Search

Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts. Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results

In this study we evaluated mitochondrial distribution of individual bovine embryos after IVF, parthenogenetic activation (PG), and somatic cell nuclear transfer (SCNT). COCs obtained from slaughterhouse ovaries were matured in vitro for at least 18 h in TCM-199 supplemented with hormones, and then divided into 3 groups. SCNT and PG oocytes were stripped by vortexing in HEPES-HECM (hamster embryo culture medium) medium (HH) containing hyaluronidase and metaphase II (MII) oocytes selected by visualization of a polar body. The PG group oocytes were exposed 24h post-maturation to 5 μM ionomycin in HH for 4 min, then rinsed 3 times in HH and allocated to a 4-h culture in 2 mM DMAP in KSOM for activation. The SCNT group oocytes were included in a nuclear transfer procedure performed as previously described (Ross et al. 2006 Biotechniques 41, 741-750). Activation was performed as described for the PG group. The IVF group COCs were co-incubated for 20 h with 106 spermatozoa/mL in IVF-TALP supplemented with heparin. To label mitochondria, 1 mM MitoTracker CMXRos Red (Molecular Probes, Eugene, OR, USA) was added to HH at a final concentration of 0.3 μM. Samples were cultured for 15 min, washed in HH, placed in a glass-bottomed 35-mm Petri dish, and then observed and live photographed by using a spinning disk confocal microscope (Nikon Eclipse TE2000-E + CARV Confocal) equipped with a Cascade 512 B camera (Roper Scientific, Tucson, AZ, USA) using a Nikon 40×, 1.3 NA oil objective lens. Z series images were taken acquiring 15 focal planes at 10-μm intervals. Analysis was performed using Metamorph software. Samples were taken at pronuclear, 4 cell, and morula stages. Each sample was classified for its mitochondrial localization in pericytoplasm, cytoplasm, and perinuclear. Data was analyzed by proc glimmix (SAS, Cary, NC, USA). Significance was set at P < 0.05. A similar pericytoplasmic distribution of mitochondria for all treatments up to the 4-cell stage was observed. At the pronucelar stage, mitochondria distribution was mostly pericytoplasmic, changing to cytoplasmic at the 4-cell stage. At the morula stage there was a significantly higher number of embryos with perinuclear distribution in IVF than in PG and SCNT embryos (Table 1). Our findings demonstrate that mitochondrial reorganization differs in fertilized more-developed embryos compared with their activated counterparts. This may have implications for further embryo development, mainly after SCNT. Table 1.Mitochondria distribution in fertilized, parthenogenetic, and cloned bovine embryos

Real-time RT-PCR can accurately quantify mRNA levels in pre-implantation embryos; however, comparisons among different embryonic stages and among embryos produced by different means often rely on a control gene, which is commonly assumed to remain constant across samples. The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine pre-implantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). Embryos were produced according to standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). Total RNA was collected from 3 pools of 10 oocyte/embryos at metaphase II (MII), PN, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and reverse-transcribed using oligo-dT primers. The cDNA was then amplified using PCR (Kocabas et al. 2006 PNAS 103, 14 027–14 032). All amplified cDNA samples were diluted to 1 ng μL–1, as determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and corroborated using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). For real-time PCR, 2 μL of cDNA was analyzed in duplicates. Absolute quantification was performed as previously described (Iager et al. 2008 Cloning Stem Cells doi:10.1089), using SYBR-green chemistry and standard curves specific for each gene. The number of RNA copies per nanogram of amplified cDNA was compared among samples using ANOVA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin A, β-actin, ribosomal protein L-15 (RPL-15), and β-glucuronidase (GUS) expression levels were similar in MII oocyte, 1-, 2-, 4-, and 8-cell embryos, while a significant increase at morula and blastocyst stages was observed (P < 0.05). A similar pattern of expression was observed for 18S ribosomal RNA, but with a significant decrease from morula to blastocyst stages (P < 0.05). Histone H2A expression was significantly higher at 1-cell stage, similar from 2-cell to morula stages and lowest at the blastocyst stage. We then compared expression between IVF and SCNT embryos at 2-, 4-, and 8-cell and blastocyst stages. GAPDH, RPL-15, GUS, and β-actin were significantly different among groups in at least 3 of the analyzed stages, which in all cases included blastocysts. 18s-rRNA was different among IVF and SCNT embryos only at the 8-cell stage, while no differences were observed at any stages for histone H2A and cyclophilin A. At the blastocyst stage, the lowest overall variability among IVF and SCNT embryos was observed for 18s-rRNA. In conclusion, none of the evaluated housekeeping genes showed consistent mRNA expression levels across developmental stages of IVF embryos. In addition, SCNT embryos, compared to IVF, had different levels of gene expression for commonly used housekeeping genes, which, if neglected, might result in data misinterpretation. In our conditions, histone H2A had similar expression levels between IVF and SCNT embryos across different stages and showed less variability than cyclophilin A. Finally, for comparisons at the blastocyst stage, 18s-rRNA had the least variability among IVF and SCNT embryos.

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.

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.

Oocyte spontaneous activation (OSA) has been reported to occur during in vitro culture of ovulated rat eggs. Approximately 1.5 h after isolation and culture, unfertilized oocytes extrude the second polar body and enter a metaphase arrest, and by 3 h individual chromatids separate and scatter throughout the egg's cytoplasm. The objective of this study was to compare the proportion of OSA and the level of maturation promoting factor (MPF) activity in oocytes from different strains. Twelve strains were selected from two commercial sources based on litter size. Mature female rats (9 to 12 weeks old) were superovulated using 20 IU eCG s.c. followed by 30 IU eCG s.c. 5 h later. After 48 h, a dose of 50 IU hCG was administered intraperitonealy and females were mated with vasectomized males. Oocytes were collected 17 h after hCG injection and cumulus cells were removed by transfer to M2 medium containing hyaluronidase (1 mg mL−1). Denuded oocytes were cultured in 50 μ­L drops of M16 medium under oil at 37°C and 5% CO2 in air. In order to assess OSA, oocytes were mounted in glycerol containing Hoechst 33342 (10 μg mL−1) on a glass slide after 6 h of in vitro culture. The proportion of activated oocytes was determined by epifluorescence microscopy. Oocytes were considered to be in metaphase II if they presented a compact group of chromosomes at the metaphase plate, and were considered activated when chromosome dispersion at different degrees was observed. Data were analyzed by ANOVA, considering each animal as an experimental unit. Significant differences were observed between strains (P < 0.01, Table). In order to determine MPF activity of each strain, 10 oocytes were removed from culture at 0, 1.5, and 3 h after oocyte collection and immediately stored in kinase buffer in LN2 for posterior analysis using an ELISA based kit (MESACUP cdc2 Kinase Assay Kit, MBL, Nagoya, Japan). The log ratio of the MPF activity at 1.5 and 3 h relative to 0 h for each animal (5 per strain) was analyzed by ANOVA. There were no MPF differences between or within strains (P > 0.3 and P > 0.05, respectively). We did not observe the expected decrease in MPF activity that allows for the exit of metaphase II arrest. This could imply that OSA is not associated with a decrease in MPF, or that MPF decreased rapidly and returned to metaphase levels by 1.5 h after culture. In conclusion, different levels of OSA were observed between strains, however, no differences in MPF activity were detected at the analyzed time points.

The high rate of gestational loss in nuclear transfer (NT) pregnancies is a major economic and animal cost, preventing the widespread use of the technique. It is currently not possible to predict which early gestation fetuses will remain viable to term. As placental abnormalties are commonly associated with nonviable fetuses, placental proteins such as pregnancy specific protein (PSP 60, Heyman et al., 2002 Biol. Reprod. 63, 1787–1794; or PSPb Hill et al., 2000 Biol. Reprod. 66, 6–13) have been investigated as possible markers of viable pregnancy. To build upon these studies, we explored the predictive value of Day-35 maternal serum PSPb value to final cloned-calf viability; PSPb profiles throughout pregnancy; and the relationship of maternal plasma estradiol levels to calf viability. Maternal serum was sampled for PSPb at the time of the first ultrasound pregnancy examination (Day 35) to determine fetal number and viability (heartbeat). PSPb values were determined using an RIA by Dr. Garth Sasser (BioTracking, Moscow, ID). Median PSPb values were compared using the Kruskal-Wallis one-way ANOVA test on ranks. PSPb levels from failed single and twin pregnancies were significantly different from those of AI bred controls (P7.5. As PSPb levels at Day 35 were not highly predictive of viable outcome, we detailed the gestational PSPb profile for a different group of cows (n=40) that carried cloned pregnancies beyond Day 90. PSPb was determined at Days 35, 50, 65, and 90, then monthly to Day 240. Although there was no clear statistical significance, PSPb levels from nonviable cloned pregnancies were consistently higher at each time point than for viable pregnancies (14 cows gave birth to live calves) and significantly higher than those of controls. At term, plasma estradiol concentrations were assessed in a group of 5 NT pregnancies. Estradiol was investigated as cloned pregnancies often show a lack of readiness for parturition (e.g. lack of udder development, prolonged gestation, dysmature calves). These 5 pregnancies produced 3 highly viable NT calves: 1 with lowered viability that survived only with a high level of intensive care, and 2 nonviable despite a high level of assistance. In this small group, estradiol levels on the day of birth rose in parallel with viability (viable, 662pg; lowered viability, 170pg; nonviable, 76pg). This is a clinical observation rather than a statistical trend that suggests further investigation may be warranted. In summary, our results suggest maternal PSPb levels have value in identifying the outliers in a population of cloned pregnancies and that term-cloned pregnancies may show abnormally low maternal estradiol concentrations at term. Table 1