131 GENE SILENCING IN BOVINE ZYGOTES: SIRNA TRANSFECTION v. MICROINJECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 224 ◽  
Author(s):  
C. M. O'Meara ◽  
J. D. Murray ◽  
J. F. Roche ◽  
S. Mamo ◽  
E. Gallagher ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Embryo Development ◽  
Relative Abundance ◽  
Gene Function ◽  
Developmental Stages ◽  
Analysis Data ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
E Cadherin

Ribonucleic acid interference (RNAi) has become an effective tool for studying gene function in a variety of cells. The objective of this study was to compare the efficiency of gene silencing when siRNA were introduced into bovine zygotes by microinjection (as done previously; Tesfaye D et al. 2007 Mol. Reprod. Dev. 74, 978-988) v. a novel method of transfection in terms of gene knockdown and embryo development. For microin-jection, in vitro-produced bovine zygotes (16 h post insemination) were randomly assigned to 1 of 3 groups over 2 experiments. In Experiment 1, E-cadherin siRNA was injected at 100 μM (n = 168) and compared with PBS-injected (n = 180) and noninjected controls (n = 152). In Experiment 2, E-cadherin siRNA was injected at 375 μM (n = 154) and compared with PBS-injected (n = 136) and noninjected controls (n = 151). Embryos were subsequently cultured in vitro until Day 7 (day of IVF = Day 0). For transfection, the zona pellucida was removed from in vitro-produced zygotes. Zona-free zygotes were randomly assigned to 1 of 4 groups (i) GAPDH (n = 67), (ii) scrambled (n = 66), (iii) E-cadherin (n = 69) siRNA treatments at 100 nM or (iv) nontransfected controls (n = 66). Zygotes were incubated in transfection medium with siRNA for 1 h at 39°C, cultured individually in the well-of-the-well system to Day 7. The proportion of zygotes undergoing cleavage and developing to the blastocyst stage was recorded, and Day 7 embryos were frozen individually for mRNA analysis. Data for mRNA expression were fitted to a general linear model, and developmental stages were tested using ANOVA. Microinjection of 100 μM E-cadherin siRNA had no effect on phenotype (P > 0.05). Injection of PBS or 375 μM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5%, 45.5%, and 62.9%, respectively) and blastocyst stage (39.0%, 32.5%, and 45%, respectively) compared with noninjected controls (P < 0.05). The mRNA abundance of the target gene was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 μM and 375 μM compared with control and PBS-injected groups (P < 0.05). Transfection with E-cadherin siRNA decreased development of 8-cell embryos (20.3 v. 53.0%, respectively) and blastocysts (7.2 v. 18.2%, respectively) compared with controls (P < 0.05). The mRNA relative abundance was not different between controls (nontransfected, or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100nM E-cadherin siRNA led to a 70% reduction in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05). Zona removal and transfection resulted in decreased embryo development compared with microinjection (P < 0.05). However, transfection yielded more efficient gene silencing of E-cadherin mRNA with reduced embryo development compared with microinjection. This technique of gene silencing could improve the efficiency of gene function studies in early bovine embryogenesis. Supported by Science Foundation Ireland.

Gene silencing in bovine zygotes: siRNA transfection versus microinjection

2011 ◽  
Vol 23 (4) ◽  
pp. 534 ◽  
Author(s):  
Ciara M. O'Meara ◽  
James D. Murray ◽  
Solomon Mamo ◽  
Emma Gallagher ◽  
James Roche ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Relative Abundance ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Sirna Transfection ◽  
Cell Stage ◽  
Transfection Medium ◽  
E Cadherin

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1 h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P < 0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P < 0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P < 0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05).

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

Acetylation and methylation profiles of H3K27 in porcine embryos cultured in vitro

Zygote ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 575-582 ◽  
Author(s):  
Luciana Simões Rafagnin Marinho ◽  
Vitor Braga Rissi ◽  
Andressa Guidugli Lindquist ◽  
Marcelo Marcondes Seneda ◽  
Vilceu Bordignon
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Developmental Stages ◽  
Methylation Status ◽  
Blastocyst Stage ◽  
Fluorescence Signal ◽  
Cell Stage ◽  
Fluorescent Signal ◽  
H3k27 Methylation

SummaryMethylation and acetylation of histone H3 at lysine 27 (H3K27) regulate chromatin structure and gene expression during early embryo development. While H3K27 acetylation (H3K27ac) is associated with active gene expression, H3K27 methylation (H3K27me) is linked to transcriptional repression. The aim of this study was to assess the profile of H3K27 acetylation and methylation (mono-, di- and trimethyl) during oocyte maturation and early development in vitro of porcine embryos. Oocytes/embryos were fixed at different developmental stages from germinal vesicle to day 8 blastocysts and submitted to an immunocytochemistry protocol to identify the presence and quantify the immunofluorescence intensity of H3K27ac, H3K27me1, H3K27me2 and H3K27me3. A strong fluorescent signal for H3K27ac was observed in all developmental stages. H3K27me1 and H3K27me2 were detected in oocytes, but the fluorescent signal decreased through the cleavage stages and rose again at the blastocyst stage. H3K27me3 was detected in oocytes, in only one pronucleus in zygotes, cleaved-stage embryos and blastocysts. The nuclear fluorescence signal for H3K27me3 increased from the 2-cell stage to 4-cell stage embryos, decreased at the 8-cell and morula stages and increased again in blastocysts. Different patterns of the H3K27me3 mark were observed at the blastocyst stage. Our results suggest that changes in the H3K27 methylation status regulate early porcine embryo development as previously shown in other species.

scholarly journals 244REAL TIME PCR EVALUATION OF EXPRESSION PROFILES OF SOME MATERNAL-SOURCED GENE TRANSCRIPTS IN IN VITRO PRODUCED PRE IMPLANTATION STAGE BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
S. Mamo ◽  
S. Ponsuksili ◽  
K. Wimmers ◽  
M. Gilles ◽  
K. Schellander
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Stage ◽  
Gene Transcripts

Gene expression profiling data collected in a time series and quality related parameters are important for understanding the developmental mechanisms carried out in a developing embryo, and are also a source to enrich the knowledge base of embryo development. However, such data are frequently constrained by limitation and handling of the sample as well as cost associated with generating such data. Cumulatively, these factors have contributed to the existing insufficient data compared to the large need stemming from a drive to control and guide optimum embryo development. In this ongoing study, with objectives to quantify and evaluate gene transcripts identified from certain developmental stages, expression profiles of two ESTs (C256 and C112), derived from an oocyte cDNA library, were analyzed from the above perspectives to understand the change in the level of these gene transcripts throughout the pre-implantation stage of embryo development. For this analysis, pools of oocytes and embryos were prepared by balancing the amount proportional to the number of cells present. mRNA was isolated separately from each pool of matured oocytes, 2-cell, 4-cell, 8-cell, and 16-cell stages, as well as morula and blastocyst stages by using Dynal beads Oligo (dt)25 (Dynabeads, Dynal Biotech, Oslo, Norway) following the manufacturer’s recommendations. These mRNAs were checked for DNA contamination and, when proved free, first-strand cDNA was synthesised by reverse transcribtion at 42°C for 2h following standard laboratory procedures. Transcript quantification was performed by real-time PCR using gene-specific primers, equal amounts of cDNA from each sample and SYBR Green universal master mix. Following this analysis, both transcripts were found to be expressed in a wave-like manner being highly expressed in mature oocytes, declining gradually as the development stage advanced, with the lowest level at the 16-cell stage, and then reviving in level thereafter until it reached blastocyst stage. Taking the 16-cell stage as calibrator for both, C256 was 26.4, 23.2, 8.5, 1.7. 2.4 and 2.7 times more expressed in oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively. Similarly, C112 was 110.7, 169.2, 9.8, 2.5, 4.1 and 7.3 times more expressed in oocyte, 2-cell, 8-cell, morula and blastocyst stages, respectively. These expression patterns suggest the probable origin of these transcripts initially to be maternal. C256 is strongly similar to human retinoid X receptor beta (RXRb) gene (NM_021976.3), which is involved in transcriptional functions and in increasing DNA binding, whereas C112 is strongly similar to TATA box-binding protein-associated factor gene (AY189986.1), which is also involved in transcriptional functions. As seen from their functions, these transcripts can be vital for developing the embryo and the variations at different developmental stages shows their most probable role as part of genes contributing to developmental competence in pre-implantation development stages.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Embryo Development ◽  
Relative Abundance ◽  
Culture Medium ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Glut 1 ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

2008 ◽  
Vol 56 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Chang-Liang Yan ◽  
Qi-En Yang ◽  
Guang-Bin Zhou ◽  
Yun-Peng Hou ◽  
Xue-Ming Zhao ◽  
...  
Keyword(s):  
Survival Rate ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Significant Loss ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Fresh Embryos

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3–100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8–89.5%) and hatched blastocyst rates (61.1–69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3–30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Fernando Henrique Biase ◽  
Robin Edward Everts ◽  
Rosane Oliveira ◽  
Weruska Karyna Freitas Santos-Biase ◽  
Giovana Krempel Fonseca Merighe ◽  
...  
Keyword(s):  
Embryo Development ◽  
Rna Binding ◽  
Blastocyst Stage ◽  
Messenger Rnas ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Expression Of Genes ◽  
Oocyte Cytoplasm ◽  
Metaphase Ii Oocytes

SummaryThe mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

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