and MPF and MAPK activity from individual cells. This allowed us to directly correlate the activity of these kinases with I<sub>SOCE</sub> levels in the same oocyte. The data is plotted over a time scale normalized to GVBD<sub>i</sub>, to determine whether there is a correlation between I<sub>SOCE</sub> inactivation and GVBD. Injection of cyclin B1 protein into oocytes induced GVBD with an average time of 2 ± 0.4 h (<italic>n</italic> = 7 donor females). As is the case after progesterone treatment, I<sub>SOCE</sub> was completely inactivated in cells that had undergone GVBD in response to cyclin B1 injection. However, the kinetics of MPF and I<sub>SOCE</sub> inactivation were more complex (<xref rid="fig3" ref-type="fig">Fig. 3</xref> A). Shortly after cyclin B1 injection, MPF activity increased to levels similar to those observed in eggs, and eventually plateaued at MPF levels two- to fourfold those in eggs (<xref rid="fig3" ref-type="fig">Fig. 3</xref> A). The time course of I<sub>SOCE</sub> inactivation lagged behind that of MPF activity (<xref rid="fig3" ref-type="fig">Fig. 3</xref> A). P-MAPK levels increased significantly only after GVBD<sub>i</sub>, when I<sub>SOCE</sub> was completely inactivated (<xref rid="fig3" ref-type="fig">Fig. 3</xref> B). I<sub>SOCE</sub> began to inactivate before P-MAPK became detectable (<xref rid="fig3" ref-type="fig">Fig. 3</xref> B), but complete inactivation of I<sub>SOCE</sub> correlated with increased P-MAPK levels.</p><p>The time delay between MPF activation and I<sub>SOCE</sub> inactivation was also evident for GVBD, which is a known target of MPF (<xref rid="bib1" ref-type="bibr">Bement and Capco, 1990</xref>). Therefore, ectopic activation of MPF by cyclin B1 injection did not faithfully reproduce the downstream effects of progesterone-induced MPF activation. In the case of progesterone, an increase in MPF levels coincided with GVBD (<xref rid="fig2" ref-type="fig">Fig. 2</xref> B). The reason for the lag period between MPF activation by cyclin B1 and GVBD is not clear to us. We suggest that it could be due to mislocalization of MPF after cyclin B1 injection, thus delaying MPF action on downstream targets.</p><p>To directly test whether MAPK activation is required for I<sub>SOCE</sub> inactivation we treated cells with the MEK inhibitor PD98059, which blocks MAPK activation. This allowed us to obtain a population of cells containing high MPF activity, without activating the MAPK cascade (<xref rid="fig3" ref-type="fig">Fig. 3, C and D</xref>). Injection of cyclin B1 into PD98059-treated oocytes induced the activation of MPF (<xref rid="fig3" ref-type="fig">Fig. 3</xref> C) with a similar time course and to comparable levels as control oocytes (<xref rid="fig3" ref-type="fig">Fig. 3</xref> A). In this case there also was a strong temporal correlation between GVBD and I<sub>SOCE</sub> inactivation. Pretreating oocytes with PD98059 delayed but did not completely block MAPK activation, as some cells at later time points showed high levels of P-MAPK (<xref rid="fig3" ref-type="fig">Fig. 3</xref> D). However, we could easily define a population of cells, between 0.9 and 1.6 GVBD<sub>i</sub>, in which MAPK activity was completely blocked by PD98059 (<xref rid="fig3" ref-type="fig">Fig. 3</xref> D, box). All of these cells had high levels of MPF activity (one- to fourfold that in eggs) (<xref rid="fig3" ref-type="fig">Fig. 3</xref> C, box). Such cells with high MPF activity and no detectable P-MAPK had completely inactivated I<sub>SOCE</sub> (<xref rid="fig3" ref-type="fig">Fig. 3, C and D</xref>). This indicates that activation of the MAPK cascade is not required for I<sub>SOCE</sub> inactivation.</p><p>
<xref rid="fig4" ref-type="fig">Fig. 4</xref> shows examples of single cells illustrating the need for MPF, but not MAPK, activation for I<sub>SOCE</sub> inactivation. In a control untreated oocyte MPF, MAPK, and Mos activities were undetectable, and I<sub>SOCE</sub> was activated in response to store depletion (<xref rid="fig4" ref-type="fig">Fig. 4</xref> A). In contrast, no I<sub>SOCE</sub> could be detected after store depletion in a cyclin B1–injected cell (<xref rid="fig4" ref-type="fig">Fig. 4</xref> B). In this case, cyclin B1 injection induced MPF and P-MAPK activation (1.6- and 0.9-fold egg levels, respectively), but little Mos protein was detected (<xref rid="fig4" ref-type="fig">Fig. 4</xref> B). This argues that Mos kinase is not required for SOCE inactivation.</p><fig id="fig4" position="float"><label>Figure 4.</label><caption><p>
<bold>Representative individual cell data from Δ87cyclin B1–injected cells.</bold> MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase in fully mature eggs (EGG). (A) I<sub>SOCE</sub> was activated in response to store depletion with ionomycin (10 μM) in an oocyte, and MPF, P-MAPK, and Mos were not detected. (B) A representative oocyte injected with Δ87cyclin B1. I<sub>SOCE</sub> was not activated in response to store depletion. MPF and P-MAPK levels were 1.6 and 0.9 times those in eggs, whereas Mos kinase was not detected in this oocyte. (C) A representative oocyte preincubated with PD98059 (100 μM) before Δ87cyclin B1 injection. I<sub>SOCE</sub> was not activated in response to store depletion, MPF levels were 2.7 times higher than those found in the control egg lysate, and both P-MAPK and Mos were not detectable.</p></caption><graphic xlink:href="0110059f4"/></fig><p>
<xref rid="fig4" ref-type="fig">Fig. 4</xref> C shows I<sub>SOCE</sub> and kinase data from an oocyte treated with the MEK inhibitor PD98059 before cyclin B1 injection. This resulted in robust activation of MPF (2.7-fold egg level), without activating either MAPK or inducing Mos protein accumulation (<xref rid="fig4" ref-type="fig">Fig. 4</xref> C). In this cell, I<sub>SOCE</sub> was almost completely inactivated, showing that SOCE inactivation can occur independently of the MAPK cascade and Mos.</p></sec><sec><title>MPF activation is necessary for I<sub>SOCE</sub> inactivation</title><p>We next examined whether MPF activation is required for SOCE inactivation. We initiated oocyte maturation by injecting Mos RNA into oocytes in the presence or absence of a specific MPF inhibitor, the Wee1 kinase (<xref rid="bib28" ref-type="bibr">Mueller et al., 1995</xref>) (<xref rid="fig5" ref-type="fig">Fig. 5</xref>). These experiments also allowed us to test whether prolonged activation of Mos and the MAPK cascade can induce I<sub>SOCE</sub> inactivation. Mos kinase is necessary and sufficient for oocyte maturation (<xref rid="bib39" ref-type="bibr">Sagata, 1997</xref>).</p><fig id="fig5" position="float"><label>Figure 5.</label><caption><p>
<bold>Correlation between I</bold>
<sub>SOCE</sub>
<bold>and Mos, P-MAPK, and MPF levels after injection of GST–Mos RNA.</bold> GST–Mos is a fusion protein between GST and <italic>Xenopus</italic> Mos, which allowed the separation between endogenous Mos (39 kD) and recombinant, injected GST–Mos (64 kD) (see Materials and methods). Oocytes were either directly injected with 10 ng GST–Mos RNA (A–C) or preinjected with 10 ng of GST–107Wee1 (Wee) 13–16 h before Mos RNA injection. GST–107Wee1 is a constitutively active form of the Wee1 kinase, which blocks MPF activation (<xref rid="bib13" ref-type="bibr">Howard et al., 1999</xref>). I<sub>SOCE</sub> levels recorded at various times after GST–Mos RNA injection. After I<sub>SOCE</sub> recording, individual cells were lysed and assayed for kinase activities. This allowed us to obtain I<sub>SOCE</sub> (squares), Mos (diamonds), P-MAPK (triangles), and MPF (circles) levels from the same oocyte. For Mos and Wee1–Mos injections, current and kinase data from 40 and 27 cells, respectively, are shown. The plotted Mos data is for GST–Mos protein levels, as endogenous Mos was not detected in these experiments. The data for individual cells are connected by drop lines to help in matching I<sub>SOCE</sub> and kinase activity from the same cell. I<sub>SOCE</sub> levels were normalized to the levels in untreated oocytes, and Mos, MAPK, and MPF levels were normalized to the levels found in fully mature eggs as described in <xref rid="fig2" ref-type="fig">Fig. 2</xref> and Materials and methods. The time scale was normalized to GVBD<sub>i</sub>, the standard deviation of which is shown in A–C (<italic>n</italic> = 5). Cells preinjected with GST–107Wee1 were plotted on the same time scale as GST–Mos-injected cells (D–F). Individual cell data were fitted with a Boltzman function to obtain a general trend of I<sub>SOCE</sub> inactivation and the activation of the different kinases.</p></caption><graphic xlink:href="0110059f5"/></fig><p>To determine the correlation between I<sub>SOCE</sub> inactivation and levels of Mos, MAPK, and MPF, we recorded I<sub>SOCE</sub> and measured the levels of the three aforementioned kinases in individual cells (<xref rid="fig5" ref-type="fig">Fig. 5</xref>). The data was plotted on a time scale normalized to GVBD<sub>i</sub>, which occurred 5.8 ± 1.4 h after Mos RNA injection (<italic>n</italic> = 5 donor females). Soon after the injection of Mos RNA (0.1 GVBD<sub>i</sub>), Mos levels were similar to those observed in eggs. Mos protein levels increased gradually over time, reaching a plateau at ∼10-fold egg levels (<xref rid="fig5" ref-type="fig">Fig. 5</xref> A). Note that we measured recombinant glutathione-<italic>S</italic>-transferase (GST)–Mos levels in these experiments, and did not detect endogenous Mos protein expression (see <xref rid="fig6" ref-type="fig">Fig. 6</xref>). I<sub>SOCE</sub> did inactivate in response to Mos RNA injection (<xref rid="fig5" ref-type="fig">Fig. 5</xref> A). The time course of I<sub>SOCE</sub> inactivation was significantly delayed compared with Mos activation. Mos protein levels reached egg levels at 0.1 GVBD<sub>i</sub>, whereas I<sub>SOCE</sub> began to inactivate around 1 GVBD<sub>i</sub> (<xref rid="fig5" ref-type="fig">Fig. 5</xref> A). This time lag translates to ∼5.2 h of high Mos activity before I<sub>SOCE</sub> inactivated. In this case we know that ectopic expression of GST–Mos was effectively targeting at least one of its downstream effectors, the MAPK cascade. This is because P-MAPK increased with a similar time course to Mos (<xref rid="fig5" ref-type="fig">Fig. 5</xref> B), reaching a plateau at ∼1.3-fold egg levels. This suggests that Mos kinase is not directly involved in I<sub>SOCE</sub> inactivation. We have shown above that activation of the MAPK cascade is not required for I<sub>SOCE</sub> inactivation. The lack of correlation between P-MAPK and I<sub>SOCE</sub> levels after Mos injection supports this conclusion.</p><fig id="fig6" position="float"><label>Figure 6.</label><caption><p>
<bold>Representative individual cell data from Wee1- and Mos-injected cells.</bold> MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase from a lysate of fully mature eggs (EGG). (A) I<sub>SOCE</sub> was activated in response to store depletion with ionomycin (10 μM) in an oocyte. MPF, P-MAPK, and Mos were not detected in this cell. (B) A representative cell injected with 10 ng GST–Mos RNA. I<sub>SOCE</sub> was not activated in response to store depletion. I<sub>SOCE</sub> was recorded at the 1GVBD<sub>i</sub> time point in this cell, i.e., at about the same time that GVBD first occurred in the population. MPF and P-MAPK levels were 1.3 and 1.6 times, respectively, higher than those in the egg lysate. GST–Mos protein (arrowhead) levels were 13.1 times higher than egg levels and no endogenous Mos protein (arrow) was detected. (C) A representative cell preinjected with 10 ng GST–107Wee1 RNA 14 h before GST–Mos RNA (10 ng) injection. We recorded I<sub>SOCE</sub> at the 1GVBD<sub>i</sub> time point. SOCE was readily activated, in response to store depletion, to levels similar to those found in oocytes. Injection of GST–107Wee1 effectively blocked MPF activation, which was detected at 0.2 times egg level. In contrast, P-MAPK and GST–Mos (arrowhead) were detected at 0.76 and 8.7 times egg levels, respectively.</p></caption><graphic xlink:href="0110059f6"/></fig><p>We have also measured MPF levels from the same cells (<xref rid="fig5" ref-type="fig">Fig. 5</xref> C). In this case I<sub>SOCE</sub> inactivation followed the same time course as MPF activation (<xref rid="fig5" ref-type="fig">Fig. 5</xref> C), indicating a strong temporal correlation between MPF levels and I<sub>SOCE</sub> inactivation. This suggests a role for MPF in I<sub>SOCE</sub> inactivation. Furthermore, in this case GVBD occurrence also mirrored MPF activation, indicating that MPF effectively targeted its downstream effectors.</p><p>If MPF is directly responsible for I<sub>SOCE</sub> inhibition, then specifically blocking MPF activation should prevent I<sub>SOCE</sub> inactivation. We accomplished this by injecting a constitutively active Wee1 kinase (GST–107Wee1), which phosphorylates MPF and blocks its activation (<xref rid="bib28" ref-type="bibr">Mueller et al., 1995</xref>; <xref rid="bib13" ref-type="bibr">Howard et al., 1999</xref>). Injection of Wee1 did not significantly alter the activation time course of either Mos or MAPK in response to Mos RNA injection (<xref rid="fig5" ref-type="fig">Fig. 5, D and E</xref>). However, Wee1 inhibited MPF activation (<xref rid="fig5" ref-type="fig">Fig. 5</xref> F) and prevented GVBD. In these cells with high Mos and MAPK levels and no MPF activity, store depletion activated I<sub>SOCE</sub> (<xref rid="fig5" ref-type="fig">Fig. 5</xref> F). This shows that MPF activation is necessary for I<sub>SOCE</sub> inactivation. Note that in Wee1-injected cells, it was possible to record from oocytes for a significantly longer period of time (up to 1.6 GVBD<sub>i</sub> or ∼10 h) because they did not undergo GVBD. Even at these latter time points (1.3–1.6 GVBD<sub>i</sub>), I<sub>SOCE</sub> was clearly detectable in response to store depletion at levels only slightly lower than those in oocytes. Oocytes injected with Wee1 alone (without Mos injection), had I<sub>SOCE</sub> levels similar to those of control oocytes (1 ± 0.09, <italic>n</italic> = 4), and no detectable MPF or MAPK activation, indicating that Wee1 does not affect I<sub>SOCE</sub>. Together, these data demonstrate that MPF activation is necessary for I<sub>SOCE</sub> inactivation.</p><p>Examples of single-cell data from Mos- and Wee1/Mos–injected cells are shown in <xref rid="fig6" ref-type="fig">Fig. 6</xref>. The oocyte had a robust I<sub>SOCE</sub> signal and no detectable Mos, MAPK, or MPF (<xref rid="fig6" ref-type="fig">Fig. 6</xref> A). In contrast, a Mos-injected cell showed complete I<sub>SOCE</sub> inactivation, and high MPF and P-MAPK levels (1.3- and 1.6-fold egg levels, respectively) (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B). Ectopic GST–Mos (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B, arrowhead) was 13.1-fold higher than endogenous Mos levels found in eggs (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B, arrow). No endogenous Mos protein was detected in this cell (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B, arrow). A representative Wee1-preinjected cell (<xref rid="fig6" ref-type="fig">Fig. 6</xref> C) showed levels of ectopic Mos and P-MAPK (8.7- and 0.76-fold egg levels, respectively) similar to a Mos-injected cell. However MPF kinase was not significantly activated (0.2-fold egg level), and I<sub>SOCE</sub> was readily induced in response to store depletion (<xref rid="fig6" ref-type="fig">Fig. 6</xref> C). This shows that despite the activation of both Mos and P-MAPK for extensive time periods, I<sub>SOCE</sub> does not inactivate in the absence of MPF activity.</p></sec><sec><title>MPF blocks the coupling between store depletion and SOCE activation</title><p>We next investigated the mechanism of MPF action in mediating SOCE inactivation. MPF could be either blocking Ca<sup>2+</sup> influx through SOCE channels, or it could be inhibiting the coupling mechanism between store depletion and SOCE. The molecular identity of the SOCE channel remains unclear, therefore we could not directly test whether MPF phosphorylates SOCE channels. However, by altering the timing of SOCE versus MPF activation, we could determine the target of MPF action. Our rational was to activate I<sub>SOCE</sub> before MPF levels increased. If MPF phosphorylates SOCE channels and blocks Ca<sup>2+</sup> influx, then preactivated I<sub>SOCE</sub> will be inhibited concurrently with MPF activation. Alternatively if MPF blocks the coupling mechanism, then preactivated I<sub>SOCE</sub> will not be affected by MPF activation.</p><p>We activated I<sub>SOCE</sub> shortly before GVBD in oocytes treated with progesterone, and continuously recorded I<sub>SOCE</sub> until, and past, GVBD occurrence (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). GVBD in this cell was apparent ∼35 min after store depletion (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). However, I<sub>SOCE</sub> remained active with no significant inhibition, even ∼20 min after GVBD (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). We confirmed that GVBD appearance was coupled to the characteristic increase in MPF activity (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B), indicating that 1,2-bis(2-aminophenoxy) ethane-<italic>N</italic>,<italic>N</italic>,<italic>N</italic>′,<italic>N</italic>′-tetraacetic acid (BAPTA) injection, which was necessary to measure I<sub>SOCE</sub>, did not interfere with MPF activation. Note that although I<sub>SOCE</sub> remained activated past GVBD in <xref rid="fig7" ref-type="fig">Fig. 7</xref> B, there was a gradual inhibition in the current over time. Recording I<sub>SOCE</sub> from a control oocyte for the same prolonged time, shows a similar gradual I<sub>SOCE</sub> inhibition (<xref rid="fig7" ref-type="fig">Fig. 7</xref> A), indicating that the gradual decline in I<sub>SOCE</sub> was not due to MPF activation. This shows that once SOCE was activated, increasing MPF levels were not able to inactivate I<sub>SOCE</sub>. This indicates that MPF does not block Ca<sup>2+</sup> influx through SOCE channels. However, if MPF is activated before store depletion, SOCE cannot be activated (<xref rid="fig1" ref-type="fig">Fig. 1</xref> F, <xref rid="fig4" ref-type="fig">Fig. 4, B and C</xref>, and <xref rid="fig6" ref-type="fig">Fig. 6</xref> B). Therefore, MPF inhibits the coupling mechanism between store depletion and SOCE activation.</p><fig id="fig7" position="float"><label>Figure 7.</label><caption><p>
<bold>Continuous recording of I</bold>
<sub>SOCE</sub>
<bold>through the GVBD transition.</bold> (A) Prolonged recording of I<sub>SOCE</sub> from a control untreated oocyte. MPF activity in this cell is also shown. (B) I<sub>SOCE</sub> was activated in a progesterone-treated oocyte before GVBD and continuously recorded until, and past, the time point at which GVBD occurred (arrow). At the end of the experiment, the cell was lysed in extraction buffer and MPF activity was measured. This cell had high MPF activity, indicating that the prolonged recording did not interfere with MPF activation at GVBD. MPF activation at GVBD is not able to block I<sub>SOCE</sub> that has been preactivated before GVBD.</p></caption><graphic xlink:href="0110059f7"/></fig><p>A Ca<sup>2+</sup> signal is required for nuclear envelope breakdown during mitosis (<xref rid="bib14" ref-type="bibr">Kao et al., 1990</xref>). This does not appear to be the case in <italic>Xenopus</italic> oocytes, because BAPTA injection did not prevent GVBD (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). However, it is possible that altering Ca<sup>2+</sup> dynamics by BAPTA injection interferes with the timing of GVBD. This would affect our interpretation of the correlation between GVBD occurrence and I<sub>SOCE</sub> inhibition. To address this issue, we activated I<sub>SOCE</sub> before GVBD by depleting Ca<sup>2+</sup> stores with thapsigargin, independent of BAPTA injection (<xref rid="fig8" ref-type="fig">Fig. 8</xref>). Thapsigargin depletes Ca<sup>2+</sup> stores by blocking the ER Ca<sup>2+</sup> ATPase, and has been shown to be effective in activating SOCE in <italic>Xenopus</italic> oocytes (<xref rid="bib33" ref-type="bibr">Petersen and Berridge, 1994</xref>; <xref rid="bib21" ref-type="bibr">Machaca and Hartzell, 1999</xref>).</p><fig id="fig8" position="float"><label>Figure 8.</label><caption><p>
<bold>Pre-activated I</bold>
<sub>SOCE</sub>
<bold>does not inactivate in response to MPF activation.</bold> Oocytes were incubated in Ca<sup>2+</sup>-free medium (L-15 with Ca<sup>2+</sup>, buffered at 50 μM), containing thapsigargin (1 μM) to deplete intracellular Ca<sup>2+</sup> store and activate I<sub>SOCE</sub>. Cells were voltage clamped in a Ca<sup>2+</sup>-free solution (70Mg; see Materials and methods) and then switched to a solution containing 30 mM Ca<sup>2+</sup> (30Ca; see Materials and methods) to induce Ca<sup>2+</sup> influx through SOCE channels. (A) I<sub>SOCE</sub> and MPF levels from a control thapsigargin–treated oocyte. (B) Oocytes were treated with progesterone (5 μg/ml) for 1 h and then thapsigargin (1 μM) was added, and the cells were incubated until GVBD occurred. I<sub>SOCE</sub> was measured ∼15 min after GVBD occurrence. I<sub>SOCE</sub> was still present in this cell, although MPF was activated at high levels, indicating that MPF is unable to block SOCE that has been preactivated before MPF induction. I<sub>SOCE</sub> (C) and MPF (D) levels in oocytes (<italic>n</italic> = 5) and cells treated with progesterone 10–15 min (<italic>n</italic> = 8) and 15–30 min after GVBD (<italic>n</italic> = 7). I<sub>SOCE</sub> levels gradually declined after GVBD and MPF levels remained relatively stable.</p></caption><graphic xlink:href="0110059f8"/></fig><p>As is the case with ionomycin, thapsigargin treatment of eggs after progesterone-induced maturation did not activate I<sub>SOCE</sub> (4 ± 2.3 nA, <italic>n</italic> = 3). In contrast, thapsigargin activated a robust I<sub>SOCE</sub> in oocytes (182.7 ± 28 nA, <italic>n</italic> = 3). To test whether preactivated I<sub>SOCE</sub> is inhibited by MPF activation, we incubated cells in progesterone plus thapsigargin (1 μM), and recorded I<sub>SOCE</sub> at different time points after GVBD occurrence. I<sub>SOCE,</sub> preactivated with thapsigargin, was readily apparent 10–15 min after GVBD occurrence (<xref rid="fig8" ref-type="fig">Fig. 8</xref> B). As expected, this cell contained high levels of MPF activity (<xref rid="fig8" ref-type="fig">Fig. 8</xref> B). A control oocyte, incubated for a similar time in thapsigargin alone, had a large I<sub>SOCE</sub> and no detectable MPF (<xref rid="fig8" ref-type="fig">Fig. 8</xref> A). These data confirm our conclusion that MPF is unable to inhibit preactivated I<sub>SOCE</sub>. This indicates that MPF activation at GVBD inhibits coupling of store depletion to SOCE activation.</p><p>Interestingly, thapsigargin-induced I<sub>SOCE</sub> levels were consistently smaller after GVBD as compared with control oocytes (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, compare A and B). I<sub>SOCE</sub> levels measured 10–15 min after GVBD in cells treated with thapsigargin, were at levels 40 ± 3% of control oocyte levels (<xref rid="fig8" ref-type="fig">Fig. 8</xref> C). If I<sub>SOCE</sub> was recorded 15–30 min after GVBD occurred, I<sub>SOCE</sub> levels declined further, to 17 ± 3% of oocyte levels (<xref rid="fig8" ref-type="fig">Fig. 8</xref> C). Therefore, preactivated I<sub>SOCE</sub> decreases gradually after GVBD.</p><p>What is the reason for this gradual downregulation of I<sub>SOCE</sub> after GVBD? In oocytes and other cell types, Ca<sup>2+</sup> store depletion results in the activation of SOCE. Therefore, a positive signal (i.e., the coupling species) is induced resulting in SOCE activation. Once the Ca<sup>2+</sup> store is refilled, SOCE is downregulated. This argues for the presence of a constitutive mechanism that downregulates I<sub>SOCE</sub> in the absence of the coupling species. As shown above, MPF activation blocks coupling between store depletion and SOCE activation. This would eliminate the positive signal to maintain active SOCE and would lead to SOCE downregulation. Therefore, the gradual inhibition of preactivated I<sub>SOCE</sub> after GVBD is likely due to the block of Ca<sup>2+</sup> store–SOCE coupling by MPF and constitutive downregulation of SOCE.</p></sec></sec><sec><title>Discussion</title><sec><title>MPF is required for SOCE inactivation</title><p>Oocyte maturation entails a complex series of biochemical and morphological adaptations that transform immature oocytes into fertilization-competent eggs, able to support embryonic development. Early electron microscopy studies have described morphological changes that occur concomitant with <italic>Xenopus</italic> oocyte maturation (for review see <xref rid="bib1" ref-type="bibr">Bement and Capco, 1990</xref>). Furthermore, extensive studies have characterized biochemical cascades that drive oocyte maturation (<xref rid="bib30" ref-type="bibr">Nebreda and Ferby, 2000</xref>). There remains much to be learned about how these signal transduction cascades regulate physiologically relevant pathways during oocyte maturation. Here we describe the relationship between the Mos–MAPK–MPF kinase cascade and SOCE. Differentiation of Ca<sup>2+</sup> signaling pathways during oocyte maturation is a prerequisite for proper egg activation after fertilization. This is because the Ca<sup>2+</sup> wave at fertilization mediates egg activation by inducing the block to polyspermy and releasing the MII block (<xref rid="bib1" ref-type="bibr">Bement and Capco, 1990</xref>). SOCE inactivates specifically at the GVBD stage of <italic>Xenopus</italic> oocyte maturation (<xref rid="fig1" ref-type="fig">Fig. 1</xref> F) (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>). This inactivation is likely to be important physiologically because <italic>Xenopus</italic> eggs can be artificially activated by pricking with a glass needle, but only in the presence of extracellular Ca<sup>2+</sup> (<xref rid="bib47" ref-type="bibr">Wolf, 1974</xref>). Therefore, Ca<sup>2+</sup> entry is sufficient to stimulate egg activation, probably due to changes in the sensitivity and organization of the Ca<sup>2+</sup> release machinery. Consequently, inactivation of I<sub>SOCE</sub> at the GVBD stage, soon after entry into meiosis, may function as a safety measure to prevent premature egg activation before fertilization.</p><p>Inactivation of ionic currents during M phase is not a peculiar property of I<sub>SOCE</sub> in <italic>Xenopus</italic> oocytes, and has been described for other membrane proteins and cell types. For example, in <italic>Xenopus</italic> oocytes, amino acid and phosphate transporters are downregulated, but not completely inactivated, during oocyte maturation (<xref rid="bib38" ref-type="bibr">Richter et al., 1984</xref>). In the same respect, surface levels of the Na<sup>+</sup>–K<sup>+</sup>–ATPase (<xref rid="bib40" ref-type="bibr">Schmalzing et al., 1990</xref>) and of β1 integrin (<xref rid="bib29" ref-type="bibr">Muller et al., 1993</xref>) decrease during <italic>Xenopus</italic> oocyte maturation. Recently, several exogenously expressed channels have been shown to be downregulated during oocyte maturation (<xref rid="bib42" ref-type="bibr">Shcherbatko et al., 2001</xref>). We have obtained similar results with exogenously expressed glutamate receptor (GluR3; unpublished data). In contrast, the levels of Ca<sup>2+</sup>-activated Cl<sup>−</sup> current (I<sub>Cl,Ca</sub>) do not decrease during maturation (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>). Therefore, there is extensive, specific regulation of membrane proteins during <italic>Xenopus</italic> oocyte maturation. Regulation of membrane proteins during M phase is not limited to <italic>Xenopus</italic> oocytes. SOCE has been shown to inactivate during mitosis of HeLa cells (<xref rid="bib34" ref-type="bibr">Preston et al., 1991</xref>), and membrane trafficking in general is inhibited during mammalian cell mitosis (<xref rid="bib46" ref-type="bibr">Warren, 1993</xref>). Therefore, determining the molecular mechanisms controlling SOCE inactivation during <italic>Xenopus</italic> oocyte maturation provides valuable insights into the regulation of plasma membrane protein levels during M phase.</p><p>Our results argue that activation of MPF leads to uncoupling of store depletion from SOCE. Several lines of evidence support this conclusion. During progesterone-induced maturation, I<sub>SOCE</sub> inactivation at GVBD coincides with MPF activation (<xref rid="fig1" ref-type="fig">Figs. 1</xref> and <xref rid="fig2" ref-type="fig">2</xref>). Furthermore, regardless of the method by which we induced oocyte maturation (progesterone, cyclin B1, or Mos injection), every cell in which I<sub>SOCE</sub> was inactivated had high MPF activity. In addition, cells with low MPF activity always activated I<sub>SOCE</sub> in response to store depletion (<xref rid="fig3" ref-type="fig">Figs. 3</xref>–<xref rid="fig6" ref-type="fig">6</xref>). Out of 155 individual cells from which we recorded I<sub>SOCE</sub> and measured kinase activity, not a single cell deviated from these criteria. Finally, Mos injection did not lead to SOCE inactivation when MPF was inhibited (<xref rid="fig5" ref-type="fig">Fig. 5</xref> F). Mos injection activates the MAPK cascade (<xref rid="fig5" ref-type="fig">Fig. 5</xref> B), and leads to the activation of other signal transduction cascades important for oocyte maturation. Specifically blocking MPF activation under these conditions with Wee1, reversed I<sub>SOCE</sub> inactivation. This demonstrates that MPF activation during oocyte maturation is necessary for I<sub>SOCE</sub> inactivation.</p><p>Is MPF activation sufficient for I<sub>SOCE</sub> inactivation? Progesterone (<xref rid="fig1" ref-type="fig">Figs. 1</xref> and <xref rid="fig2" ref-type="fig">2</xref>) and Mos RNA injection data (<xref rid="fig5" ref-type="fig">Fig. 5</xref> C) argue that it is. Under these conditions, the time course of MPF activation paralleled that of I<sub>SOCE</sub> inhibition. In contrast, rapid activation of MPF with cyclin B1 injection revealed a delay before GVBD and SOCE inactivation (<xref rid="fig3" ref-type="fig">Fig. 3</xref> A). As discussed above this time lag could be an artifact of ectopic MPF activation, and not a true reflection of endogenous MPF activity. Two pools of p34<italic><sup>cdc2</sup></italic> kinase exist in the immature <italic>Xenopus</italic> oocyte, an inactive preMPF pool and a 10-fold excess-free p34<italic><sup>cdc2</sup></italic> protein pool (<xref rid="bib17" ref-type="bibr">Kobayashi et al., 1991</xref>). Progesterone and cyclin B1 likely activate the two p34<italic><sup>cdc2</sup></italic> pools differentially, which would explain the delay in GVBD after cyclin B1 injection. Nonetheless, after progesterone, Mos, or cyclin B1 injection, I<sub>SOCE</sub> inactivation correlates temporally with GVBD. As GVBD is a target for MPF, we argue that MPF activation is sufficient for I<sub>SOCE</sub> inactivation.</p><p>In contrast to the MPF requirement for SOCE inactivation, we did not observe any correlation between I<sub>SOCE</sub> levels and P-MAPK and Mos levels (<xref rid="fig3" ref-type="fig">Figs. 3</xref>–<xref rid="fig6" ref-type="fig">6</xref>). This indicates that neither Mos kinase nor the MAPK cascade plays a direct role in I<sub>SOCE</sub> inactivation.</p></sec><sec><title>MPF blocks coupling of store depletion to SOCE activation</title><p>Activation of MPF before Ca<sup>2+</sup> store depletion blocks SOCE activation. This was true whether MPF was activated by progesterone treatment (<xref rid="fig1" ref-type="fig">Figs. 1</xref> and <xref rid="fig2" ref-type="fig">2</xref>), Mos RNA injection (<xref rid="fig5" ref-type="fig">Figs. 5</xref> and <xref rid="fig6" ref-type="fig">6</xref>), or cyclin B1 protein injection (<xref rid="fig3" ref-type="fig">Figs. 3</xref> and <xref rid="fig4" ref-type="fig">4</xref>). In contrast, when MPF was induced after SOCE activation, it was unable to block I<sub>SOCE</sub> (<xref rid="fig7" ref-type="fig">Figs. 7</xref> and <xref rid="fig8" ref-type="fig">8</xref>). This indicates that MPF blocks SOCE activation and not the current through SOCE channels. MPF could uncouple store depletion from SOCE activation by inhibiting the coupling species. Alternatively, MPF could target SOCE channels and prevent them from responding to the coupling species, without inhibiting the coupling species. In either case, our results demonstrate that MPF blocks coupling of Ca<sup>2+</sup> store depletion to SOCE activation. Further investigations of the downstream targets of MPF will provide critical insights into the identity of the elusive coupling mechanism between store depletion and SOCE activation.</p><p>MPF is a member of the cyclin-dependent kinase family or cell cycle regulators. Cyclin-dependent kinases are master regulators of entry and progression through both mitosis and meiosis (<xref rid="bib27" ref-type="bibr">Morgan, 1997</xref>). In that capacity, MPF controls various cellular processes crucial for meiotic progression. For example, MPF induces GVBD and stabilizes Mos kinase, which is a component of CSF (<xref rid="bib31" ref-type="bibr">Palmer and Nebreda, 2000</xref>). CSF is required to maintain metaphase II arrest until fertilization (<xref rid="bib24" ref-type="bibr">Masui, 1991</xref>). Our results indicate that SOCE inactivation can now be added to the list of cellular processes driven by MPF. Interestingly, MPF has recently been shown to regulate Ca<sup>2+</sup> oscillations at fertilization in ascidian eggs (<xref rid="bib20" ref-type="bibr">Levasseur and McDougall, 2000</xref>). The differentiation of Ca<sup>2+</sup> signaling pathways is an important element of oocyte maturation. The fact that MPF regulates both Ca<sup>2+</sup> release and Ca<sup>2+</sup> influx in oocytes from different species strongly supports the idea that Ca<sup>2+</sup> signaling is tightly controlled and functionally relevant during meiosis.</p></sec></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Oocyte isolation and enucleation</title><p>
<italic>X. laevis</italic> females (<italic>Xenopus</italic> Express) were anesthetized by immersion in Tricaine (1 g/liter, pH 7) for 5–10 min. Ovarian follicles were removed and digested in Ca<sup>2+</sup>-free Ringer's solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl<sub>2</sub>, 5 mM Hepes, pH 7.6), containing 2 mg/ml collagenase type IA (Sigma-Aldrich) for 2 h at room temperature. The oocytes were extensively rinsed with Ringer's solution and incubated in 0.5× L-15 medium (GIBCO BRL) at 18°C. Oocytes were used for up to 4 d after isolation. Oocyte maturation was induced by incubating cells in L-15 containing 5 μg/ml progesterone. In some experiments oocytes were enucleated. This was accomplished by poking a hole on the animal pole using a glass needle, followed by the application of gentle pressure on the oocyte. This results in the expulsion of the germinal vesicle (nucleus). Oocytes were allowed to recover for at least 30 min before electrophysiological recordings.</p></sec><sec><title>Electrophysiological methods</title><p>The I<sub>SOCE</sub> was measured as previously described (<xref rid="bib22" ref-type="bibr">Machaca and Haun, 2000</xref>). In brief, SOCE was activated by depletion of intracellular Ca<sup>2+</sup> stores with either ionomycin (10 μM) or thapsigargin (1 μM for ≥3 h). Oocytes were injected with 7 nmoles BAPTA before store depletion, to buffer intracellular Ca<sup>2+</sup> rise and block the endogenous Ca<sup>2+</sup>-activated Cl<sup>−</sup> current (I<sub>Cl,Ca</sub>), which would otherwise mask the I<sub>SOCE</sub>. Assuming an oocyte volume of 1 μl, this would result in a final concentration of ∼7 mM BAPTA, which completely blocks I<sub>Cl,Ca</sub>. I<sub>SOCE</sub> was typically measured at the hyperpolarizing potential of −140 mV. This is because we wanted to maximize I<sub>SOCE</sub>, to most accurately determine the level of inactivation. <italic>Xenopus</italic> oocytes or eggs were voltage clamped with two microelectrodes by the use of a GeneClamp 500 (Axon Instruments, Inc.). Electrodes were filled with 3 M KCl and had resistances of 0.5–2 MΩ. Voltage stimulation and data acquisition were controlled using Curcap30 (Bill Goolsby, Emory University, Atlanta, GA). Current data was filtered at 10 kHz, digitized, and analyzed using Origin<sup>®</sup> software (Microcal Software, Inc.). Some experiments used 30Ca or 70Mg solutions (30Ca: 55 mM NaCl, 30 mM CaCl<sub>2</sub>, 10 mM Hepes, pH 7.4; 70Mg: 70 mM MgCl<sub>2</sub>, 10 mM Hepes, pH 7.4).</p></sec><sec><title>MPF kinase assay</title><p>MPF kinase activity was measured by lysing an oocyte in 50 μl extraction buffer (80 mM β-glycerophosphate, 20 mM Hepes, pH 7.5, 20 mM EGTA, 15 mM MgCl<sub>2</sub>, 1 mM sodium vanadate, 50 mM NaF, 1 mM DTT, 10 μg/ml aprotinin, 50 μg/ml leupeptin, 1 mM PMSF). Lysates were centrifuged at 16,000 <italic>g</italic> for 5 min, and the supernatant was stored at −70°C. MPF activity was measured using the SignaTECT<sup>TM</sup> cdc2 kinase assay system (Promega), according to manufacturer's recommendations. This assay measures MPF activity in terms of the phosphorylation of a specific substrate, i.e., a biotinylated peptide derived from histone H1 (PKTPKKAKKL).</p></sec><sec><title>Western blotting</title><p>P-MAPK, total MAPK, and Mos protein levels were measured by Western blotting. Lysates were prepared as for the MPF assay, and protein concentration was measured using the Bradford assay (Bio-Rad Laboratories). SDS-PAGE gels were loaded with equal amounts of protein, transferred onto Immobilon P-PVDF membranes (Millipore), and probed with the appropriate primary antibody: anti–phospho-MAPK and anti–total MAPK (Cell Signaling Technology) or anti-Mos antibodies (Santa Cruz Biotechnology, Inc.). This was followed by the appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) and detected using the ECL Plus Western blot detection system (Amersham Pharmacia Biotech). Westerns were visualized using a STORM™ system (Molecular Dynamics), which allowed a linear response to be measured from the band intensity on the Western blot. This was crucial for accurate quantification of P-MAPK and Mos protein levels. To control for variability between different trials, a large batch of lysate from mature eggs was prepared for both P-MAPK and Mos. This control lysate was loaded on each gel used for quantifying P-MAPK and Mos levels. The same lysate was used for all P-MAPK and Mos gels, respectively, which provided a consistent internal control for all the experiments. Using this method we could faithfully reproduce (with <15% error margin) P-MAPK and Mos protein levels from the same sample in different experiments.</p></sec><sec><title>Reagents</title><p>The following reagents were used: ionomycin and PD98059 (Calbiochem); thapsigargin (Molecular Probes Inc.); progesterone, salts, and other reagents (Sigma-Aldrich). The following constructs were provided by A.M. MacNicol (University of Arkansas Medical Science): GST–Mos; GST–107Wee1, which encodes a constitutively active Wee1 kinase (<xref rid="bib28" ref-type="bibr">Mueller et al., 1995</xref>; <xref rid="bib13" ref-type="bibr">Howard et al., 1999</xref>); and a His<sub>6</sub>-tagged Δ87cyclin B1 protein (<xref rid="bib19" ref-type="bibr">Kumagai and Dunphy, 1995</xref>; <xref rid="bib13" ref-type="bibr">Howard et al., 1999</xref>). Oocytes were injected with 10 ng of the GST–Mos and GST–107Wee1 RNAs and ∼200 pg Δ87cyclin B1 protein.</p></sec></sec></body><back><ack><p>We are grateful to Angus MacNicol for various clones and helpful discussion. We would like to thank David Clapham, Angus MacNicol, and Larry Suva for comments on the manuscript.</p><p>This work was supported by a grant from the National Institutes of Health (GM-61829).</p></ack><fn-group><fn id="fn1"><label>*</label><p>Abbreviations used in this paper: BAPTA, 1,2-bis(2-aminophenoxy) ethane-<italic>N</italic>,<italic>N</italic>,<italic>N</italic>′,<italic>N</italic>′-tetraacetic acid; CSF, cytostatic factor; GST, glutathione-<italic>S</italic>-transferase; GVBD, germinal vesicle breakdown; GVBD<sub>i</sub>, time of initial appearance of GVBD; GVBD<sub>50</sub>, time at which 50% of the oocyte undergo GVBD; I<sub>SOCE</sub>, SOCE current; I–V, current–voltage; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MII, second meiotic division; MPF, maturation-promoting factor; P-MAPK, phospho-MAPK; SOCE, store-operated Ca<sup>2+</sup> entry.</p></fn></fn-group><ref-list><ref id="bib1"><mixed-citation publication-type="journal">Bement, W.M., and D.G. 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