The difference between early pregnancy and delivery rate is quite large in assisted reproduction techniques (ARTs), including animal cloning. However, it is not clear why the implanted fetuses aborted after the early pregnancy stage. In the present study, we tried to evaluate the developmental and morphological characteristics of porcine parthenogenetically activated (PA) embryos or fetuses by electric stimulation during the early pregnancy period. The implanted PA and artificially inseminated (AI) embryos and fetuses were collected at day 26 and 35 after embryo transfer, respectively. The developmental and morphological parameters in the PA embryos at day 26 were similar to the AI embryos. The size, weight, formation of major organs, and apoptotic cells were not statistically different in both embryos at day 26. However, the PA fetuses at day 35 showed ceased fetal development and degenerated with abnormal morphologies in their organs. The day 35 PA fetuses showed significantly higher apoptotic cells and lower methylation status in three differentially methylated regions of the H19 gene compared to their comparators. Therefore, the normal development of PA embryos and fetuses during early gestation could lead to these pregnancies being misinterpreted as normal and become one of the main reasons for the gap between early pregnancy and delivery rate.
Mitochondria play important roles in cell survival, including energy production, storage of proapoptotic factors, and mitochondria-related responses, which regulate the intracellular redox potential of porcine embryos in vitro (Dumollard, Duchen,
Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced
pluripotent stem cells (piPSCs) is very low. The present study was performed to
investigate the effect of cell cycle inhibitors on the cell cycle
synchronization of piPSCs. piPSCs were generated using combination of six human
transcriptional factors under stem cell culture condition. To examine the
efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel
coated plate with stem cell media and they were treated with staurosporine (STA,
20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO,
200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were
in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells
at G1 in DAI group was significantly higher than that in control, while STA,
ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of
viability and apoptosis were affected by STA and ROSC treatment, but there were
no significantly differences between control and DAI groups. Real-Time qPCR and
FACs results revealed that DAI treatment did not affect the expression of
pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and
apoptosis, but Oct4 expression was significantly decreased. Our results suggest
that DAI could be used for synchronizing piPSCs at G1 stage and has any
deleterious effect on survival and pluripotency sustaining of piPSCs.
To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal
stabilizer on an appropriate time is one of the possible approaches. However,
the exact treatment timing and effect of cytoskeletal stabilizer such as
cytochalasin B (CB) is not well known during oocyte vitrification procedures.
Thus, the present study was conducted to determine optimal treatment timing of
CB during vitrification and warming procedures. In experiment 1, the survival
rates of the post-warming pig oocytes were analyzed by fluorescein diacetate
(FDA) assays with 4 classifications. In results, post-warming oocytes showed
significantly (p<0.05) decreased number of alive oocytes
(31.8% vs. 86.4%) compared to fresh control. In detail, the significant
difference (p<0.05) was found only in strong
fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6%
vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after
(Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly
(p<0.05) higher (91.6%) survival rates compared to
group of CB-Vitri (83.7%), significantly (p<0.05) and
comparable with group of Vitri Control (88.7%) by morphological inspection. In
FDA assay results, group of Vitri-CB showed significantly
(p<0.05) higher (44.2%) survival rates compared to
groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the
increased survival rates of post-warming pig oocyte treated with Vitri-CB method
are firstly described here. The main finding of present study is that the CB
treatment during recovery could be helpful to refresh the post-warming pig
oocyte resulting its improved survival rates.
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