Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin. PA/plasmin system playa role in mammalian fertilization and motility and acrosome reaction of sperm. The present study was undertaken to identify PAs in porcine gametes and investigate a possible role of plasminogen in in vitro fertilization in the pig. When boar spermatozoa were preincubated in a fertilization medium (mTBM) for 0, 2, 4 or 6 h, the activity of tPA-PAI (110~117 kDa), tPA (62~70 kDa), and uPA (34~38 kDa) was observed in the sperm incubation medium and sperm sample. PA activities in the sperm incubation medium significantly (p<0.05) increased according to increasing incubation times, while PA activities in sperm significantly (p<0.05) decreased at the same times. In addition, the rate of acrosome reaction in spermatozoa increased by increasing culture times. When oocytes were separated from porcine cumulus-oocytes complexes at 0, 22 or 44 h of maturation culture, no PA activities were observed in cumulus free-oocyte just after aspiration from follicles. However, the activity of tPA-PAI (108~113 kDa) and tPA (75~83 kDa) was observed at 22 h of in vitro culture and significantly (p<0.05) increased as the duration of the culture increased. On the other hand, when porcine oocytes were activated by sperm penetration or calcium ionophore, plasminogen significantly (p<0.05) increased ZP dissolution time (sec) in activated oocytes by sperm penetration. These results suggest that supplementation of plasminogen to fertilization medium may playa positive role in the improvement of in vitro fertilization ability in the pig.

The present study was undertaken to identify changes of plasminogen activators (PAs) in porcine oviductal epithelial cells (POECs) during the estrous cycle classified with post-ovulatory stages (Post-Ov), early to mid-luteal stages (Early-mid L) and pre-ovulatory (Pre-Ov) stages. The urokinase-type plasminogen activator (uPA) was only observed on day 5 and day 7 of culture in the POECs on all the estrous cycles and gradually increased according to increasing culture times, but not Early-mid L. In POECs-conditioned medium, uPA, tissue-type (tPA) and tPA-PA inhibitor (tPA-PAI) activity were observed at all culture times during estrous cycles. The uPA activity of POECs-conditioned medium on Post-Ov stage were significantly (p<0.05) decreased during prolonged cultures. On the other hand, the tPA activity of POECs-conditioned medium at Post-Ov stage was significantly (p<0.05) higher on day 5 than compared to the other days. Although was higher on day 1 at Post-Ov stage, the tPA-PAI activity of POECs-conditioned medium was significantly (p<0.05) higher on day 7 at all stage than that of day 5 of the culture. Taken together, these results suggest that uPA, tPA and tPA-PAI are produced by POECs, and the variations of the PAs activity are regulated in the different stages of the estrous cycle.

Production of transgenic animals for studying specific gene has been limited due to a low efficiency, lack of skilled researchers and the need for expensive equipment. Currently, the boar spermatozoa as a vector to deliver exogenous DNA into the oocyte were used to improve the efficiency of transfection rate. In this study, we revealed that the optimal conditions for DNA uptake in spermatozoa by liposome were to 90 min of incubation, 17'C, 10' spermatozoa, 4 ng/ml of exogenous DNA and 0.5% (v/v) liposome, without damage to fertility. In addition, the developmental rate to the blastocyst stage of embryo in control group was significantly higher than those embryos with exogenous DNA and liposome, whereas there were no significant differences in embryo development between the liposome and type of DNA. The transfection rates of embryo using treated spermatozoa with both liposome and circular DNA were higher than those using linear DNA. These findings raise the possibility thattreated spermatozoa with liposome/DNA complexes could be used in in vitro fertilization, and the exogenous DNA transferred into the oocytes. Taken together, we demonstrated that liposome a vector for the uptake of exogenous DNA in boar spermatozoa could improve the efficiency of sperm-mediated gene transfer in creating transgenic pig and the other domestic transgenic animals.

This study was conducted to investigate the effect of periods of sperm preincubation, concentrations and storage periods of miniature pig sperm on in vitro penetration of porcine follicular oocytes. High concentration (1×105, 2.5×105, 5×105, 1×106, 5×106 and 1×107) did support sperm penetration than low concentrations (P<0.05). However, polyspermic oocyte rates were increased with high concentrations of sperm. On the other hand, sperm preincubated during 1, 2 or 5h could be penetrated than sperm preincubated during 0, 3 or 4h (P<0.05). When sperm were storaged with different periods, in vitro pentration rates were significantly higher 0～2 days than 3～4 days of sperm storage (p<0.05). These results indicate that sperm treatment factors can effect in vitro penetration in miniature pig.

본 연구는 0.5 ml straw를 이용한 정자 동결융해 시 융해 온도가 동결정자의 성상에 미치는 영향을 파악하고 미니 돼지의 동결정액에 최적화된 적정 융해 조건을 찾기 위하여 실시하였다. 정액동결 straw를 37, 50 및 70℃에서 각각 5초, 10초 및 45초간 융해하여 생존율(SYBR-14/PI staining), 정자원형질막기능검사 (Hypoosmotic Swelling Test) 및 첨체반응율 (CTC : chlorotetracycline staining)을 검사 한 결과 70℃에서 5초간 융해한 정자의 생존율과 CTC 결과가 37℃와 50℃에서 10초 및 45초간 융해한 처리구보다 유의적(p<0.05)으로 높은 생존율과 낮은 비율의 첨체 반응율을 얻었다. 따라서 미니 돼지의 동결 정액은 고온에서 단시간 융해를 하는 것이 정자의 성상에 유리한 것으로 사료된다.

The purpose of this study was to determine effects of oxytocin and interleukin-1α on in vitro development of bovine embryo cultured with endometrial epithelial and stromal cells isolated from bovine uterus. The expressions of COX-2 mRNA in bovine endometrium were also studied. When embryos were cultured with epithelial cells, the rate of blastocysts was significantly (p<0.05) higher in embryos treated with oxytocin than that of control group. The rate of hatched blastocysts was also significantly (p<0.05) higher in embryos treated with oxytocin than those of two control groups. On the other hand, when the embryos were cultured with stromal cells, the rate of blastocysts were significantly (p<0.05) higher than those of groups treated with IL-1α, oxytocin and control with stromal cells than that of control group without stromal cells. The rate of blastocysts hatched were also significantly (p<0.05) higher in group treated with IL-1α than those of control group without stromal cells and oxytocin group. In another experiment, COX-2 gene was expressed in embryo group treated with oxytocin during the co-culture of embryos with epithelial cells. In contrast, COX-2 mRNA was expressed in group treated with IL-1α when the embryos were cultured with stromal cell. This result shows that oxytocin and IL-1α were stimulate embryo development in vitro when embryos were cultured with epithelial and stromal cells, and can affect the development of bovine embryos in the uterus.