Collection and culture of oocytes

Animal procedures complied with the Animals (Scientific Procedures) Act, 1986. Wild-type mouse (Mus musculus) strains were bred from C57BL/6 or DBA/2 stocks in-house or otherwise supplied by Charles River (L'Arbresle, France). Mixed C57BL/6 and B6D2F1 background hybrid lines containing the transgenes pOct4-mCherry, pPrm2-Prm2-mCherry, pCAG-Plcz-ires-Venus20, Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo(mT/mG)22, pPGK-Cre or pNanog-eGFP55 were generated in-house or were kind gifts. Oocytes were collected from 8- to 12-week-old females that had been super-ovulated by standard serial intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG) followed 48 h later by 5 IU human chorionic gonadotropin (hCG)56. Oviductal metaphase II (mII) oocytes were collected in M2 medium (EMD Millipore, UK)57 ∼15 h post-hCG injection and cumulus cells dispersed by hyaluronidase treatment56. After repeated washing in M2, denuded oocytes were incubated in kalium simplex optimized medium (KSOM; Millipore)58 under mineral oil in humidified 5% CO 2 (v/v air) at 37 °C, until required. Where appropriate (for example, for diagnosis of fertilization by second polar body extrusion), mII oocytes with a degenerate first polar body were selected; by 16 h post-hCG, 71.0±2.0% (n=2,294) of first polar bodies had degenerated. Activation of B6D2F1 or mT/mG oocytes to produce parthenogenetic haploid embryos was in Ca2+-free CZB medium supplemented with 10 mM SrCl 2 , for 2.5 h in humidified 5% CO 2 (v/v air) at 37 °C (refs 59, 60). SrCl 2 treatment was initiated 16–17.5 h post-hCG; times are given after the start of SrCl 2 treatment. For phICSI and controls, embryos were chosen that possessed a second polar body and a single pn at 5–7 h. For phICSI, SrCl 2 -activated haploid parthenogenotes were injected with sperm at the times indicated after commencement of SrCl 2 treatment. Spontaneously arising parthenogenotes containing a single pn were selected from pCAG-Plcz-ires-Venus females20 following brief culture in vitro. For maternal genome labelling with 5-bromo-2'-deoxyuridine (BrdU), haploid B6D2F1 parthenogenotes were cultured in KSOM containing 5 μM BrdU (Sigma) for 5 h, starting 4 h after SrCl 2 treatment was initiated. S-phase was determined by culturing 2-cell ph embryos for 1 h at the appropriate times in KSOM containing 100 μM BrdU.

Sperm preparation and microinjection

For sperm preparation, cauda epididymidal sperm from 8- to 12-week-old males were triturated for 45 s in nuclear isolation medium (NIM; 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 3.0 mM EDTA; pH-7.0) containing 1.0% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at room temperature (25 °C)56,58,59. Sperm were washed twice in NIM and pelleted (1,890 g) at ambient temperature; head-tail detachment was enhanced by trituration during pellet resuspension. Finally, sperm were resuspended in ice-cold NIM (∼0.5 ml per epididymis) and stored at 4 °C for up to 3 h until required. ∼50 μl of each suspension was typically mixed with 20 μl of polyvinylpyrrolidone (PVP, average M r ≈360 000; Sigma-Aldrich) solution (15% (w/v)) and sperm injected (ICSI) into oocytes in droplet of M2 within ∼60 min, essentially as described56. Injected oocytes were transferred to KSOM under mineral oil equilibrated in humidified 5% CO 2 (v/v air) at 37 °C. For phICSI, SrCl 2 -activated haploid parthenogenotes were injected with sperm at the times indicated after commencement of SrCl 2 treatment. For phICSI-13, this was 13 h after the initiation of SrCl 2 treatment of B6D2F1 oocytes, just after pnMBD. Where parthenogenotes were from pCAG-Plcz-ires-Venus, sperm were injected soon after pnMBD. In some experiments (Supplementary Fig. 9d,e), zygotes produced by mating were injected with a sperm immediately after pnMBD, referred to as zygotic ICSI, zICSI.

Ablation of single 2-cell embryo blastomeres

One of the two blastomeres in 2-cell embryos was destroyed by suction of nuclear material into a piezo-actuated pipette (inner diameter, 7–8 μm). In the case of phICSI-13, embryos of the 2+1 nuclear type (Fig. 1b) were selected and the blastomere with a single (maternal) nucleus was destroyed.

Pronuclear transfer and cytoplast transfer

For paternal pn and zygotic cytoplast transfer60,61, the zona pellucida of donor and recipient embryos was cut in M2 medium with a fine microneedle. Donor zygotes produced by ICSI (B6D2F1 oocytes and B6D2F1 or mtdT sperm) were held for 10–15 min in KSOM containing cytochalasin B (5 μg ml−1) and nocodazole (1 μg ml−1) just before manipulation in M2 medium similarly supplemented with cytochalasin B (5 μg ml−1) and nocodazole (1 μg ml−1). Part of the zygote containing the male pn was aspirated into a micropipette (outer diameter, 15 μm) and introduced with inactivated sendai virus (HVJ) envelope (GenomeONE-CF, Ishikawa Sangyo Kaisya, Japan) diluted 10x from a stock solution, into the space between the blastomere and zona pellucida of a 2-cell parthenogenetic haploid BDF1 embryo. Male pronuclear transfer into male pn-enucleated zygotes and zygotic cytoplast transfer into 2-cell blastomeres of biparental ICSI embryos were performed as controls.

ROSI and phROSI

ROSI was performed by injecting B6D2F1 mII oocytes with round spermatid nuclei (from 8 to 10-week-old B6D2F1 or mtdT) in M2 medium, followed by activation with 10 mM SrCl 2 in calcium-free CZB-G medium for 2.5 h (ref. 13). phROSI was performed by applying the ROSI procedure to parthenogenetic haploid embryos entering the first mitosis as nucleus recipients.

Generation of recombinant fusion constructs

Recombinant constructs were used for the expression of cRNA and/or transgenes encoding fusion proteins. To generate the backbone vector, pCI-neo-mKO2-FLAG, an mKO2 SmaI/EcoRV fragment was generated by PCR from the Kusabira Orange-encoding plasmid, pmKO2-SI (Medical & Biological Laboratories, UK) and cloned into the mammalian expression vector, pCI-neo (Promega, UK). Into this construct, we inserted an EcoRV/NotI fragment derived from p3XFLAG-CMV-14 (Sigma-Aldrich, UK) to generate an mKO2-FLAG 3 fusion. H3.3 was cloned into pCI-Neo-mKO2-FLAG as an XbaI/SalGI fragment from cDNA generated by mII oocyte RT-PCR. The Prm2 gene was cloned following PCR amplification of tail tip genomic DNA as an XhoI/EcoRI fragment that includes ∼1 kb upstream of the Prm2 start codon, and inserted into pCI-neo-mCherry1. Expression of mVenus-hGeminin and mCherry-hCdt1 (the kind gifts of the RIKEN Brain Science Institute, Wako, Japan) was after they had been cloned as PCR-amplified EcoRI/XbaI fragments into pCI-neo. To generate the transgene construct in which the Oct4A promoter drives mCherry expression, a fragment including the Oct4A start codon and ∼5 kb of upstream promoter sequences62 was generated by PCR and cloned with an mCherry reporter fragment into the NotI site of vector pGEM-T (Promega).

Embryo transfer

E1.5 (2-cell) embryos (the day following activation) were transferred to the oviductal ampullae of pseudo-pregnant CD-1 females at day 0.5 (that is, plugged females that had been mated with vasectomized males the previous night). Pups were delivered by natural birth and where appropriate, fetuses, pups and placentae collected by Caesarian section at the desired time point. Pups and placentas collected by caesarian section at term (E19.5) were weighed immediately. Newborn pups were fostered by CD-1 females.

Genotyping

Mouse tissue samples were digested at 55 °C for 3 h in 25–100 μl of a lysis buffer containing 10% (w/v) sodium dodecyl sulfate and with 2 mg ml−1 proteinase K (Sigma). 1 μl of a 1:10 dilution of each sample was used for genotyping by PCR in a 10 μl reaction volume. PCR primer sequences are given in Supplementary Table 2. Primers for microsatellite analysis were selected from the Mouse Microsatellite Data Base of Japan63.

Preparation and injection of cRNA

5'-capped and polyadenylated cRNA was synthesized from linearized plasmid template DNA in a T7 mScript Standard mRNA Production System (Cellscript, USA) according to the recommendations of the manufacturer46,64. cRNAs were dissolved in nuclease-free water, quantified on a Nanophotometer and stored in aliquots at −80 °C until required. cRNA solutions were diluted as appropriate with sterile water and injected (typically at concentrations of 0.01 to 1 μg μl−1) within 1 h of thawing via a piezo-actuated micropipette into mII oocytes or embryos in M2 medium. Following cRNA injection, oocytes or embryos were cultured for at least 3 h prior to subsequent manipulation.

Immunocytochemistry

5′-Methylcytosine (5mC), hydroxymethylcytosine (5hmC) and BrdU were detected in embryonic DNA following fixation in 4% (w/v) paraformaldehyde and treatment with 2 M HCl for 30 min. Fixed embryos were processed as soon as possible but were stored where necessary at 4 °C. For primary antibody labelling, samples were incubated overnight at 4 °C with mouse anti-5mC antibody (1:200 (v/v); EMD Millipore, UK), for 1.5 h at 37 °C with rabbit anti-5hmC antibody (1:200; Active Motif, USA) or for 1.5 h with rat anti-BrdU antibody (1:100; Abcam, UK). Additional primary antibodies recognized Oct4 (1:100; Santa Cruz, USA), Cdx2 (1:100; BioGenex Laboratories, USA), H3K4me3 (1:250; Abcam, UK), H3K9me2 (1:50; Abcam), H3K27me3 (1:50; Abcam) and H4K12ac (1:250; Abcam). Primary antibody incubation was followed by a 1 h incubation at 37 °C with the appropriate secondary antibody (1:250; Life Technologies, UK) conjugated to Alexa 350, Alexa 488 and/or Alexa 594. DNA was stained by incubating samples at 37 °C for 20 min in propidium iodide (1:200; Sigma, USA) or Hoechst 33342 (1:1,000; Sigma).

Fluorescence imaging

Images of live oocytes or embryos following cRNA injection were captured on an Olympus IX71 stand equipped with an Andro Zyla sCMOS camera and OptoLED illumination system (Cairn Research, UK) and processed using Metamorph software (Molecular Devices, LLC, USA). Excitation at 587 nm in combination with an ET-mCherry filter system was used for mCherry fluorescence detection and at 484 nm with an ET-EYFP filter system to detect Venus epifluorescence. Fluorescence of fixed samples was visualized on an Eclipse E600 (Nikon, Japan) microscope equipped with a Radiance 2100 laser scanning confocal system (BioRad, USA)46. Images were processed with ImageJ (imagej.nih.gov/ij/) or MetaMorph (Molecular Devices, USA) analysis software. Quantitative analyses subtracted background from subject area fluorescence intensities, which can produce negative results in beads experiments in which background levels from latex are lower than those of oocytes. Mouse fetus fluorescence stereomicrographs (Supplementary Fig. 6c) were captured by a Leica MZ16 FA fluorescence stereomicroscope, with LAS AF 4.0 imaging software (Leica Microsystems GmbH, Germany).

Nuclear volume estimation

Nuclear volumes were estimated in zygotes and 2+1 type phICSI-13 2-cell embryos respectively 9 h post-ICSI or 30 h after initial SrCl 2 exposure. These times correspond to approximately the same period after the paternal genome has completed its first S-phase respectively in ICSI and phICSI. Maternal nuclei were distinguished by comparatively low zygotic 5hmC or by BrdU labelling in phICSI. Propidium iodide staining was determined in z-stacked confocal images using ImageJ.

Blastocyst cell counting

Blastocyst cell counting46 was performed by fixing blastocysts in 4% (w/v) paraformaldehyde and incubating them at 4 °C overnight in rabbit anti-Oct4 antibody (1:100; Santa Cruz) or for 1 h at 37 °C in mouse anti-Cdx2 antibody (1:100; BioGenex, USA), followed by 1 h at 37 °C in Alexa 488-conjugated anti-rabbit IgG (Invitrogen) or Alexa 594-conjugated anti-mouse IgG (Invitrogen) respectively. Cells stained with Alexa 488 were scored as Oct4-positive (pluriblasts) and those with Alexa 594, as Cdx2-positive (trophoblasts).

DNA-conjugated latex microbeads

We adapted a method using streptavidin latex microbeads (Dynabeads; Invitrogen, MA, USA) conjugated to biotinylated DNA, so that we could perform high-resolution physiological analysis of chromatin and spindle function in living oocytes and embryos, as previously performed for spindles30,31, chromatin29,30,31 and mouse oocytes32. DNA fragments (2,605 bp) were amplified using LA Taq DNA polymerase (TAKARA BIO, Japan) from plasmid pCI-Neo (Promega Corp., WI, USA) using the primer pair (5'→3'): sense, CTGGCGTAATAGCGAAGAGG; antisense, ATAATACCGCGCCACATAGC. The sense primer was pre-labelled with biotin at its 5' end (Invitrogen). Following agarose gel electrophoresis, DNA fragments were gel-purified using Wizard SV Gel and PCR Clean-Up System (Promega Corp., WI, USA). For methyl-DNA bead conjugation (Fig. 7c–e), a portion of the DNA amplimer was methylated using the CpG methyltransferase, M.SssI (New England BioLabs, MA, USA) according to the recommendations of the manufacturer. Methylation reaction products were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and the reaction efficiency was assessed by gel electrophoresis following challenge with the methylation-sensitive restriction enzyme, HpaII (New England BioLabs). Streptavidin-coupled beads (2.8 μm diameter) were decorated with DNA using Dynabeads kilobaseBINDER Kit (Life Technologies, USA) according to the recommendations of the manufacturer. Briefly, magnetic latex microbeads from 4 μl of suspension were mixed on a rotator for 3 h at room temperature (25 °C) with 3 μg of biotinylated DNA fragment in binding buffer in 40 μl of binding reaction. The beads were harvested magnetically, washed, resuspended in nuclease-free water and stored in aliquots at -20 °C until required. Immediately prior to injection, beads were mixed with 3% (w/v) PVP (average M r , 360,000) in M2 medium. Microinjection was performed as for ICSI56, coinjecting 5–7 beads per oocyte or embryo instead of sperm heads.

To validate the method in our system, we injected DNA-beads into mII oocytes and evaluated microtubule formation and histone accumulation by DNA staining and immunofluorescence microscopy (Supplementary Fig. 12a–c). In all experiments, we showed that DNA-beads injected into mII oocytes were stained with Hoechst 33342 or propidium iodide, in a DNA-dependent manner (Supplementary Fig. 12a). Beads in mII oocytes stained with an antibody against acetylated H4K12ac in a DNA-dependent manner that was independent of DNA methylation (n≥10; Supplementary Fig. 12b). As previously reported32, DNA-beads induced microtubule nucleation regardless of 5mC status (n≥11), whereas beads lacking DNA failed to do so (n=11) (Supplementary Fig. 12c). The kinetics of cRNA-encoded histone H3.3 deposition were similar for DNA-beads and ICSI controls (Supplementary Fig. 10).

Statistical analysis

Experiments were performed on ≥2 days. The number of samples (n) per experiment reflects power calculation and oocyte and embryo survival after manipulation. All samples were randomly collected; that is, we did not knowingly select different classes of healthy oocytes or embryos except where stated, and no data are selectively excluded. Data analysis was performed with or without blinding. Statistical differences between pairs of data sets were analysed by a two-tailed unpaired t-test. One-way ANOVA followed by a Tukey–Kramer post hoc test was used for multiple comparisons. The log-rank test was used to compare mouse longevity plots created by the Kaplan–Meier method. Values of P<0.05 were considered statistically significant. For model-assessment of possible ICSI-phenotype contribution to developed embryos, we assumed that two embryonic phenotypes I and II were characterized by respectively an elevation or diminution of certain modifications (for example, 5mC and 5hmC). The estimated probability for development to term is q I =0.103 for phenotype-I (according to 24/232 for phICSI-derived embryos) and q II =0.451 for phenotype-II (according to 107/237 for ICSI-derived embryos) (Supplementary Table 1). A nominal phenotype-I population may contain an unknown phenotype-II fraction, ρ. Subsequently, the model combines (1) the probability for observing phenotype-I associated histone states in k out of k experiments in the mixed population, and (2) the cumulative probability for the event that at least n=1,2,... out of N=232 individuals developing to term derive from phenotype-II.

Ad (1) The probability of observing k histone-unmodified cases (phenotype-I) in k experiments is binomial p(k|k, 1-ρ) distributed.

Ad (2) The probability of observing m phenotype-II and (N—m) phenotype-I cases in N experiments is binomial p(m|N, ρ). The probability that respectively n and (N—n) cases develop to term is the product of binomial p(n|m, q II ) and p(N—n|N—m, q I ). Marginalization with respect to m

results in the probability for the combined two-step process

Finally, the cumulative probability for observing at least n individuals is

Taking (1) and (2) as independent evidence and assuming ρ to be the same in both types of experiments, the overall probability is the product

Supplementary Figure 13 shows P as a function of ρ for different values of n and k=20 (5hmC). Accordingly, the production of 9 or more phenotype-II-derived embryos out of a total of 24 can be excluded on the 5% significance level, that is, P(9)≤0.0465, while the probability that all embryos derive from phenotype-II is P(24)≤9.84e−17. For k=9 (5mC), the same is true for 14 or more embryos, that is, P(14)≤0.0493, and for all embryos P(24)≤1.05e−16. Regarding both evidences for k=9 and k=20 as independent and multiplying both binomials results in significance starting from n=7, that is, P(7)≤0.0465, and P(24)≤9.32e−17. The simplifying assumption of only two phenotypes limits the model applicability to either only one phenotype-determining modification or (equivalently) multiple but 100%-correlated modifications. Allowing for two independent modifications A and B, would require a more involved four-phenotype-model. However, our data suggest high correlation of A and B.

Single-cell whole transcriptome amplification

ICSI and phICSI 2-cell embryos were produced by injecting B6D2F1 sperm respectively into B6D2F1 oocytes or parthenogenotes. For phICSI-13, blastomeres were separated from 2+1 embryos (Fig. 1e). In all cases, a random sample of the embryos was cultured to confirm preimplantation developmental viability (Supplementary Table 1). For transcriptome amplification, embryos were exposed to acid Tyrode’s solution (pH2.5; Sigma) to dissolve the zona pellucida and allowed to recover for 1 h in KSOM in humidified 5% CO 2 (v/v air) at 37 °C. Embryos were then gently triturated in Ca2+-free, Mg2+-free M2 medium to separate the blastomeres and the extant polar body if there was one. ICSI blastomeres were collected 27 h post-ICSI and the binuclear blastomeres of phICSI 2+1 embryos 27 h after the initiation of SrCl 2 exposure. One blastomere per tube was collected in a minimal volume into 5.4 μl of lysis buffer (Active Motif) containing 10 ng of tRNA (Roche), 1 μg of protease (Active Motif), and 1 μl of 37.5 μM biotinylated oligo-dT peptide nucleic acids (PNAs; Active Motif) and flash-frozen in liquid N 2 until extraction, reverse transcription and global amplification of blastomere mRNA36,65. Proteolytic digestion was performed by incubating samples for 10 min at 45 °C, followed by inactivation of protease at 75 °C for 1 min, and annealing of PNA to poly(A) tails of mRNAs, at 22 °C for 15 min. PNA-mRNA complexes were precipitated in magnetic force field using streptavidin-conjugated metal beads. While precipitating, bead pellets were washed with 10 μl of wash buffer 1 (50 mM Tris-HCl, 75 mM KCl, 10 mM DTT, 0.25% [v/v] Igepal), 20 μl of wash buffer 2 (50 mM Tris-HCl, 75 mM KCl, 10 mM DTT, 0.5% [v/v] Tween-20), and again with 20 μl of wash buffer 1 (supernatant was removed after each washing step). Solid phase reverse transcription was performed for 45 min under rotation at 44 °C, in a 20 μl reaction containing 0.5 mM of each dNTP (GE Healthcare), 200 U of SuperScript II reverse transcriptase (Invitrogen), 0.25% (v/v) Igepal, 5 mM DTT, 30 μM of C 15 GTCTAGAN 8 primer, 15 μM of C 15 GTCTAGACTTGAGT 24 VN primer (Metabion), and 1 × first strand buffer (Invitrogen). Primers were annealed at room temperature for 10 min, before addition of the enzyme. Following reverse transcription, beads were precipitated in magnetic racks and washed in 20 μl of wash buffer 3 (50 mM KH 2 PO 4 , 1 mM DTT, 0.25% (v/v) Igepal) and resuspended in 10 μl of buffer for tailing (4 mM MgCl 2 , 0.1 mM DTT, 0.2 mM dGTP, 10 mM KH 2 PO 4 ). Reaction mixtures were overlaid with 40 μl of mineral oil, and the cDNA single strands released from beads by heating to 95 °C for 5 min followed by incubation on ice for 3 min. Addition of dGTPs on 5′ termini of single stranded cDNAs was performed by adding 10 U of terminal dNTP transferase (TdT; USB-Affymetrix) and incubating the mixture for 60 min at 37 °C. After inactivation of TdT at 70 °C for 5 min, we added 35 μl of whole transcriptome amplification (WTA) reaction mix 1 (4 μl of buffer I (Expand Long Template, Roche) and 3% (v/v) deionized formamide). Hotstart PCR was performed by incubating the sample at 78 °C and adding 5.5 μl of WTA reaction mix 2 (3.2 mM each dNTP, 12 mM TCAGAATTCATGC 15 primer, and 7.5 U of PolMix (Expand Long Template, Roche)). WTA consisted of 40 cycles in an MJ Research PCR cycler: 20 cycles of 15 s at 94 °C, 30 s at 65 °C, and 2 min at 68 °C followed by 20 cycles with an increase of elongation step of 10 s per cycle, followed by a final cycle with 7 min of elongation.

For quality control36,65, 0.5 μl of each WTA product was used as a template for end-point PCR to amplify each of the three transcripts: β2-microglobulin, β-actin and GAPDH. The primers used were: β-actin forward, CAGCTTCTTTGCAGCTCCTT); β-actin reverse, CTCGTCACCCACATAGGAGTC; β2-microglobulin forward, TGGTGCTTGTCTCACTGACC; β2-microglobulin reverse, CCGTTCTTCAGCATTTGGAT; GAPDH forward, GAAGGGCATCTTGGGCTAC; GAPDH reverse, GCCTCTCTTGCTCAGTGTCC. PCR reactions consisted of 1 × PCR buffer (PAN-Biotech GmbH, Germany), 0.4 μM each primer (Eurofins), 5 μg BSA (Roche), 0.5 U Taq polymerase (PAN-Biotech), and 0.1 mM each dNTP (GE Healthcare). PCR products were visualized on 1.5% (w/v) agarose gels stained with ethidium bromide. Only samples that were positive for all three transcripts were used for microarray analysis36,65.

Sample labelling and microarray hybridization

Labelling of primary whole transcriptome amplified (WTA) product was by PCR with Cy5-labelled primers. Reaction mix contained 5 μl of buffer I (Expand Long Template, Roche), 3% (v/v) deionized formamide, 0.35 mM each dNTP, 2.5 μM 5'-U*CAGAAU*TCAU*CCC*CCCC*CCCC*CCCC*-3' primer (*denotes nucleotides conjugated with Cy5 fluorophore; Metabion), 7.5 U of PolMix (Expand Long Template, Roche) and 1 μl of primary WTA product in a final volume of 49 μl. PCR parameters were: one cycle with 1 min at 95 °C, 11 cycles with 15 s at 95 °C, 1 min at 60 °C, and 3 min 30 s at 65 °C, 3 cycles where the elongation time was increased 10 s per cycle, and finally one cycle with an elongation time of 7 min. Labelled products were purified using a PCR purification kit (Qiagen) according to the instructions of the vendor. Purified Cy5-labelled DNA was denatured by incubation for 5 min at 95 °C followed by incubation on ice. Hybridization solution was prepared by mixing 42 μl of denatured Cy5-labelled DNA, 55 μl of 2x HiRPM hybridization buffer (Agilent), 11 μl of 10 × GE Blocking agent (Agilent), 4 μl of 25% (v/v) Tween-20, and 4 μl of 25% (v/v) Igepal. Four 100 μl samples of hybridization mix were overlaid on 4 hybridization fields of Agilent Whole Mouse Genome (4x44K) Oligo Microarray with SurePrint (G4122F) microarray slides and incubated for 17 h at 65 °C under constant rotation. After hybridization, slides were washed in Agilent Wash buffer 1 for 1 min on a shaker, in darkness, and incubation continued in Agilent Wash buffer 2 pre-warmed to 37 °C. Slides were dried by washing for 30 s in acetonitrile and scanned on a GenePix 4400 A scanner (Molecular Devices). Numerical readouts of fluorescence intensities (GPR files) were generated using GenePixPro 7 (Molecular Devices).

Bioinformatics

The quality of whole transcriptome amplification was assessed by control PCR. Gene expression data were quality assessed by inspection of chip raw images and gene expression frequency distributions. All experiments were of sufficiently high quality for further bioinformatic analysis (16 and 8 expression profiles for ICSI and phICSI, respectively). Raw gene expression data were background corrected (limma R-package, normexp method)66,67, log 2 -transformed and normalized by quantile normalization. Technically replicated probes (identical Agilent IDs) were replaced by their median per sample. Gene ranking was performed according to moderated t-statistics (limma R-package). For graphical display and functional annotation probes targeting the same gene were disambiguated by retaining only the probe with the lowest P-value. The normalized heatmap (Fig. 8c) employed Euclidean distance and complete linkage for agglomerative clustering. Enrichment for biological process annotation associated with genes most differentially expressed between ICSI and phICSI was obtained by querying the DAVID database68 for GO BP/5, GO BP/FAT and KEGG, Reactome, BBID, BioCarta, and Panther pathway annotation using the R-package RDAVIDWebService69 for submitting stepwise prolonged gene-lists starting from the top 50, 100, 150, 200, 250 and so on. top-ranking genes up to a length of 550 corresponding to an FDR-adjusted P-value of 0.15. If identical annotation terms were returned for different lists only the one with the lowest EASE P-value was retained. For assessing randomness of gene ranking in the ICSI_1 versus ICSI_2 comparison, all 6,435 possible 8+8 array combinations were enumerated and P-values calculated (limma R-package). The resulting curves were kernel smoothed in two-dimensional and graphically displayed as density. From the 128 possible combinations respecting the experimental 2-cell pairing, the one with the highest number of FDR-adjusted P-values <0.05 (corresponding to the largest difference between ICSI_1 and ICSI_2) was selected for graphical display. In the ICSI versus phICSI comparison, 10,000 of the 735,471 possible 16+8 array combinations were analogously calculated to obtain the corresponding graphical display. Array data are deposited at the Gene Expression Omnibus (GEO) with the accession number GSE60595.

Ratiometric PCR (qPCR)

Relative transcript levels were quantified by transferring single embryos (1 per tube) in a minimal volume (<0.5 μl) of 0.1% (w/v) Sarkosyl (Teknova, Hollister, CA, USA) containing 10 ng μl−1 tRNA (Hoffmann-La Roche, Ch), heated at 65 °C for 5 min and used to programme cDNA synthesis primed with oligo(dT) 20 and random 8-mers (each at 30 mM) in a 21 μl reaction volume containing 200 U SuperScript III reverse transcriptase (Invitrogen Corp., CA, USA). Real-time qPCR was in an ABI 7500 Real Time PCR System (Applied Biosystems, CA) in reactions (20 μl total) containing 1–2 μl of the template cDNA, forward and reverse primers (Supplementary Table 2; each at 5 nM) and 12.5 μl of Power SYBR (ABI), using the parameters: 10 min at 95 °C, followed by 45 cycles of (15 s at 95 °C, 1 min at 58–60 °C and 35 s at 72 °C). Each sample was assayed in triplicate and given sample sets collected on at least two days. Primer sets were non-dimerizing under the conditions employed and steady state levels of transcripts were normalized against internal controls36.

Data availability

Microarray data have been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE60595. The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information Files or from the corresponding authors upon reasonable request.