Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 μm2, 113 μm2, and 86 μm2 respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

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Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽

10.1530/rep.1.0966 ◽

2007 ◽

Vol 133 (1) ◽

pp. 231-242 ◽

Cited By ~ 43

Author(s):

Craig Smith ◽

Debbie Berg ◽

Sue Beaumont ◽

Neil T Standley ◽

David N Wells ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Ectopic Expression ◽

Cell Number ◽

Transcript Levels ◽

Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

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Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

Molecular and Cellular Biology ◽

10.1128/mcb.01188-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3123-3130 ◽

Cited By ~ 26

Author(s):

Klaus Fortschegger ◽

Bettina Wagner ◽

Regina Voglauer ◽

Hermann Katinger ◽

Maria Sibilia ◽

...

Keyword(s):

Matrix Protein ◽

Replicative Senescence ◽

Inner Cell Mass ◽

Umbilical Vein ◽

Cell Mass ◽

Preimplantation Embryos ◽

Proliferative Potential ◽

Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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186 LASER CAPTURE MICRODISSECTION FOR GENE EXPRESSION ANALYSIS OF INNER CELL MASS AND TROPHOBLAST FROM BOVINE BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab186 ◽

2011 ◽

Vol 23 (1) ◽

pp. 194

Author(s):

M. Filliers ◽

W. de Spiegelaere ◽

L. J. Peelman ◽

K. Goossens ◽

C. Burvenich ◽

...

Keyword(s):

Gene Expression ◽

Laser Capture Microdissection ◽

Gene Expression Analysis ◽

Inner Cell Mass ◽

Cell Mass ◽

Gene Expression Pattern ◽

Serial Sections ◽

Trophoblastic Cells ◽

Keratin 18 ◽

Laser Capture

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.

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The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage

Reproduction ◽

10.1530/rep.0.1260161 ◽

2003 ◽

pp. 161-169 ◽

Cited By ~ 44

Author(s):

GA Thouas ◽

GM Jones ◽

AO Trounson

Keyword(s):

Culture System ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Control Culture ◽

High Density ◽

Preimplantation Embryos ◽

Mouse Zygotes

A novel system of in vitro culture termed the 'glass oviduct' or 'GO' culture system is described. Mouse zygotes were cultured in pairs to the blastocyst stage in open-ended 1 microl glass capillaries. 'GO' culture supported the development of significantly more hatching or hatched blastocysts than did a standard microdroplet (10 zygotes per 20 microl) control culture (48.3 versus 3.3%, respectively). 'GO' bslastocysts contained significantly larger populations of cells (92+/-3 versus 75+/-3), and inner cell mass (25+/-1 versus 21+/-1) and trophectoderm (68+/-2 versus 53+/-3) subpopulations, compared with microdroplet-derived blastocysts. Before blastulation, 'GO'-derived morulae were found to contain significantly more cells than microdroplet-derived morulae (27+/-0.7 versus 14+/-0.5). After implantation, 'GO' blastocysts formed fetuses at a similar rate to microdroplet-derived blastocysts (55 versus 62%), but at a lower rate than blastocysts derived in vivo (80%). 'GO'- and microdroplet-derived fetuses were similar in wet weight to each other (0.412 and 0.415 g, respectively) but were heavier than fetuses derived from flushed blastocysts (0.390 g). An additional experiment investigated whether the beneficial effect of 'GO' culture was due to the significantly increased embryo density. Proportions of hatching or hatched blastocysts after 'GO' culture (50%) were higher than after standard microdroplet culture (7.6%), but were not different from culture in high embryo density microdroplets (20 zygotes per 10 microl; 42%). 'GO' blastocysts contained more cells (79.6+/-2.1) than did standard microdroplet-derived blastocysts (68.7+/-2.0), but were similar to high density microdroplet-derived blastocysts (85.8+/-2.7). Similarly, 'GO' blastocysts contained more trophectoderm cells (62.2+/-2.0) than did standard microdroplet-derived blastocysts (52.7+/-1.7), but were similar to the high density microdroplet blastocysts (68.8+/-2.5). Numbers of inner cell mass cells ('GO', standard microdroplet and high density microdroplet culture) were not different from each other (17.4+/-0.5, 16+/-0.5 and 17+/-0.4, respectively). In conclusion, the 'GO' culture system represents an alternative method to the microdroplet system for small numbers of preimplantation embryos, without detriment to implantation potential.

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Patterns of lactic dehydrogenase isozymes in mouse embryos over the implantation period in vivo and in vitro

Development ◽

10.1242/dev.36.3.653 ◽

1976 ◽

Vol 36 (3) ◽

pp. 653-662

Author(s):

Marilyn Monk ◽

John Ansell

Keyword(s):

Specific Activity ◽

Inner Cell Mass ◽

Cell Mass ◽

Lactic Dehydrogenase ◽

Giant Cells ◽

Preimplantation Embryos ◽

Blastocyst Implantation ◽

Trophoblast Giant Cells

Following blastocyst implantation, or outgrowth in vitro, the LDH isozyme pattern changes from that of the maternally inherited B subunit isozyme form (LDH-1) to a pattern dominated by A subunits (Auerbach & Brinster, 1967, 1968). In preimplantation embryos we have also observed additional isozyme bands, as yet unidentified. An analysis of the pattern of newly synthesized LDH isozymes and specific activity of LDH in different regions of early post implantation embryos suggests that there is a sequential activation of A and B subunits, and that activity first appears in ICM- (inner cell mass) derived tissues and then in trophoblast-derived tissues. In vitro, in the absence of ICM cells, the transition of LDHisozyme pattern does not occur in outgrowing trophoblast giant cells. This suggests a possible inductive interaction between ICM and trophoblast.

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Inner cell mass (ICM) and trophectoderm (TE) cell gene expression of murine IVF preimplantation embryos is different from in vivo embryos

Fertility and Sterility ◽

10.1016/j.fertnstert.2008.07.1581 ◽

2008 ◽

Vol 90 ◽

pp. S344

Author(s):

G. Giritharan ◽

L. Delle Piane ◽

A. Donjacour ◽

F.J. Esteban ◽

J.A. Horcajadas ◽

...

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Preimplantation Embryos ◽

Cell Gene Expression ◽

Cell Gene

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Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice

Reproduction ◽

10.1530/rep-08-0265 ◽

2009 ◽

Vol 137 (2) ◽

pp. 271-283 ◽

Cited By ~ 46

Author(s):

Raúl Fernández-González ◽

Juan de Dios Hourcade ◽

Irene López-Vidriero ◽

Alberto Benguría ◽

Fernando Rodríguez De Fonseca ◽

...

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Epigenetic Mechanisms ◽

Long Term Effects ◽

Long Term Consequences

We have reported thatin vitroculture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos culturedin vitroin KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared within vivoderived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos culturedin vitroorin vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

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Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

Zygote ◽

10.1017/s0967199414000677 ◽

2014 ◽

Vol 24 (1) ◽

pp. 18-30 ◽

Cited By ~ 10

Author(s):

Alejandra E. Velasquez ◽

Fidel O. Castro ◽

Daniel Veraguas ◽

Jose F. Cox ◽

Evelyn Lara ◽

...

Keyword(s):

Gene Expression ◽

Survival Rate ◽

In Vitro Culture ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryo Morphology ◽

Developmental Potential ◽

Bovine Blastocyst

SummaryEmbryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

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