The aim of this study was to investigate the role of Src homology 2-containing phosphotyrosine phosphatase SHP2 in intricate signaling network invoked by oocyte to achieve cytoplasmic maturation and also blastocyst development. Activation of SHP2 regulates multicellular differentiation, proliferation and survival through numerous signal pathways. The most prominent pathway is RAS/PI3K and p-AKT signaling cascade, as a result mitogenic effect become enhanced. Oocytes were cultured in cisplatin an anticancer drug, but selective activator of SHP2 and our grouping were SOF medium alone, SOF + EGF, SOF + CISPLATIN 0.3 μM, and SOF + EGF + CISPLATIN 0.3 μM. We evaluated that EGF neutralizes the apoptotic effect of cisplatin as well as maintain the high expression of SHP2, as a result blastocyst development become boosted up. We also found that inhibition of SHP2 with its specific inhibitor PHPS1 5 μM decreases the blastocyst development and neutralizes growth factors effect. The developmental ability and quality of bovine embryos were determined by assessing their cell number, gene expression, immunofluorescence, and immunoblot. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. Our results show that SHP2 have significant effect on MAP kinase pathways which expand the cumulus cells during oocyte maturation and blastocyst development as compare to inhibition of SHP2 with PHPS1. SHP2 not only transduce the signaling of epidermal growth factor but it also has a role in signal transduction of FGF and IGF. The expression of ERK, PI3K/p-AKT and mTOR was increased with EGF, but with the treatment of SHP2 inhibitor the expression of these genes become drop done. So we can conclude from these results that SHP2 is important for oocyte maturation as well as for blastocyst development.

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5ug/mL R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; 53.29~72.94%, 6 h; 21.40~58.90%, 12 h; 8.26~25.93%, 24 h; 1.00~13.78%, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging 70.8~77.8% and 52.1~84.5%, respectively, were not significantly different. However, in vitro development rates of the same groups ranging 25.7~40.0% and 12.9~1.8%, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

Lipid metabolites involved in cellular regulation as signaling mediators. Prostaglandins (PGs), metabolites of lipid are involved to pregnancy at the time of implantation but the functional roles of PGs on embryo development are still controversy and largely unknown. In previous report, the levels of and at embryos of morula stage and blastocyst stage were explored (Cheon et al., 1998). In this study, the previous suggestion was confirmed and the possible downstream mediator of prostaglandin and prostaglandin on the expansion and hatching of mouse embryo was examined. As expected, developmental rate of the blastocyst to expanded stage was a concentration-response curve that showed the highest expansion rate at 10 , but at 100 , the rate was decreased. In contrast to the , stimulated expansion without toxicity at highest concentration. Cotreatment of PGs with indomethacin overcame the inhibitory effects of indomethacin in expansion. Exogenous PGs also improved the development of expanded embryos to the hatching stage. Besides, PGs receptors' transcripts detected at blastocyst. was caused of calcium fluctuation in the blastocyst but did not. The changes of intracellular calcium concentration were different between indomethacin pretreated embryos and non-treated embryos. Based on these results it is suggested that PGs work as paracrine and/or autocrine factors through calcium and the others which were not identified in this study.