Control of Sperm Penetration In Vitro by Cumulus Cells in Porcine Oocytes

돼지의 체외수정시 난구세포에 의한 정자침입의 조절

The functional role of cumulus cells on the penetration and polyspermy during in vitro fertilization in porcine was examined. The penetration rate was significantly higher (P＜0.01) in oocytes with (61%) than without (25%) cumulus cells, but significant differences in polysper-my rates were not observed. When hyaluronidase was added to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than in oocytes without cumulus cells at 0 (61% vs 34% ; P＜0.05), 0.01 (56% vs 35% ; P＜0.05), 0.1 (66% vs 30% ; P＜0.05) and 1.0mg/$m\ell$ (39% vs 27%). The polyspermy rates were lower in oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocyte culture was prolonged. At 16 and 20 h after insemination, the penetration rates were significantly higher (P＜0.05) in oocytes with (48 and 62% for 16 and 20 h) than without (25 and 31% for 16 and 20 h) cumulus cells in medium containing hyaluronidase. Polyspermy rates were significantly (P＜0.05) lower in oocytes without (13% and 16%) than with (37% and 48%) cumulus cells at 16 and 20 h after insemination. In cumulus-free oocytes inseminated in medium containing different concentrations of cumulus cells, the penetration rates were significantly (P＜0.05) higher in medium with than without hyaluronidase. The proportion of polyspermy was lower in medium without than with hyaluronidase at 0 (10% vs 0%), 10$^2$(25% vs 0%), 10$^4$(24% vs 14%) and 10$^{6}$ (29% vs 10% ; P＜0.05) cumulus cells/$m\ell$. These results suggest that cumulus cells can have a positive influence on sperm penetration, its action on polyspermy control does appear to function primarily on zona pellucida by co-culture of cumulus cells and oocytes in medium without hyaluronidase.

본 연구는 체외에서 돼지정자의 난자 내 침입에 있어서 난구세포의 기능적인 역할을 검토하기 위하여 실시하였다. 체외수정 시 정자침입율은 난구세포 부착 (61%) 난자가 제거 (25%) 된 난자에 비해 높았으나 (P＜0.001), 다정자침입에는 영향을 미치지 않았다. 한편 체외수정시 hyaluronidase 를 0, 0.01, 0.1 및 1.0mg/$m\ell$ 농도로 첨가된 배양액내에서 난구세포가 부착된 난자내의 정자침입률은 각각 61, 56, 66 및 39%로 이들 세포를 제거한 난자에서의 침입률 34, 35, 30 및 27%에 비해 높았다. 그러나 다정자침입률은 hyaluronidase의 농도에 관계없이 난구세포를 제거한 난자에서 낮았으며, hyaluronidase의 농도가 높아지면서 다정자침입률이 낮아지는 경향을 나타냈다. 또한 hyaluronidase를 첨가한 배양액내에서 수정후 16 및 24시간에서의 정자침입률은 난구세포를 제거한 경우 (25 및 31%) 보다 난구세포가 부착된 난자 (48 및 62%)에서 높았으며 (P＜0.05), 다정자침입률은 난구세포 제거 시 유의적으로 낮게 나타났다 (P＜0.05). 한편, 난자로부터 채취한 난구세포를 여러 농도로 첨가한 후 난구세포 제거난자를 이용하여 체외수정한 결과 hyal uronidase 첨가보다는 무첨가시 정자의 침입률과 다정자 침입률이 낮게 나타났다. 본 연구의 결과로부터 난구세포는 정자의 침입에 효과적으로 작용하였으며, hyaluronidase의 첨가와 난구세포수의 조절이 정자의 침입과 다정자침입에 영향을 미치는 것으로 추측되었다.