2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A211 CLONING, TRANSGENESIS AND STEM CELLS EFFECT OF TRICHOSTAIN A ON PRODUCTION OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER AND SUBSEQUENT GESTATIONS TIONS Luiz Sérgio Almeida Camar amargo go, Car arolina Cap apobiango R. Quin uintão tão, Anna Car arolina Denic enicol, Lilian Tam amy Iguma, Bruno Camp ampos Car aravalho alho, Luiz Gusta ustavo Bruno Siqueira & João Henrique Moreira Viana EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. Histone deacetylation is associated to a compact and less permissive chromatin, which may impair an efficient nuclear reprogramming of somatic cell nucleus. The inhibition of histone deacetylation can be achieved by histone deacetylases inhibitors, as Trichostatin A. This study evaluated the effect of zygotes treatment with trichostatin A on production and quality of nuclear-transferred bovine embryos. Oocytes were matured in vitro, denuded and exposed to Hoechst 33342 (Sigma, St Louis, USA) and cytochalasin (Sigma) before enucleation. Zygotes were reconstructed with somatic cells from adult Gyr cow, fused with double electric pulse of 2.4 kV/cm for 30 µsec and activated with ionomycin (Sigma) for 5 min. After ionomycin activation the zygotes were randomly separated in two groups: Trichostatina (TRICHO, n = 132) - zygotes were cultured for 4h in 6-DMAP followed by 7h in CR2aa medium plus with 2.5% fetal calf serum, both supplemented with 50nM Trichostatin A (Sigma); Control (CONT, n = 116) - zygotes were cultured in the same conditions described above but without Thichostatin A supplementation. Parthenogenetic embryos (PART group, n = 287) were used as control for activation and embryo culture. Embryos from all groups were cultured in CR2aa supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5%O2 and 90%N2. Embryos at 168h postactivation (hpa) from TRICHO and CONT groups were transferred to synchronized recipients. Analyses of cleavage at 72hpa, blastocyst at 168hpa, total cell number and apoptotic cell index were performed by ANOVA and means compared by SNK test. Data are shown as mean±SEM. Gestation and birth rates were analyzed by Fischer’s Exact Test. No difference on cleavage (87.7±4.1%, 83.3±4.0% and 79.3±4.1%) and blastocysts (38.4±4.7%, 34.2%±6.0% and 30.5±3.7) rates was found among TRICHO, CONT and PART groups, respectively. Embryos from TRICHO group presented lower (P < 0.05) index of apoptotic cells (0.065±0.009; n = 17) than embryos from CONT group (0.129±0.021; n = 13), but similar (P > 0.05) to those from PART group (0.083±0.011; n = 19). No difference (P > 0.05) on total cell number was found among groups. Although it is not statistically significant (P > 0.05), pregnancy rate at 60 days with embryos from TRICHO group (64.2%; 9/14) was numerically higher than those ones from CONT group (45.4%; 5/11). One pregnancy from TRICHO group went to term (7.1%; 1/14) but the calf died in the second day after birth. No offspring was obtained with embryos from CONT group. In conclusion, exposure of clone zygotes to 50 nM of trichostatin A decreases apoptosis in embryos, which may favor pregnancy rate. [Financial support: Embrapa Project 01.07.01.002, CNPq 403019/2008-7 and Fapemig]. Keywords: cloning, histone deacetylases inhibitor, apoptosis. A212 CLONING, TRANSGENESIS AND STEM CELLS EFFICIENCY OF NUCLEAR TRANSFER IN EQUINE: DIRECT INJECTION OF THE DONOR CELL NUCLEUS INTO THE RECIPIENT CYTOPL OPLAST ASTS Claudia Barbosa Fernandes 1 , Camila Gabriela Pereira Gonçalves 1 , Lilian Rigatto Martins 2 & Fernanda da Cruz Landim Alvarenga 3 1 USP FMVZ, SAO PAULO, SP, BRAZIL. 2 UFMT, MATO GROSSO, MT, BRAZIL. 3 UNESP FMVZ, BOTUCATU, SP, BRAZIL. The aim of this study was to evaluate the efficiency of NT with donor cell nucleus direct injection in equine recipient cytoplasts. There were used 150 equine compact and expanded oocytes in vitro matured (IVM) for 30h or kept for 24h under the roscovitine action before the period of IVM. After 30 h of IVM equine oocytes were denuded and incubated during 30 min in H-MEM medium, with FBS, Hoechst 33342 and cytochalasin B. The oocytes enucleation and the recipient cytoplasts reconstitution with equine fibroblasts were performed between 32 and 34 h after the beginning of IVM. The metaphaseI (MI) and metaphaseII (MII) oocytes enucleated and reconstituted successfully were incubated for one hour until the artificial activation, using Roscovitine + Ionomycin (I+R) and Ionomycin + 6 DMAP (I+6DMAP). After the activation period, the oocytes were cultured in DMEM/F12 with FBS, myoinositol and gentamicin at 39ºC in 5% CO2, 5% O2 and 90% N2 during 3 days. The assessment of the activation and nuclear decondensation formation rates were performed with Hoechst 33342 in inverted microscopy. There was used the Analysis of Deviance to select the best logistic regression model to explain the percentage of equine oocytes with nuclear decondensed formation after NT. The equine oocytes classified as compact that were exposed or not to roscovitine, in MI and MII at the time of enucleation, had 0%, 20% and 37.5%, 25% of nuclear decondensation rate after activation with I+R and 0% 30% and 22.2%, 50% after I+6DMAP, respectively. The equine oocytes classified as expanded that were exposed or not to roscovitine in MI and MII at the time of enucleation, had 14.2%, 30% and 0%, 14.2% of nuclear decondensation rate after activation with I+R and 12.5 %, 20% and 20%, 33.3% after I+6DMAP respectively. We can observe that the only significant effect in chromatin decondensation rates was the stage of nuclear maturation, regardless of the oocyte classification, the artificial activation protocol and the exposure or not to roscovitine, the percentage of oocytes with the decondensed nucleus formation on the third day of culture was significantly higher for MII (P = 0.033) compared to MI. There was no significant interaction among any other factors, however, for the equine the technique when the donor cell nucleus was directly injected into the recipient cytoplast resulted in unsatisfactory cloned embryos production. [Acknowledgement: FAPESP]. Keywords: cloning, artificial activation, nuclear maturation. s442
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A213 CLONING, TRANSGENESIS AND STEM CELLS NEW STRATEGY TEGY TO INDUCE TRANSGENESIS IN IN VITRO FERTILIZA TILIZATION TION (IVF) BOVINE EMBRYOS Natalia Gabriela Canel, Romina Jimena Bevacqua, María Inés Hiriart & Daniel Salamone FACULTAD DE AGRONOMÍA U.B.A., BUENOS AIRES, ARGENTINA. Direct plasmid injection into the cytoplasm of IVF zygotes is a simple and efficient method to induce transgenesis in mammals, however it still gives transient expression from extrachromosomal plasmid DNA. To solve this problem, we employed a new transgenesis method (Ogino H et al., Nat. Prot., 1, 1703-10) only employed in X. laevis, consisting in intracytoplasmic injection of I-SceI meganuclease (Mega) plus a plasmid that contains its recognition sites flanking Pax6 promoter-GFP cassette (IS-EGFP). IS-EGFP (gifted by Dr. Ogino H) was digested by 0.5 UI of Mega at 37ºC for 45 min. Fragment digestion was confirmed by agarose electrophoresis. Digestion mixture or plasmid alone were transferred to 10% PVP at a final concentration of 25 or 50 ng/µL IS-EGFP (groups IS-EGFP 25/50 +Mega, for digestion mixture, and IS-EGFP 25/50 for plasmid alone) and were microinjected into IVF presumptive zygotes. A control group injected with 50 ng/µL of pCX- EGFP plasmid was included. Embryos were cultured in SOF medium and rates of blastocysts were evaluated on day 7. Expression of egfp was determined at days 4, 5 and 7. Data was analyzed by Fisher’s test. All treatments showed no differences in blastocyst rates [from 8/40 (20%) to 19/51 (37.3%)]. Expression of egfp at day 4 was lower (P < 0.05) for all treatments compared to control group (from 0 to 8.2% vs. 30.2%), except for IS-EGFP 50 (22.5%). The same pattern was observed at day 5 for IS-EGFP 25 +Mega or alone groups (8.2 and 7.9% respectively), but not (P < 0.05) for IS-EGFP +Mega or alone groups (13.7 and 47.5% respectively), which did not differed from control (30.2%). At day 7, all groups showed egfp expression levels not different from control (from 23.8 to 35% vs. 32.6%), except for IS-EGFP 25 +Mega, which showed lower (P < 0.05) expression levels (14.3%). Embryos with IS-EGFP 25 +Mega or alone and IS-EGFP 50 +Mega tended to show lower expression rates than those with IS-EGFP 50 alone or control. Blastocyst rates of embryos expressing egfp did not differed among groups. However, at all evaluations, groups with Mega showed a tendency to express egfp at lower levels than the groups treated with plasmid alone. Conclusions: IS-EGFP has a delayed expression dynamics compared with pCX-EGFP. The presence of the enzyme keeps the EGFP fragment released, possibly preventing concatemer formation. Our results show that the use of IS-EGFP + Mega influences the transient transgene expression in IVF bovine embryos. Keywords: transgenesis, ivf, bovine. A214 CLONING, TRANSGENESIS AND STEM CELLS EXPRESSION OF XIST, G6PD AND HSPA1A GENES IN BOVINE BLAST ASTOCY OCYST STS S RECONSTITUTED BY NT FROM RECIPIENT OOCYTES PRODUCED BY CHEMICALL ALLY ASSISTED ENUCLEATION N aiara a Zo ccal Sar araiv aiva 1 , Clar lara a Slade Oliv liveir eira 1 , Ta tiane Almeida Dr ummond Tetzner 1 , Mar arina Ragagnin de Lima 1 , Simone Cr istina Méo 2 & Joaquim Mansano Garcia 1 1 FCAV-UNIVERSIDADE ESTADUAL PAULISTA - UNESP, JABOTICABAL, SP, BRAZIL. 2 EMBRAPA PECUÁRIA SUDESTE-CPPSE, SÃO CARLOS, SP, BRAZIL. N The aim of this work was to study the effect of enucleation (EN) technique on the expression of XIST, G6PD and HSPA1A genes on bovine blastocysts produced by IVF, parthenogenetic activation and reconstitution by NT of somatic cells after conventional EN or chemically assisted EN. Bovine oocytes were in vitro matured in TCM 199 + FCS + hormones for up 24 h. IVF embryos were produced with female sexed sperm because the cell line of donor nuclei for NT was female. Parthenogenetic embryos were produced by activation with 5 ìM ionomycin for 5 min and 10 µg/mL cycloheximide for 4 h. In conventional EN, after IVM for 18 h, MII oocytes were denuded, incubated with Hoechst 33342, and enucleated by removing the 1 st PB and the adjacent cytoplasm. In chemically assisted EN, after 19 h of IVM, the oocytes were denuded, exposed to 0.05 ìg/mL demecolcine for 2 h and underwent to enucleation, through the removal of the protrusion formed in the cortical region. After in vitro culture in SOFaa with 2.5% FCS and 0.5% BSA for seven days at 38.5°C, 5% CO 2 in air under saturated humidity, blastocysts were individually submitted to RNA extraction with Trizol and reverse transcription with Reverse Transcriptase ImPron-II kit. Relative transcript quantification of XIST, G6PD e HSPA1A genes was performed in real-time PCR with SYBR Green ® (Invitrogen), employing GAPDH as endogenous control, in triplicate, with five to ten samples per group and final volume of 20 ìL. The measn amplification efficiency was estimated using a linear regression on the log fluorescence per cycle. After applying the comparative CT method (ÄÄCT) with the SDS software v1.4, data was analyzed by the pairwise fixed reallocation randomization test. All expression ratios were calibrated by IVF group and shown in log 10 scale. No significant differences in XIST and G6PD genes were detected between control groups (IVF: 0 and 0, and parthenogenetic: 4.088 and 1.398, respectively) and reconstituted groups. However, a higher expression (P < 0.05) of these genes was seen in chemically assisted EN group (6.690 and 3.851) compared to conventional EN (-0.643 and -0.794). HSPA1 gene expression was similar (P > 0.05) between groups. Tthe expression of the G6PD gene has been associated with high embryo respiratory activity rates, suggesting that the increased expression of this gene is indicative of embryo quality. Also, higher levels of ROS generated by increased oxygen consumption may trigger G6PD activation. The expression pattern of more genes related to stress response should be investigated to conclude if the difference in expression between the EN techniques relates to the quality of embryos obtained. [Funded by FAPESP 06/51481-5 and 07/55969-5]. Keywords: demecolcine, enucleation, gene expression. s443
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