Supporting Information Bermejo-Alvarez et al. 10.1073/pnas.0913843107 SI Material and Methods lysis buffer was extracted with phenol/chloroform treatment and In Vitro Embryo Production. Selected cumulus–oocyte complexes finally suspended in 16 μL of milliQ water. Eight microliters of each (COCs) obtained from ovaries collected at slaughter were ma- sample were used to perform embryo sexing by PCR as described in tured for 24 h in TCM-199, supplemented with 10% (vol/vol) FCS ref. 1. After embryo sexing, the individually stored poly(A) RNA (FCS) and 10 ng/mL epidermal growth factor at 39 °C under an from 10 (in vitro) or 5 (in vivo) embryos of the same sex or par- atmosphere of 5% CO2 in air with maximum humidity. In vitro thenotes were pooled and RNA extraction continued. Immediately fertilization was performed with X- or Y-sorted sperm from three after extraction, the RT reaction was carried out following the different bulls or unsorted semen from one of the bulls (Bull 1) as manufacturer's instructions (Bioline, Ecogen) using poly(T) primer, described in ref. 1. Matured COCs were inseminated with 12.5 μL random primers, and MMLV reverse transcriptase enzyme in a of frozen-thawed, percoll-separated sperm added to 25-μL total volume of 40 μL to prime the RT reaction and to produce droplets under mineral oil (15–20 oocytes per droplet) at a final cDNA. Tubes were heated to 70 °C for 5 min to denature the sec- concentration of 1 × 106 spermatozoa/mL. Gametes were co- ondary RNA structure and then the RT mix was completed with the incubated at 39 °C under an atmosphere of 5% CO2 in air with addition of 100 units of reverse transcriptase. They were then in- maximum humidity. At ≈20 h postinsemination, presumptive zy- cubated at 42 °C for 60 min to allow the reverse transcription of gotes were denuded and transferred to 25-μL culture droplets (1 RNA, followed by 70 °C for 10 min to denature the RT enzyme. embryo per microliter) under mineral oil. Blastocysts were pro- The quantification of all mRNA transcripts was carried out by duced in 12 independent experiments (equals days of ovary col- real-time qRT-PCR; five groups of cDNA per experimental lection). In all cases, X- and Y-sperm from an individual bull were group, each obtained from 10 embryos, were used with two used on the same day and were never split across days. Depending repetitions for all genes of interest. PCR was performed by adding on the availability of oocytes, one, two, or all three bulls were a2-μL aliquot of each sample to the PCR mix containing the processed on a given day. Details for developmental rates and specific primers to amplify histone H2Az (H2a.z), brain-ex- sexing accuracy of the bulls used in this study can be found in ref. pressed X-linked 1 (BEX1), calpain 6 (CAPN6), GST Mu3 2. Parthenogenetic activation was achieved by incubation of ma- (GSTM3), fragile-X mental retardation 1 neighbor (FMR1NB), ture oocytes in 5 μM ionomycin for 5 min followed by 3 h in- spermidine/spermine N1-acethyltransferase 1 (SAT1), brain-ex- cubation in 2 mM 6-DMAP. Embryo culture took place in SOF + pressed X-linked 2 (BEX2), sushi-repeat-containing protein X- 5% FCS. Plates were incubated for 7 days at 39 °C under an at- linked 2 (SRPX2), progesterone receptor membrane component mosphere of 5% CO2, 90% N2, and 5% O2 with maximum hu- 1 (PGRMC1), ubiquitin-conjugating enzyme E2A (UBE2A), midity. Day-7 blastocysts produced from X- and Y-sorted sperm DEAD box polypeptide 3 Y-linked (DDX3Y), eukaryotic were snap-frozen in groups for microarray analysis and those translation initiation factor 2, subunit 3, structural gene Y-linked produced with unsorted sperm and parthenogenetic activation (EIF2S3Y), laminin alpha 1 (LAMA1), DNA-methyltransferase had their zona pellucida removed with 0.2% pronase and were 3 alpha (DNMT3A), and three nonannotated transcripts present then individually snap-frozen for array validation. Oocytes and in the array and named X24112, YZRSR2 and Y2467. Primer embryos collected at different stages were snap-frozen in groups sequences and the approximate sizes of the amplified fragments of 10 to analyze the evolution of transcription during pre- of all transcripts are shown in Table S6. For quantification, real- implantation stages of different genes presented in the array. time PCR was performed as previously described (3). qPCR conditions were 94 °C for 3 min followed by 35 cycles (94 °C 10 s, In Vivo Embryo Production. In vivo bovine blastocysts were produced 56 °C 30 s, 72 °C 10 s, and 10 s of fluorescence acquisition – using standard superovulation protocol. Briefly, the estrous cycles SYBR-) (Rotor Gene 6000, Corbett Research). Each pair of of crossbred beef heifers were synchronized using a combination of primers were tested to achieve efficiencies close to 1 and then an intravaginal progesterone device (CIDR) and administration of the comparative cycle threshold (CT) method was used to a prostaglandin F2α analog to ensure luteolysis. Beginning on day quantify expression levels as described (4). To avoid primer- 10 of the synchronized cycle, each animal received twice daily in- dimmers artifacts, fluorescence was acquired in each cycle at a jections of Folltropin (Bioniche Animal Health) for 4 days, with temperature higher than the melting temperature of primer- prostaglandin given with the sixth injection. Animals were in- dimmers (specific for each product, 80–86 °C). Then, the seminated at estrus and embryos were recovered by flushing the threshold cycle or the cycle during the log-linear phase of the reproductive tract at slaughter. Blastocysts were snap-frozen in- reaction at which fluorescence increased above background was dividually in liquid nitrogen for subsequent analysis.All ex- determined for each sample. When the efficiency is close to 1, perimental procedures involving animals were licensed by the within this region of the amplification curve, a difference of one Department of Health and Children, Ireland, in accordance with cycle is equivalent to doubling of the amplified PCR product. the Cruelty to Animals Act (Ireland 1897) and the European According to the comparative CT method, the ΔCT value was Community Directive 86/609/EC and were sanctioned by the determined by subtracting the endogenous control (H2a.z) CT Animal Research Ethics Committee of University College Dublin. value for each sample from each gene CT value of the sample. Calculation of ΔΔCT involved using the highest sample ΔCT Independent Verification of Array Data Using Rea-lTime RT-PCR. Poly value (i.e., the sample with the lowest target expression) as a (A)RNAwasextractedfromindividualblastocystsproducedinvitro constant to subtract from all other ΔCT sample values. Fold- with unsorted semen or by parthenogenetic activation or derived in changes in the relative gene expression of the target were de- vivo following the manufacturer’s instructions, using the Dynabeads termined using the equation 2–ΔΔCT. mRNA Direct Extraction KIT (Dynal Biotech) with minor mod- ifications. After 5 min incubation in lysis buffer with Dynabeads, Expression Analysis on Preimplantation Stages and Tissues. RNA poly(A) RNA attached to the Dynabeads was extracted with a extraction and DNA synthesis of denuded oocytes and pre- magnet, suspended in washing buffer A and stored at 4 °C while implantation embryos was performed as described above. Male and DNA extraction and sexing was performed. The DNA present in femaleadulttissues(brain,medulla,testes,spleen,kidney,lung,and Bermejo-Alvarez et al. www.pnas.org/cgi/content/short/0913843107 1of7 muscle) were collected from a local slaughterhouse and imme- by DNase treatment following the protocol described in ref. 4 with diately snap-frozen in liquid nitrogen. RNA extraction was per- minor modifications. PCR was performed and products were vi- formedwithULTRASPEC(Ecogen)avoidingDNAcontamination sualized in 2% ethidium bromide stained gels under UV light. 1. Bermejo-Alvarez P, Rizos D, Rath D, Lonergan P, Gutierrez-Adan A (2008) Can bovine in 3. Bermejo-Alvarez P, Rizos D, Rath D, Lonergan P, Gutierrez-Adan A (2008) Epigenetic vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? Biol differences between male and female bovine blastocysts produced in vitro. Physiol – Reprod 79:594 597. Genomics 32:264–272. 2. Bermejo-Alvarez P, Lonergan P, Rath D, Gutierrez-Adan A, Rizos D (2010) 4. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) Developmental kinetics and gene expression in male and female bovine embryos method. Nat Protoc 3:1101–1108. produced in vitro with sex-sorted spermatozoa. Reprod Fertil Dev 22:426–436 . A B C D F M Fig. S1. Analysis of microarray data for male and female bovine blastocysts between the three individual bulls. Venn diagrams showing the number of transcripts common or specific to each bull comparison using three statistical corrections: (A) False-discovery rate (FDR) at P < 0.05; (B) FDR at P < 0.01; and (C) Bonferroni at P < 0.05. (D) Principal component analysis mapping of the 18 analyzed samples showing the aggregation of the samples according to the sex and to the bull. Bermejo-Alvarez et al. www.pnas.org/cgi/content/short/0913843107 2of7 Fig. S2. Chromosome distribution of the expressed X-linked transcripts. The position along the X-chromosome is shown on a Megabase vertical scale. For rep- resentation, the chromosome was divided into 30 3-Mb length sections and a scatter chart on the right shows the percentage of X-linked transcripts up-regulated in females located on each section compared with the total X-linked transcripts up-regulated in females with known location. The location of the genes analyzed for putative imprinting plus XIST are noted by red arrows (Right); gene names in bold letters are preferentially or totally paternally expressed.