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The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P < 0.01 and a fold change >2-fold or <0.5 fold as cutoffs, there were 617 differentially expressed genes (DEG) between embryos that survived to day 30 of gestation vs those that did not, 470 DEG between embryos that survived to day 60 and those that did not, 432 DEG between embryos that maintained pregnancy from day 30 to day 60 vs those where pregnancy failed after day 30, and 635 DEG regulated by CSF2. Pathways and ontologies in which DEG were overrepresented included many related to cellular responses to stress and cell survival. It was concluded that gene expression in the blastocyst is different between embryos that are competent to establish and maintain pregnancy vs those that are not. The relationship between expression of genes related to cell stress and subsequent embryonic survival probably reflects cellular perturbations caused by embryonic development taking place in the artificial environment associated with cell culture.

The purpose of this work is to address the relationship between transcriptional profile of embryos and the pregnancy success based on gene expression analysis of blastocyst biopsies taken prior to transfer to recipients. Biopsies (30–40% of the intact embryo) were taken from in vitro-produced day 7blastocysts (n= 118), and 60–70% were transferred to recipients after reexpansion. Based on the success of pregnancy, biopsies were pooled in three groups (each 10 biopsies) namely: those resulting in no pregnancy (G1), resorbed embryos (G2), and those resulting in calf delivery (G3). Gene expression analysis of these groups was performed using home-made bovine preimplantation-specific cDNA array (219 clones) and BlueChip (with ∼2,000 clones). Microarray data analysis results revealed a total of 52 and 58 genes were differentially regulated during comparison between G1 vs. G3 and G2 vs. G3. Biopsies resulted in calf delivery were enriched with genes necessary for implantation (COX2 and CDX2), carbohydrate metabolism (ALOX15), growth factor (BMP15), signal transduction (PLAU), and placenta-specific 8 (PLAC8). Biopsies from embryos resulting in resorption are enriched with transcripts involved protein phosphorylation (KRT8), plasma membrane (OCLN), and glucose metabolism (PGK1 and AKR1B1). Biopsies from embryos resulting in no pregnancy are enriched with transcripts involved inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes which may play an important role in determining the fate of the embryo after transfer.

The aim of the present study was to explore whether the blastocyst diameter and the zona thickness at 168 h after fertilisation are useful parameters to predict quality and viability of bovine in-vitro-produced (IVP)-embryos. Although significant (P < 0.05), the blastocyst diameter at 168 h correlated only poorly with the total number of cells (R2 = 0.13) and with the number of trophectoderm (TE) cells (R2 = 0.17). Hatched blastocysts (n = 66) at 216 h had a significantly greater mean diameter at 168 h (194.8 16.8 m) compared with either blastocysts that had started but not finished hatching at 216 h (n = 26, 178.4 16.7 m) or failed to commence hatching (n = 136, 162.7 12.9 m). Transfer of 101 IVP blastocysts to synchronised recipients resulted in the birth of 38 calves (38%). There were significantly more bull calves born than cow calves (P < 0.05), but this was not correlated with blastocyst diameter or zona thickness at 168 h. There was also no correlation between the diameter of blastocysts or the zona thickness at 168 h and parameters of subsequent developmental characteristics, including rates of pregnancy, resorptions and abortions, pregnancy duration, delivery to term and birthweight. Overall, the present results indicate that the blastocyst diameter and the zona thickness at 168 h are good predictors for subsequent hatching ability in vitro, but not for the number of TE cells, inner cell mass cells or total cells and neither for subsequent developmental characteristics after transfer to recipients.

Large-scale expression profiling of micro-RNAs (miRNAs) in bovine granulosa cells from dominant and subordinate follicles on Day 19 of the estrous cycle revealed enriched micro-RNA-183-96-182 cluster miRNAs in preovulatory dominant follicles that coordinately regulate the forkhead box protein O1 (FOXO1) gene. However, little is known about the role of this cluster in bovine granulosa cell function. We used an in vitro granulosa cell culture model to investigate this role. Granulosa cells aspirated from small growing follicles (3–5 mm in diameter) were cultured in Dulbecco modified Eagle medium/F-12 medium supplemented with fetal bovine serum and transfected with locked nucleic acid-based miRNA mimics, inhibitors, and corresponding negative controls. Overexpression of the miRNA cluster resulted in suppression of FOXO1mRNA and protein, whereas inhibition of the cluster increased expression of FOXO1mRNA. Overexpression also increased the relative rate of cell proliferation, whereas inhibition slowed it down. Similarly, the proportion of cells under G0/G1arrest declined, whereas the ratio of cells in S phase increased in response to miR-183-96-182 overexpression. Selective knockdown of FOXO1mRNA using anti-FOXO1 small interfering RNA increased the rate of granulosa cell proliferation, decreased the proportion of cells under G0/G1arrest, and increased the proportion of cells in the S phase of cell cycle. Our data suggest that miR-183-96-182 cluster miRNAs promote proliferation and G1/S transition of bovine granulosa cells by coordinately targeting FOXO1, suggesting a critical role in granulosa cell function. MicroRNA-183-96-182 cluster regulates bovine granulosa cell function by targeting FOXO1gene.

There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13–14 mm delivered significantly (P< 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9–10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P< 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first time we were able to show that IFOT is a feasible technique to generate bovine blastocysts by transferring in vitro-matured oocytes derived from slaughterhouse ovaries. These IFOT-derived blastocysts closely resemble in vivo-derived blastocysts in terms of lipid content and freeze survival. Thus, the present study laid the groundwork for newly created scientific experiments enabling novel analytical possibilities. Nevertheless, IFOT-derived embryos still reached lower pregnancy rates by trend compared with in vivo-derived embryos, also implicating an important role for the maturational environment in further developmental characteristics.

After a porcine–to–primate xenotransplantation, hyperacute rejection (HAR) destroys the transplanted organ within minutes. The HAR can be overcome either by a knockout of the gene for α–1,3–galactosyltransferase or by overexpression of human complement regulatory proteins such as hCD59 and DAF. When HAR can be controlled, the next hurdle is acute vascular rejection (AVR) which is primarily due to an incompatibility of human protein C and porcine thrombomodulin, both of which are important factors in the coagulation cascade. This incompatibility leads to thrombosis and finally to a disseminated intravascular coagulation (DIC), causing rejection of the xenotransplant. Human thrombomodulin is a good candidate gene for improving survival time of porcine organs after xenotransplantation and for overcoming AVR. Here, we transfected adult fibroblasts obtained from a double transgenic boar (hCD59/DAF) with a construct for human thrombomodulin (hTM). After selection with 800 μg/mL G418 for 14 days, cells were analyzed for integration of the construct by PCR and were visually selected for expression of the hTM–GFP fusion protein under ultraviolet light with a fluorescein isothiocyanate (FITC) filterset. A total of 39 positve clones were obtained, of which two were used in somatic nuclear transfer. Ovaries were collected from a local slaughterhouse, and follicles of 2–5 mm in diameter were aspirated. After 38–42 h of in vitro maturation oocytes were denuded and enucleated. For cell cycle synchronization, the donor cells were serum–starved for 48 h, subsequently trypsinized and placed into the perivitelline space of the enucleated oocytes. The complexes were fused and activated electrically followed by an incubation in DMAP for 3 h. Puberal gilts were synchronized by treatment with 5 mL Regumate® (Intervet UK, Ltd., Milton Keynes, Buckinghamshire, UK) for 13 days. At the end of treatment, the animals received 1000 IU pregnant mare serum gonadotropin (PMSG) intramuscularly followed by an injection of 500 IU hCG 72 h later. Cloned embryos were transferred surgically 20 h after hCG injection. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of the pregnancy. Out of 1409 reconstructed complexes, 1161 were fused (82.4%) successfully. In total, 1040 embryos were transferred to 8 recipients (∼130 embryos/gilt, range 70–162). Five of the eight recipients (62.5%) became pregnant as determined by ultrasound on Days 25, 36, and 51 and will farrow within the next weeks. These results show that cloning with triple transgenic adult donor cells is compatible with high pregnancy rates. Porcine multitransgenic organs will be used in perfusion experiments to test the effectiveness of this novel approach to overcoming the incompatibilities between the porcine and the human coagulation systems.This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535). The hTM–construct was a gift of Dr. Wu which is gratefully acknowledged.

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

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Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2mM glutamine, 1% non-essential amino acids, 0.1mM mercaptoethanol, 100UmL-1 penicillin, 100mgmL-1 streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24h, 48h and 72h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456-461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24h, 48h and 72h, respectively). After nuclear transfer (for each CI group n=336-384 reconstr. embryos; SD n=215) there was a statistically significant difference in the fusion rate between 48h CI and SD cells (74.8% v. 87.5%, t-test P&lt;0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24h CI and 48h CI (17.4%; 9.1%, 9.6%, t-test P&lt;0.050), there was no difference between SD and 72h CI and within the CI groups (72h CI 10.6%). Four transfers of reconstructed embryos (72h CI, n=138-163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n=96-110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).

Despite intensive research, porcine nuclear transfer is still characterized by low success rates. To determine the effect of maturation period of porcine oocytes on subsequent development following nuclear transfer, we investigated fusion rate, induction of activation and development to blastocyst stage of somatic cells. For this we used MII-oocytes after 38, 40, and 42h of maturation culture as recipients. Oocytes surrounded by a compact cumulus mass were selected and placed into North Carolina State University (NCSU) 37 oocyte maturation medium supplemented with 0.1mgmL-1 cysteine, 10ngmL-1 epidermal growth factor, 10% porcine follicular fluid, 50m 2-mercaptoethanol, 0.5mgmL-1 cAMP, 10 IU each of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) for 22h in humidified air with 5% CO2 at 38.5C. Subsequently the oocytes were moved to fresh NCSU37 maturation medium without cAMP, eCG and hCG and incubated for an additional 16, 18, and 20h. In the first experiment, a total of 878 MII-arrested oocytes were enucleated, fused with pig fetal fibroblasts in calcium-free medium and activated approximately 3h later with an electrical stimulus. This was followed by incubation in 6-dimethylaminopurine for 3h and subsequent analysis of development in vitro. Maturation period had no effect on the frequencies of fusion (87% v. 75% v. 84%, respectively), and cleavage (82% v. 81% v. 87%, respectively), but when MII-oocytes recovered at 40h of maturation were used as recipients, 41/279 (14,8%) the numbers of cloned embryos developing to the blastocyst stage on Day 7 of culture was significantly (ANOVA followed by multiple pairwise comparisons using Tukey test, 6 replicates, P&lt;0,05) higher than those of embryos reconstituted with oocytes collected at 38h (27/285, 9.6%) and 42h (16/314, 4.9%). In the second experiment, reconstructed embryos derived from oocytes matured for 40h were surgically transferred to the oviducts of synchronized German Landrace gilts. Transfers were made on the first day of standing oestrus within 3h of activation to assess their development in vivo. Synchronization was achieved by injections of 1500IU eCG followed by 500IU hCG 3 days later. Of 4 recipients receiving an average of 150 zygotes (range, 136 to 163), 2 became pregnant as determined by ultrasound between Days 25 and 35 of gestation. Of the two pregnant recipients, one subsequently farrowed 4 piglets on Day 115 of pregnancy. These results indicate that the maturation period is critical and affects development of porcine nuclear transfer embryos. This study was funded by the Deutsche Forschungsgemeinschaft (DFG; SFB265).