Control Blastocysts Research Articles

Ovarian stimulation retards postimplantation development and alters global gene expression profile of blastocysts in mouse

This study compares the quality, developmental capacity and global gene expression profile of embryos generated from superovulated and natural cycles. We found that ovarian stimulation impaired quality of blastocysts (cell number: 68.9 +/- 6.9 vs. 76.8 +/- 7.9; diameter of blastocysts: 145.3 +/- 76.9 microm vs. 152.6 +/- 65.5 microm), fetus development (rate of development to term: 45.5% vs. 69.1%; weight of 18.5-dpc fetuses: 1.23 +/- 0.03 g vs. 1.34 +/- 0.03 g) and caused 92 genes differentially expressed.

Heat shock memory in preimplantation mouse embryos

To investigate the consequences of possible physiologic stress to embryos caused by in vitro fertilization procedures, we used heat shock response in preimplantation mouse embryos as a model. A heat shock "memory" was discovered that renders cleavage-stage embryos more responsive at the transcriptional level to secondary perturbation with very low doses of heat, even several cell cycles after the initial stress has occurred.

Abnormal Levels of Transcript Abundance of Developmentally Important Genes in Various Stages of Preimplantation Bovine Somatic Cell Nuclear Transfer Embryos

Based on microarray data comparing gene expression of fibroblast donor cells and bovine somatic cell nuclear transfer (SCNT) and in vivo produced (AI) blastocysts, a group of genes including several transcription factors was selected for evaluation of transcript abundance. Using SYBR green-based real-time polymerase chain reaction (Q-PCR) the levels of POU domain class 5 transcription factor (Oct4), snail homolog 2 (Snai2), annexin A1 (Anxa1), thrombospondin (Thbs), tumor-associated calcium signal transducer 1 (Tacstd1), and transcription factor AP2 gamma (Tfap2c) were evaluated in bovine fibroblasts, oocytes, embryos 30 min postfusion (SCNT), 12 h postfertilization/activation, as well as two-cell, four-cell, eight-cell, morula, and blastocyst-stage in vitro fertilized (IVF) and SCNT embryos. For every gene except Oct4, levels of transcript were indistinguishable between IVF and SCNT embryos at the blastocyst stage; however, in many cases levels of these genes during stages prior to blastocyst differed significantly. Altered levels of gene transcripts early in development likely have developmental consequences downstream. These results indicate that experiments evaluating gene expression differences between control and SCNT blastocysts may underestimate the degree of difference between clones and controls, and further offer insights into the dynamics of transcript regulation following SCNT.

160 ATTEMPTS TO CULTURE BIOPSIED CELLS FROM IN VITRO BOVINE BLASTOCYSTS FOR GENOTYPING

Genomic tools have now become available for most livestock species and are being used routinely for marker-assisted selection in cattle. One major challenge in bovine selection is the possibility to detect multiple markers from biopsies of pre-implantation stage embryos which allows to transfer only selected embryos following genotyping. Preliminary studies have shown that 2 ng of DNA collected from 200 embryonic cells (hatched blastocyst) may be sufficient for genotyping based on few markers (<100). However, the present genotyping techniques are much more demanding in terms of DNA. The aim of this work was to test different in vitro culture conditions of biopsied cells issued from bovine blastocysts to produce a large number of cells for genotyping. Bovine embryos were produced in vitro according to a standard protocol (Menck M et al. 1997 Reprod. Nutr. Dev. 37, 141-150). Only grade 1 embryos were biopsied using a microblade under a stereomicroscope. Biopsies had from 5 to 10 cells. Biopsied embryos were in vitro cultured in B2 + 2.5% FCS seeded with VERO cells for 48 h to assess the survival rate. Individual biopsies were cultured in vitro in 4-well culture dishes (Nunc) coated with collagen type 1 at 39°C in a humidified air atmosphere and 5% CO2 under 3 medium conditions. Intact hatched Days 8 to 10 blastocysts were cultured under the same conditions as controls. In condition 1, 43 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 10% FCS and 0.25% ITS (insulin, rransferrin, selenium). In condition 2, 30 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 20% FCS supplemented with 1 mM sodium pyruvate, 1 μg mL-1 of heparin, and 1 μg mL-1 of FGF4. In condition 3, 30 biopsies and 43 control blastocysts were cultured in a complex medium composed of 30% of [DMEM/F12 + 20% FCS] and 70% [DMEM/F12 + 20% FCS conditioned medium using mitomycined VERO cells] supplemented with 1 mM sodium pyruvate, 1.5 μg mL-1 of heparin, and 1.5 μg mL-1 of FGF4 (adapted from Oda et al. 2006 Methods Enzymol. 419, 387-400). Medium was replaced every 3 days. Outgrowths were physically detached and isolated cells were cultured using condition 3. For further passages, monolayers were trypsinized (0.025%) and cells were analyzed by immunofluorescence using anti-cytokeratin 1-8 antibodies. After biopsy and 48 h of in vitro culture, 97.1% (100/103) of embryos survived. For all culture conditions, none of the biopsied cells attached to the coated dishes and no colony were observed after culture. Control intact blastocysts adhered and formed significantly lower rate of outgrowths for condition 1 v. 2 and 3: 77.1% v. 85.7% and 93%, respectively (P < 0.05). After several passages, 3 cell lines were produced and we observed a network of cytokeratin filaments by immunofluorescence suggesting an epithelial cell type for this network. These results show that production of a large number of cells from biopsies was not efficient enough for genotyping. However, the 3 tested culture conditions are favorable for the production and multiplication of cells from intact bovine blastocysts and condition 3 seems to be a suitable medium condition for embryonic cell culture.

244 CHANGES IN THE TRANSCRIPTOME OF THE BOVINE PRE-IMPLANTATION EMBRYO INDUCED BY COLONY-STIMULATING FACTOR-2

Colony-stimulating factor-2 (CSF-2) is a cytokine expressed in bovine oviduct and endometrium that has been reported to improve the proportion of embryos that become blastocysts in vitro and survive after transfer to recipients. One effect of CSF-2 that might be related to increased embryonic survival is a preferential increase in the number of cells in the inner cell mass. The objective of the current study was to determine changes in the embryo transcriptome caused by CSF-2 that promote blastocyst formation and establishment and maintenance of pregnancy after transfer. Bovine embryos were produced in vitro and cultured in KSOM-BE2 +10 ng/mL recombinant BoCSF-2 added at Day 5 after insemination. On Day 6 (24 h after treatment), embryos at the morula and early blastocyst stage were harvested and stored in groups of 50 at -80°C. A total of 4 pools of GM-colony-stimulating factor treated blastocysts and 4 control blastocysts were subjected to transcriptional profiling using the Bos taurus 2-color Agilent chip (4 × 44 K format). Before labeling, total RNA starting sample was spiked with control genes (artificial clones) of known concentration provided by Agilent. Labeling was done simultaneously with complimentary RNA (cRNA) amplification. Two rounds of linear RNA amplification were employed. Images were extracted using the Agilent Feature Extraction Software (Agilent Technologies, Santa Clara, CA, USA) and normalized within arrays by the Lowess method. Statistical analysis was performed using the JMP Genomics program (SAS Inst., Cary, NC, USA). The normalized data were log2 transformed, and the quantile normalization method was used to normalize data between arrays. Differences in gene expression were determined using PROC ANOVA (fixed false discovery rate = 0.01). Only genes with a 1.5-fold difference and P < 0.05 were considered differentially expressed. A total of 216 genes were differentially expressed between CSF-2 and control embryos. Of these, 141 could be annotated (61 genes up-regulated and 80 genes down-regulated by CSF-2). These included 13 genes involved in Wnt pathways, including 5 inhibitors of Wnt signaling (FRP, MAB21L2, PCDH24, PDE7, PPPR23A) that were up-regulated by CSF-2 and 5 genes involved in transmission of Wnt signals (WNT16, ROR2, CSNK2B, CELSR2, DTX3) that were down-regulated by CSF-2. Several other genes associated with differentiation were down-regulated by CSF-2 including CXCL12, FEZF1, PLD2, and RGS12. Expression of 1 gene that inhibits apoptosis (PRKAR2B) was increased by CSF-2, whereas expression of 6 genes involved in apoptosis pathways (DAPK1, MADD, NOD2, PIK3IP1, RIPK3, RNF7) were down-regulated. Results indicate that CSF-2 promotes pluripotency and decreases apoptosis in bovine pre-implantation embryos. This research was supported by USDA-AFRI. B. Loureiro andL. Oliveira were supported by a CAPES (Brazil)/Fulbright Fellowship.

Hatching and Distribution of Actin Filaments in Mouse Blastocysts Whose Activities of Protein Kinase A were Suppressed by H-89

The role of actin filaments and contractions in hatching was determined in mouse blastocysts whose actin filament bundling abilities had been suppressed by H-89, an inhibitor of protein kinase A. The hatching rate of blastocysts developed from morulae in a medium containing H-89 at a concentration of 4.0 microM was 17.2%, which was significantly lower than the 76.7% of the control blastocysts developed from morulae in a medium without H-89. The rates of blastocysts starting hatching and forming a slit in the zona pellucida were significantly lower in H-89-treated blastocysts (84.4 and 21.9%) than in control blastocysts (100.0 and 90.6%). The lengths of time needed for slit formation in the zona pellucida and for completion of hatching were significantly longer in the H-89-treated blastocysts (27.4 and 43.3 h) than in the control blastocysts (6.5 and 18.8 h). Over the course of 32 h after blastocoel formation, the number of strong contractions was similar in the H-89-treated and control blastocysts, but the number of weak contractions was significantly fewer in the H-89-treated blastocysts (2.41 times) than in the control blastocysts (4.19 times). Although the distribution of actin filaments was similar in the H-89-treated and control blastocysts in the pre-hatching, hatching and post-hatching periods, the rate of H-89-treated blastocysts in which most trophectoderm cells possessed the fluorescence of actin filaments (12.7%) was significantly lower than the 95.0% of the control blastocysts in the pre-hatching period. These results suggest that actin filament-mediated movements of trophectoderm cells contribute to hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and by enlarging the protrusion. We also suggest that the low hatching ability of the treated blastocysts is related to weak contractions with a low frequency and to strong contractions requiring a longer time for re-expansion.

Highly significant improvement in embryo implantation and increased live birth rate achieved after comprehensive chromosomal screening: implications for single embryo transfer

OBJECTIVE: To improve the identification of viable embryos for uterine transfer with a view to increasing implantation and birth rates. DESIGN: Prospective clinical trial. MATERIALS AND METHODS: Comparative genomic hybridization (CGH) was used to screen all 23 pairs of chromosomes for aneuploidy in over 500 blastocysts derived from 115 patients. The patients were of advanced maternal age (mean 39 years) and in most cases had at least one previous failed IVF attempt (mean failed cycles 2). RESULTS: The CGH approach underwent extensive validation prior to clinical application, confirming concordance between the biopsied trophectoderm (TE) cells and the inner cell mass. A full chromosome screen was obtained for 93% of blastocysts tested. Almost all patients (96%) had at least one normal embryo available for transfer. Currently, 42 patients have had their blastocysts transferred, with the remaining patients scheduled to receive transfers in the coming weeks. The clinical pregnancy rate stands at 86% (36/42). Fifteen cycles have now gone to term, giving a birth rate per cycle of 80% (versus 60% in control blastocyst transfer cycles). Even more impressive was the implantation rate (fetal heart detected) which was increased from 28% in controls to 66% (54/82) in cases with CGH screening (2.4 fold increase, P<0.0003). CONCLUSIONS: We report the first births following comprehensive chromosome screening of blastocyst stage embryos. The sampling of several TE cells led to a robust diagnosis, less susceptible to problems such as chromosomal mosaicism than previous screening methods. Implantation and birth rates were exceptional (66% per embryo and 80% per cycle started respectively). These results were especially good given the poor reproductive history of the patients and were significantly better than cycles without chromosome screening. The dramatic improvement in implantation rate provides a clear indication that methods such as this will allow high pregnancy and birth rates in cycles involving single embryo transfer.

Aberration of blastocyst microRNA expression is associated with human infertility

To examine human blastocyst microRNA (miRNA) expression in correlation with human infertility. MicroRNAs are small, noncoding RNA molecules that regulate gene expression via mechanisms such as degradation and translational suppression of targeted messenger RNAs. Recent data has pointed to the importance of miRNAs in disease states and during mouse embryo development. Descriptive study. Nonprofit research foundation. Transferable quality human blastocysts donated with consent to research (n = 40). Quantitative real-time PCR. MicroRNA expression profile. Morphologically similar blastocysts derived from patients with polycystic ovaries or male factor infertility exhibited a significant decrease in the expression of six miRNAs in comparison with donor fertile control blastocysts (P < 0.05). Annotation of predicted gene targets for these differentially expressed miRNAs included gene ontology (GO) biological processes involved in cell growth and maintenance and transcription as well as GO molecular functions implicated in nucleic acid binding and signal transducer activity. Three predicted miRNA target genes were selected for analysis and demonstrated significant altered expression consistent with aberrant miRNA profiles. This study describes for the first time that transferable quality blastocysts derived from infertile patients (male factor infertility and polycystic ovaries) possess aberrant miRNA profiles. With growing evidence indicating the importance of miRNAs during development, an association may exist with human infertility.

101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P &lt; 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.

70 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP: EFFECT OF CYTOCHALASIN B PRETREATMENT ON SUBSEQUENT DEVELOPMENT

Oocyte cryopreservation is still a challenge in most mammalian species because of their extreme sensitivity to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw survival and subsequent development; however, a previous study in immature [germinal vesicle (GV) stage] oocytes from prepubertal lambs reported that pretreatment with cytochalasin B (CB) did not improve maturation (Silvestre MA et al. 2006 Anim. Reprod. Sci. 93, 176–182). We previously reported that GV oocytes from mature ewes can be vitrified using a cryoloop with high survival, maturation, and subsequent in vitro fertilization (Moawad AR and Campbell KHS 2008 Reprod. Fertil. Dev. 20, 122). The aim of this study was to evaluate the effects of CB pretreatment prior to vitrification of GV oocytes (from mature ewes) on subsequent development. Cumulus–oocyte complexes obtained at slaughter were randomly divided into 2 groups and incubated with or without 7.5 μg mL–1 CB for 60 min before vitrification. Oocytes from each group were vitrified or used as toxicity and controls. For vitrification, oocytes were equilibrated in 10% ethylene glycol (EG) and 0.25 m trehalose (T) in HEPES/TCM-199 plus 10% fetal bovine serum (BM) for 3 min. Oocytes were then exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the cryoloop within 1 min, and immersed in liquid nitrogen. Oocytes were warmed by exposure to (1) 10% EG and 1 m T in BM, (2) 0.5 m T in BM, and (3) BM for 3 min in each solution at 39°C. Oocytes were then matured, fertilized, and cultured in vitro as previously described. The frequency of cleavage 24 and 48 h post-insemination (pi), development to morula (5 days pi), blastocyst (7 days pi), and total cell numbers of blastocyst-stage embryos were evaluated. Cleavage was significantly lower (P &lt; 0.001) in vitrified and CB-vitrified groups at both 24 h pi (15.2 v. 14.7%) and 48 h pi (27.3 v. 23.5%) than in other groups. Development to morula stage was significantly lower (P &lt; 0.001) in vitrified and CB-vitrified oocytes (12.1 v. 17.7%) than in toxicity, CB-control, and control groups (43.1, 42.6, and 52.8%, respectively); however, no significant difference (P &gt; 0.05) was observed between CB-vitrified and CB-toxicity groups. There was a significant decrease (P &lt; 0.01) in development to blastocyst in CB-vitrified (2.9%) compared with CB-control (22.9%) and control (24.5%), but this did not differ significantly (P &gt; 0.05) from toxicity and CB-toxicity groups (9.8 v. 11.4%). No blastocysts developed from the vitrified group. Hatched blastocysts were observed only in CB-control and control groups (8.2 v. 5.7%). Total cell numbers were significantly greater (P &lt; 0.05) in control blastocysts than in toxicity, CB-vitrified, and CB-control (143 v. 87.25, 73, and 82, respectively). However, this did not differ significantly from the CB-toxicity group (115). These results support our previous data and suggest that pretreatment of GV-stage ovine oocytes with CB prior to vitrification has a positive effect on subsequent development.

Effects of phenazine ethosulfate during culture of bovine embryos on pregnancy rate, prenatal and postnatal development

The objective was to evaluate the use of phenazine ethosulfate (PES) during culture of embryos on fetal and postnatal calf development. Oocytes collected from abbatoir-derived ovaries were matured and fertilized, and the resulting embryos were cultured in vitro by standard procedures, in a chemically defined medium plus BSA. Day 0 of culture was 18 ± 2 h after the onset of IVF. From 2.5 to 6.5 d, half of the eight-cell embryos served as controls, and the remainder were exposed to 0.3 μM PES, which decreases lipid content of embryos. Good-quality blastocysts exposed to PES ( n = 38) or control ( n = 35) blastocysts were transferred nonsurgically to synchronized recipients in estrus 6–7.5 d earlier, resulting in 9 calves in each group. These in vitro-produced pregnancies were evaluated weekly between 35 and 98 d postestrus by ultrasonography, and postnatal development of the calves was monitored for 1 month. Based on a limited number of transfers, use of PES during in vitro culture did not affect pregnancy rates compared to the control at 35 or 98 d ( P > 0.1). Transfer at 7–7.5 d after estrus resulted in higher 98-d pregnancy rates than at 6–6.5 d (34% vs. 10%; P < 0.05). In vitro-derived fetuses that aborted had retarded fetal and placental development compared to those that went to term, but there was no difference in fetal loss between the PES treatment and controls. One calf in the PES group weighing 36 kg was born dead at 252 d of gestation, and another calf in this group was dead some hours after birth and weighed 22.2 kg when parturition was induced at 310 d of gestation. It is unclear whether these two abnormal calves were caused by the PES treatment, or were due to in vitro procedures in general. In conclusion, the use of PES during in vitro culture had no effect ( P > 0.1) on pregnancy rates, conceptus losses between Days 35 and 98 of pregnancy, nor fetal postnatal development in calves born normally.

Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts

The purpose of this study was to systematically examine blastocoelic fluid reduction prior to vitrification and its potential benefits. In addition, we compared artificial collapse (AC) by laser pulse to a mechanical method. Mouse and dicarded human blastocysts were used in this study. Blastocysts were collapsed using either a 10 ms pulse with a laser (LAC) or else mechanical puncture with a microneedle (MAC). Blastocysts were vitrified on cryoloops using a two-step ethylene glycol/dimethyl sulfoxide protocol. We examined the effects of AC on specific outcome parameters such as overall survival, reexpansion, cell proliferation, and DNA damage. Unlike others, we report overall high survival rates with expanded blastocysts even without fluid reduction. We did detect a significant increase in blastomeres showing signs of DNA damage in the control group (13%) in comparison to blastocysts AC prior to vitrification (LAC 3%, MAC 5%; p < 0.001). Control blastocysts exhibited a lower rate of reexpansion. Within 3 h of warming, 73% and 81%, respectively of mechanically or laser collapsed blastocysts were fully reexpanded as compared to only 53% of control blastocysts (p < 0.001). Overall blastomere count was also significantly lower in control blastocysts (CT 103+32, MAC 121 + 37, LAC 134 + 35; p < 0.0001). Early blastocysts with smaller blastocoelic volumes did not benefit from any further reduction of fluid volume. AC can reduce DNA damage and enhance postwarming reexpansion and cell proliferation in expanding blastocysts. The laser method also appeared to be effective and may offer some advantages over mechanical collapse.

Effect of retinoic acid on apoptosis and expression of Fas proteins in mouse blastocysts cultured in vitro

Mouse blastocysts were exposed to doses of 0, 1 and 10 micromol/L retinoic acid (RA) for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10 micromol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo

BackgroundEnvironmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation.ResultsWe performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 μg/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts.ConclusionWe have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.

81 INDUCED BLASTOCOEL COLLAPSE IMPROVES SURVIVAL RATES OF PORCINE BLASTOCYSTS AFTER VITRIFICATION

Cryopreservation of porcine blastocysts is regarded as a demanding task because of the high chilling sensitivity and low survival rates. Ultrarapid vitrification techniques combined with the previous artificial induction of collapse of the blastocoel have been successfully applied in mouse and human. The purpose of our work was to use the Cryotop (Kitasato Supply Co., Tokyo, Japan) vitrification method combined with induced blastocyst collapse for cryopreservation of porcine blastocysts produced with parthenogenetic activation of in vitro-matured oocytes. A total of 1200 slaughterhouse-derived porcine oocytes were matured for 41–42 h. Zona-intact or zona-free oocytes (after pronase digestion) were activated with a single electric impulse of 63 or 43 V cm–1, respectively, and incubated in 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide for 4 h (Du et al. 2007 Cryobiology 54, 181–187). Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2, and 90% N2 and maximum humidity. On Day 6, collapse was induced manually in approximately half of the blastocysts with the use of a fire-polished holding pipette and injection pipette (Cook Australia, Brisbane, Queensland, Australia). Ten min after the manipulation, collapsed and control (not manipulated) blastocysts were vitrified with the Cryotop, and then warmed and diluted in 1, 0.5, and 0 m sucrose, respectively (Kuwayama et al. 2005, Reprod. BioMed. Online 11, 300–308). Reexpansion of blastocysts was evaluated under a stereomicroscope after 16–18 h of culture under conditions described above. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical calculations; values with P &lt; 0.05 were regarded as significant. Reexpansion rates of blastocysts are shown in Table 1. Our results indicate that manually induced collapse of the blastocoel increases reexpansion rates of both zona-intact and zona-free porcine blastocysts after Cryotop vitrification. The combination of these simple techniques may help to improve the efficiency of cryopreservation of pig embryos for both experimental and practical applications. Table 1. Reexpansion rates after vitrification of blastocysts with or without induced blastocoel collapse The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen, and ClausWillemoes for their help and excellent technical assistance.

Role of nonessential amino acids on porcine embryos produced by parthenogenesis or somatic cell nuclear transfer

Amino acids play a multitude of roles during early embryonic development and have been demonstrated to facilitate improved development of in vivo or in vitro fertilized and parthenogenetic embryos in several species. However, review of emerging literatures, shows that culture milieu of cloned embryos might be different from those of in vitro fertilized embryos. This study therefore, evaluated the effect of nonessential amino acids (NEAA) on yield and quality of porcine embryos produced by somatic cell nuclear transfer (SCNT) and compared them with parthenogenetic embryos as control. Analysis showed that, supplementation of NEAA to culture medium significantly improved the blastocyst rate of parthenogenetic (38.9 +/- 8.8 vs. 27.5 +/- 9.0%) as well as SCNT (22.5 +/- 2.2 vs. 13.8 +/- 3.4%) embryos although cleavage rates were not different (P < 0.05). These blastocysts also had higher hatching ability and contained higher cell number than control blastocysts (P < 0.05). TUNEL labeling revealed that blastocysts cultured in the presence of NEAA were less predisposed to biochemical apoptosis and showed lower indices of TUNEL, fragmentation, and total apoptosis than those cultured in the absence of NEAA (P < 0.05). Real-time qRT-PCR analysis further revealed that NEAA decreased the expression ratio of BAX:BCL-xL and enhanced the relative abundance of IGF2 transcripts. Therefore, our study suggests that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion, hatching, and total cell number and decreasing the apoptosis by positively modulating the expression of embryo survival related genes, similar to those reported for in vivo or in vitro fertilized embryos. Nonessential amino acids improve the yield and quality of cloned and parthenogenetic porcine embryos and modulate the expression of embryo survival related genes.

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 microm(2), 113 microm(2), and 86 microm(2) 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.

AMP Kinase Activation Increases Glucose Uptake, Decreases Apoptosis, and Improves Pregnancy Outcome in Embryos Exposed to High IGF-I Concentrations

Women with polycystic ovarian syndrome are at increased risk of miscarriage. Although evidence exists that metformin reduces this risk, the mechanism is unknown. This study tests the hypothesis that AMP kinase (AMPK) activation with metformin directly improves insulin signaling within the blastocyst, leading to improved pregnancy outcomes. Murine embryos were exposed to 200 nmol/l IGF-I, similar to the concentrations that can occur during polycystic ovary syndrome (PCOS). Resulting blastocysts were compared with embryos cocultured with excess IGF-I plus metformin and embryos cultured in control medium for the following: AMPK phosphorylation, insulin-stimulated glucose uptake, and apoptosis. Study and control blastocysts were also transferred into control animals. On embryonic day (E) 14.5, resulting fetuses were examined for size and rates of fetal implantation and resorption. Compared with control blastocysts, blastocysts exposed to high concentrations of IGF-I showed a decrease in AMPK activation and insulin-stimulated glucose uptake and an increase in the number of apoptotic nuclei. Blastocysts cocultured in metformin and excess IGF-I performed as well as controls in all studies. 5-Aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside, another AMPK activator, also prevented the effects of excess IGF-I on blastocysts. Implantation rates and fetal size at day 14.5 were significantly lower among IGF-I-exposed embryos transferred into control mothers compared with control embryos transferred into control mothers. Both of these parameters were reversed by co-incubation with metformin and IGF-I before transfer. Activation of embryonic AMPK may be the mechanism responsible for the improved pregnancy outcomes seen in PCOS patients taking metformin.

Vitrification of in vitro cultured porcine two-to-four cell embryos

The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master ® technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows ( n = 11) on day 2 (D0 = onset of estrus). Some embryos ( N = 63) were vitrified within 3 h after collection, warmed and cultured for 120 h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96 h in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24 h (Group VB; N = 65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N = 70) but were cultured in vitro for 120 h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6 ± 0.7% and 3.2 ± 0.5%, respectively). The survival and hatching rates of VB embryos (75.0 ± 0.69% and 33.6 ± 0.13%) were lower ( p < 0.001) than those obtained with control embryos (89.1 ± 0.8% and 47.5 ± 0.12%). Hatched VB embryos had a lower ( p < 0.01) total cell number than hatched control embryos (70.3 ± 4.5 versus 90.6 ± 3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master ®.

Differential but beneficial effect of phytohemagglutinin on efficiency of in vitro porcine embryo production by somatic cell nuclear transfer or in vitro fertilization

In vitro production of porcine embryos has become routine in most laboratories but the yield and quality of the resultant blastocysts remains sub-optimal. Phytohemagglutinin (PHA) is an N-acetylgalactosamine/galactose sugar-specific lectin with a wide variety of biological activities including mitogenesis, mediation of cell recognition and agglutination of cells. This study was therefore, designed to investigate the effect of PHA on the preimplantation development and quality of parthenogenetic, somatic cell nuclear transferred (SCNT) and in vitro fertilized (IVF) porcine embryos cultured in the absence or presence of PHA. Analysis showed that, supplementation of PHA significantly improved the blastocyst rate of parthenogenetic (70.6 +/- 0.2 vs. 51.4 +/- 0.6%) and SCNT (27.7 +/- 1.7 vs. 12.5 +/- 0.3%) embryos but not IVF embryos (25.0 +/- 14.3 vs. 20.1 +/- 12.7%). Nonetheless, PHA-treated blastocysts had higher hatching ability and contained higher cell number than control blastocysts in all the groups (P < 0.05). TUNEL labeling revealed that blastocysts cultured in the presence of PHA were less predisposed to biochemical apoptosis and showed lower indices of TUNEL, fragmentation and total apoptosis than those cultured in the absence of PHA (P < 0.05). Real time qRT-PCR analysis of parthenogenetic blastocysts revealed that PHA decreased the expression ratio of BAX:BCL-xL transcripts. Therefore, our study suggests that PHA improves the blastocyst yield and quality by enhancing blastocyst expansion, hatching and total cell number and decreasing the apoptosis. However, PHA has a differential effect on development rate of IVF derived embryos. These results may represent an approach towards achieving better preimplantation development of porcine embryos in vitro.