Focus on ART Effects of Embryo Culture on Global Pattern of Gene Expression in Preimplantation Mouse Embryos
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REPRODUCTIONRESEARCH Focus on ART Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos Paolo Rinaudo1,2 and Richard M Schultz1 1Department of Biology and 2Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104, USA Correspondence should be addressed to R Schultz; Email: [email protected] Abstract Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM 1 amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s med- ium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression. Reproduction (2004) 128 301–311 Introduction (Doherty et al. 2000). We elected to assess the effect of embryo culture on global patterns of gene expression in Preimplantation embryos can develop in media of compo- embryos cultured in these two media using oligonucleo- sition ranging from simple balanced salt solutions and tide microarrays, because of these aforementioned differ- carbohydrates to very complex constituents (e.g. Ham’s ences in maintaining or not maintaining appropriate F-10) with the further addition of serum or a feeder layer monoallelic maternal H19 expression. of somatic cells (Lane 2001, Natale et al. 2001, Niemann Until recently, quantitative analysis of high-resolution, two-dimensional protein gels (Latham et al. 1991), mRNA & Wrenzycki 2000). The blastocysts that develop after differential display (Ma et al. 2001), analysis of expressed culture are known to be developmentally competent sequence tags derived from libraries of various preimplan- because, after embryo transfer, live offspring are born tation stages (Ko et al. 2000, Sharov et al. 2003) and ana- (Ecker et al. 2004). Nevertheless, embryo culture can per- lysis of selected genes (Ho et al. 1995) have been used. turb gene expression (Ho et al. 1995). Of particular note Although these approaches have shed some light on the is that expression of imprinted genes appears particularly molecular basis underlying preimplantation development, sensitive to culture conditions. For example, appropriate they offer limited insight because only a small number of maternal monoallelic expression of H19 is observed after genes can be readily analyzed. Microarray techniques pro- culture in K modified simplex optimized medium (KSOM) vide a powerful approach to study patterns of gene þ amino acids (KSOM/AA), whereas biallelic expression expression on a global scale (Hamatani et al. 2004, Wang is found after culture in Whitten’s medium (WM) et al. 2004). The ability to amplify the small amounts of (Doherty et al. 2000). Moreover, biallelic expression is mRNA present in preimplantation mouse embryos, which accompanied by loss of DNA methylation of cytosine resi- can only be isolated in limited numbers, makes it feasible dues on the paternal allele in the differentially methylated to generate enough material for microarray analysis. We domain essential for repression of the paternal allele used Affymetrix oligonucleotide arrays (MOE430A chip) q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00297 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/26/2021 01:19:08PM via free access 302 P Rinaudo and R M Schultz containing more than 22 000 transcripts and variants, hybridization. Total RNA yield was 74–133 ng per repli- together with a T7-based linear double amplification cate pool. This total RNA was used for linear, two-round method, and report here changes in the global patterns of amplification by in vitro transcription (Affymetrix Small gene expression that occurred during preimplantation Sample Target Labeling Assay version II, www.affymetrix.- development in mouse blastocysts cultured in WM com). cRNA yield after the first amplification was 1.5– or KSOM/AA from the one-cell stage. We report that, 5 mg, and 0.5 mg of each replicate was used as input tem- after culture in WM, 114 genes were mis-expressed, plate for the second amplification. Final yield of biotiny- whereas only 29 were mis-expressed after culture in lated cRNA was 74–138 mg, of which 15 mg per replicate KSOM/AA. Of note is that 14 common genes were mis- was fragmented and hybridized to Affymetrix GeneChips. expressed in either medium and are involved in ion and cRNA samples were hybridized to MOE430A GeneChip, water transport. then washed and stained on fluidics stations and scanned at 3 mm resolution according to the manufacturer’s instructions (GeneChip Analysis Technical Manual, Materials and Methods www.affymetrix.com). Collection of preimplantation mouse embryos and RNA extraction Analysis of the microarrays Mouse CF-1 £ B6D2F1/J embryos were isolated from Microarray Analysis Suite 5.0 (MAS; Affymetrix, Santa superovulated mice as previously described (Zeng & Clara, CA, USA) was used to quantify microarray signals Schultz 2003). Briefly, CF-1 female mice were injected with default analysis parameters and global scaling to tar- with 5 IU equine chorionic gonadotrophin and 42–46 h get mean ¼ 150. Quality control parameters for all later with 5 IU human chorionic gonadotrophin (hCG); samples were within the following ranges: scale factor the females were then mated to B6D2F1/J males over- 1–2.7, background 42–88, percent genes detected night. The next morning, zygotes were obtained from the 37–44% on MOE430A, actin 30/50 signal ratio 1.5–2.8, ampullae and cultured in either WM (Whitten 1971) or and GAPDH 30/50 signal ratio 2.3–7.5. The MAS metrics KSOM/AA (Ho et al. 1995) to the blastocyst stage in 5% output was loaded into GeneSpring v5 (Silicon Genetics, CO2 –5% O2 –90% N2 at 37 8C. Cultures were initiated www.silicongenetics.com) with per-chip normalization with more embryos when WM was used, to compensate to the 50th percentile and per-gene normalization to for the poorer development in WM compared with that in the median. A filtered list was created of all genes KSOM/AA. After culture in either medium, late-cavitating detected (MAS ‘P’ call) in at least five of six replicates of blastocysts of similar morphology were harvested at 96 h the in vivo group. post-hCG. Embryos that developed in vivo were harvested Independent analyses were applied to identify genes 96 h post-hCG and those that looked morphologically with statistically significant differences in any of the similar to embryos that developed in vitro were collected two culture conditions (WM or KSOM/AA). The Gene- and used for analysis. Cell numbers were determined by Spring pairwise comparison (Welch t-test with ANOVA, differential labeling of inner cell mass and trophectoderm P ¼ 0.05, Benjamini and Hochberg multiple testing cor- cells as previously described (Handyside & Hunter 1984). rection) was conducted between all the possible pair The numbers of total cells and of inner cell mass cells combinations (in vivo, WM, KSOM/AA). The Gene- after culture in WM were 70.6 ^ 4.3 (mean^ S.E.M.) Spring multi-class analysis was applied to the entire and 16.6 ^ 1.2 respectively. Corresponding numbers after sample set (Welch t-test with Welch ANOVA, P ¼ 0.05, culture in KSOM/AA were 81.8 ^ 3.8 and 23.9 ^ 1.7 Benjamini and Hochberg multiple testing correction). respectively. All animal experiments were approved by A one-way ANOVA for microarrays (lgsun.grc.nia.nih. the Institutional Animal Care and Use Committee and gov/ANOVA/index.html, default parameters) was also were consistent with NIH guidelines. conducted. A non-redundant list was compiled contain- Total RNA was extracted from pools of 80 embryos ing candidate genes called significantly different in at (120 ng total RNA) using Trizol containing 2 ml Pellet Paint least one analysis. (Novagen, Madison, WI, USA) according to the manufac- Genes exhibiting altered expression after culture were turer’s instructions (Invitrogen). Total RNA was dissolved imported to Expression Analysis Systematic Explorer in 10 ml sterile water and stored at 280 8C. RNA mass (EASE) to test for over-representation of annotation classes and size distribution were determined using the Agilent (Hosack et al. 2003). EASE is a program that provides stat- Bioanalyzer with RNA 6000 Nano LabChips (Palo Alto, istical methods (reported as an EASE score) for discovering CA, USA). biological themes within gene lists, using previously pub- lished annotation databases. Over-representation does not refer to abundance of gene expression, but rather cDNA preparation for microarray analysis describes a class of genes that have similar functions – for Total RNA samples were submitted to the Penn Micro- example, transcription factors – regardless of their array Facility for target preparation and GeneChip expression level, and appear more often in a list of interest Reproduction (2004) 128 301–311 www.reproduction-online.org Downloaded from Bioscientifica.com at 09/26/2021 01:19:08PM via free access Gene expression in in vitro cultured embryos 303 Figure 2 Differential expression profiles of genes after culture in either Whitten’s medium (WM) (A) or KSOM/AA (KAA) (B).