Supporting Information

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 s- Hepes-PVA), and cumulus cells were removed by a brief hyaluronidase treatment. For Gd 3+ 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 MgCl 2,11mM glucose, and 20 mm Hepes at ph 7.4) containing 2 mm CaCl 2 (HBSS/Ca). For Gd 3+ treatments between 10 and 200 μm, Gd 3+ was dissolved in HBSS/Ca to avoid formation of precipitates. We refer to HBSS/Ca containing 200 μm Gd 3+ as HBSS/Ca/LGd. Ca 2+ insulation was initially carried out in HBSS containing 2 mm Gd 3+. This medium was effective at causing Ca 2+ insulation as shown by repetitive Ca 2+ oscillations in the absence of extracellular Ca 2+, 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 Gd 3+ for Ca 2+ insulation are the MPF and MAPK assays shown in Fig. 1. For all other experiments, we modified the Ca 2+ insulation medium to HBSS containing 37 mm sodium lactate and 5 mm Gd 3+ (HBSS/HGd); the addition of sodium lactate prevented precipitate formation at concentrations up to 20 mm Gd 3+. We confirmed experimentally that the addition of 37 mm sodium lactate to HBSS, HBSS/ Ca, or HBSS/Ca/LGd did not affect Ca 2+ oscillatory patterns or development of ICSI eggs. For experiments testing embryo development, eggs were washed free of HBSS/Ca or Gd 3+ -containing media and cultured in KSOM (Millipore, catalog no.mr- 106-D). For the BAPTA/AM experiments, eggs were loaded for 60 min with varying concentrations of BAPTA/AM in Ca 2+ -free CZB medium (81.6 mm NaCl, 4.8 mm KCl, 1.2 mm MgSO 4, 0.11 mm EDTA, 5.6 mm glucose, 37 mm sodium lactate, 1.2 mm KH 2 PO 4, 25.1 mm NaHCO 3, 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 CaCl 2 ) or Ca 2+ -free CZB. Eggs were cultured at 37 C in a humidified atmosphere of room air (HBSS) or 5% CO 2 (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 <5 min after initiating injections into the appropriate culture medium. In vitro fertilization (IVF). IVF of zona pellucida-free eggs was performed as described (6) with the following modifications. Epididymal sperm from a B6SJLF1 male was collected into 500 μl of HTF medium containing 3 mg/ml BSA (HTF/BSA) and allowed to capacitate for 1.5 2 h before use. Groups of eggs were inseminated for 10, 20, or 30 min in a 100-μL drop of HTF/BSA containing 2 10 6 sperm per ml. After the specified insemination time, the eggs were quickly washed through three drops of Ca 2+ -free CZB medium by using a thin bore pipette to remove unbound sperm. Half of the eggs were then transferred to the control medium and half to the treatment medium and cultured for an additional 1.5 h. For Gd 3+ experiments, the control medium was HBSS/Ca containing 37 mm lactate and the treatment medium was HBSS containing 5 mm Gd 3+ and 37 mm lactate (HBSS/HGd). For BAPTA/AM experiments, the control medium was CZB and the treatment medium was Ca 2+ -free CZB. After 1.5 h in the various treatments, eggs were fixed and stained for DNA, tubulin, and actin. Chromatin configuration was analyzed only from control and treated eggs matched for insemination time and that contained a single decondensing sperm head; these eggs are referred to as fertilized eggs throughout the manuscript. Unfertilized and polyspermic eggs were not counted. Ca 2+ Imaging and Mn 2+ Quenching Techniques. Before undergoing ICSI or IVF, eggs were incubated for 20 min in Ca 2+ -free CZB containing 2 mm fura-2, acetoxymethyl ester (Invitrogen) and 0.025% Pluronic-F127 (Invitrogen). Intracellular Ca 2+ concentration ([Ca 2+ ]i) was measured by using an InCyt Im2 ratio imaging system (Intracellular Imaging) attached to an Olympus CKX41 inverted microscope with an Okolabs stage microenvironmental chamber enclosed in a CO 2 microscope cage incubator (Warner Instruments). The objective was an Olympus 20 UPlanSApo, 0.75 NA. Fluorescence ratio images were generated by using excitation filters of 340 and 380 nm. All measurements were performed at 37 C in a humidified atmosphere of room air (HBSS) or 5% CO 2 (CZB). Area under the curve of the Ca 2+ oscillation tracings was calculated by using the trapezoid rule (7). The Mn 2+ quenching technique was performed essentially as described (8). After observation of the first Ca 2+ transient, 1 mm Mn 2+ was added to the culture medium during continuous fluorescence monitoring. An algebraic summation of the fluorescence values with excitation at 340 nm and 380 nm (Fiso = 3 F340 + F380) was used to obtain a measure of Ca 2+ -independent fluorescence. 1of7

Fluorescence and Immunofluorescence Microscopy. For spindle staining, eggs were fixed in 4% (wt/vol) paraformaldehyde, washed in blocking buffer (PBS containing 0.3% BSA, 0.01% Tween-20, and 0.01% NaN 3 ), and then permeabilized in PBS containing 0.3% BSA, 0.1% Triton X-100, and 0.01% NaN 3. The eggs were incubated in primary anti α-tubulin antibody (Sigma) diluted 1:100 in blocking buffer, washed, then incubated in Alexa Fluor 633 goat anti-mouse IgG1 (Invitrogen) diluted 1:1,000 in blocking buffer. In some experiments, F-actin was stained by incubating the eggs in 10 μg/ml Phalloidin tetramethylrhodamine B isothiocyanate (Sigma). Samples were mounted in Vectashield containing 1 mg/ml DAPI (Vector Laboratories). For CG staining, zonae pellucidae were removed by brief incubation in acid Tyrode s solution. The eggs were fixed in 4% (wt/vol) paraformaldehyde in PBS, blocked in PBS containing 0.3% BSA and 100 mm glycine, and then permeabilized in PBS containing 0.1% Triton X-100. CGs were labeled in 100 μg/ml FITC-conjugated peanut agglutinin in PBS. The eggs were washed in blocking buffer and mounted as above. Slides were scanned by using a Zeiss confocal microscope (Zeiss LSM 510 UV). Immunoblotting. Lysates of 12 eggs per group were separated by SDS/PAGE using 4 12% (wt/vol) Tris-glycine gels (Invitrogen) and then transferred to polyvinylidene fluoride membranes (Invitrogen). Membranes were blocked overnight in TBST containing 5% (wt/vol) BSA, then incubated in rat anti-mouse ZP2 antibody (9) diluted 1:1,000 in TBST containing 5% (wt/vol) BSA for 1 h at room temperature. Membranes were washed in TBST and then incubated with horseradish peroxidase-conjugated anti-rat secondary antibody (GE Healthcare) diluted 1:100,000 in TBST for 1 h at room temperature. Detection was performed by using Super Signal West Femto Maximum Substrate Kit (Thermo Fisher Scientific). 1. 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. 2. Cruzalegui FH, et al. (1992) Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase. Proc Natl Acad Sci USA 89:12127 12131. 3. Igarashi H, Knott JG, Schultz RM, Williams CJ (2007) Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization. Dev Biol 312:321 330. 4. 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. 5. 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. 6. Evans JP, Schultz RM, Kopf GS (1995) Mouse sperm-egg plasma membrane interactions: Analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) beta. J Cell Sci 108:3267 3278. 7. Hildebrand FB (1987) Introduction to Numerical Analysis (Dover Publications, Mineola, NY), 2nd Ed. 8. 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. 9. Rankin TL, et al. (2003) Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs. Dev Cell 5:33 43. Fig. S1. Effects of different concentrations of Gd 3+ on Ca 2+ oscillatory patterns in ICSI eggs. Eggs were placed immediately after ICSI into HBSS/Ca containing the indicated concentrations of Gd 3+ and observed for their Ca 2+ oscillatory patterns: (A) 0μM Gd 3+ ;(B) 5μM Gd 3+ ;(C) 10μM Gd 3+ ;(D) 50μM Gd 3+ ;(E) 100 μm Gd 3+ ;(F) 200 μmgd 3+. Fractions on tracings indicate number of ICSI eggs/total to exhibit indicated pattern. At least three independent experiments were done. 2of7

Fig. S2. Examples of Ca 2+ oscillatory patterns in ICSI eggs cultured in control medium or under Ca 2+ -insulation conditions. Eggs were placed immediately after ICSI into HBSS/Ca (A C) or HBSS/HGd (D F) and observed for their Ca 2+ oscillatory patterns. Patterns shown are representative of 14 ICSI eggs per group monitored in at least three independent experiments. 3of7

Fig. S3. Effects of inhibiting Ca 2+ fluxes on cortical granule (CG) exocytosis. ICSI eggs were cultured in the indicated treatment conditions for 1 h after ICSI and then stained for CGs. Representative confocal sections across the cortex (Left), equatorial region (Center), and magnified view of the boxed regions to highlight subcortical CGs (Right) are shown for each treatment. Images are representative of 13 eggs per group. (A) MII eggs. (B) ICSI eggs cultured in HBSS/Ca. (C) ICSI eggs cultured in HBSS. (D) ICSI eggs cultured in HBSS/Ca/LGd. (E) ICSI eggs cultured in HBSS/HGd. (Scale bar: 20 μm.) (F) Groups of 12 eggs were treated as indicated for 1 h after ICSI and then lysed and immunoblotted for ZP2. MII eggs were used as controls. A representative blot from three independent experiments is shown. Ca, HBSS/Ca; ØCa, HBSS; H Gd, HBSS/HGd; L Gd, HBSS/Ca/LGd; MII, MII eggs. 4of7

Fig. S4. Effects of BAPTA/AM and extracellular Ca 2+ on Ca 2+ oscillatory pattern, cell cycle resumption, and MPF and MAPK activities in ICSI eggs. MII eggs were loaded with 0, 1, 2, or 5 μm BAPTA/AM for 60 min. The eggs then underwent ICSI and were observed for Ca 2+ oscillatory patterns, cell cycle resumption, and MPF and MAPK activities in CZB or Ca 2+ -free CZB. ICSI eggs were fixed and stained for actin, tubulin, and DNA either 1 (B, E, H, K, N, and Q) or6h(c, F, I, L, O, and R) after ICSI. (A C) ICSI eggs cultured in CZB; (D F) 1μM BAPTA/AM-treated ICSI eggs cultured in CZB; (G I) 1μM BAPTA/AM-treated ICSI eggs cultured in Ca 2+ -free CZB; (J L) 2μM BAPTA/AM-treated ICSI eggs cultured in CZB; (M O) 2μM BAPTA/AM-treated ICSI eggs cultured in Ca 2+ -free CZB; (P R) 5μM BAPTA/ AM-treated ICSI eggs cultured in CZB. (S) 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, c, and d) are significantly different, P < 0.05, ANOVA. (T and U) MPF and MAPK assays of MII eggs and BAPTA/AM-treated ICSI eggs. Data expressed as the mean ± SEM of four experiments. (Scale bars: 20 μm.) 5of7

Fig. S5. Effects of blocking SOCE and altering extracellular Ca 2+ on egg activation events. (A) Blocking SOCE does not affect ICSI-induced Ca 2+ oscillations. (a c) MII eggs were incubated in thapsigargin to deplete Ca 2+ stores, then were placed into HBSS (a), HBSS + 10 μm Gd 3+ (b), or HBSS + 2 μm Synta66 (c) and monitored for Ca 2+ uptake when 5 mm Ca 2+ was added to the medium (arrows). (d f) ICSI eggs were placed into HBSS/Ca (d), HBSS/Ca + 10 μm Gd 3+ (e), HBSS/ Ca + 2 μm Synta66 (f) and observed for their Ca 2+ oscillatory pattern. Representative tracings are shown; fractions on tracings indicate number of eggs/total to exhibit the indicated pattern. (B) Blocking SOCE does not prevent ICSI-mediated Ca 2+ influx. ICSI eggs were monitored simultaneously for Ca 2+ oscillations and fluorescence quenching in HBSS/Ca containing 10 μm Gd 3+.(a) NoMn 2+ added; (B) 1μM Mn 2+ added after first Ca 2+ transient (arrow). Fractions on tracings indicate number of eggs to exhibit the pattern shown/total observed. (C) Effect of extracellular Ca 2+ on CA-CaMKIIγ induced egg activation. MII eggs were injected with crna encoding EGFP or CA-CaMKIIγ, cultured for 6 h in CZB or Ca 2+ -free CZB and then evaluated for second polar-body (PB2) emission and pronucleus (PN) formation. Data expressed as mean ± SEM of three experiments. *P < 0.05 compared with EGFP, ANOVA. Table S1. Oscillation characteristics of ICSI eggs cultured under control and Ca 2+ -insulation conditions Characteristic Control (n =17) Ca 2+ -insulation (n =14) Peak amplitude, ΔF340/380 1.04 ± 0.04 1.55 ± 0.04** Duration of first oscillation, min 3.4 ± 0.17 7.08 ± 0.45** Frequency, min 1 13.8 ± 1.1 18.7 ± 1.6* AUC, arbitrary units 5.0 ± 0.26 12.7 ± 0.86** n, no. of eggs observed; AUC, area under the curve. *P < 0.05, **P < 0.0001, Mann Whitney test. Table S2. Summary of effects of blocking Ca 2+ influx on chromatin configuration after IVF Experiment Treatment Total fertilized eggs MII (%) AII (%) PB2 (%) Gd HBSS/Ca 33 1 (3.0) 2 (6.1) 30 (90.9) HBSS/HGd 56 2 (3.6) 34 (60.7)* 20 (35.7)* BAPTA/AM CZB 30 5 (16.7) 4 (13.3) 21 (70.0) Ca 2+ -free CZB 29 16 (55.2) 6 (20.7) 7 (24.1) AII, anaphase II; MII, metaphase II; PB2, second polar body emitted. *Significant difference in number of eggs to have specific chromatin configuration in Gd experiment, Fisher s exact test, P < 0.0001. Significant difference in number of eggs to have specific chromatin configuration in BAPTA/AM experiment, Fisher s exact test, P < 0.0001. 6of7

Table S3. Numbers of eggs represented by images or data shown in experiments presented in Figs. 1 3 Option Fig, Treatment 1 2 3 1 HBSS/Ca B: 20/21 C: 20/20 HBSS E: 24/24 F: 21/21 HBSS/Ca/LGd H: 23/26 I: 18/20 HBSS/HGd K: 20/21 L: 20/23 2C Ctr a: 14/16 b: 18/19 c: 17/19 1.5 h wash d: 19/19 e: 17/19 f: 18/20 3 h wash g: 14/15 h: 15/16 i: 13/15 3 Ø BAPTA-AM, CZB B: 21/23 C: 21/21 1 μm BAPTA-AM, CZB E: 18/22 F: 20/23 1 μm BAPTA-AM, Ca 2+ -free CZB H: 22/23 I: 18/22 5 μm BAPTA-AM, CZB K: 21/22 L: 18/22 3M Ø BAPTA-AM, CZB 25 23 1 μm BAPTA-AM, CZB 26 20 1 μm BAPTA-AM, Ca 2+ -free CZB 13 0 5 μm BAPTA-AM, CZB 27 2 5 μm BAPTA-AM, Ca 2+ -free CZB 14 0 For Figs. 1 and 3, option 1 and 2 are 1 h and 6 h, respectively. For Fig. 2, options 1, 2, and 3 are fixed before washing, fixed 3 h after wash, and fixed 6 h after wash, respectively. For Fig. 3M, options 1 and 2 are the number of ICSI eggs and formed 2PN, respectively. Panel labels from each figure indicated before colon; fractions indicate number of ICSI eggs that images represent/total number of ICSI eggs in that group. 7of7