Calcium influx-mediated signaling is required for

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Mar 13, 2012 - signaling at fertilization, the embryo will fail to implant and/or develop to term (1). .... polar body by 1 h after ICSI, whereas the Ca2+-insulated ICSI eggs remained .... 2PN, two pronuclei; 3PN, three pronuclei. Miao et al.
Calcium influx-mediated signaling is required for complete mouse egg activation Yi-Liang Miaoa, Paula Steinb, Wendy N. Jeffersona, Elizabeth Padilla-Banksa, and Carmen J. Williamsa,1 a Reproductive Medicine Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and bDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca2+) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores. We tested the hypothesis that Ca2+ influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca2+ source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca2+ influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca2+ occurred, in the absence of Ca2+ influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca2+ chelator, BAPTA/ AM, demonstrated that Ca2+ influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca2+ transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca2+-free medium remained arrested in metaphase II. Inhibition of store-operated Ca2+ entry had no effect on ICSI-induced egg activation, so Ca2+ influx through alternative channels must participate in egg activation signaling. Ca2+ influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca2+. These results suggest that Ca2+ influx at fertilization not only maintains Ca2+ oscillations by replenishing Ca2+ stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

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universal feature of fertilization in mammals is that the fertilizing sperm evokes a series of repetitive calcium (Ca2+) oscillations in the egg that persist for several hours and terminate around the time of pronucleus formation. This pattern of Ca2+ oscillations is essential for events of “egg activation,” the complex series of events that occurs between the time of sperm-egg plasma membrane fusion and cleavage to the two-cell stage (1). Successful egg activation accomplishes conversion of these two gametes into a single embryo capable of implantation and fullterm development. In mammals, the pattern of Ca2+ oscillations that occurs at fertilization is responsible for driving the early developmental program, and in the absence of appropriate Ca2+ signaling at fertilization, the embryo will fail to implant and/or develop to term (1). The sperm protein responsible for initiating Ca2+ oscillations at fertilization is a testis-specific phospholipase C, PLCζ, which is released from the sperm head after sperm-egg plasma membrane fusion (2). The first Ca2+ transient experienced by the egg is a result of PLC-mediated generation of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated Ca2+ release from the endoplasmic reticulum (ER). The intracellular cytoplasmic Ca2+ level then rises to 1–3 μM and persists close to this level for several minutes before returning to baseline. The return to baseline is likely mediated by the combined actions of sarcoplasmic/ER Ca2+-ATPase (SERCA) pumps that move Ca2+ back into the ER and plasma membrane Ca2+ ATPase (PMCA) pumps that move Ca2+ out of the cell. Once initiated, repetitive Ca2+ oscillations persist for several hours. Persistence of the Ca2+ oscillations depends on Ca2+ influx to replenish Ca2+ stores www.pnas.org/cgi/doi/10.1073/pnas.1112333109

(3, 4). There is an absolute requirement for Ca2+/calmodulindependent protein kinase II gamma (CaMKIIγ) signaling downstream of Ca2+ oscillations to accomplish egg activation in vivo (5). CaMKIIγ activity leads to proteasome-mediated degradation of cyclin B and decreases in maturation promoting factor (MPF), and mitogen-activated protein kinase (MAPK) activities that trigger resumption of meiosis (1). Although it has been clear for many years that Ca2+ oscillations are important for initiating mammalian development, less is known regarding how the Ca2+ oscillations are transduced into actions by downstream effectors that are responsible for development. In mouse eggs, CaMKIIγ activity oscillates with only a slight lag period after initiation of each Ca2+ oscillation, suggesting that the Ca2+ transients each generate discrete waves of CaMKIIγ activity (6). Similarly, protein kinase C (PKC) translocation to the egg plasma membrane, indicative of PKC activation, occurs with a temporal pattern similar to the timing of Ca2+ oscillations (7, 8). The intermittent elevations in Ca2+ provide a digital mechanism for generating graded responses by downstream effectors at the same time as avoiding down-regulation due to hyperactivation. There is now evidence from somatic cell culture systems that alterations in subplasma membrane Ca2+ levels via store-operated Ca2+ entry (SOCE) are responsible for triggering specific Ca2+-dependent signaling pathways (9). These signaling pathways are not activated in response to alterations in bulk intracellular cytoplasmic Ca2+ in the absence of Ca2+ influx. These observations led us to hypothesize that Ca2+ influx across the plasma membrane contributes to downstream signaling required for egg activation. In this study, we used both intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) of zona pellucida-free eggs as methods of fertilization in conjunction with biochemical manipulations of Ca2+ influx and efflux to document a critical role for Ca2+ influx through plasma membrane channels in the initiation of development at fertilization. Results Effects of Modulating Plasma Membrane Ca2+ Fluxes on Events of ICSI-Induced Egg Activation. In somatic cells, SOCE can be

inhibited specifically by incubation in medium containing 1 μM gadolinium (Gd3+) (10). This treatment results in a gradual rundown of induced Ca2+ oscillations despite the presence of extracellular Ca2+ because depleted intracellular stores cannot be refilled. At higher micromolar concentrations, Gd3+ is no longer specific for SOCE and inhibits Ca2+ influx via additional channel types, whereas millimolar Gd3+ also inhibits PMCA pumps and simultaneously prevents Ca2+ efflux and influx. This

Author contributions: Y.-L.M. and C.J.W. designed research; Y.-L.M., P.S., and E.P.-B. performed research; Y.-L.M., P.S., W.N.J., and C.J.W. analyzed data; and Y.-L.M., P.S., W.N.J., and C.J.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1112333109/-/DCSupplemental.

PNAS | March 13, 2012 | vol. 109 | no. 11 | 4169–4174

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Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved February 1, 2012 (received for review July 28, 2011)

technique, known as “Ca2+ insulation,” causes induced Ca2+ oscillations to persist indefinitely, even in the absence of extracellular Ca2+ (11, 12). Of note, Gd3+ is not soluble in the presence of phosphate or bicarbonate, so these experiments are generally carried out in Hepes-buffered saline solution (HBSS) or HBSS supplemented with 2 mM Ca2+ (HBSS/Ca). To determine the effects of modulating Ca2+ influx and efflux on egg activation, we treated eggs that had undergone ICSI (ICSI eggs) with various concentrations of Gd3+. Of note, ICSI was used because HBSS does not support standard IVF and because ICSI allowed us to control the timing of fertilization within a precise time window. Similar to eggs fertilized by IVF, untreated ICSI eggs exhibited repetitive low frequency Ca2+ oscillations (Fig. 1A). These eggs resumed meiosis and emitted the second polar body by 1 h after ICSI (Fig. 1B) and reached the pronuclear stage by 6 h after ICSI (Fig. 1C). As expected, ICSI eggs that were placed immediately after microinjection into Ca2+-free medium (HBSS) generated only one or two Ca2+ transients (Fig. 1D). Although these eggs completed anaphase, they failed to undergo spindle rotation or polar body emission (Fig. 1E). In addition, neither maternal nor paternal DNA formed normal-appearing pronuclei (Fig. 1F). In the presence of extracellular Ca2+, ICSI eggs required treatment with 200 μM Gd3+ (HBSS/Ca/LGd) to completely block Ca2+ entry required for subsequent Ca2+ oscillations, although there was a small increase in baseline Ca2+ over time and the oscillation frequency was somewhat reduced at 50–100 μM Gd3+ (Fig. 1G and Fig. S1). Only one or two sperm-induced Ca2+ oscillations were observed in these treated ICSI eggs, and 1 h after ICSI, they had completed anaphase but failed to undergo spindle rotation and enter telophase (Fig. 1H). Even after continued culture for 6 h, the DNA remained condensed and no pronuclei formed (Fig. 1I). Insulating ICSI eggs from extracellular Ca2+ using 5 mM Gd3+ (HBSS/HGd) significantly increased the amplitude and lengthened the time of the initial Ca2+ transient, likely due to inhibitory effects on Ca2+ efflux via PMCA pumps (Fig. 1J, Table S1, and Fig. S2). The initial transient was followed by repetitive intracellular Ca2+ oscillations of somewhat lower oscillation frequency compared with controls, but the mean area under the Ca2+ curve was increased in Ca2+-insulated eggs (Table S1). Ca2+-insulated ICSI eggs completed anaphase by 1 h after ICSI (Fig. 1K); however, despite the repetitive Ca2+ oscillations, they failed to undergo spindle rotation and emit a second polar body and, as a result, formed three rather than two pronuclei by 6 h after ICSI (Fig. 1L). At fertilization, rapid inactivation of MPF activity is followed by a slower decline in MAPK activity; reduction in activity of both kinases is required for completion of meiosis and pronuclear formation (13). To determine whether Ca2+ influx influenced the activities of these kinases, we used a dual kinase assay to measure MPF and MAPK activities in individual ICSI eggs in which Ca2+ fluxes were modulated. As expected, in control ICSI eggs MPF activity declined within 1 h after ICSI, whereas MAPK activity was reduced by 4 h after ICSI (Fig. 1 M and N). Neither MPF nor MAPK activities were significantly different from ICSI egg controls when ICSI eggs were cultured without extracellular Ca2+ or were cultured in Gd3+ to block Ca2+ influx and/or efflux, at either time point (Fig. 1 M and N). To determine whether modulating Ca2+ influx or efflux affected cortical granule (CG) exocytosis, ICSI eggs were cultured for 1 h under the conditions described above and then stained for CGs. The extent of CG exocytosis was similar in all groups, although there was a subtle difference in the distance of some CGs from the plasma membrane in the groups where Ca2+ influx was prevented (Fig. S3 A–E). There was no difference among the treatment groups in alterations of the zona pellucida in response to CG exocytosis as indicated by the degree of proteolytic cleavage of zona pellucida protein ZP2 to ZP2f (Fig. S3F). 4170 | www.pnas.org/cgi/doi/10.1073/pnas.1112333109

To rule out the possibility that the abnormalities in egg activation under Ca2+ insulation conditions was a result of Gd3+ toxicity, we washed the ICSI eggs free of Gd3+ after 1.5 h and placed them into standard embryo culture medium. The majority (13/21; 62%) of the ICSI eggs continued to exhibit Ca2+ oscillations (Fig. 2 A and B). Almost all control eggs emitted a second polar body by 1 h after ICSI, whereas the Ca2+-insulated ICSI eggs remained arrested in anaphase 1.5 h after ICSI. If washed free of Gd3+ after treatment for 1.5 h, the ICSI eggs emitted a second polar body and formed two pronuclei (Fig. 2 C and D). These embryos progressed to the blastocyst stage almost as well as controls (Fig. 2D). However, if the ICSI eggs were washed free of Gd3+ after treatment for 3 h, they failed to emit a polar body, formed three pronuclei, and cleaved (Fig. 2 C and D). These embryos failed to progress beyond the two-cell stage; this finding was expected because of their abnormal chromosome complement. We also tested for Gd3+ toxicity by parthenogenetically activating eggs in the presence of Gd3+-containing media. MII-

Fig. 1. Effects of inhibiting Ca2+ fluxes on Ca2+ oscillations, cell cycle resumption, and MPF and MAPK activities after ICSI. ICSI eggs were observed for alterations in intracellular Ca2+ for 1 h after ICSI; spindle morphology/DNA configuration and MPF and MAPK activities were determined 1 h and 6 h after ICSI. Fractions on tracings indicate the number of ICSI eggs/total to exhibit indicated pattern; see also Table S3. (A–C) HBSS/Ca. (D–F) HBSS. (G–I) HBSS/Ca/ LGd. (J–L) HBSS/HGd. (M and N) MPF and MAPK assays on single MII eggs and ICSI eggs cultured 1 or 4 h in the indicated media. For these kinase assays, HBSS/HGd contained no lactate and only 2 mM Gd3+. Data expressed as mean ± SEM of four experiments; one egg evaluated per group in four independent experiments. *P < 0.05 compared with MII, ANOVA. (Scale bar: 20 μm.)

Miao et al.

arrested eggs were microinjected with cRNA encoding a constitutively active form of CaMKIIγ (CA-CaMKIIγ), which can activate mouse eggs in the absence of Ca2+ oscillations (5, 14). More than 90% of all injected eggs (n ≥ 40 per group) cultured for 6 h in HBSS/Ca/LGd or HBSS/HGd formed pronuclei, suggesting that the Gd3+ did not have toxic side effects. Effects of Buffering Intracellular Ca2+ on Events of ICSI-Induced Egg Activation. Based on the above findings, we predicted that even if

repetitive cytoplasmic Ca2+ oscillations were blocked, second polar body emission would occur after ICSI as long as Ca2+ influx from the extracellular medium was allowed. The Ca2+ chelator 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid acetoxymethyl ester (BAPTA/AM) has been used to manipulate the extent of Ca2+ oscillations that occur after IVF of zona pellucida-free mouse eggs (15, 16). We first tested various concentrations and times of exposure to BAPTA/AM to determine the treatment that would limit the egg to having a single Ca2+ transient after ICSI. The single transient pattern was almost always observed in ICSI eggs loaded with 1 or 2 μM BAPTA/AM for 60 min, whether or not there was Ca2+ in the extracellular medium, whereas only a single extremely low amplitude Ca2+ transient was observed in ICSI eggs loaded with 5 μM BAPTA/AM (Fig. 3 A, D, G, and J and Fig. S4). Staining of the spindles and F-actin demonstrated that a single Ca2+ transient was sufficient to induce meiosis resumption followed by polar body emission and pronucleus formation as long as extracellular Ca2+ was present (Fig. 3 D–F). In contrast, BAPTA/ AM-treated ICSI eggs that exhibited a single Ca2+ transient but were cultured in the absence of extracellular Ca2+ failed to resume meiosis and instead formed a second spindle in the region of the sperm DNA (Fig. 3 G–I). The spindle morphology observed in these ICSI eggs was identical to that in ICSI eggs treated with 5 μM BAPTA/AM (Fig. 3 J–L). Despite preloading with 1 μM BAPTA/AM to inhibit subsequent Ca2+ oscillations, >60% of the ICSI eggs placed into Ca2+-containing medium after ICSI emitted a second polar body and formed two pronuclei, whereas none of the ICSI eggs placed into Ca2+-free medium did so (Fig. 3M). ICSI eggs in the 5 μM BAPTA/AM group rarely formed pronuclei; pronuclei formed only if they were cultured in Ca2+-containing medium (Fig. 3M). Measurements of MPF and MAPK activities were consistent with these findings. One hour after ICSI, MPF levels decreased Miao et al.

significantly in control ICSI eggs and were slightly lower than in MII eggs in both of the BAPTA/AM-treated groups (Fig. 3N). Six hours after ICSI, MPF levels were significantly reduced in control ICSI eggs and in eggs treated with 1 μM BAPTA/AM then cultured in Ca2+-containing medium, whereas in BAPTA/ AM-treated ICSI eggs cultured in Ca2+-free medium, the MPF levels had returned to a level similar to that in MII eggs (Fig. 3O). At the 6-h time point, MAPK levels in BAPTA/AM-treated eggs cultured in Ca2+-containing medium were slightly lower than in MII eggs but not as low as in control ICSI eggs, whereas MAPK levels in BAPTA/AM-treated eggs cultured in Ca2+-free medium were similar to MII eggs (Fig. 3O). Taken together with the data presented regarding effects of controlling Ca2+ fluxes with Gd3+, the results of the BAPTA/AM experiments support the idea that movement of Ca2+ across the plasma membrane is required for spindle rotation and second polar body emission. In addition, assuming there is at least one ICSI-induced Ca2+ transient, Ca2+ influx can substitute for subsequent Ca2+ oscillations to drive cell cycle resumption during egg activation. Ca2+ Influx and Events of IVF-Induced Egg Activation. Although most

aspects of ICSI-induced and IVF-induced events of egg activation are essentially identical, there are subtle differences in the Ca2+ oscillatory patterns observed (17) and an obvious difference in events that occur during IVF in the egg’s outer cortex during sperm-egg fusion. To determine whether Ca2+ influx was required to support polar body emission after standard IVF, we performed a series of experiments in which zona pellucida-free eggs were fertilized quickly by using a high sperm concentration and then observed under different culture conditions for their ability to resume meiosis and emit a polar body. Ca2+-oscillatory patterns of eggs placed after sperm-egg fusion into HBSS/Ca or HBSS/HGd (Ca2+ insulation) were similar to those observed after ICSI, with the caveat that the first Ca2+ transient occurred before imaging could be started (Fig. 4 A and C). More than 90% (30/33) of fertilized eggs cultured in Ca2+-containing medium emitted a second polar body after 1 h in culture (Fig. 4B and Table S2). Significantly fewer (20/56; 36%) of the Ca2+-insulated fertilized eggs emitted a second polar body, whereas the majority (34/56; 61%) remained arrested in anaphase II (Fig. 4D). In a second set of IVF experiments, eggs were loaded with 1 μM BAPTA/AM before IVF and then cultured in the presence or absence of extracellular Ca2+. These eggs exhibited a single Ca2+ transient PNAS | March 13, 2012 | vol. 109 | no. 11 | 4171

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Fig. 2. Ca2+ oscillation patterns and development of ICSI eggs after washing out 5 mM Gd3+. (A and B) Representative tracings of ICSI eggs cultured in HBSS/HGd for 1.5 h and then transferred to HBSS/Ca. Fraction indicates number of ICSI eggs/total to exhibit the indicated pattern. (C) Chromatin configuration after washing ICSI eggs free of 5 mM Gd3+ after 1.5 or 3 h of treatment; see Table S3 for egg numbers. ICSI eggs were cultured in (a–c) HBSS/Ca for 1.5 (a), 3 (b), or 6 h (c); HBSS/HGd for 1.5 h (d); (e and f) HBSS/HGd were cultured for 1.5 h then HBSS/Ca for 1.5 h (e) or 3 h (f); (g) HBSS/HGd for 3 h; (h and i) HBSS/HGd for 3 h then HBSS/Ca for 1.5 (h) or 3 h (i). PB2, second polar body; Sp, sperm DNA. (Scale bar: 20 μm.) (D) Effects of washing out 5 mM Gd3+ after 1.5 or 3 h on embryo development. Graph shows the percentage of ICSI eggs to reach each embryo stage at the specified times after ICSI. n = 37–40 per group from three independent experiments. *P < 0.0001, Fisher’s exact test. Ctr, no Gd3+ treatment; 2PN, two pronuclei; 3PN, three pronuclei.

Fig. 4. Effects of blocking Ca2+ influx on chromatin configuration after IVF. (A–D) Fertilized eggs were observed for alterations in intracellular Ca2+ for 1 h and spindle morphology was determined 1.5 h after IVF. (A and B) HBSS/ Ca. (C and D) HBSS/HGd. (E–H) One micromolar BAPTA/AM-loaded fertilized eggs cultured in CZB (E and F) or Ca2+-free CZB (G and H). Tracings are representative of 18–23 eggs per group. Images shown represent the most common spindle morphology in that group; see also Table S2.

Fig. 3. Effects of BAPTA/AM and extracellular Ca2+ on Ca2+ oscillatory pattern, cell cycle resumption, and MPF and MAPK activities in ICSI eggs. MII eggs were loaded with 0, 1, or 5 μM BAPTA/AM for 60 min. The eggs then underwent ICSI and were observed for Ca2+ oscillatory patterns, cell cycle resumption, and MPF and MAPK activities in CZB medium (contains 2 mM Ca2+) (33) or Ca2+-free CZB. ICSI eggs were stained for actin, tubulin, and DNA either 1 (B, E, H, and K) or 6 h (C, F, I, and L) after ICSI; see Table S3 for egg numbers. (A–C) ICSI eggs cultured in CZB; (D–F) 1 μM BAPTA/AM-treated ICSI eggs cultured in CZB; (G–I) 1 μM BAPTA/AM-treated ICSI eggs cultured in Ca2+-free CZB; (J–L) 5 μM BAPTA/AM-treated ICSI eggs cultured in CZB. (M) ICSI eggs were cultured for 6 h under the indicated conditions and then evaluated for pronucleus (PN) formation. Data expressed as mean ± SEM of three experiments. Groups with different letters (a, b, and c) are significantly different, P < 0.05, ANOVA. (N and O) MPF and MAPK assays of MII eggs and BAPTA/ AM-treated ICSI eggs. Data expressed as the mean ± SEM of four experiments. *P < 0.05 compared with MII eggs, ANOVA. (Scale bar: 20 μm.)

(Fig. 4 E and G). The majority (21/30; 70%) of the BAPTA/AMloaded eggs cultured with Ca2+ emitted polar bodies, whereas 4172 | www.pnas.org/cgi/doi/10.1073/pnas.1112333109

55% (16/29) of those cultured without Ca2+ remained arrested in metaphase II (Fig. 4 F and H), and a small number either entered anaphase or emitted a second polar body (Table S2). These experiments were limited by our inability to precisely time transfer of the fertilized eggs after sperm-egg fusion but before the first Ca2+ transient, so we could not be certain that Ca2+ influx was completely inhibited in all cases in the relevant groups. However, both experiments demonstrated that the majority of the fertilized eggs exhibited essentially the same responses as ICSI eggs cultured under the same conditions and suggest that the requirement for Ca2+ influx applies to both IVF and ICSI. Store-Operated Calcium Entry and Calcium Signaling at Fertilization.

Mouse eggs express STIM1, the ER Ca2+ sensor, and there is evidence that STIM1 could mediate SOCE at fertilization (18). ORAI1, a Ca2+ pore-forming protein that mediates SOCE, is also expressed in mouse eggs (19). To test whether egg activation signaling was initiated specifically by Ca2+ entry via SOC channels, we first documented the efficacy of two different SOCE inhibitors in preventing Ca2+ entry into mouse eggs in response to Ca2+ store depletion. Thapsigargin, a potent and specific inhibitor of SERCA pumps, is widely used to cause depletion of ER Ca2+ stores by preventing reuptake from the cytoplasm. Freshly ovulated metaMiao et al.

Calcium Influx and CaMKIIγ Signaling at Fertilization. To determine whether Ca2+ influx across the plasma membrane is upstream or downstream of CaMKIIγ during egg activation signaling, we tested whether extracellular Ca2+ was required for CACaMKIIγ–induced parthenogenetic activation. MII eggs were injected with cRNA encoding CA-CaMKIIγ and then cultured for 6 h in the presence or absence of extracellular Ca2+. More than 90% of the CA-CaMKIIγ cRNA-injected eggs formed a second polar body and pronucleus, whether or not Ca2+ was present in the culture medium, whereas controls did not (Fig. S5C). Because injection of this cRNA elicits normal activation, bypassing the need for Ca2+ influx, we conclude that Ca2+ influx across the plasma membrane is not required for egg activation provided there is sufficient CaMKIIγ activity. These findings suggest that CaMKIIγ activation is positioned downstream of Ca2+ influx during sperm-induced egg activation.

Discussion In this study, we used the Ca2+ insulation technique to demonstrate that bulk oscillations in cytoplasmic Ca2+ alone are not sufficient to fully activate eggs in the absence of Ca2+ influx. Instead, cytoplasmic Ca2+ oscillations fully support pronucleus formation, but Ca2+ influx via Gd3+-sensitive plasma membrane channels is required to mediate spindle rotation and second polar body emission at fertilization. These data indicate an absolute requirement for Ca2+ influx to transmit signals required for complete egg activation rather than simply serving as a source of Ca2+ to refill depleted intracellular stores. This observation is important because it provides information regarding spatially restricted control of egg activation signaling in the subplasma membrane region that will alter the prevailing view that global cytoplasmic calcium changes directly drive all downstream Ca2+-dependent signaling molecules to carry out egg activation. A schematic indicating how this information extends the current view of signaling during egg activation is shown in Fig. 5. The second method used for separating the effects of Ca2+ influx from the effects of sperm-mediated Ca2+ release from IP3sensitive intracellular stores was chelating intracellular Ca2+ levels with BAPTA/AM. These experiments showed that one Ca2+ transient in response to ICSI was sufficient to cause cell cycle resumption but only if extracellular Ca2+ was present, indicating that Ca2+ influx can substitute for global cytoplasmic Ca2+ oscillations to drive cell cycle resumption. This finding is consistent with a previous report that chelating intracellular Ca2+ levels sufficiently to allow only a single Ca2+ transient during standard IVF causes resumption of meiosis in a high percentage of the eggs (16). Lawrence et al. (15) demonstrated that at least two Ca2+ Miao et al.

transients are required to induce pronuclear formation in eggs treated after fertilization with BAPTA/AM to block subsequent Ca2+ transients. This slight discrepancy from our findings is likely a result of the high concentration of BAPTA/AM (5 μM) used in these experiments, which in our hands effectively suppressed intracellular Ca2+ alterations and prevented cell cycle resumption, even with extracellular Ca2+ present. Addition of the BAPTA/AM after the oscillations began, allowing for normal initial responses to Ca2+ influx, probably explains the successful pronucleus formation despite the high BAPTA/AM concentration used in their study. The results presented here shed light on previous observations that antibody-mediated ligation of an extracellular mouse egg membrane-associated protein resulted in reorganization of cortical actin and cell cycle resumption in the absence of measurable Ca2+ oscillations (22). Furthermore, the results are consistent with experiments in which MPF and MAPK inhibitors were used to induce mouse egg activation (23). Our data suggest that in both cases, Ca2+ influx from the culture medium across the plasma membrane supported the observed activation events. Our findings are also consistent with the previous observation that eggs can be activated by Ca2+ influx induced by repetitive cell membrane electroporation in Ca2+-containing medium in the absence of Ca2+ release from internal stores (24). Studies in which this electroporation method was used to show that more than one Ca2+ transient was required for complete parthenogenetic egg activation appear to conflict with our results (25). However, the electroporation experiments were done in the absence of sperm, and our interpretation of the different findings is that the sperm provides a unique signal that is more effective in inducing downstream signaling cascades important for egg activation than Ca2+ influx via electroporation. Numerous types of regulated Ca2+ channels support Ca2+ influx across plasma membranes (26). Although some Ca2+ influx via SOCE channels probably occurs during egg activation, it is not likely to be required based on our findings that two different SOCE inhibitors could not block Ca2+ oscillations or egg activation in response to sperm. This finding is consistent with previous observations that SOCE is inactivated during meiosis in Xenopus oocytes (27, 28). Instead, our data suggest that one or more alternate Ca2+-permeable channels are opened in response to the initial Ca2+ transient, either directly in response to Ca2+

Fig. 5. Working model of Ca2+ influx signaling during egg activation. Ca2+ oscillations activate CaMKIIγ, causing decreases in MPF and MAPK activities that promote cell cycle resumption. The first Ca2+ transient increases CaMKIIγ activity enough to drive the metaphase to anaphase transition and causes exocytosis of sufficient CGs to induce the block to polyspermy. The first Ca2+ transient also triggers Ca2+ influx, providing Ca2+ to refill ER stores necessary for persistence of the oscillations. This Ca2+ influx activates subplasma membrane signaling required to support spindle rotation and second polar body (PBII) emission, and may help retain CGs at the plasma membrane (PM). Additional Ca2+ oscillations or Ca2+ influx are required for pronucleus (PN) formation. CaMKIIγ activity can bypass the need for Ca2+ oscillations and Ca2+ influx to support PBII emission and PN formation, but not CG exocytosis. Solid arrows indicate well-documented direct or indirect connection; dashed arrows indicate less well characterized connection.

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phase II-arrested eggs were depleted of Ca2+ by incubation in 10 μM thapsigargin in Ca2+-free medium for 1 h, and then SOCE was observed after adding 5 mM Ca2+ into the extracellular medium. In somatic cells, 1–2 μM Gd3+ is sufficient to inhibit SOCE; however, 10 μM Gd3+ was required for complete inhibition of SOCE in Ca2+-depleted eggs under these conditions (Fig. S5A). A second SOCE inhibitor, Synta66 (20), also completely blocked SOCE in Ca2+-depleted eggs when used at 2 μM (Fig. S5A). To test whether SOCE was required for egg activation signaling, we treated ICSI eggs with 10 μM Gd3+ or 2 μM Synta66 and recorded alterations in cytoplasmic Ca2+. These SOCE inhibitors did not prevent ICSI-induced Ca2+ influx as indicated by the occurrence of persistent Ca2+ oscillations (Fig. S5A) and by quenching of the fura 2 signal when 1 mM Mn2+ was added to the incubation medium after the first Ca2+ transient (Fig. S5B) (21). In addition, ICSI eggs incubated in 10 μM Gd3+ and 2 μM Synta66 emitted a second polar body and formed pronuclei to a similar extent as controls. Taken together, these observations indicate that although SOCE may occur at fertilization, it is not required for egg activation and that alternate Ca2+ influx channels can support the requisite Ca2+ entry.

sensitization or indirectly in response to an activated signaling cascade, (e.g., one involving PLC or PKC activation). There are many candidate Ca2+ channels that could serve this function. It is not likely that Ca2+ influx occurs via voltage-gated channels because mouse eggs only experience very small changes in membrane potential at fertilization (29). Transient receptor potential (TRP) channels are good candidates because they are widely expressed in nonexcitable cells and have important roles in modulating Ca2+ influx (30). Of note, some TRP channels can be sensitized by Ca2+ or by PKC-dependent phosphorylation. In addition, canonical TRPC channels can be activated by diacylglycerol (31), which is generated by PLC activity at fertilization and can be detected at the egg plasma membrane (8). In summary, the results presented herein indicate that Ca2+ influx not only maintains Ca2+ oscillations by replenishing Ca2+ stores, but also provides an important spatially restricted Ca2+ signal required for complete egg activation at fertilization. These studies provide evidence that bulk intracellular Ca2+ oscillations do not directly activate all downstream signaling pathways required for egg activation. Instead, the Ca2+ oscillations trigger Ca2+ influx back into the egg, likely by activating Ca2+ channels in addition to those normally activated by Ca2+ store depletion. This Ca2+ influx is required to activate the downstream signaling molecules including CaMKIIγ that are essential for sperm-induced resumption of meiosis and embryo development and are particularly important in driving the cortical actin-based functions of spindle rotation and polar body emission. Understanding how Ca2+ influx affects egg activation is important for improving laboratory practices in clinical assisted reproductive technologies such as standard IVF and ICSI. In addition, this knowledge is particularly relevant to the growing field of fertility preservation 1. Ducibella T, Schultz RM, Ozil JP (2006) Role of calcium signals in early development. Semin Cell Dev Biol 17:324–332. 2. Swann K, Saunders CM, Rogers NT, Lai FA (2006) PLCzeta(zeta): A sperm protein that triggers Ca2+ oscillations and egg activation in mammals. Semin Cell Dev Biol 17: 264–273. 3. Igusa Y, Miyazaki S (1983) Effects of altered extracellular and intracellular calcium concentration on hyperpolarizing responses of the hamster egg. J Physiol 340: 611–632. 4. Kline D, Kline JT (1992) Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev Biol 149:80–89. 5. Backs J, et al. (2010) The gamma isoform of CaM kinase II controls mouse egg activation by regulating cell cycle resumption. Proc Natl Acad Sci USA 107:81–86. 6. Markoulaki S, Matson S, Abbott AL, Ducibella T (2003) Oscillatory CaMKII activity in mouse egg activation. Dev Biol 258:464–474. 7. Halet G, Tunwell R, Parkinson SJ, Carroll J (2004) Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs. J Cell Biol 164: 1033–1044. 8. Yu Y, Halet G, Lai FA, Swann K (2008) Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation. Biol Cell 100:633–643. 9. Di Capite J, Ng SW, Parekh AB (2009) Decoding of cytoplasmic Ca(2+) oscillations through the spatial signature drives gene expression. Curr Biol 19:853–858. 10. Luo D, Broad LM, Bird GS, Putney JW, Jr. (2001) Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells. J Biol Chem 276: 5613–5621. 11. Bird GS, Putney JW, Jr. (2005) Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells. J Physiol 562:697–706. 12. Kwan CY, Takemura H, Obie JF, Thastrup O, Putney JW, Jr. (1990) Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. Am J Physiol 258:C1006–C1015. 13. Ducibella T, Fissore R (2008) The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol 315:257–279. 14. Knott JG, et al. (2006) Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations. Dev Biol 296: 388–395. 15. Lawrence Y, Ozil JP, Swann K (1998) The effects of a Ca2+ chelator and heavy-metalion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases. Biochem J 335:335–342. 16. Gardner AJ, Williams CJ, Evans JP (2007) Establishment of the mammalian membrane block to polyspermy: Evidence for calcium-dependent and -independent regulation. Reproduction 133:383–393.

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because it is essential to design egg cryopreservation methods that prevent even low levels of Ca2+ influx from occurring prematurely and leading to egg activation in the absence of the fertilizing sperm. Materials and Methods Generation of Camk2g Mutant Construct and in Vitro Transcription. Detailed procedures are described in SI Materials and Methods. Animals and Chemicals. Detailed information is provided in SI Materials and Methods. Egg Collection, Treatments, and Imaging. Detailed protocols are described in SI Materials and Methods. MPF and MAPK Assays. MPF and MAPK activities were determined in a single assay as described (32). Immunoblotting, Fluorescence, and Immunofluorescence Microscopy. Detailed protocols are described in SI Materials and Methods. Data Analysis. Data were analyzed by using Prism software (Graphpad). Analyses used are indicated in figure legends. ACKNOWLEDGMENTS. We thank Jurrien Dean (National Institute of Diabetes and Digestive and Kidney Diseases) for the mZP2 antibody and Glaxo Smith Kline for the gift of Synta66; Jim Putney and Gary Bird (National Institute on Environmental Health Sciences) for critical reading of the manuscript and advice throughout this project; and Grace Kissling (National Institute on Environmental Health Sciences) for assistance with statistical analyses. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences, Grant Z01-ES102985.

17. Sato MS, Yoshitomo M, Mohri T, Miyazaki S (1999) Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26: 49–58. 18. Gómez-Fernández C, et al. (2009) Relocalization of STIM1 in mouse oocytes at fertilization: Early involvement of store-operated calcium entry. Reproduction 138: 211–221. 19. Lopez-Guerrero AM, Pozo-Guisado E, Gomez-Fernandez C, Alvarez IS, MartinRomero FJ (2012) Calcium signalling in mouse oocyte maturation: The roles of STIM1, ORAI1 and SOCE. Mol Hum Reprod, 10.1093/molehr/gar071. 20. Ng SW, di Capite J, Singaravelu K, Parekh AB (2008) Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels. J Biol Chem 283:31348–31355. 21. Chiavaroli C, Bird G, Putney JW, Jr. (1994) Delayed “all-or-none” activation of inositol 1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes. J Biol Chem 269:25570–25575. 22. Tutuncu L, Stein P, Ord TS, Jorgez CJ, Williams CJ (2004) Calreticulin on the mouse egg surface mediates transmembrane signaling linked to cell cycle resumption. Dev Biol 270:246–260. 23. Phillips KP, et al. (2002) Inhibition of MEK or cdc2 kinase parthenogenetically activates mouse eggs and yields the same phenotypes as Mos(-/-) parthenogenotes. Dev Biol 247:210–223. 24. Ozil JP, Swann K (1995) Stimulation of repetitive calcium transients in mouse eggs. J Physiol 483:331–346. 25. Ducibella T, et al. (2002) Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. Dev Biol 250:280–291. 26. Parekh AB, Putney JW, Jr. (2005) Store-operated calcium channels. Physiol Rev 85: 757–810. 27. Machaca K, Haun S (2000) Store-operated calcium entry inactivates at the germinal vesicle breakdown stage of Xenopus meiosis. J Biol Chem 275:38710–38715. 28. Arredouani A, Yu F, Sun L, Machaca K (2010) Regulation of store-operated Ca2+ entry during the cell cycle. J Cell Sci 123:2155–2162. 29. Jaffe LA, Cross NL, Picheral B (1983) Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians. Dev Biol 98:319–326. 30. Gees M, Colsoul B, Nilius B (2010) The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2:a003962. 31. Hardie RC (2007) TRP channels and lipids: From Drosophila to mammalian physiology. J Physiol 578:9–24. 32. Svoboda P, Stein P, Hayashi H, Schultz RM (2000) Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127: 4147–4156. 33. Chatot CL, Lewis JL, Torres I, Ziomek CA (1990) Development of 1-cell embryos from different strains of mice in CZB medium. Biol Reprod 42:432–440.

Miao et al.

Supporting Information Miao et al. 10.1073/pnas.1112333109 SI Materials and Methods Generation of Camk2g Mutant Construct and in Vitro Transcription.

The following primers were used to amplify nucleotides 124–997 of Camk2g (GenBank accession no. NM_172171.2) from mouse brain cDNA: 5′-AATTCTGCAGGCCAGTATGGCCAC-3′ and 5′-AATGGGATCCTTACTATAAGCACTCTACCGT-3′. These nucleotides encode the first 291 amino acids of CaMKIIγ but lack the autoinhibitory region, so the translated protein should be constitutively active (1, 2). The PCR product was inserted into the PstI and BamHI sites of the in vitro transcription vector, pIVT (3). The final construct was linearized and transcribed in vitro by using the T7 mMESSAGE mMACHINE kit (Ambion). For use as a control, cRNA encoding EGFP was similarly transcribed from a modified version of pIVT containing the EGFP sequence. The cRNA was purified by using the RNeasy kit (Qiagen), eluted in RNase-free water, resuspended to a final concentration of 20 μg/ mL, and stored in small aliquots at −80  C until use. Animals and Chemicals. CF-1 female mice (6 wk of age; Harlan Laboratories) and B6SJLF1 male mice (8-12 wk of age; The Jackson Laboratories) were housed in a temperature-controlled environment under a 12 h light:12 h dark cycle. All animal procedures complied with National Institutes of Health/ National Institutes of Environmental Health Sciences animal care guidelines. All chemicals were purchased from Sigma Chemical unless otherwise indicated. Egg Collection, Culture Media, and Treatments. Egg collection and treatments. Female mice were superovulated by using 5 IU

equine CG followed 48 h later with 5 IU human CG (hCG) (both from Calbiochem). Metaphase II-arrested (MII) eggs were collected 13–14 h after hCG administration into Whitten’s-Hepes medium (4) containing 0.01% polyvinyl alcohol (Whitten’sHepes-PVA), and cumulus cells were removed by a brief hyaluronidase treatment. For Gd3+ experiments, control eggs were cultured after ICSI or IVF in Hepes-buffered saline solution (HBSS; 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 11 mM glucose, and 20 mM Hepes at pH 7.4) containing 2 mM CaCl2 (HBSS/Ca). For Gd3+ treatments between 10 and 200 μM, Gd3+ was dissolved in HBSS/Ca to avoid formation of precipitates. We refer to HBSS/Ca containing 200 μM Gd3+ as HBSS/Ca/LGd. Ca2+ insulation was initially carried out in HBSS containing 2 mM Gd3+. This medium was effective at causing Ca2+ insulation as shown by repetitive Ca2+ oscillations in the absence of extracellular Ca2+, but precipitates formed and ICSI eggs died when they were washed free of this medium after 4–6 h of treatment for long-term culture. The only experiments in this report using 2 mM Gd3+ for Ca2+ insulation are the MPF and MAPK assays shown in Fig. 1. For all other experiments, we modified the Ca2+ insulation medium to HBSS containing 37 mM sodium lactate and 5 mM Gd3+ (HBSS/HGd); the addition of sodium lactate prevented precipitate formation at concentrations up to 20 mM Gd3+. We confirmed experimentally that the addition of 37 mM sodium lactate to HBSS, HBSS/ Ca, or HBSS/Ca/LGd did not affect Ca2+ oscillatory patterns or development of ICSI eggs. For experiments testing embryo development, eggs were washed free of HBSS/Ca or Gd3+-containing media and cultured in KSOM (Millipore, catalog no.MR106-D). For the BAPTA/AM experiments, eggs were loaded for 2+ 60 min with varying concentrations of BAPTA/AM in Ca -free CZB medium (81.6 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 0.11 mM EDTA, 5.6 mM glucose, 37 mM sodium lactate, 1.2 mM Miao et al. www.pnas.org/cgi/content/short/1112333109

KH2PO4, 25.1 mM NaHCO3, 0.27 mM pyruvate, 1 mM glutamine, and 5 mg/mL BSA at pH 7.4) (5). After ICSI or IVF, the culture medium was either CZB (which contains 2 mM CaCl2) or Ca2+-free CZB. Eggs were cultured at 37  C in a humidified atmosphere of room air (HBSS) or 5% CO2 (CZB or KSOM). Intracytoplasmic sperm injection (ICSI). Cauda epididymal sperm were collected from B6SJLF1 males into HTF medium (Millipore, catalog no. MR-070-D). Sperm tails were removed by repeated freezing and thawing, and the sperm heads were stored in HTF at −80  C. To perform ICSI, a single sperm head was microinjected into an MII egg by using a Leica DMI 6000B inverted microscope equipped with a XenoWorks Micromanipulator system and a PrimeTech PMM-150FU Piezo drill (Sutter Instruments). The injection medium was Whitten’s-Hepes-PVA, and groups of 6–8 eggs were injected and moved within