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

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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Faculty Opinions recommendation of In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.14266986.15779098 ◽

2012 ◽

Author(s):

Phyllis Leppert

Keyword(s):

Gene Expression ◽

In Vitro Culture ◽

Differential Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Mouse Embryos ◽

Differential Gene

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The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7971-7976.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7971-7976

Author(s):

L M Whyatt ◽

A Düwel ◽

A G Smith ◽

P D Rathjen

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Es Cells ◽

Expression System ◽

Cell Mass ◽

Embryonic Stem ◽

Expression Vectors ◽

Developmental Control ◽

Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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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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15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab15 ◽

2016 ◽

Vol 28 (2) ◽

pp. 137

Author(s):

Y. Liu ◽

A. Lucas-Hahn ◽

B. Petersen ◽

R. Li ◽

D. Hermann ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Culture ◽

Histone Acetylation ◽

Mrna Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Cell Numbers ◽

Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

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127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab127 ◽

2004 ◽

Vol 16 (2) ◽

pp. 186

Author(s):

J.O. Gjørret ◽

P. Maddox-Hyttel

Keyword(s):

Production Systems ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Embryo Cell ◽

Developmental Potential ◽

Cell Numbers ◽

Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to<0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P<0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

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94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab94 ◽

2014 ◽

Vol 26 (1) ◽

pp. 161

Author(s):

A. Velasquez ◽

D. Veraguas ◽

F. O. Castro ◽

J. F. Cox ◽

L. l. Rodriguez-Alvarez

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Gene Expression Pattern ◽

Blastocyst Stage ◽

Embryo Morphology ◽

Bovine Embryo ◽

Gene Markers ◽

Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

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145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

Reproduction Fertility and Development ◽

10.1071/srb09abs145 ◽

2009 ◽

Vol 21 (9) ◽

pp. 63

Author(s):

L. Ganeshan ◽

C. O'Neill

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Early Embryo ◽

Pluripotent Cells ◽

Drug Induced ◽

Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

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2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab2 ◽

2004 ◽

Vol 16 (2) ◽

pp. 123 ◽

Cited By ~ 2

Author(s):

D.O. Brandão ◽

G. Vajta ◽

P. Maddox-Hyttel ◽

D. Stringfellow ◽

P. Lövendahl ◽

...

Keyword(s):

In Vitro Culture ◽

Bovine Serum ◽

Culture System ◽

Culture Medium ◽

Inner Cell Mass ◽

Fetal Bovine Serum ◽

Cell Mass ◽

Embryo Morphology ◽

Fetal Bovine

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: >1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: <0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P<0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P<0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P<0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P<0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.

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

Reproduction ◽

10.1530/rep-06-0247 ◽

2007 ◽

Vol 134 (1) ◽

pp. 63-72 ◽

Cited By ~ 114

Author(s):

Gnanaratnam Giritharan ◽

Said Talbi ◽

Annemarie Donjacour ◽

Francesca Di Sebastiano ◽

Anthony T Dobson ◽

...

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Gene Expression Pattern ◽

Preimplantation Embryos ◽

Global Pattern ◽

Vitro Fertilization ◽

Trophoblastic Cells

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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