CDK2 inhibition plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or MII arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage as demonstrated by the formation of distinct γH2AX foci in nuclei of day 3- and day 5-embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activating of the ATM-P53-P21 pathway. However, the mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for double strand break (DSB) repair reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in day 7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after H2O2 treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups (76.8±0.3 vs 69.1±0.4; 0.01 mM, 55.7±1.0; 0.1 mM, 38.2±1.6%; 1 mM, P<0.05). The expressions of ST3GAL5 in H2O2 treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 5% . The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin- like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy; 2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cells; and 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA- transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1; by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processes; however, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.