Control Blastocysts Research Articles

Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta1 subunit mRNAs and protein were not detectable in beta1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase alpha1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

26 NUCLEAR REPROGRAMMING AND EMBRYONIC AND FETAL DEVELOPMENT OF CLONED OVINE EMBRYOS PRODUCED USING CAFFEINE-TREATED OOCYTES AS CYTOPLAST RECIPIENTS

We have previously reported that treatment of ovine oocytes with 10 mM caffeine prior to their use as cytoplast recipients for somatic cell nuclear transfer (SCNT) increased the activities of both MPF and MAPK kinases, increased the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC), increased total cell numbers at the blastocyst stage, and altered the expression profiles of a number of developmentally regulated genes including Oct-4 and Oct-4-regulated genes and the stress response genes (HSP27 and HSP70). In addition, an increase in nuclear translocation of HSP27 was observed (Lee and Campbell 2006 Biol. Reprod. 74, 691–698; Choi et al. 2006 Reprod. Fertil. Dev. 18, 122; Choi et al. 2007 Reprod. Fertil. Dev. 19, 134). The objectives of the present study were to determine the potential mechanisms by which caffeine increased cell numbers and improved nuclear reprogramming. We previously hypothesized that caffeine treatment increased the degree of PCC and removed chromatin-bound somatic factors, allowing access of oocyte factors involved in reprogramming. With the use of a TUNEL assay, the accessibility of DNaseI (determined by fluorescence intensity) was increased in caffeine-treated embryos (P < 0.05), supporting the hypothesis. The timing of pronuclear formation, onset of DNA synthesis, and first cleavage were also studied. At 6 h post-activation (hpa), no difference in frequency of pronuclear formation was observed between groups but higher numbers of Brdu-labelled nuclei were detected in caffeine-treated embryos (67.6% v. 43.5%; P < 0.01). In caffeine-treated SCNT embryos, 40.5%, 54.3%, and 65% were cleaved at 20, 22, and 24 hpa as compared to 27.3%, 38.9%, and 48.4% in non-treated controls. On Day 7 there were no significant differences in the frequency of development to the blastocyst stage between treatment and control groups (26.06% v. 22.88%); however, treated embryos had increased total cell numbers (98.5 v. 76.6; P < 0.05) and lower percentages of apoptotic nuclei (11.27% v. 20.3%; P < 0.05). A total of 106 treated and 120 control blastocysts were transferred to the uteri of 43 and 49 recipient ewes, respectively. Five and six ewes established pregnancy, respectively. In the treatment group, three ewes maintained pregnancy for over 100 days and one live lamb was born; in contrast, all controls aborted within 100 days. Taken together, these results suggest that the increased cell numbers and improved reprogramming in SCNT embryos produced using caffeine-treated oocytes as cytoplast recipients may be mediated by increased NEBD and PCC facilitating chromatin access. In addition, increased MAP kinase activity may result in translocation of HSP27 to the nucleus, resulting in reduced apoptosis. Furthermore, caffeine-treated embryos maintained pregnancies for a longer period, supporting our previous observations (Lee et al. 2006 Reprod. Fertil. Dev. 18, 135–136) and suggesting that in sheep the use of caffeine to increase oocyte kinase activities results in embryos with a greater developmental competence.

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1™ ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2™ ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P < 0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7 ± 2.1, polarized monolayer 61.9 ± 2.1) than the control culture (42.8 ± 2.6; P < 0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems ( P < 0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6 ± 0.9; P < 0.05) than in non-polarized (1.7 ± 0.3) or polarized (1.5 ± 0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments ( P < 0.05), whereas, the relative abundance of Bcl-2 mRNA to the β-tubulin was lower than co-culture treatments ( P < 0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.

Phosphatidylinositol 3-Kinase Activity Is Critical for Glucose Metabolism and Embryo Survival in Murine Blastocysts

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.

Gel-Loading Tip Vitrification of In Vitro-Matured Bovine Oocytes and Subsequent Embryo Production by IVF and Nuclear Transfer

The purpose of this study was to cryopreserve bovine oocytes for subsequent blastocyst production by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). A vitrification procedure using gel-loading tips as containers was applied to cryopreserve in vitro-matured and denuded oocytes. In Experiment 1, oocytes were vitrified-warmed in vitrification solution (VS) containing 25, 28, 31, or 40% ethylene glycol (EG) and 1.0 M sucrose. The proportions of survived oocytes that appeared to be morphologically normal after warming, and cleaved oocytes after IVF were lower with 25% EG-based VS when compared with 28–40% EG-based VS. Blastocyst yields 8 days after IVF of oocytes vitrified-warmed in 28 and 31% EG-based VS (12 and 17%, respectively) were not significantly different from those of the fresh control group (32%). Day-7 blastocysts derived from vitrified oocytes were composed of a smaller number of inner cell mass (ICM) and trophectoderm cells than the fresh Day-7 blastocysts. In Experiment 2, oocytes vitrified-warmed in 31% EG-based VS were subjected to enucleation and SCNT. The proportions of oocytes fused and cleaved in the vitrified group were comparable to those in the fresh control group. Blastocyst yields 6 and 7 days after SCNT of vitrified oocytes were lower than those of control oocytes, but 8 days after the SCNT, the difference became statistically comparable (45 versus 58% in control group). ICM and trophectoderm cell numbers in Day-7 blastocysts derived from vitrified oocytes were smaller than those of control blastocysts due to a slower developmental rate. In conclusion, bovine oocytes cryopreserved by vitrification in gel-loading tip were capable of developing into blastocysts after conventional IVF and SCNT, with slightly smaller cell numbers and a slower developmental rate.

Analysis of DNA Fragmentation of Porcine Embryos Exposed to Cryoprotectants

The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.

154 EFFECTS OF BLASTOMERE BIOPSY AND OXYGEN CONCENTRATION DURING CULTURE ON THE DEVELOPMENT AND INTERFERON-TAU SECRETION OF BOVINE EMBRYOS

Pre-implantation genetic diagnosis has become a powerful tool in human in vitro fertilization. Yet, for ethical reasons, controlled experiments assessing the effects of embryo biopsies on human embryos are difficult to perform. Therefore, a series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos cultured under either atmospheric or low (5%) oxygen. Embryos were generated by IVF of in vitro-matured oocytes and cultured in CR1aa medium containing 5% bovine fetal serum. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed from each of a large number of embryos at either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% (10/58) of embryos proceeding to the blastocyst stage, which was significantly lower than when biopsies were performed at 72 h (37.5% (33/88), P &lt; 0.05). In the second experiment, embryos were cultured under either atmospheric or 5% O2 following blastomere removal. Biopsies at 72 h had no effect on rate of blastocyst formation with 38.5% (110/286) of controls and 33.7% (60/178) of biopsied embryos proceeding to the blastocyst stage. However, culture under 5% O2 significantly increased the number of blastocysts from 29.9% (69/231) to 43.3% (101/233). This effect was significant in both biopsied and control embryos. In contrast, staining of blastocysts for cell numbers did not reveal any effects of biopsies or culture conditions. In the final experiment, biopsies were again performed at 72 h and embryos were cultured as previously. Blastocysts were collected and cultured individually for 48 h in 50-μL-medium droplets in their respective O2 concentrations after which time the medium was assayed for concentration of interferon-tau (IFN-τ). Previous work has shown that IFN-τ secretion can be affected by genetic and environmental factors. Reduced O2 concentration again significantly increased blastocyst formation from 24.9 to 36.9% (49/197 vs. 75/203, P &lt; 0.05), while blastomere removal had no effect on development. IFN-τ secretion did not differ between biopsied and control blastocysts, although culture under atmospheric O2 resulted in significantly increased IFN-τ concentration in medium droplets (12,690.1 ± 2715.6 vs. 4726.8 ± 2488.5 pM, P &lt; 0.05). This work was supported by a grant (HMK) from the National Institutes of Health (HD 36421).

230 EFFECT OF IN VITRO CULTURE TREATMENT AND DONOR EWE DIET ON IGF2R EXPRESSION IN FETAL TISSUES

In separate experiments, elevation of donor ewe plasma urea levels and IVC of embryos in serum have been associated with occurrence of large offspring syndrome (LOS). Fetuses displaying LOS also have abnormal expression of the imprinted gene IGF2R. The present study, therefore, examined the effect of variation in donor ewe nitrogen metabolism and embryo culture on fetal development. Zygote-donor ewes were offered a diet with either a 0% (LN) or 3% urea supplement (HN) for two weeks during which superovulation and artificial insemination (AI) took place. Zygotes were recovered 36 h after AI and cultured for 5 days in synthetic oviductal fluid (SOF) to which either steer serum (10%; SOFS) or albumin (0.4%; SOFA) was added. In vivo control (C) blastocysts were recovered (Day 5) from superovulated ewes offered diet LN. All embryos were transferred singly to recipient ewes; fetal tissues were recovered and snap-frozen at Day 125 of gestation. IGF2R expression was measured by semi-quantitative PCR in fetal heart and kidney and expressed as the ratio to 18S mRNA. Data were analyzed by ANOVA with donor ewe plasma urea-N prior to (preUN) and mean plasma urea-N during (meanUN) feeding as covariates. Correlation analysis was used to test relationships between variables. Heart IGF2R expression (see Table 1) was lower in SOFA than in SOFS fetuses (P = 0.003; preUN, P = 0.007), while kidney IGF2R expression was lower in HN than in LN fetuses (P = 0.04; preUN, P = 0.015; meanUN, P = 0.032). Overall the range of both heart and kidney IGF2R expression was greater for fetuses from cultured (1st/3rd quartiles; heart, 0.87–1.05; kidney, 0.77–1.10) than from C embryos (heart, 1.03–1.16; kidney, 0.84–1.00). In IVC fetuses, heart IGF2R expression was negatively associated with fetal body (r = −0.31; P = 0.003) and heart weight (r = −0.35; P = 0.008) whereas kidney IGF2R expression was not associated (r = −0.23; P = 0.089) with fetal body weight and not related to kidney weight. Heart and kidney IGF2R expression were only weakly associated (r = 0.26; P = 0.054). In conclusion, changes in IGF2R expression in both fetal heart and kidney were associated with donor ewe N status and IVC conditions. In this study, decreases in heart IGF2R expression were reflected in increased heart weight, but kidney IGF2R expression was not related to kidney weight. Table 1. Fetal (kg), organ weights (g), and IGF2R expression This study was funded by Defra.

93 BLASTOCYST PRODUCTION FROM BOVINE OOCYTES VITRIFIED IN A CLOSED (BIOSECURE) SYSTEM FOLLOWING IN VITRO MATURATION IN THE PRESENCE OR ABSENCE OF VITAMIN E

The value of assisted reproductive technologies intended for conservation of livestock genetics ultimately will depend on their effectiveness in both sustaining gamete/embryo viability and ensuring stringent biosecurity. This study investigated bovine oocyte survival following vitrification in a sealed system prior to storage in liquid nitrogen. It also tested the effect of supplementary vitamin E on tolerance of oocytes to vitrification procedures. Healthy COCs from abattoir-derived ovaries were matured in TCM-199 supplemented with 10% v/v fetal calf serum (FCS) in the absence (control) or presence (VitE) of 100 μM α-tocopherol (Sigma, Poole, UK) in humidified atmosphere (5% CO2 in air; 38.5°C). Between 22 and 24 h after commencement of IVM, COCs were pipetted to remove excess cumulus cells, and then equilibrated at room temperature in 7.5% DMSO plus 7.5% ethylene glycol (EG) in HEPES-buffered Holding Medium (HM; Irvine Scientific, Santa Ana, CA, USA) for 7 min before transfer to vitrification solution (15% DMSO, 15% EG, and 0.5 M sucrose in HM; Irvine Scientific). Loading of oocytes (n = 78 control and 85 VitE) into CryoTips (Irvine Scientific) and heat-sealing (each end) was achieved within 90 s, with tips then plunged into liquid nitrogen. Subsequent warming and cryoprotectant removal were at room temperature in HM with 1 M sucrose for 2 min, 0.5 M sucrose for 4 min, and HM alone for 6 min. Oocytes were allowed recover for approximately 3 h in TCM-199 with 20% FCS (5% CO2 in air; 38.5°C), and then fertilized in vitro (single sire). After 22 h (Day 1) presumptive zygotes were transferred to SOF containing fatty acid-free BSA (4 mg mL−1) and incubated for up to 8 days (5% O2, 5% CO2, 90% N2; 38.5°C). Cleavage data (Day 2) and blastocyst yields (Days 7 to 9) were analyzed by chi-square test. In addition to those that were vitrified, some oocytes (n = 9 per treatment) were observed via video to permit analysis (ImageJ; NIH, USA) of volume excursions during the 7 min immediately following initial exposure to HM with 7.5% DMSO plus 7.5% EG. Data were compared using ANOVA. Overall incidence of cleavage by Day 2 was 45% (range: 36 to 51%) and 35% (31 to 43%) for control and VitE, respectively, (NS). Day 7 and total control blastocyst yields were 7.4% and 18.5%, respectively; corresponding yields for VitE were 19% and 25% (control vs. VitE, NS). Video evidence indicated that although Control oocytes invariably reached minimal volume later than VitE oocytes (30 vs. 20 s), in each case this was 52% of initial size. By 7 min, both had similar volumes, the respective means being 94% and 92% of initial size. In the present study provision of vitamin E during IVM did not significantly enhance the subsequent resilience and development of oocytes subjected to a vitrification protocol. However, this protocol achieved efficient and biosafe bovine gamete storage. This work was funded by SEERAD; CryoTips and vitrification solutions were donated by Irvine Scientific; VCM was supported by MLC, UK.

88 IS STEREOMICROSCOPY AN EFFICIENT METHOD FOR EVALUATING THE VITRIFIED PORCINE EMBRYO QUALITY?

The development of the open pulled straw vitrification has provided excellent results of in vitro porcine embryo development. Embryo quality evaluation after vitrification has been traditionally focused on morphological assessment performed by stereomicroscopy. The objective of this experiment was to evaluate the efficiency of the stereomicroscopic evaluation of vitrified-warmed (V) porcine blastocysts. Unhatched blastocysts were obtained after slaughter from Large-White gilts (n = 9). Blastocysts (n = 75) were vitrified and warmed using the protocol described by Cuello et al. (2004 Theriogenology 61, 353-361). After warming, vitrified blastocysts were cultured for 24 h. Then blastocysts were morphologically assessed for their progression and morphology by stereomicroscopy. Blastocysts that reformed their blastocoelic cavities showing an excellent appearance were considered viable. Some of the viable blastocysts kept their zonae pellucidae (V viable expanded blastocysts) and others hatched during the in vitro culture (V viable hatched blastocysts). The remaining blastocysts were classified as degenerated embryos. A group of fresh blastocysts was not vitrified and cultured in vitro for 24 h (control group). All of the control blastocysts were considered viable by stereomicroscopy. Some fresh, V viable expanded, V viable hatched, and V degenerated blastocysts (n = 13, n = 19, n = 9, and n = 9, respectively) were processed for ultrastructural study by light and transmission electron microscopy or stained with Hoechst-33342 and TUNEL for cell death evaluation (n = 16, n = 21, n = 11, and n = 6, respectively). All V hatched blastocysts showed ultrastructure similar to that of control hatched blastocysts. However, 26.3% of the V viable expanded blastocysts revealed important ultrastructural alterations in comparison with control expanded blastocysts. These observations suggest that stereomicroscopic evaluation was not efficient enough for V expanded blastocysts. As expected, degenerated blastocysts showed ultrastructural disintegration and disorganization. Hatched V blastocysts did not differ (P &lt; 0.05) from control hatched blastocysts with regard to the total cell number and ratio of death cells (173 � 4.8 vs. 202.1 � 10.9 and 2.8 � 0.5% vs. 1.9 � 0.3%, respectively). However, V expanded blastocysts a had higher (P &lt; 0.01) cell death level (4.3 � 3.4%) than that observed in the control expanded blastocysts (1.1 � 0.3%). Degenerated embryos showed the lowest (P &lt; 0.01) total cell number (45.7 � 4.0). The 66.7% of the degenerated blastocysts exhibited wide TUNEL-labeled areas, and the remaining 33.3% showed TUNEL label over 19.4 � 6.3% of the cells. In conclusion, the hatching rate assessed by stereomicroscopy is a more efficient parameter than assessing the in vitro viability (ratio of blastocysts that reformed their blastocoelic cavities after warming) for estimating the quality of V blastocysts. This work was supported by CICYT (AGL2004-07546) and S�neca (01287/PD/04).

24 GLOBAL TRANSCRIPT PROFILING OF CLONED BOVINE BLASTOCYSTS USING AFFYMETRIX GENECHIP TECHNOLOGY

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.

The effect of mechanically collapsing human blastocysts prior to cryopreservation

Cryoprotectants and sucrose are used during embryo freezing to decrease the amount of water in cells and prevent ice crystal formation and subsequent damage. However, the blastocyst stage embryo contains a large fluid-filled central cavity called the blastocoele. If the fluid in the blastocoele is not lost during cryopreservation, damage may occur to the embryos. The objective of this study was to determine if artificially collapsing blastocysts prior to the addition of the cryoprotectant may decrease the amount of damage and result in a higher survival and developmental rate following cryopreservation. Embryos used in this study were donated by patients who had consented to the use of their surplus embryos for research. On Days 5 or 6, blastocysts demonstrating a blastocoele cavity comprising at least 50% of its diameter, were randomized to be mechanically-collapsed (MC) or served as controls. After cryopreservation and thaw, embryos were evaluated for survival and reexpansion of the blastocoele cavity at 6 hours and hatching after 24 hours. Blastocysts in the MC group (n=12) were held in place with a holding pipette (Humagen, Charlottesville, VA) and an ICSI pipette inserted at 3:00, opposite the inner cell mass. The blastocyst collapsed within seconds and was immediately cryopreserved. Control blastocysts (n=12) were not manipulated prior to freezing. Cryopreservation was performed using a two-step addition of glycerol (5 and 10%) in modified HTF-Hepes (Irvine Scientific, Irvine, CA) containing 2.0M sucrose. Cooling began at room temperature and proceeded at a rate of 2oC/min to -7oC where seeding occurred. Cooling continued at a rate of 0.3oC/min to -38oC, at which time the vials were plunged in liquid nitrogen. Thawing was performed by removing the vials from liquid nitrogen and letting them incubate in a 30oC water bath until all ice crystals had dissolved. Blastocysts were put through a 7-step dilution series to remove the glycerol and sucrose (10%Gly + 0.4M sucrose; 5% + 0.4M; 2.5% + 0.4M; 0.4M sucrose; 0.2M sucrose; 0.1M sucrose), rinsed in fresh medium and then cultured for an additional 24 hours. Upon thaw, 8/12 (67%) of the control embryos survived the procedure with 6/8 (75%) re-expanding after 6 hours in culture. None of the control embryos underwent hatching in vitro. Of the manipulated embryos, 9/12 (75%) survived the procedure with 8/9 (89%) re-expanding and 2/9 (22%) hatching. The rate of survival and reexpansion were not significantly different between the two groups. However, the performance of a likelihood ratio showed that the rate of hatching in the MC group was increased (p<0.05). These preliminary results suggest that artificially reducing the blastocoele cavity may improve the post-thaw development of blastocysts, possibly by reducing cryo-damage. We find these results promising and will continue the evaluations with additional embryos as they become available for research.

Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts.

The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.

Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

To establish a protocol for production of bovine in-vitro produced (IVP) blastocysts that were sex-identified and cryopreserved, we examined the sexing efficiency and accuracy of Day-3 and Day-4 embryos by polymerase chain reaction (PCR), the development of the biopsied embryos into Day-7 blastocysts and the freezability of these blastocysts by vitrification in gel-loading tips. One or two blastomeres were isolated from IVP embryos at either the 8-cell or 16-cell stage (Days 3 and 4, respectively) by a pressing-out method, and were then subjected to primer extension preamplification (PEP)-PCR. The successful sex-identification rate of biopsied samples amplified, purified and analyzed for sex by a second PCR (88.9%) was higher than that of those amplified and analyzed without purification (32.0%). Developmental rates into Day-7 blastocysts of biopsied embryos (Day-3, 65.5%; Day-4, 70.8%) were similar to those of non-biopsied control embryos (Day-3, 74.5%; Day-4, 65.1%). Total cell numbers and the inner cell mass (ICM) ratio of blastocysts derived from biopsied embryos were also comparable with those of control embryos. Blastocysts were vitrified-warmed in the presence of 20% DMSO, 20% ethylene glycol and 0.6 M sucrose using gel-loading tips as containers. The proportions of biopsied blastocysts that were hatched or hatching rates after warming were high, regardless of the biopsy time (Day-3, 94.1%; Day-4, 91.9%), similar to the rates for control blastocysts (Day-3, 97.5%; Day-4, 96.9%). In conclusion, a protocol that allows sexing of Day-3 and Day-4 bovine embryos without compromising either the developmental ability to the blastocyst stage or freezability of Day-7 blastocysts was developed.

A morphological and chromosomal study of blastocysts developing from morphologically suboptimal human pre-embryos compared with control blastocysts.

IVF laboratories performing embryo transfer at day 2 or 3 after fertilization are currently discarding pre-embryos considered suboptimal using morphological criteria. The objective of this study was to investigate whether blastocysts, cultured from such pre-embryos (surplus), were chromosomally and morphologically normal. As a control group we used morphologically good quality embryos (GQE), cultured to the blastocyst stage. Human pre-embryos considered suboptimal were cultured to the blastocyst stage. As a control group, frozen-thawed pre-embryos of good quality were cultured under identical conditions. The chromosomal status of the blastocysts obtained was studied by multi-colour fluorescence in-situ hybridization for chromosomes 13, 16, 18, 21, 22, X and Y. There is, on average, a significantly higher degree of chromosomal aberrations in blastocysts derived from surplus pre-embryos compared to blastocysts derived from GQE, and the chromosomal aberrations are generally found in a higher number of blastomeres per blastocyst. In addition, blastocysts from surplus pre-embryos had significantly poorer morphology compared to GQE. Improvement in morphology and/or developmental rate in surplus pre-embryos between day 2 and day 3 did not predict a morphologically/chromosomally normal blastocyst. However, this study shows that close to half of the surplus pre-embryos that reach the blastocyst stage can be considered chromosomally normal when assessed for these seven chromosomes. Furthermore, we found that chromosomal aberrations were more concentrated in a particular cell population within blastocysts derived from GQE, compared with surplus blastocysts. The study suggests that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded.

Retinoic acid decreases the viability of mouse blastocysts in vitro

This study was designed to examine the cytotoxic effect of retinoic acid on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryo development in vitro. Mouse blastocysts were exposed for 24 h to doses of 0, 0.1 micromol/l and 10 micromol/l all-trans retinoic acid and observed for their capacity to implant and develop during the early postimplantation period in vitro. When retinoic acid-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to reach a later stage of embryo development. Compared with the findings for the control blastocysts, exposure to retinoic acid resulted in a significant reduction in the average number of total cells in blastocysts and the trophectoderm/inner cell mass lineage. The effect was associated with a significant increase in the frequency of cells identified as being engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and Annexin V techniques. This is the first evidence that retinoic acid induces cell death (apoptosis) and inhibits cell proliferation in mouse blastocysts. This results in the retardation of early postimplantation blastocyst development and subsequent blastocyst death.

Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation ( n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1) medium only control; (2) activin (10 ng/ml); (3) epidermal growth factor (EGF) (10 ng/ml); (4) insulin 5 μg/ml and (5) GH (100 ng/ml). There was an increase ( P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher ( P<0.05) survival rates with a mean blastocyst survival of 95.0±1.5 and 93.1±3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3±3.4 and 62.0±3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7±3.3%. With the exception of insulin, all growth factors and GH tested showed higher ( P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.

Mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation does not improve blastocyst survival rates

Objective: Mechanical reduction of the blastocoelic cavity has been recently reported by Vanderzwalmen and co-workers (Fertil Steril O-125, S48; Vol. 74:3, Suppl. 1, 2000) to increase blastocyst viability after vitrification. The purpose of our study was to evaluate the effectiveness of this novel technique in our ART laboratory’s slow cooling protocol for blastocyst cryopreservation. Design: Mouse blastocysts (B6C3F1/B6D2F1) with or without mechanical removal of the blastocoelic cavity’s fluid were cryopreserved using a two-step slow cryopreservation protocol, followed by a rapid thawing and a four-step dilution at room temperature. Blastocysts were maintained in culture for 24 hours to assess viability and further development. Materials/Methods: One hundred forty-six mouse blastocysts (n=3) were randomly allocated into two groups. Blastocysts in group A (n=72) were transferred into micro-droplets containing freeze-thaw diluent (modified human tubal fluid with hepes and supplemented with 20 mg/ml human serum albumin) for mechanical reduction of the blastocoelic cavity. For the procedure, each blastocyst was placed with the inner cell mass at either 12 or 6 o’clock and firmly secured with a holding pipet. A micro-needle positioned at 3 o’clock, was slowly inserted through the cellular junction of the trophectoderm into the blastocoele cavity until complete blastocyst compaction was observed. Control blastocysts in group B (n=74) were incubated in freeze-thaw diluent for 15 min. before cryopreservation. The freezing solutions for both groups of embryos were 5% glycerol (10 min.), followed by 9% glycerol and 0.2 M sucrose (10min.). Blastocysts were transferred into cryovials and placed into the freezing machine at 20 °C, then cooled at 2 °C/min. to -6 °C, seeded and held for 10 min., followed by cooling at 0.3 °C/ min. to -36 °C and plunged into liquid nitrogen for storage. Blastocyst thawing was accomplished by placing the cryovials in a 35 °C water bath, then transferring the blastocysts into 6% glycerol with 0.4 M sucrose, 3% glycerol with 0.2 M sucrose, 0.1 M sucrose, and freeze-thaw diluent at five minutes intervals. Embryos were rinsed with G2.2 medium and incubated overnight in micro-droplets overlayed with mineral oil. Data were analyzed using ANOVA and chi-square. Results: Post-thaw survival was 73.6% for blastocysts in group A and 68.91% in group B. At 24 hours, complete blastocyt expansion occurred in 86.7% of the blastocysts with mechanical shrinkage and in 70.5% in the control group. Blastocyst hatching was observed in 52.8% of the surviving embryos in protocol A and 47% in protocol B. No significant statistical differences between group A and B were observed for any of the parameters evaluated. Conclusions: The observations reported here suggest that mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation neither appears to increase blastocyst viability nor has a deleterious effect. Supported by: Center for Reproductive Health.

Increased cell death in mouse blastocysts exposed to high D-glucose in vitro: implications of an oxidative stress and alterations in glucose metabolism.

Signs of apoptosis have been observed in rodent blastocysts exposed to high d-glucose concentrations in vitro. The mechanism underlying the detrimental influence of glucose remains to be identified. It has been postulated that high d-glucose concentrations induced oxidative stress in rat post-implantation embryos in vitro. A decreased glucose uptake has also been implicated in the embryotoxicity of glucose in pre-implantation mouse embryos. We examined whether the high incidence of cell death in high d-glucose-treated embryos was associated with a disrupted redox status and with alterations in glucose transport and metabolism. After blastocysts were incubated in different concentrations of d-glucose for 24 h, they were examined for the proportion of nuclei showing signs of chromatin degradation using the TUNEL technique, for the generation of reactive oxygen species and for the mitochondrial membrane potential using specific fluoroprobes and the confocal microscopy. Glucose transport and metabolism were assessed using radiolabelled 3-O-methylglucose and glucose, respectively. Compared to the control blastocysts, high d-glucose-treated embryos showed a higher incidence of TUNEL-positive nuclei and reactive oxygen species generation principally in the inner cell mass cells. Decreased glucose transport and glycolytic activity but unmodified pentose phosphate pathway activity were detected in these embryos. Incubation in high d-glucose concentrations in vitro increased cell death, induced oxidative stress and decreased glucose transport and metabolism in mouse blastocysts. As only glycolysis was affected, however, we suggest that metabolic inhibition occurred downstream glucose transport and glucose-6-phosphate formation.

In vitro formation of tetraploid rat blastocysts after fusion of two‐cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.