Supporting Online Material for

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1 Supporting Online Material for Autophagy Is Essential for Preimplantation Development of Mouse Embryos Satoshi Tsukamoto, Akiko Kuma, Mirei Murakami, Chieko Kishi, Akitsugu Yamamoto, Noboru Mizushima* *To whom correspondence should be addressed. Published 4 July 2008, Science 321, 117 (2008) DOI: /science This PDF file includes: Materials and Methods Figs. S1 to S9 References Additional material: Movie S1

2 Materials and Methods Animals Mice carrying a transgene encoding LC3 fused to enhanced green fluorescent protein (GFP-LC3 mice) were used to monitor autophagosome formation in oocytes and embryos (S1). Atg5 +/- mice (S2), Atg5 flox/flox mice (S3) and C57BL/6-Tg(Zp3-cre)93Knw/J (Jackson Laboratories) (S4) were used to produce oocyte specific Atg5-deficient mice (Atg5 flox/- ;Zp3-Cre mice). Wild-type C57BL/6 mice were obtained from Japan SLC, Inc. Most mice used in this study were between 8 and 12 weeks old. All animal experiments were approved by the institutional committee of Tokyo Medical and Dental University. Collection and culture of oocytes and embryos Female mice were superovulated by intraperitoneal injection of 5-IU of pregnant mare serum gonadotropin (PMSG; Teikoku Zouki, Japan) followed 48 h later by intraperitoneal injection of 5 IU of human chorinonic gonadotropin (hcg; Teikoku Zouki). MII-oocytes and embryos were flushed from the oviducts or uteri in FHM (HEPES-buffered KSOM) medium and then cultured in KSOM (supplemented with amino acids) medium with BSA (1 mg/ml, Sigma) (S5). When it was necessary, hyaluronidase (150 U ml -1, Sigma) was added to the FHM medium to remove the cumulus cells. Medium was covered with mineral oil (Sigma) and equilibrated at 37 C in a humidified atmosphere of 5% CO 2, 95% air. Fertilized embryos were cultured in KSOM until the control embryos reached the blastocyst stage at E When indicated, E2.5 embryos were cultured in the presence of cycloheximide (Sigma). Fluorescence microscopy Oocytes and embryos were collected and immediately fixed in 4.0% paraformaldehyde, ph 7.5, for 30 min at room temperature, washed three times in phosphate buffer and analyzed under a fluorescence microscope (Olympus IX81) equipped with a CCD camera (CoolSNAP HQ2, Nippon Roper). To detect lysosomes, embryos were collected and immediately stained with Lysotracker Red for 5-10 min in the presence of lysosome protease inhibitors (E64d and Pepstatin, Sigma). Quantification of GFP-LC3 dots was performed using the Top Hat algorithm of MetaMorph version 7.0 (Molecular Devices) as previously described (S2).

3 Electron microscopy For conventional electron microscopy, 2-cell embryos were fixed for 2 h in 2.5% glutaraldehyde in 0.5 mg/ml polyvinylpyrrolidone (PVP) (Sigma) M sodium phosphate buffer (PB), ph 7.4. After washing, cells were embedded into 1.3% low-melting point agarose (Sigma) in PVP-PB, and postfixed in 1.5% OsO 4 in 0.1 M PB, ph 7.4, for 1 h. The cells were further dehydrated with a graded series of ethanol and were embedded in epoxy resin. Ultrathin sections were doubly stained with uranyl acetate and lead citrate and observed using a Hitachi 7100 electron microscope (Hitachi, Tokyo, Japan). Immuno-electron microscopy analysis of endogenous LC3 was performed using a pre-embedding gold enhancement immuno-gold method as previously described with slight modification (S6). Fertilized embryos were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), ph 7.5, for 30 min at room temperature. After washing with the same buffer three times for 5 min, the fixed embryos were embedded in agar. The specimens were permeabilized in PB containing 0.25% saponin for 30 min. The cells were washed with PB, blocked by incubating for 30 min in PB containing 0.1% saponin, 10% BSA, 10% normal goat serum, and 0.1% cold water fish skin gelatin, and exposed overnight to anti-lc3 rabbit polyclonal antibody (kindly provided by Dr. Mitsunori Fukuda, Tohoku University) (1/500 dilution) (S7) in blocking solution. After washing with PB containing 0.005% saponin, the specimens were incubated with colloidal gold (1.4 nm in diameter, Nanoprobes Inc.)-conjugated goat anti-rabbit IgG in blocking solution for 2 h. The specimens were then washed with PB and fixed with 1% glutaraldehyde in PB for 10 min. Specimens were washed with phosphate buffered saline (PBS) containing 50 mm glycine, PBS containing 1% BSA, and milliq water, and the gold labeling was intensified with a gold enhancement kit (GoldEnhance EM, Nanoprobes Inc.) for 5 min at room temperature according to the manufacturer s instructions. The specimens were washed with distilled water, post-fixed in 1% OsO4 containing 1.5% potassium ferrocyanide in PB for 60 min at room temperature, and washed again with distilled water. The specimens were dehydrated in a series of graded ethanol solutions and embedded in epoxy resin. Ultra-thin sections were observed under an H7600 electron microscope (Hitachi, Japan). Genotyping of neonates and embryos For genotyping of newborn mice, routine PCR was performed using tail genomic DNA as previously described (S3). For genotyping of embryos, individual embryos were lysed by incubation at 55 C for 2 h in 4 µl of lysis buffer (10 mm Tris-HCl, ph8.8, 50 mm KCl, 0.45% NP-40, 0.45% Tween-20, 2.5 mm MgCl 2 ) containing proteinase K

4 (200 µg/ml). After heating at 95 C for 5 min, an aliquot of the lysate was directly used as a template for PCR. The following primers were used for genotyping: primer I (5L2); Primer II (Short2); Primer III (NRI, 5 -CCTCTTGCAAAACCACACTGCTCGACATTG-3 ); Primer IV (5WT3, 5 -CAGTTTGTGGCTGCCCTGGTCAATATATC-3 ). Nucleotide sequences of primers I and II were previously described (S3). PCR reactions were carried out using KOD-Plus FX DNA Polymerase (Takara) according to the manufacturer s instructions. The positions of the primers are provided in fig. S8. In vitro fertilization (IVF) and parthenogenetic activation For IVF, MII-oocytes were inseminated with sperm from week-old wild-type male mice in mhtf medium as previously described (S8). Fertilization was evaluated by the formation of the second polar body and both male and female pronuclei at 6 h post-insemination. Inseminated embryos were cultured in KSOM medium. For parthenogenetic activation, MII-oocytes were collected and transferred to Ca 2+ -free CZB medium containing 10 mm SrCl 2 (Sigma) and 5 µg/ml cytochalasin B (Sigma) for 2-4 h as previously described (S9). Activated oocytes with two pronuclei were cultured in vitro under the same conditions as for the IVF experiments. RT-PCR Pools of ten MII oocytes or embryos were transferred to 3 µl of PB and immediately stored in liquid nitrogen. Cells were ruptured by three freeze/thaw cycles and directly used as a template for cdna preparation. After treatment with RQ RNase-Free DNase (Promega), the following reaction mixture was used: 5x first-strand buffer, 1 mm dntps, 12.5 pmol random primer, 20 U RNaseOUT, and 2 U RevTrase (Takara). PCR was carried out using 1 µl of cdna. The following primers were used for amplification of Atg5: mapg5-d, 5 - TTGCTTTTGCCAAGAGTCAGC-3 ; and mapg5-f, 5 -ATCAGCTTCTTTCATACACGAC-3 ; and for β-actin amplification: 86b-actin, 5 -CTGGGTATGGAATCCTGTGG-3 ; and 87b-actin, 5 -GTACTTGCGCTCAGGAGGAG-3. Immunoblot analysis MII-oocytes were collected from wild-type female mice at h post hcg injection. Two- and eight-cell stage embryos were collected at 36 h and 55 h post insemination, respectively. For analysis of LC3 conversion, total cell extracts from 300 oocytes and 2-cell embryos were subjected to immunoblot analysis using anti-lc3 antibody as

5 previously described (S10). The amount of LC3-II correlates with autophagosome number. For analysis of mtor activity, total cell extracts from 500 oocytes, 2-cell and 8-cell embryos were subjected to immunoblot analysis. Signals were detected with Immobilon Western reagents (Millipore). Anti-phospho-S6K1 (phosphorylated at threonine 389) and anti-s6k1 antibodies were purchased from Cell Signaling Technology, and anti-tubulin antibody was purchased from Sigma. Ovarian histology Ovaries from Atg5 flox/+ ;Zp3Cre or Atg5 flox/- ;Zp3Cre female mice were dissected, washed in PBS, fixed in 4% paraformaldehyde, and processed for the production of paraffin sections. Sections (5 µm) were stained with hematoxylin and eosin. Total protein synthesis assay Oocytes and 2-cell embryos were collected, and half of the 2-cell embryos were subsequently cultured in vitro until the 4- and 8-cell stages. At the end of each culture period, oocytes and embryos were incubated for 2 h in amino acid-free KSOM supplemented with BSA, including 500 µci/ml of [ 35 S]-methionine (>1,000 Ci/mmol; Amersham Bioscience). When indicated, cycloheximide was added to the medium. After incubation, embryos were washed three times with FHM medium without BSA and placed in 10 µl of 2x Laemmli sample buffer, boiled for 2 min, and stored at 4 C until use. Total proteins were separated on 10% SDS-polyacrylamide gels. Following electrophoresis, the gels were dried and exposed for 4-5 days to an imaging plate (Fujifilm). Scanning and quantification were performed using a BAS 3000 analyzer (Fujifilm) and ImageGauge software. Time-lapse microscopy For time-lapse recording of GFP-LC3 dots after fertilization, MII-oocytes from GFP-LC3 transgenic females were inseminated with sperm from wild-type males as described above. Three hours after IVF, inseminated embryos were briefly treated with hyaluronidase, washed several times in protein-free KSOM medium, and attached to a glass-bottom dish (Matsunami). Time-lapse recording was performed using a humidified CO 2 incubator system (Tokai-Hit). Images were captured every 10 min.

6 A DIC GFP-LC3 Fig. S1 B GFP-LC3 LysoTracker Red Merge C 0h 6h 12h 22h 38h 54h 72h 96h E 12h 18h 24h 30h 36h F D lysosome autophagosome LC3 autolysosome Fig. S1. Induction of autophagy after fertilization. (A) A higher magnification image of the 1-cell embryo shown in Fig. 1A. Scale bar, 20!m. (B) Partial co-localization of GFP-LC3 (left) with LysoTracker Red (middle). Embryos were treated with LysoTracker Red and lysosome protease inhibitors (pepstatin and E64d) for 1 h. In the merged image (right), green and red signals indicate GFP-LC3 and LysoTracker Red, respectively. The arrow and arrowhead indicate structures positive for only GFP-LC3 and for both GFP-LC3 and LysoTracker, respectively. (C) Total GFP-LC3 signals were reduced during early embryogenesis. In vitro fertilized embryos were analyzed by fluorescence microscopy. Times (h) after IVF are indicated. Images were taken with the same exposure time and are displayed with the same contrast adjustment. Scale bar, 20!m. (D) Schematic representation of LC3 degradation in autolysosomes. LC3 associated with the inner autophagosomal membrane is degraded in autolysosomes, whereas LC3 on the outer membrane dissociates from autolysosomes. (E) Autophagy is transiently suppressed from the late 1-cell to the middle of the 2-cell stage. Embryos were analyzed as in panel C with shorter interval times. (F) E1.5 embryos were collected from oviducts of GFP-LC3 transgenic females mated with WT males. Left: an unfertilized oocyte, Right: a fertilized embryo. Scale bar, 20!m.

7 Fig. S2 L L L M Fig. S2. Conventional electron microscopic analysis of 2-cell stage embryos. An autophagosome (arrow), autolysosome (arrowhead), lysosomes (L), and mitochondria (M) are shown. A similar autolysosome is shown in an immuno-electron microscopic image in Fig. 1C. Scale bar, 500 nm.

8 Fig. S3 MII-oocyte 2-cell 8-cell p-s6k1 (Thr 389) S6K1 Tubulin Fig. S3. Phosphorylation of p70s6-kinase 1 (S6K1), an mtor substrate, in oocytes and developing embryos. Total cell extracts from 500 oocytes and embryos were subjected to immunoblot analysis using anti-phosphothr389-s6k1 (p-s6k1), anti-s6k1, and anti-tubulin antibodies.

9 Fig. S4 -/- +/- Fig. S4. Induction of autophagy in Atg5 -/- embryos derived from Atg5 +/- males and females. Two-cell embryos collected from Atg5 +/- ;GFP-LC3 females mated with Atg5 +/- males were observed by fluorescence microscopy, and then were subjected to genotyping. Active autophagy was observed even in Atg5 -/- embryos derived from Atg5 +/- parents. Scale bar, 20!m.

10 A Atg5 flox/+ ;Zp3-Cre Atg5 flox/- ;Zp3-Cre Fig. S5 CL CL B C D No. of ovulated eggs (4) (3) (5) :WT :Atg5 flox/+ ;Zp3-Cre :Atg5 flox/- ;Zp3-Cre Pronucleus formation (%) (108) (97) :WT :Atg5 flox/- ;Zp3-Cre kb M Atg5 +/- father Atg5 flox/- ;Zp3-Cre mother Offspring - flox ! - Cre Fig. S5. Embryonic lethality of autophagy-deficient embryos despite normal oogenesis and fertilization. (A) Normal histology of ovaries from oocyte-specific Atg5-deficient female mice. Ovaries from Atg5 flox/+ ;Zp3-Cre (control) and Atg5 flox/- ;Zp3-Cre female mice were fixed, embedded, and stained with hematoxylin and eosin. Arrows indicate secondary follicles. CL, Corpus luteum. Scale bar, 100!m. (B) Average number of MII-oocytes after superovulation of indicated females. The total number of superovulated females examined is shown in parentheses. Each value represents the mean ± s.d. (C) Normal fertility of Atg5 flox/- ;Zp3-Cre female mice. Superovulated WT or Atg5 flox/- ;Zp3-Cre females were mated with WT males overnight. E0.5 (1-cell) embryos were collected and pronucleus formation was morphologically evaluated. Each value represents the mean ± s.d. from three females. The total number of eggs examined is shown in parentheses. (D) Embryonic lethality of autophagydeficient embryos. Atg5 +/- males (father), Atg5 flox/- ;Zp3-Cre females (mother), and their offspring were genotyped by PCR analysis. A representative result is shown. The positions of the floxed (flox), KO (-), WT (+), and deleted ("; generated by recombination of the floxed allele) alleles, as well as the Cre transgene, are indicated. Data are summarized in Fig. 2C.

11 A MII-oocyte 2-cell 4,8-cell B 2-cell Atg5 4-cell flox/+ ;Zp3-Cre;GFP-LC3 8-cell Fig. S6 Atg5 flox/+ ;Zp3-Cre x WT Atg5 Actin Atg5 flox/- ;Zp3-Cre x WT Atg5 Actin Fig. S6. Expression of zygotic Atg5 and restoration of autophagy in Atg5-depeleted embryos. (A) RT-PCR analysis of Atg5 and actin mrna levels in MII-oocytes, 2-, and 4, 8-cell stage embryos derived from Atg5 flox/+ ;Zp3-Cre and Atg5 flox/- ;Zp3-Cre female mice mated with wild-type males. (B) Restoration of GFP-LC3 dot formation in Atg5- deficient embryos by wild-type sperm. Embryos from Atg5 flox/- ;Zp3-Cre;GFP-LC3 females crossed with wild-type males were isolated at the stages indicated, fixed and analyzed by fluorescence microscopy. Scale bar, 20 µm.

12 Fig. S7 E4.5 Atg5 +/- # x Atg5 +/- $ Atg5 flox/- ;Zp3-Cre# x Atg5 +/- $ Fig. S7. Developmental defects of autophagy-deficient embryos. The E3.5 embryos shown in Fig. 3A were cultured for 24 h in vitro. Arrowheads indicate normal blastocysts at E4.5, whereas arrows indicate developmentally retarded embryos. Scale bar, 100!m.

13 A WT allele ex3 B kb M Blastocysts Fig. S8 5L2 short floxed allele 1160bp Neo 0.5- " Cre deleted allele 5L2 short ex2 300bp ex3 +/- "/+ +/- "/+ +/- "/+ "/+ +/- WT allele KO allele WT3 Neo 180bp short2 C # oocyte flox%! - flox%! - $ sperm No. of blastocysts NRI short2 130bp Fig. S8. Genotyping of rescued blastocysts. (A) Diagram of Atg5 WT, floxed, deleted (generated by recombination of the floxed allele), and KO alleles. Genotyping was performed by PCR using four primers designated 5L2, short2, WT3, and NRI. (B) An example of blastocyst genotyping is shown. Embryos were collected from Atg5 flox/- ;Zp3-Cre females mated with Atg5 +/- males. The upper gel shows the WT (+) and deleted (") alleles dectected using the 5L2 and short2 primers, and the lower gel shows the WT and KO (-) alleles detected using the WT3, NRI, and short2 primers. (C) Genotypic distribution of blastocysts developed in vitro from E1.5 embryos obtained from Atg5 flox/- ;Zp3- Cre females crossed with Atg5 +/- males.

14 A B Fig. S9 CHX (!g/ml) Lane E3.5 Non-treated CHX (0.1!g/ml) CHX (1.0!g/ml) No. of CHX treated eggs No. of nontreated eggs E4.5 Protein synthesis (%) Fig. S9. Inhibition of protein synthesis causes a developmental block. (A) E2.5 embryos were treated with the indicated concentrations of cycloheximide (CHX) for 2 hours. The CHX-treated embryos were mixed with nontreated embryos as indicated (because the experiments shown in Fig. 4 also include some embryos rescued by wild-type sperm). Total protein synthesis rates of ten embryos were measured as in Fig. 4 and shown as % of nontreated embryos (lane 1). (B) E2.5 embryos were treated with CHX as in panel A and cultured for two days in vitro. Scale bar, 100!m.

15 Supporting References S1. N. Mizushima, A. Yamamoto, M. Matsui, T. Yoshimori, Y. Ohsumi, Mol. Biol. Cell 15, 1101 (2004). S2. A. Kuma et al., Nature 432, 1032 (2004). S3. T. Hara et al., Nature 441, 885 (2006). S4. W. N. de Vries et al., Genesis 26, 110 (2000). S5. J. D. Biggers, L. K. McGinnis, M. Raffin, Biol. Reprod. 63, 281 (2000). S6. H. Luo et al., Cell Struct. Funct. 31, 63 (2006). S7. T. Itoh et al., Mol. Biol. Cell, 19, 2092 (2008). S8. S. Kito et al., Comp. Med. 54, 564 (2004). S9. S. F. Ma et al., Theriogenology 64, 1142 (2005). S10. N. Hosokawa, Y. Hara, N. Mizushima, FEBS Lett. 580, 2623 (2006). Movie S1. Induction of autophagy after fertilization. Time-lapse recording of GFP-LC3 dots in inseminated embryos. Time-lapse images were taken every 10 min beginning 3 h after IVF. Times after IVF are indicated in the upper right corner. (QuickTime; 1.3 MB)