The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM CaCl2 ․ 2H2O), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM CaCl2 ․ 2H2O) supplemented with 100, 200 or 300 μg/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 μsec. The activated embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 μg/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM CaCl2 ․ 2H2O (T1), 1.0 mM CaCl2 ․ 2H2O (T2) and 0.1 mM CaCl2 ․ 2H2O with 100 μg/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-α/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

In this study I report that in vitro development rates of bovine nuclear transfer embryos activated either with boar sperm cytosolic factor (SCF) or with ionomycin followed by cycloheximide (CHX) and subsequent in vivo developmental rates after embryo transfer are related to blastocyst quality as evaluated by apoptosis analysis. SCF was extracted from porcine semen then purified for post-activation injection after nuclear transfer. The optimal timing for SCF injection was determined to be at least 22 h post-IVM for parthenogenetic activation of bovine oocyies. A total of 364 oocytes were successfully enucleated and 268 (73.6%) fused and were injected with SCF. The survival rate of fused and injected embryos was 109/113 (96.5%) after 2 h. The cleavage rates of nuclear transfer embryos after 3 d of culture in the ionomycin/CHX treated group were significantly higher than those of the SCF-activated group (93.3% vs 81.7%, p<0.01, respectively). However, at 7 d and 9 d there was no significant difference between the total developmental rates to blastocyst for either treatment group. Total blastocyst cell numbers were also not significantly different between the two activation treatments (ionomycin/CHX: 149.57.7 vs. SCF: 139.34.4 cells). In contrast, the apoptotic levels in the SCF blastocysts were higher than those produced after the chemical treatment (28.25.1% vs. 8.80.6%, respectively). A total of 18 expanded or hatching blastocysts was transferred to nine synchronized recipients in each activation group; 5/9 (55.5%) and 2/9 (22.2%) were pregnant at 40 d in the ionomycin/CHX treatment and SCF activated group, respectively. However, only one went to term in the ionomycin/CHX treatment while none of the pregnancies from the SCF group were maintained by 90 d. In conclusion, these results suggest that SCF derived from different species is a limited activator to be used for activation after bovine nuclear transfer in lieu of a chemical activation protocol.