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The present study investigated the global pattern of two histone modifications and methylation of DNA during in vitromaturation of bovine oocytes retrieved from follicles of two different sizes (<2 mm and 2–8 mm). The methylation status of histone H3 at position lysine K9 (H3K9 me2), the acetylation status of histone H4 at position lysine K12 (H4K12ac) and the methylation of DNA were assessed by immunocytochemistry. In parallel, the relative abundance of mRNAs coding for proteins specifically involved in reprogramming, including HLA-B associated transcript 8 (G9A), suppressor of variegation 3-9 homolog 1 (SUV39H1), the somatic isoform of DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and zygote arrest 1 (ZAR1) was determined by RT-PCR. The α-H3K9 me2 signal was present in the GV stage and remained detectable until the end of the maturation period. α-H4K12ac antibody gave a stronger signal in GV and GVBD oocytes and markedly decreased after GVBD. The signal showing the methylation of DNA was present during the entire maturation period. The five transcripts showed a gene-specific expression profile. Results revealed the global patterns of H3K9 me2, H4K12ac, DNA methylation and the mRNA pool profiles of genes critically involved in epigenetic modifications during bovine oocyte maturation and their possible relationship with the acquisition of oocyte developmental competence and follicular development. [ABSTRACT FROM AUTHOR]

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2–8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis. [ABSTRACT FROM AUTHOR]

Episomal plasmids have emerged as useful tools to achieve stable transgenesis in mammalian cell cultures. Here, the suitability of scaffold/matrix attachment region (S/MAR) carrying episomal plasmids and conventional plasmids for the generation of transgenic murine and bovine embryos was assessed. Bovine zygotes were produced from slaughterhouse ovaries, and murine zygotes were isolated from superovulated and mated NMRI females. Zygote stages were microinjected with approximately 10 pl of plasmid solution. The S/MAR encoding plasmids pEPI or minicircle preparations (gift of J. Bode, Braunschweig, Germany), devoid of most of the plasmid backbone, were used as episomal plasmids. Both plasmids carry an enhanced green fluorescent protein (EGFP) gene driven by the cytomegalovirus promoter (CMV). The plasmids peGFP (CMV-eGFP), pdsRED encoding red fluorescent protein (CMV-RFP), pOct4-GFP (germ line-specific Oct-4 promoter-EGFP), and pgAChR-GFP (muscle-specific γAChR promoter) were used as conventional plasmids. To study the effects of DNA methylation at cytosine/guanine dinucleotids (CpG), plasmid DNA was treated with CpG-methylase in the presence of S-adenosyl-methionin, and in some experiments, completeness of DNA methylation was verified by methylation-sensitive restriction endonucleases. Embryos were analyzed during in vitroculture up to blastocyst stage by fluorescence microscopy, and selected stages were harvested for RT-PCR analysis or DNA recovery. Microinjection of circular plasmids with ubiquitous CMV (n= 505) or germ line-specific Oct-4 promoter (n= 176) driven transcription in bovine zygotes resulted in 159 and 44 blastocysts, of which 94 and 27 showed expression of EGFP. Microinjection of bovine zyotes (n= 179) with S/MAR plasmids yielded a total of 18 blastocysts of which 12 were green fluorescent protein-positive. On average, >50% of the blastocysts were EGFP-positive, irrespective of whether S/MAR carrying episomal plasmids or conventional plasmids had been injected. In contrast, injection of the γAChR (muscle-specific) driven construct did not give rise to EGFP expression (n= 20), suggesting that promoter specificity was maintained. Injection of murine zygotes (n= 126) with CMV or Oct-4 promoter constructs was less successful, about 10 to 20% of the obtained blastocysts expressed EGFP. In the case of unmethylated pOct4-GFP plasmid, the onset of EGFP expression was found to coincide with the time point of major embryonic genome activation [i.e. late 1-cell stage in murine (n= 25) and 4- to 8-cell stages in bovine (n= 75) embryos]. In contrast, injection of CpG-methylated plasmids (murine n= 33; bovine n= 101) delayed the onset of EGFP expression for a further 30 to 40 h. Recovery of plasmid sequences from blastocyst stages and bisulfite sequencing indicated that the majority of plasmids are maintained in an episomal status. Thus, plasmid-mediated transgenesis is a robust method to express foreign DNA in a promoter-specific manner in mammalian embryos and can be employed to analyze reprogramming events.The excellent technical support by E. Lemme and K. Korsawe is acknowledged. Funded by DFG. [ABSTRACT FROM AUTHOR]

Despite considerable progress in the understanding of ovarian follicular growth in cattle, the variable and unpredictable superovulatory response of donor animals is still a limiting factor to the success in the embryo transfer industry. One of the main factors affecting the outcome is the presence or absence of a dominant follicle at the time of gonadotropin treatment. It has been shown that the removal of the dominant follicle using ultrasound-guided aspiration before superovulation increases the number of ova and transferable embryos (Bungartz and Niemann 1994 J. Reprod. Fertil. 101, 581–593). Transrectal color Doppler sonography has been used to evaluate the blood flow in the ovarian artery and to test the ovarian blood flow in relation to ovarian response upon hormonal treatment for superovulation. It has been determined that blood flow parameters (BFV: blood flow volume, PI: pulsatility index) have only limited diagnostic value to predict the outcome of a superovulation treatment (Honnens et al.2008 doi.org/10.1016/j.anireprosci.2008.05.077). The objective of the present study was to investigate the effect of dominant follicle ablation on the ovarian blood flow during superovulation related to the outcome. At Day 8 of the estrous cycle, the dominant follicle was removed in 16 animals (Group 1). In 12 animals, the dominant follicle was left intact (Group 2). All animals received an eCG (3000 I.U.) injection on Day 10, a PGF2α injection on Day 12 and were inseminated twice starting 48 h later. Ovarian blood flow was determined in all animals by investigating the left and right ovarian arteries on Days 8, 10 and 12 of the estrous cycle and on Day 6 p.i. using a Logiq Book XP (General Electrics, UK) equipped with a 7.5 MHz probe. Doppler analysis was carried out using Pixelflux software (Chameleon Software, Germany). The PI was measured as described recently (Honnens et al.2008 doi.org/10.1016/j.anireprosci.2008.05.077). On Day 7 p.i., ova/embryos were collected by standard non-surgical uterine flushing and classified according to the IETS guidelines. Similar results were obtained for the PI in group 1 on Day 8 (1.13 ± 0.08) and Day 10 (1.35 ± 0.09). A decrease (P≤ 0.05) was measured at Day 12 (0.89 ± 0.07) and Day 6 p.i. (0.60 ± 0.06). In group 2, the PI remained constant until Day 12 (Day 8: 1.34 ± 0.11, Day 10: 1.43 ± 0.14, Day 12: 1.44 ± 0.12). At Day 6 p.i., the PI was significantly reduced (0.66 ± 0.09; P≤ 0.05). Between both groups, a significant difference (P≤ 0.05) was only detected at Day 12. More ova/embryos and transferable embryos (P≤ 0.05) were recovered from group 1 (11.3 ± 1.6, 4.7 ± 0.9) compared to group 2 (4.8 ± 1.2, 1.9 ± 0.6). However, no significant relation (P> 0.05, Spearman) between the PI and the number total ova/embryos or the number of transferable embryos could be determined. These results show that removing the dominant follicle increases the number of embryos after superovulation. However, PI in general could not be used to predict the outcome of a superovulatory treatment. [ABSTRACT FROM AUTHOR]

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situhybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells.Supported by DFG and Goyaike SAACIYF. [ABSTRACT FROM AUTHOR]

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence. [ABSTRACT FROM AUTHOR]

Somatic cloning has emerged as a valuable tool for the production of transgenic animals. However, the technology still suffers from low efficiencies (<1?). In the present study, we used 2 different recipient synchronization protocols and assessed the effects on the cloning efficiency in pigs using triple transgenic donor cells within a bigger study on xenotransplantation. Double transgenic adult fibroblasts (hCD59/DAF) were transfected with a construct coding for human thrombomodulin (hTM). After selection with 800 g mL-1 G418 for 14 days, cells were analyzed for integration of the construct by PCR and visually selected for expression of the hTM-GFP fusion protein. One positive cell clone was used for all nuclear transfers. Nuclear transfer (NT) was performed using in vitro-matured oocytes, as previously described (Hoelker et al. 2005 Cloning Stem Cells 7, 35?44). Peri-puberal German Landrace gilts were used as recipients in 2 different synchronization protocols. Group 1 received 1200 IU pregnant mare serum gonadotropin (PMSG) IM 117 h prior to embryo transfer, followed by 500 IU human chorionic gonadotropin (hCG) 72 h later. Nuclear transfer complexes were transferred surgically 46 h later, approximately 6 h post-ovulation. Group 2 was synchronized by administration of 5 mL Altrenogest for 12 days (20 mg gilt/day). At the end of treatment, the animals received 1000 IU PMSG IM, followed by an injection of 500 IU hCG 80 h later. Cloned embryos were transferred surgically approximately 20 h prior to ovulation. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of gestation. Animals were checked for pregnancy by ultrasound 25 days after transfer (Table 1). In group 1, only one pregnancy could be maintained, leading to the birth of one piglet after caesarean section. In contrast, 12 pre-ovulatory transfers resulted in 9 pregnancies with a total of 47 cloned piglets born. All offspring had normal birth weights (1.0?1.5 kg) and showed no malformations. Average litter size in group 2 was 5.2, and the overall efficiency was 3.1? and 4.4? when related to pregnant recipients. The results show that the transfer of NT-complexes into oviducts of pre-ovulatory recipients significantly improved the success rates of porcine cloning. This is an important feature for the production of transgenic pigs for biomedical and agricultural application. This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535). [ABSTRACT FROM AUTHOR]

The development of a functional nucleolus accompanying the major embryonic genome activation (EGA) is considered a marker for embryo quality and viability. However, the use of this marker is limited by the lack of accurate knowledge of the biology of embryonic nucleologenesis. The objective of this study was to elucidate the role of RNA polymerase I (RPI) and total transcriptional activity, reflecting EGA, for nucleologenesis in in vivo-developed porcine embryos. Late 4-cell-stage embryos were cultured in the absence (control) or presence of actinomycin D (AD; 0.2 g mL-1, 3 h for RPI inhibition; 2.0 g mL-1, 3 h for total transcriptional inhibition). Late 2-cell-stage embryos were cultured to the late 4-cell stage with 0.2 g mL-1 AD (long-term inhibition) to prevent EGA. Embryos were fixed at the late 4-cell stage and processed for RT-PCR (de novo synthesized rRNA), autoradiography (ARG, following culture with 3H-uridine for the last 20 min before fixation), TEM, FISH (probe-labeling rRNA and rDNA), silver staining of nucleolar proteins, and immunofluorescence for RPI. Control embryos displayed typical extranucleolar and nucleolar ARG labeling, fibrillo-granular nucleoli, and focal RPI localization signaling de novo rRNA synthesis in functional nucleoli, confirmed by RT-PCR. All nuclei showed large FISH clusters (rRNA and rDNA) that in 88% of the cases were co-localized with large foci of silver-stained nucleolar proteins. After RPI inhibition, only extranucleolar ARG labeling was detected and, instead of fibrillo-granular nucleoli, a segregated dense-fibrillar component and a granular component, but no fibrillar centers, were observed. RPI was dispersed in all nuclei, the number of nuclei presenting large FISH clusters decreased to 40%, and only 42% of nuclei showed nucleolar proteins localized to large foci. After total transcriptional inhibition and long-term inhibition, the nuclei did not display any ARG labeling and presented inactive nucleolus precursor bodies indicating lack of rRNA (RT-PCR) and total RNA synthesis. However, 40% of the nuclei in both groups presented large FISH clusters of rRNA. This rRNA is considered as partially processed residues of maternally inherited molecules, and their clustering is most likely independent of EGA. Inhibition of transcriptional activity at the time of EGA caused the dispersion of RPI (de novo synthesized) but did not influence the localization of silver-stained nucleolar proteins to large foci (41%). On the other hand, EGA inhibition caused the lack of RPI labeling and hampered the localization of nucleolar proteins to foci. Differences between these 2 groups could be due to the activation of RNA polymerase II before the 3-h AD treatment. In conclusion, RPI transcription and de novo protein synthesis are required for formation of functional nucleoli. However, the clustering of maternally inherited nucleolar transcripts is independent on transcriptional activity at the time of EGA. Failure in constituent RNA polymerase activation during EGA leads to pattern-specific changes in nucleologenesis, which may serve as a marker for early embryo quality. [ABSTRACT FROM AUTHOR]

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 10.5% (mean SD) vs. 83.7 9.2%; fetal fibroblasts: 64.8 17.3% vs. 82.5 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 3.5% vs. 24.9 8.6%). In the second experiment, FSSCs of 3 different sizes (<14 m, 15–20 m, >21 m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 3.1 vs. 91.3 10.1 vs. 92.3 5.1; cleavage rates: 83.5 7.9 vs. 83.1 1.6 vs. 83.2 5.8; blastocyst rates: 15.3 7.9 vs. 17.6 6.8 vs. 10.4 2.7, respectively). In the third experiment, FSSCs 15–20 m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3 7.3% vs. 86.8 5.3%; cleavage rates: 86.1 6.7% vs. 80.7 5.9%; blastocyst rates: 21.4 5.6% vs. 18.4 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL-1 human bFGF and 2 μg mL-1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK, and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and sub-cultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC, and KLF4, as evaluated by immunocytochemistry, and expression of NANOG, SSEA-1, and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR, and 150 ng mL-1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project "PartnErS" (218205; 204, 523) and EU FP7 Stem Cell Project "PluriSys" (223485). [ABSTRACT FROM AUTHOR]

It has been hypothesized that high non-physiological IGF-1 levels are partially responsible for the recurrent pregnancy loss observed in women with the polycystic ovary syndrome (Eng GS et al.2007 Diabetes 56, 2228–2234). The aim of this study was to determine the effect of supraphysiological concentrations of IGF-1 on blastocyst production and the occurrence of apoptosis in bovine embryos, which are a good model for human embryo development (Baumann CG et al.2007 Mol. Reprod. Dev. 74, 1345–1353). COC obtained by slicing from abattoir ovaries were matured (TCM-199, Sigma) for 24 h and fertilized (Fert-TALP) for 18 h (Day 0) in vitro. Two different IGF-1 (Recombinant human IGF-1, R&D Systems GmbH, Wiesbaden, Germany) concentrations (supraphysiological = 1000 ng mL-1and physiological = 100 ng mL-1) were added to the culture media (Synthetic oviduct fluid/BSA) and compared with a control group (no IGF-1 supplementation). On Day 8, blastocyst rates (22 replicates) were recorded and DNA degradation was detected in blastocyst nuclei using a cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany) based on the terminal deoxinucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) principle. Embryos (n= 27 [control], n= 29 [both IGF-1 groups]) from 4 replicates were examined by confocal laser scanning microscopy. Data were analyzed by ANOVA and the Fisher exact test using the SigmaStat 2.0 software package (Jandel Scientific, San Rafael, CA). Cleavage was numerically improved by both, 1000 (59.1 ± 1.8) and 100 (58.2 ± 2.8) ng IGF-1 over controls (53.5 ± 2.2), but the differences did not reach statistical significance (P= 0.22). The proportion of hatched blastocysts was enhanced by 100 (5.8 ± 1.0, P= 0.03) and 1000 (5.1 ± 0.7, P= 0.03) ng IGF-1 compared to controls (2.8 ± 0.6). Total blastocyst rate was increased by 100 ng IGF-1 (34.4 ± 1.9, P= 0.02) over controls (28.3 ± 1.7), but not by 1000 ng IGF-1 (29.1 ± 1.6 P= 0.75). The 100 ng IGF-1 group (38.5 ± 3.7) had fewer degenerated embryos (P= 0.01) compared to 1000 ng IGF-1 (49.7 ± 3.3). The proportion of embryos displaying at least one apoptotic cell was greater in the 1000 ng IGF-1 group over controls (96% v. 77% P= 0.04). The number of blastomeres with TUNEL-positive nuclei per embryo was higher in the supraphysiological group (5.5 ± 0.6, P< 0.001) compared with the control (2.3 ± 0.4) and the physiological group (2.5 ± 0.3). There were no significant differences between the control and the 100 ng IGF-1 group in this regard (P= 0.49). In conclusion, supraphysiological concentrations of IGF-1 do not increase blastocyst production but increase levels of apoptosis in bovine embryos produced in vitro.M. A. V. is in the PhD program of the University of Veterinary medicine, Hannover, Germany, and is supported by the German Academic Exchange Service (DAAD) [ABSTRACT FROM AUTHOR]

DNA methylation accounts, beside the modifications of histones, for the major epigenetic reprogramming event during embryonic development and is an important mechanism of transcriptional regulation. DNA methyltransferase 1 (DNMT1) is the main enzyme responsible for maintenance of DNA methylation, whereas DNA methyltransferase 3a (DNMT3a) catalyzes de novomethylation. Recently, it has been shown that in vitroproduction (IVP) of bovine embryos significantly influences the relative abundance of DNMT1 and DNMT3a transcripts throughout bovine pre-implantation development (Höffmann K et al.2006 Reprod. Fertil. Dev. 18, 231–232 abst).The objective of the present study was to examine the influence of IVP on the amount and localization of DNMT1 and DNMT3a protein during early pre-implantation development by immunostaining and confocal laser microscopy. In vivo-derived embryos were collected from synchronized and superovulated heifers by transvaginal endoscopic flushing of oviducts (zygotes to 8-cell stages; Besenfelder U et al.2001 Theriogenology 55, 837–845) or by uterine flushing (16-cell stages to blastocysts). For in vitroproduction of bovine embryos, standard protocols of IVP were used (Wrenzycki C et al.2001 Biol. Reprod. 65, 323–331). Zona-free embryos of both origins were wholemount fixed and permeabilized. Nonspecific immunoreactions were blocked. Embryos were stained with primary antibodies at the appropriate dilutions (anti-DNMT1 at 1:50, anti-DNMT3a at 1:25). After washing, the samples were incubated with secondary Alexa 647-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) at dilutions of 1:100 (DNMT1) or 1:50 (DNMT3a). After additional washing, embryos were incubated in Sytox Green (Molecular Probes) and mounted on slides. Controls were produced using only the secondary antibody or by omission of both antibodies. Embryos were analyzed using a confocal scanning laser microscope (LSM 510, Zeiss, Hamburg, Germany). Fluorescence intensity of individual embryos was assessed on central optical sections using ImageJ software (National Institutes of Health, Bethesda, MD). The amount of DNMT1 protein was increased (P< 0.05) in in vivo-derived embryos at the 4-cell, 10 to 16-cell stage, morulae, and blastocysts compared with their in vitro-derived counterparts. Expression of DNMT3a protein was significantly upregulated in IVP-derived 2-cell embryos, whereas a significant increase was seen in 4-cell embryos and blastocysts of in vivoorigin. DNMT1 is localized in the cytoplasm of the pre-implantation embryos of both origins and is translocated to the nucleus at the early morula stage. DNMT3a can be found in the cytoplasm of all pre-implantation stages. For the first time, protein expression patterns of DNMT1 and DNMT3a have been analyzed in in vivo- and in vitro-produced bovine embryos throughout pre-implantation development. These results indicate that IVP alters protein expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.We acknowledge HW Schaumann Stiftung for financial support. [ABSTRACT FROM AUTHOR]

Xenotransplantation is considered a solution to diminish the acute shortage of human organs. Although the hyperacute rejection occurring instantly after xenotransplantation can already be reliably controlled, the following immunological defense like the acute vascular rejection (AVR) remains a major hurdle for long-term survival of xenografts in porcine-to-primate organ transplantation. AVR is primarily characterized by endothelial cell activation with severe consequences on coagulation. The human A20 (hA20) gene exhibits antiapoptotic and anti-inflammatory properties in endothelial cells (Ferran C 2006 Transplantation 82, 36–40) and could thus prevent endothelial cell activation leading to AVR and xenograft destruction. Here, hA20-transgenic pigs were produced by somatic cell nuclear transfer (SCNT) in order to examine the ability of hA20-expressing tissues and organs to modulate the AVR. Two hA20-expression vectors, containing the promoters CAGGS or EF1-α in addition to a neomycin resistance cassette, were transfected into porcine fetal fibroblasts. Transfection was accomplished by electroporation, and the cell clones were selected with G418 (800 μg mL-1) for 14 days. Resistant cell clones were screened in PCR with hA20-specific primers. SCNT was performed as previously described (Hölker M et al.2005 Cloning Stem Cells 7, 35–44). After 8 SCNT sessions with pCAGGSEhA20-transgenic cell clones, embryo transfer was carried out to 16 peripuberal recipients resulting in 12 pregnancies (75%). Sixteen fetuses were isolated after sacrificing the recipient sows, and 45 piglets were born. Six of 16 fetuses (37.5%) and 15 of 45 (33.3%) piglets were transgenic. Four SCNT sessions were completed with pEF1hA20-transgenic cell clones following embryo transfer to 6 sows and 5 pregnancies were established (83.3%). Five fetuses were isolated and 14 piglets born. Five of 5 fetuses (100%) and 9 of 14 (64.3%) piglets were transgenic. Expression analysis (RT-PCR and Northern blots) showed transcription of the hA20 gene in heart, muscle, and cultivated porcine aortic endothelial cells of the pCAGGSEhA20-transgenic animals. No transcription was detected in pEF1hA20-transgenic animals. Current results show that hA20-transgenic pigs develop physiologically compared to wildtype counterparts. Expressing tissues and organs were only found in pCAGGSEhA20-transgenic animals, which are now being tested regarding functionality of the hA20 transgene.We would like to thank Prof. Beyaert from the University of Ghent, Belgium, for providing us with the A20-expression vector pCAGGSEhA20. [ABSTRACT FROM AUTHOR]

The process of maturation encompasses a complex series of molecular and structural events. Completion of the nuclear changes to produce a metaphase II (MII) oocyte does not reflect the molecular and structural maturity of an oocyte, which is sometimes termed cytoplasmic maturation. The Golgi apparatus phosphorylates, fragments, and changes the localization during oocyte maturation. GM130 and phospho-GM130 are used as markers for the Golgi apparatus and phosphorylated Golgi apparatus, respectively. The goal of this study was to analyze the dynamics of the Golgi apparatus and its association with microtubules in bovine oocytes at different stages of in vitromaturation [IVM; i.e. germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and MII]. The roles of CDC2A kinase (also known as p34cdc2) and cytoplasmic-dynein ATPase on Golgi dynamics were studied by using specific inhibitors. The distribution of the markers was assessed by immunocytochemistry and laser confocal microscopy. To unravel the role of CDC2A and cytoplasmic dynein ATPase on the dynamics of the Golgi apparatus, the inhibitors roscovitine (ROS) and sodium-orthovanadate (SOV), respectively, were used. In the first experiment, the nuclear maturation rate was checked in the presence of the inhibitors at different times and for different incubation times to explore whether oocytes were able to reach the MII stage. At the GV and GVBD stages, the Golgi apparatus is observed as fragments named mini-Golgies and at the MI and MII stages as punctate foci throughout the cytoplasm. Our results showed 2 well-defined movements of the Golgi apparatus toward opposite directions, depending on the maturation stage. The first movement was observed between 5 and 9 h of IVM (i.e. the GVBD stage), when the Golgi apparatus relocalized from the ooplasm to the periphery. The second movement was observed between 9 and 15 h of IVM (i.e. the MI stage), when the Golgi apparatus moved from the cortex to throughout the cytoplasm and remained there up to the MII stage. The use of inhibitors on CDC2A and cytoplasmic-dynein ATPase at selected time points revealed that CDC2A played a crucial role on the distribution of this organelle during the first movement, whereas the final localization at the GVBD stage was dependent on cytoplasmic-dynein transport. The second movement of the Golgi apparatus was disturbed by the SOV treatment, but not by the use of ROS, suggesting a role of cytoplasmic-dynein-dependent transport during the distribution and organization of the punctate foci at the MI stage. The phosphorylation status of Golgi was not affected at the different incubation times with inhibitors, except in those oocytes incubated with ROS for 24 h, suggesting a role of CDC2A. In conclusion, we describe the involvement of CDC2A during the first movement of the Golgi apparatus and the importance of cytoplasmic-dynein ATPase activity in the first and second relocalization of Golgi during bovine oocyte maturation.DAAD. [ABSTRACT FROM AUTHOR]

This study was designed in order to evaluate the global transcription and morphology of reprogramming during somatic cell nuclear transfer (SCNT). Following 20–22 h of IVM, couplets of MII cytoplasts and starved bovine fibroblasts were, after 2 h co-culture, electrically fused, chemically activated by 5 µm ionomycin for 5 min, followed by a 3–4 h incubation in 2 mm 6-DMAP, and fixed at 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 16 h post-activation (hpa). The reconstructed embryos were processed for lacmoid staining, autoradiography following [3H]uridine incubation, transmission electron microscopy (TEM), and immunofluorescence (4 and 12 hpa) in order to evaluate chromatin dynamics, transcriptional activity, nuclear and nucleolar ultrastructure, and localization of nucleolar proteins (upstream binding factor (UBF) and fibrillarin), respectively, during the first cell cycle. Likewise, starved fibroblasts were fixed and processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillo-granular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. During first 3 hpa, the majority of the embryos displayed a single block of condensed chromatin surrounded by a more or less complete nuclear envelope (NE) and an abundant population of elongated somatic cell mitochondria. This somatic cell complex was located peripherally in the ooplasm. At the subsequent time points, the embryos displayed pronuclear-like structures, which from 8 hpa were located centrally in the ooplasm. From about 4 hpa, the somatic cell complex had dispersed and the elongated mitochondria could no longer be tracked. The first nucleolus-related structures were observed at 1.5 hpa and only in nuclei with a complete NE. At 1.5 to 4 hpa, the nucleolus-related structures appeared either as bodies presenting a large fibrillar center and presumptive dense fibrillar component, but no granular component, or as electron-dense nucleolus precursor bodies (NPBs). From 4 hpa and onward, only compact NPBs were observed. At 4 and 12 hpa, UBF was localized into small discrete clusters of foci enclosed in a shell-like structure labeled by fibrillarin in the nucleus. In conclusion, at SCNT, the somatic cell cytoplasm remains structurally organized in a somatic cell complex over the initial 3–4 hpa. During the same period, the somatic cell chromatin undergoes condensation and the NE is partially dissolved. Subsequently, pronucleus-like euchromatic nuclei with typical NPBs are formed, although somatic cell nucleolar components may be temporarily seen. Throughout the process, transcription is repressed.This work was supported by Marie Curie Intra-European Fellowships (MEIF-CT-2006-021629), and by grant VEGA 1/3255/06 and DFG. [ABSTRACT FROM AUTHOR]

The hyperacute rejection after porcine-to-human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors or by knocking out the gene for α1,3-galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase-I (hHO-I) is known to have anti-apoptotic and cell-protective properties. Thus, the expression of hHO-I on porcine endothelial cells could have beneficial effects in a xenotransplantation setting to prevent the formation of AVR. Here we describe the first transgenic pigs with functional expression of hHO-I. Fibroblasts were obtained by an ear punch from a decay accelerating factor (DAF)-transgenic female pig and were cultured in vitro (Kues WA et al. 2005 Biol. Reprod. 72, 1020–1028). Cells reaching confluency of 70 to 80% were detached with EDTA/trypsin and subsequently transfected by electroporation at 450V/350 µF with a vector coding for hHO-I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 µg mL–1) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone that showed strong expression of the transgene, as determined by immunostaining, was selected for somatic nuclear transfer (Hoelker M et al. 2005 Cloning Stem Cells 7, 35–44). In total, 205 reconstructed embryos were transferred to 2 synchronized peripuberal German Landrace gilts, which gave birth to 9 live piglets, all with normal birth weights. PCR and Southern blot analyses revealed that all of the offspring had integrated the vector into their genome. Two transgenic animals were sacrificed for further characterization and ex vivo perfusion experiments. In these animals, Northern blotting showed weak transcription of the transgene in all xenorelevant organs such as heart, kidney, and liver. Immunostaining of the kidneys revealed that expression of the transgene was confined to the endothelial cell layer. These hHO-I-transgenic porcine kidneys were subjected to an ex vivo perfusion assay and survived ex vivo perfusion for 240 min with AB-pooled human blood, whereas perfusion of 2 non-transgenic controls had to be terminated after 60 min due to problems attributed to immunological rejection. Histology revealed that perfused hHO-I-transgenic kidneys exhibited no indication for xenogenic activation of the human coagulation system. These preliminary results show that functional hHO-I can be expressed in transgenic pigs and that transgenic expression of hHO-I might improve long-term survival of porcine xenografts. These results are encouraging and warrant further studies on endothelial cell activation and the biological function of hemeoxygenase-I in the context of xenotransplantation.This study was funded by the Deutsche Forschungsgemeinschaft Ni 256/22-1, -2, -3. [ABSTRACT FROM AUTHOR]

The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo.We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20. [ABSTRACT FROM AUTHOR]

Recently we discovered a novel type of stem cells which can be derived from primary cultures of fetal connective tissue using a high-density culture (HD) system (Kues et al. 2005 Biol. Reprod. 70, 1020–1028). This cell type shows most of the characteristics of true embryonic stem cells and is a promising alternative for developing therapeutically useful cells. The goal of the present study was to analyze and characterize stem cell populations obtained from five different tissues, i.e., brain, heart, lung, liver, and adrenal gland, using this HD culture system. Explant cultures of the double transgenic strain OG2/Rosa26 were established from mouse fetuses at Day 13.5 post-conception. In this strain, cells with pluripotent properties are characterized by the green fluorescence of the green fluorescent protein (GFP) which is under the transcriptional control of the Oct-4 promoter. In a total of five replicates, cells were subcultivated and reseeded at a concentration of 25 000 cells/cm–2. Cell culture conditions were as recently reported for fetal somatic stem cell derivation (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). After colony formation and/or observation of green fluorescence under UV illumination, mRNA was isolated for RT-PCR analysis of the expression of genes known to be involved in pluripotency, i.e., Oct-4, Nanog, Stat3, TNAP, Rex1, and Sox2. Under these culture conditions, 12.5% of the liver and 80% of the brain explants showed senescence and could not be seeded at the required concentration. No colony formation was observed in brain and heart cultures. Cells from heart explants showed a three-dimensional growth over the whole surface of the attached cells and were difficult to separate. On average, colony formation was observed after 11 days 8 (mean SD) (fetal fibroblasts), 10 days 7 (lung), 18 days 11 (liver), and 4 days 1 (adrenal gland). Green fluorescence was detected after 17 days 9 (fetal fibroblasts), 17 days 9 (lung), 35 days 13 (heart, randomly distributed), 26 days 10 (brain), 26 days 8 (liver), and 8 days 5 (adrenal gland). A single large clump of stem-like cells formed in 43% of the fetal fibroblast, 35% of the lung, and 41% of the liver derivatives. Oct-4 expression was not observed in any of the cultures. Nanog and Stat3 were expressed in all cell cultures, and TNAP was expressed in all cultures from heart, brain, lung, and adrenal gland. Rex1 was expressed in all cultures from brain and adrenal gland, and in 25% of the heart cultures and in 16.6% of the fetal fibroblasts. Sox2 was expressed in all cultures from brain and in 16.6% of fetal fibroblast cultures. These preliminary results show that cells with some of the typical features of stem cells can be derived from various tissues of the body, but that the efficiency is different among tissues. Only cells derived from the adrenal gland exhibited better colony formation than the original fibroblasts.This work was funded by DFG grant Ni 256/28-1. [ABSTRACT FROM AUTHOR]

Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034–8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage up to the morula stage, telomerase is not active or only found at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from local abattoir followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2–4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells.Supported by DFG and Goyaike S.A.A.C.I. Y.F. [ABSTRACT FROM AUTHOR]

A well-orchestrated expression of genes is required to ensure mammalian oocyte progression from the first meiotic arrest to the metaphase II stage to achieve full developmental competence. DYNLL1 (cytoplasmic dynein light chain LC8), PMSB1 (proteasome beta subunit 1), and DYNC1I1 (cytoplasmic dynein 1 intermediate chain) are crucial genes for nuclear and cytoplasmic maturation. DYNLL1 and DYNC1I1 are constituents of the cytoplasmic dynein 1 complex, the main transport system of the cell. PMSB1 is a subunit of proteasome 20S that is the catalytic core of 26S proteasomes and belongs to the ubiquitin-dependent proteolytic system required for protein degradation. The goal of this study was to compare the relative abundance (RA) of the transcripts of the above genes in oocytes collected from different size follicles at different stages of IVM: germinal vesicle (GV), GV breakdown (GVBD), MI, and MII. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and were matured in M199, supplemented with 1% BSA-FAF, Suigonan (10 UI PMSG/5 UI HCG), and 100 M cysteamin, at 39C in 5% CO2. First, meiotic progression and developmental competence were evaluated in both groups of oocytes by Hoechst staining and IVF/IVC (TALP medium/SOF-system), respectively. Denuded oocytes were frozen at -80C for RNA analysis. Time points selected for freezing were 0 h for GV; 8 h for GVBD in oocytes from <2 mm follicles and 9 h for oocytes from 2-8 mm follicles; 15 h for MI; and 24 h for MII. The RA of the mRNAs were determined by semiquantitative RT-PCR. One-way and two-way analysis of variance was used for statistical analysis. Cleavage and blastocyst formation rates were significantly higher in the embryos from oocytes recovered from follicle size 2–8 mm; maturation rates were not statistically different between the two groups. The RA of DYNLL1 and DYNC1I1 were significantly higher in oocytes from follicle size 2–8 mm compared to oocytes from the small follicles. No significant difference in PMSB1 expression was found between the two groups. DYNLL1, DYNC1I1, and PMSB1 decreased significantly during IVM in oocytes from 2–8 mm follicles. Only DYNLL1 decreased significantly in the oocytes isolated from the small follicles. When comparing size and stages, DYNLL1 RA was significantly higher in GVBD and MI in the oocytes from follicle size 2–8 mm compared to the other group. A decreased expression of these genes is expected during maturation because the proteins are involved in transport into the cell and because of protein degradation. De novo synthesis of RNA would not be possible after GVBD. The higher mRNA expression of DYNLL1 and DYNC1I1 in oocytes recovered from the bigger follicles could be related to the higher proportion of blastocysts after IVF. The results provide insight into regulatory mechanisms of oocyte maturation and could serve as a marker to assess the developmental potency of bovine oocytes. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4?8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2?3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) plays a critical role in the elimination of reactive oxygen species (ROS) by acting as substrate for GSH peroxidase (GPX) which results in the oxidation of GSH. Glutathione reductase (GSR) regenerates GSH from oxidized glutathione (GSSG). In bovine oocytes, GSH concentration increases and is thought to have beneficial effects on embryo development. The goal of the present study was to analyze the GSH content in bovine blastocysts during blastocoel expansion and to determine the effects of tertiary-butyl hydroperoxide (tBH) in causing oxidative stress. It is metabolized by GPX with conversion of GSH to GSSG. Blastocysts were generated with standard protocols of in vitro production (IVP) using SOF medium supplemented with FAF-BSA and were collected at Days 7/8 of culture (Day 0 = IVF). Diameters of blastocysts were measured and embryos were allocated to 4 groups: early blastocysts (ebla, less than 135 m), blastocysts (bla, 135–180 m), expanded blastocysts (exbla, greater than 180 m), and hatched blastocysts (hbla). Pools of the different categories (3 replicates, 36 embryos in total) were frozen at -80C until assayed for GSH by HPLC. Total cell numbers were determined using the Hoechst 33342 stain. Blastocysts from the 4 groups were cultured together for 30 min in tBH (20 M) containing SOF medium to induce oxidative stress. This was followed by another culture period for 1 or 8 h. Samples of blastocysts were frozen at -80C at 0 h, 30 min, 1 h, and 8 h to assess GSH levels (Cereser et al. 2001 J. Chromatogr. B. Biomed. Sci. Appl. 752, 123–132) and transcript abundance. Semi-quantitative real-time RT-PCR was used to determine the relative abundance (RA) of GPX and GSR transcripts. Differences between groups were tested using ANOVA followed by a Tukey or Student''s test (P ? 0.05). The grade of expansion significantly increased GSH concentration in bovine blastocysts. Expanded blastocysts (0.38 ± 0.04 ng) and hatched blastocysts (0.46 ± 0.05 ng) had a higher GSH content than early blastocysts (0.21 ± 0.01 ng) and blastocysts (0.27 ± 0.02 ng). However, GSH concentration remained constant in blastomeres during expansion and hatching as the number of cells increased from 54.9 ± 3.5 (ebla) to 152.3 ± 4.7 (hbla). After induction of oxidative stress, blastocysts showed a significant decrease in GSH content after a 1-h recovery period, followed by an increase in their GSH content after a period of 8 h (0 h: 0.26 ± 0.01 ng; 30 min: 0.25 ± 0.01 ng; 1 h: 0.20 ± 0.01 ng; 8 h: 0.32 ± 0.01 ng, respectively). This was accompanied by a significant increase of the RA of GSR transcripts in blastocysts collected after the 8-h recovery period compared to embryos at the other time points of the treatment. GPX mRNA abundance remained stable during the entire culture period. In the present study, GSH concentration was determined in bovine embryos during blastocoel expansion for the first time. The increased GSH content might to some extent be due to de novo GSH synthesis. GSSG is reduced to GSH most likely by GSR. Results show that bovine IVP embryos may serve as a new model for the study of oxidative stress. [ABSTRACT FROM AUTHOR]