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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A219 CLONING, TRANSGENESIS AND STEM CELLS
THE EXPRESSION OF PLURIPOTENCE MARKERS IN DONOR CELLS INCREASED CLONING EFFICIENCY IN BOVINE
José Reinaldo Manriquez, Alejandra Estela Velasquez, Fidel Ovidio Castro & Lleretny Rodriguez
UNIVERSIDAD DE CONCEPCION, CHILLÁN, CHILE.
Nuclear transfer (NT) in mammals is a complex process in which a differentiated cell is reprogrammed to induce embryo
development. NT is associated with different degrees of nucleus epigenetic reprogramming of the donor cells. Expression of certain genes in
these cells facilitates its expression in the embryo produced after NT. We observed that certain somatic cell lines express pluripotency genes (e.g
OCT4 and SOX2). Based on these facts we postulate that expression of pluripotency genes in donor cells increase cloning efficiency expressed
as good quality blastocysts. To test this, we correlate the developmental potential (DP) of NT bovine embryos created from 5 different cells lines
(termed CWA, HAR, HCH, HB12, HAN) with the expression level of OCT4 and SOX2 in each cell line. DP was assessed on the basis of
blastocysts rate; grading and total cell counts at day 7. Donor fibroblast cell lines were isolated from adult cattle after collagenase digestion of
tissue biopsies. For NT cells (passages 3-7) were used at least 1d after reaching confluence. Cloned embryos were produced by zona-free NT,
cultured individually for 7d in SOFaci in 5%O2; 5%CO2 and 90%N, 100% humidity at 39ºC in sealed aluminium foiled bags, in well of the well
system. Gene expression analysis: total RNA was extracted (4 biological replicates per cell line), converted to cDNA and subjected to real time
PCR with specific primers; semiquantification was conducted using the standard curve method. Data were analysed with non-parametric test
using Infostat (Buenos Aires, Argentina). Day-7 embryos were produced from all cell lines (CWA:64.3%; HAR:62.2; HCH:57.2; HB12:49.4;
HAN:27.9). The total percentage of blastocyst, including grade I and total cell number obtained with HAN was significantly lower compared
with the other 4 cell lines, while HAR-derived embryos showed the best grading and cell number counts. OCT4 and SOX2 expression was
evaluated in all cell lines (only OCT4 was quantified). The highest expression was obtained in HAR and the lowest in HAN. There was a
correlation between expression level of OCT4 in the cell lines and the blastocysts rate (r = 0,92; P = 0,02), rate of grade I blastocysts (r = 0,96;
P = 0,01) and total cell number (r = 0,98; P = 0,002). The expression of SOX2 was determined in three of the cell lines, but it did not correlated
with any of the parameters mentioned earlier. Conclusions: expression of OCT4 in the cell lines used for NT might increase embryo quality at
blastocyst stage and could be a useful marker to select cell lines and to improve cloning efficiency. [This work was partially supported by
Fondecyt grant No. 11100082 from the Ministry of Education of Chile].
Keywords: nuclear transfer, oct4, cell line.
A220 CLONING, TRANSGENESIS AND STEM CELLS
IN VITRO CULTURE OF BOVINE EMBRYOS IN MURINE ES CELL CONDITIONED MEDIA NEGATIVEL
TIVELY AFFECTS
EXPRESSION OF PLURIPO
URIPOTENCY RELATED MARKERS OCT4, SOX2 AND SSEA1
C lara a Slade Oliv
liveir
eira,
Mar
arc ela Mar
aria Souza,
Naiar
aiara a Zo ccal Sar
araiv
aiva,
a, Tatiane Almeida Dr ummond Tetzner
etzner, Mar
arina Ragagnin de Lima,
Flavia Lombardi Lopes & Joaquim Mansano Garcia
UNESP, JABOTICABAL, SP, BRAZIL.
Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that
culture conditions for in vitro produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their
undifferentiated state. Therefore, the aim of this work was to evaluate the effect of culture medium on pluripotency related markers expression
of IVP blastocysts, in order to produce suitable ICM for further culturing. We tested KSR (5%) and FCS (5%) supplements in SOF 0.5% BSA
medium, and ES cell conditioned medium (CM) on IVC (Groups: KSR, KSR CM, FCS and FCS CM). After, the expression of pluripotencyrelated
markers was assessed by immunocytochemistry of OCT3/4 (Sigma), NANOG (Sigma), SOX2 (Sigma) and SSEA1 (Invitrogen), and
mean fluorescence intensity from nuclei area was measured using Adobe Photoshop CS3 software (0 to 255 scale). No difference was detected
between KSR and FCS groups. On FCS supplemented groups, we detected down-regulation of pluripotency markers in the inner cell mass
(OCT3/4: FCS 70.40a, FCS CM 49.29b; SOX2: FCS 132.43a, FCS CM 93.72b; SSEA1: FCS 59.39a, FCS CM 49.19b), except for NANOG
(FCS 29.00, FCS CM 24.24) (One way ANOVA and Tukey post test, P = 0.05). On KSR supplemented groups, no statistic difference was
observed, although there was a numeric decrease in KSR CM group (NANOG: KSR 32.80, KSR CM 29.60; OCT3/4: KSR 66.82, KSR CM
56.87; SOX2: KSR 135.11, KSR CM 113.29; SSEA1: KSR 65.8, KSR CM 54.21). Then we evaluated SOX2 gene expression by real-time
PCR, using SYBR Green detection system (Applied Biosystems). SOX2 relative gene expression revealed lower levels on KSR CM
blastocysts (KSR 0.44±0.08a, KSR CM 0.27±0.04b), and a remarkable variation in SOX2 mRNA levels on FCS supplemented blastocysts
(SFB 0.53±0.25, SFB CM 0.47±0.29) (Unpaired T test, P = 0.05). In conclusion, pluripotency-related markers tend to decrease after
supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not
differ from FCS, but FCS replacement by KSR would be interesting in order to produce blastocysts with stable SOX2 gene expression levels.
[Financial Support: FAPESP 07/58506-6, 08/58370-0 and 09/54510-4].
Keywords: pluripotency, in vitro fertilization, embryonic stem cells.
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