Investigation of Imprinted Gene Expression for In Vitro Fertilized Mouse Embryos

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

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Analysis of imprinted gene expression and implantation in haploid androgenetic mouse embryos

Andrologia ◽

10.1111/and.12222 ◽

2014 ◽

Vol 47 (1) ◽

pp. 102-108 ◽

Cited By ~ 6

Author(s):

M. Hu ◽

Z. Zhao ◽

L.-C. TuanMu ◽

H. Wei ◽

F. Gao ◽

...

Keyword(s):

Gene Expression ◽

Mouse Embryos ◽

Imprinted Gene

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Effect of vitrification on in vitro development and imprinted gene Grb10 in mouse embryos

Reproduction ◽

10.1530/rep-16-0480 ◽

2017 ◽

Vol 154 (3) ◽

pp. 197-205 ◽

Cited By ~ 10

Author(s):

Jianfeng Yao ◽

Lixia Geng ◽

Rongfu Huang ◽

Weilin Peng ◽

Xuan Chen ◽

...

Keyword(s):

Dna Methylation ◽

Cpg Island ◽

Formation Rate ◽

Mouse Embryos ◽

Major Type ◽

Hatching Rate ◽

Preimplantation Embryo Development ◽

Imprinted Gene

Vitrification of embryos is a routine procedure in IVF (in vitrofertilization) laboratories. In the present study, we aimed to investigate the effect of vitrification on mouse preimplantation embryo developmentin vitro, and effect on the epigenetic status of imprinted geneGrb10in mouse embryos. The blastocyst formation rate for vitrified 8-cell embryos was similar to the non-vitrified 8-cell embryos, whereas the blastocyst hatching rate was lower than that of the non-vitrified group. The expression level ofGrb10major-type transcript decreased significantly in vitrified blastocysts compared with non-vitrified andin vivoblastocysts. Moreover, the global DNA methylation level in 8-cell embryos and blastocysts, and the DNA methylation at CpG island 1 (CGI1) ofGrb10in blastocysts were also significantly decreased after vitrification.In vitroculture condition had no adverse effect, except for on the DNA methylation inGrb10CGI1. These results suggest that vitrification may reduce thein vitrodevelopment of mouse 8-cell embryos and affect the expression and DNA methylation of imprinted geneGrb10.

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20 NUCLEAR TRANSFER ALTERS EXPRESSION AND HISTONE MODIFICATIONS OF THE IMPRINTED GENE PHLDA2 IN THE BOVINE PLACENTA

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab20 ◽

2015 ◽

Vol 27 (1) ◽

pp. 102

Author(s):

J. C. T. Penteado ◽

D. R. Arnold ◽

R. C. Gaspar ◽

C. V. da Rocha ◽

J. R. Sangalli ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Fertilization ◽

Promoter Region ◽

Nuclear Transfer ◽

Proximal Promoter ◽

Paternal Allele ◽

Vitro Fertilization ◽

Histone Marks ◽

Imprinted Gene

Proper implantation and placental formation are crucial for the continuity of mammalian species. Embryonic and placental developments are under intense genetic and epigenetic control, such as the regulation of differentiation of pluripotent cells into highly specialised fetal and placental cells. In the present study the objectives were to evaluate expression and epigenetic control of the imprinted gene PHLDA2, a maternally expressed gene that appears to be a regulator of placental growth, in cotyledonary and inter-cotyledonary tissues of bovine placentas on Day 60 of pregnancies produced by embryo transfer (ET; n = 3), in vitro fertilization (IVF; n = 5), and nuclear transfer (NT; n = 6), by real time PCR (qPCR). In vitro culture of IVF and NT embryos was performed in SOF medium supplemented with 2.5% fetal bovine serum, at 39°C in a humidified atmosphere of 5% CO2 and 5% O2 for 7 days. For evaluation of gene expression, gene-specific standard curves were used, and results were analysed as a ratio to 2 separate housekeeping controls (GAPDH and β-actin). Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR; precipitated/total input DNA) was also performed on the proximal promoter region of PHLDA2, with antibodies against H3K4me2 (permissive histone modification) and H3K9me2 (inhibitory histone modification) in these samples. Products of the ChIP-qPCR for PHLDA2 were digested with a restriction enzyme (AciI) that recognises a specific sequence of the maternal allele (Bos indicus), separating it visually on a gel, from the paternal allele (Bos taurus). Digestion products were separated on a 3% agarose gel, and ethidium bromide was used for visualisation. ImageJ (NIH, Bethesda, MD, USA) was used to analyse band intensity. Gene expression, ChIP, and digestion data were analysed using the least-squares ANOVA and the general linear model procedures (SAS Institute Inc., Cary, NC, USA). Further comparison of means was performed using Duncan's multiple range test (P < 0.05 was considered significant). Expression of the imprinted gene PHLDA2 was 11 times higher in samples produced by NT and, interestingly, also in samples produced by IVF (P < 0.05) compared with the samples produced by ET. ChIP-qPCR for the histone marks, followed by allelic analysis, showed a significant increase of the permissive mark H3K4me2, especially in the silenced paternal allele (P < 0.05), and a reduction of the inhibitory H3K9me2 mark, in the promoter region of the PHLDA2 gene, in clones. The differences observed for these 2 histone marks corroborated with the pattern of gene expression for these samples (elevated in TN placentas). In conclusion, the reproductive biotechnologies of nuclear transfer and in vitro fertilization induce changes in placental expression of the imprinted gene PHLDA2, and nuclear transfer also affects the pattern of histone marks on the proximal promoter region of the imprinted gene PHLDA2.

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Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos

Theriogenology ◽

10.1016/j.theriogenology.2009.01.011 ◽

2009 ◽

Vol 71 (9) ◽

pp. 1408-1416 ◽

Cited By ~ 25

Author(s):

A. Dhali ◽

V.M. Anchamparuthy ◽

S.P. Butler ◽

R.E. Pearson ◽

I.K. Mullarky ◽

...

Keyword(s):

Gene Expression ◽

Mouse Embryos ◽

Droplet Vitrification

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187 IMPRINTED GENE EXPRESSION IN IN VIVO-AND IN VITRO-PRODUCED BOVINE FETUSES AND PLACENTAS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab187 ◽

2008 ◽

Vol 20 (1) ◽

pp. 173

Author(s):

F. Perecin ◽

S. C. Méo ◽

W. Yamazaki ◽

C. R. Ferreira ◽

F. H. Biase ◽

...

Keyword(s):

Gene Expression ◽

Expression Patterns ◽

Rna Extraction ◽

Imprinted Genes ◽

Total Rna ◽

Imprinted Gene ◽

Bovine Fetuses ◽

Low Efficiency

Some gestational alterations associated with bovine somatic cell nuclear transfer (SCNT) are presumably consequences of abnormal imprinted gene expression. This work aimed to evaluate the expression patterns of imprinted genes IGF2 and IGF2R in bovine fetuses and chorioallantoic membranes derived from in vivo- and in vitro-produced embryos. Fetuses were produced by AI (in vivo group, n = 3), IVF (n = 3), parthenogenesis (n = 3), or SCNT (n = 2). Cows with positive pregnancy diagnosis after ultrasonographic examination were slaughtered between Days 33 and 36 of gestation. The reproductive tract was transported on ice to the laboratory, where fetuses and chorioallantoic fragments were collected and stored in liquid nitrogen. Total RNA extraction was performed using TRIzol, according to manufacturer's instructions, and the reverse transcription reaction was carried out with 1 µg of total RNA, 6.75 µm oligo pd(T)12–18, and 50 U of reverse transcriptase (Improm-II, Promega, Madison, WI, USA). The relative quantification of IGF2 and IGF2R transcripts was done using real-time PCR with SYBR Green dye. The average efficiency of PCR amplifications was estimated for each gene using a linear regression on the logarithm of fluorescence per cycle (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66), and the expression ratios were calculated according to the method described previously by Livak and Schmittgen (2001 Methods 25, 402–408). To verify statistical differences, a pair-wise fixed reallocation randomization test (Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) was used. All expression ratios were normalized by glyceraldehyde 3-phosphate dehydrogenase expression and calibrated by the in vivo group (expression assumed as 1.00 for all genes and tissues). The analysis of relative differences on transcript levels of imprinted genes in fetuses revealed IGF2 down-regulation (P < 0.05) in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to the in vivo group and IVF fetuses (2.02). In chorioallantois, IGF2 was down-regulated (P < 0.001) in parthenotes (0.001) when compared to the in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was down-regulated (P < 0.001) in SCNT chorioallantois (0.25) when compared to the in vivo group. Low expression of IGF2 in parthenogenetic fetuses and chorioallantois confirms its imprinted status in bovine. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in bovine SCNT-derived fetuses and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes for the low efficiency of SCNT procedures in this species. Such alterations suggest modifications in DNA methylation patterns at IGF2 and IGF2R imprinting centers.

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260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab260 ◽

2007 ◽

Vol 19 (1) ◽

pp. 246

Author(s):

A. Baji Gal ◽

S. Mamo ◽

S. Bodo ◽

A. Dinnyes

Keyword(s):

Gene Expression ◽

Real Time ◽

Standard Error ◽

Real Time Pcr ◽

Housekeeping Gene ◽

Mouse Embryos ◽

Cell Stage ◽

The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

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