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

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.

260 COMPREHENSIVE IDENTIFICATION OF RELATIVE GENES CAUSING ABNORMAL DEVELOPMENT OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 237
Author(s):  
J. Park ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Differentially Expressed Genes ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Differentially Expressed ◽  
Vitro Fertilization

Developmental failure of a cloned animal using somatic cell nuclear transfer (SCNT) procedures is considered to be the result of abnormal expression of developmentally important genes caused by incomplete reprogramming of the donor cell nuclei. However, there are few reports about stage-specific gene expression during cleavage progression of cloned embryos. The aim of this study was to identify using fluorescein differential display method, the differentially expressed genes in cloned embryos at early developmental stages compared with those produced by in vitro fertilization. Bovine cumulus-oocytes complexes (COCs) were aspirated from follicles (2-8 mm in diameter) of slaughterhouse ovaries and cultured in TCM-199 supplemented with 10% fetal calf serum (FCS) for 18 h for somatic cell nuclear transfer (NT) or 24 h for in vitro fertilization (IVF) at 39�C. Removal of oocyte nuclei for NT was performed by squeezing out a small amount of the cytoplasm laying beneath the first polar body by means of a glass needle. Donor cells for NT were obtained from skin cells of an adult cow and cultured in DMEM supplemented with 10% FCS. After the transfer of somatic cell into enucleated oocytes, DC electric pulses at 200 V/mm for 2 � 10 �s were used for fusion, and the reconstructed embryos were treated with 10 �g/mL cycloheximide for 6 h. The embryos were then cultured for 120 h (morula stage) or 168 h (blastocyst stage) in modified SOF medium under 5% CO2, 5% O2 and 90% N2 at 39�C. Total RNA obtained from NT and IVF embryos were analyzed by differential display RT-PCR (DDRT-PCR) as previously described (Minami et al. 2001 Biol. Reprod. 64, 30-35). We obtained several differences in gene expression patterns between NT and IVF embryos at the morula and blastocyst stage. A total of 52 cDNA fragments were isolated and analyzed. Semiquantitative analysis revealed that some genes (NADH dehydrogenase subunit 1, SR rich protein, KIAA0107, ribosomal protein L19) were highly expressed in IVF embryos compared with NT embryos, whereas other genes (CASK) were highly expressed in NT embryos compared with IVF embryos. These results indicate that the differentially expressed genes observed in NT embryos may be representative of marker genes for the production of normal NT offspring and DDRT-PCR procedure is quite useful for identification of several genes that are differentially expressed between NT and IVF embryos.Although the detailed function of the genes and their products remains to be determined, it is likely that the reprogramming mechanisms can be elucidated genetically by the analysis of differentially expressed genes in the future.

scholarly journals 219 METHYLATION STATUS OF A DIFFERENTIALLY METHYLATED REGION (DMR) WITHIN THE BOVINE Igf2 GENE IN PREIMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 260
Author(s):  
C. Gebert ◽  
C. Wrenzycki ◽  
D. Herrmann ◽  
R. Reinhardt ◽  
D. Gröger ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Vitro Fertilization ◽  
Igf2 Gene ◽  
Donor Cells ◽  
Methylation Patterns

Specific DNA regions within imprinted genes become differentially methylated on the maternal and paternal chromosomes during germ cell development. These DMRs play a crucial role in the regulation of imprinted gene expression. The murine insulin-like growth factor2 gene (Igf2) is imprinted and contains an intragenic DMR within the last exon. Recently it became known that the bovine Igf2 gene is also imprinted (Dindot et al. 2004 Biol. Reprod. 71, 470–478) where we have now identified an intragenic DMR in the last exon with the paternal allele being methylated. Aberrant methylation patterns within the bovine Igf2 gene could result in deregulated gene expression and could therefore be involved in the development of fetal abnormalities such as the large offspring syndrome (LOS) in cattle. We have studied the methylation status of 27 CG dinucleotides within this DMR in bovine pre-implantation embryos of different origin by bisulfite sequencing. DNA was isolated from expanded blastocysts collected in vivo and generated by in vitro fertilization (IVF), somatic nuclear transfer (NT), and parthenogenesis (PA). Additionally, DNA was obtained from fibroblasts derived from a female and a male adult animal and used as donor cells for NT and from zygotes and 4-cell embryos both produced by IVF. After PCR amplification of the bisulfite-treated DNA, PCR products were cloned and sequenced. Methylation percentages were calculated for each individual clone by division of the 27 CpGs with the number of methylated CpGs per sample. The methylation levels (%) from each sample were then used to obtain the global methylation levels of the analyzed region. Methylation decreased during the transition from the zygote (28.4% ± 3.8 SEM) to the 4-cell embryo (6.3% ± 2.2 SEM) indicating that the DMR is demethylated after fertilization. An increased methylation level was observed in expanded blastocysts (in vivo: 10.2% ± 1.2 SEM; IVF: 10.1% ± 0.7 SEM; female NT: 12.4% ± 1.4 SEM). Thus, remethylation starts before the blastocyst stage. The higher methylation level of male NT blastocysts (22% ± 1.9 SEM) in comparison to their in vivo and IVF counterparts could be due to an insufficient reprogramming of the donor cells after nuclear transfer. Female and male donor cells were both heavily methylated (77% ± 2.2 SEM; 72% ± 2.9 SEM, respectively). Parthenogenetic expanded blastocysts were less methylated (2.3% ± 1 SEM), probably due to their diploid maternal genome. Results show for the first time that the methylation status at this DMR is associated with the origin of the embryo. Analysis of methylation patterns in pre-implantation embryos could provide a diagnostic tool to unravel mechanisms involved in fetal malformations often observed after the use of in vitro fertilization and/or nuclear transfer.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals 106 GENOMIC IMPRINTING OF IGF2R IN TISSUES OF BOVINE FETUSES GENERATED BY ARTIFICIAL INSEMINATION OR IN VITRO FERTILIZATION

2005 ◽  
Vol 17 (2) ◽  
pp. 204 ◽  
Author(s):  
S. Hiendleder ◽  
D. Bebbere ◽  
S. Bauersachs ◽  
M. Stojkovic ◽  
H. Wenigerkind ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Artificial Insemination ◽  
Expression Patterns ◽  
Allelic Expression ◽  
Paternal Allele ◽  
Specific Gene ◽  
Vitro Fertilization ◽  
Bovine Fetuses ◽  
Parent Of Origin

The insulin-like growth factor 2 receptor gene (IGF2R) is involved in fetal growth regulation. A study in sheep associated fetal overgrowth after in vitro embryo culture with abnormal DNA methylation and expression of IGF2R (Young et al. 2001 Nat. Genet. 27, 153–154). This suggested that abnormal IGF2R imprinting is a major cause of fetal overgrowth. To test this hypothesis in bovine fetuses, we developed a microsatellite marker for IGF2R from cDNA sequence data and screened 45 Day-80 fetuses generated in vivo, by artificial insemination (AI), or in vitro, by in vitro fertilization (IVF) procedures, for parent-of-origin-specific gene expression. A total of 17 fetuses were heterozygous, but available parental DNA samples showed that only 12 (8 AI, 4 IVF) allowed unambiguous discrimination of parental alleles. Parent-of-origin-specific allelic expression patterns indicated that bovine IGF2R was expressed predominantly from the maternal allele and thus imprinted in fetal heart, kidney, liver, lung, muscle, and cotyledon tissue. However, the relative amount of expression from the paternal allele was tissue-specific and ranged from 6.4 ± 0.8% in skeletal muscle up to 27.4 ± 0.9% in cotyledon (SPSS or 11.5, ANOVA, P < 0.001). Tissues that originated from the same germ layer showed similar allelic expression ratios whereas significantly different expression ratios (P < 0.05) were observed between tissues originating from different germ layers. Contrary to expectations from sheep data, there was no evidence for gross abnormalities in IGF2R imprinting in tissues from overgrown (n = 2) or normal sized (n = 2) IVF fetuses. However, relative paternal expression levels in several tissues showed significant relationships (P < 0.05–0.001) with growth parameters and pointed to subtle changes in paternal IGF2R expression in overgrown IVF fetuses. We thank W. Scholz and M. Weppert for excellent technical assistance.

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