Abstract
This study has evaluated effect of the spermatozoa incubation on the glycosidase activity and fertilizing ability in vitro in the pig. To identify sperm glycosidases specific for sugar residues found in the zona pellucida of pig oocytes, the spermatozoa were treated experimentally and assayed for activities of $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase and N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase). The glycosidases activity were higher in spermatozoa incubated for 2h than without incubation. The $\beta$-GlcNAc'ase activity was at least two-fold higher than other glycosidase regardless of spermatozoa incubation. In the same glycosidases, the activity had a tendency to increase as time of spermatozoa incubation was prolonged, but there were no differences in spermatozoa incubated during the various periods (4~24h). The percentages of spermatozoa that reached acrosome reaction were affected by glycosidases in the medium (P<0.05, for mannosidase), and were higher in spermatozoa with that than without incubation. On the other hand, the spermatozoa motility were decreased with incubation periods, but no effects by different glycosidases on the change of sperm motility during the various periods of incubation. In other experiment, the binding and penetration of pig spermatozoa were tested with oocytes matured in vitro in the presence of various glycosidase. The penetration rates were decreased with incubation of spermatozoa when oocytes were inseminated in medium with different glycosidases. These rates were higher in spermatozoa non-incubated than with incubation for 2h (P<0.05 for GlcNAc'ase; P<0.01 for control group). The sperm-zona binding rate in control group were higherthan in medium with glycosidases. In addition, the highest binding rate were obtained in medium with GlcNAc'ase. In all glycosidases, the sperm-zona binding rate in spermatozoa without incubation were higher than incubation for 2h. The significant differences were obtained in spermatozoa treated with $\alpha$-D-mannosidase (P<0.05). These results suggest that $\beta$-GlcNAc'ase is present mainly in the plasma membrane of pig spermatozoa. It was also shown that the glycosidase activity were increased in all glycosidases in spite of low sperm-zona binding rate and penetration rates by spermatozoa incubation.
본 연구는 돼지 동결-융해 정자의 배양에 의한 체외수정능력과 glycosidase activity를 평가하기 위하여 chlortetracycfine fluorescence분석법을 이용하여, glycosidase가 체외수정능력과 정자침입에 미치는 영향에 대하여 검토하였다. 또한 돼지난자의 투명대내에서 발견된 당잔기에 대한 정자의 glyco-sidase 특이성을 확인하기 위하여 $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase 및 N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase)의 activity를 분석하였다. 그 결과 glycosidase activity는 동결정자의 응해 후 배양하지 않았을 때보다 2시간 배양했을 때 더 높게 나타났다. $\beta$-GlcNAc'ase의 activity 는 정자 배양 유무에 관계없이 다른 glycosidase 처리시보다 최소한 2배 이상 높게 나타났다. 또한 첨체반응이 유기된 정자의 비율은 glycosidase ($\alpha$-D-mannosidase; P<0.05)에 의해 영향을 받았으며 자를 배양하지 않은 경우보다는 배양된 정자에서 높게 나타났다. 그러나 배양시간에 따른 정자의 존성에 대해 glycosidase의 종류에 따른 유의차는 인정되지 않았다. 한편 투명대내 정자의 접착과 입에 대한 또 다른 실험에서, 서로 다른 glyco-sidase가 첨가된 배양액내에서 수정된 정자가 배양시간이 길어짐에 따라 정자의 침입율은 낮아졌다 $\beta$-GlcNAc'ase; P<0.05). 투명대내의 정자접착정도는 glycosidase의 첨가시에 무첨가시보다 접착정도가 더 높았으며, 가장 높은 접착율은 $\beta$-GlcNAc'ase 첨가시 나타났다. 또한 모든 glycosidase 처리시 2시간 배양한 정자보다는 배양하지 않은 정자에서 투명대에 대한 접착정도가 높게 나타났으며, $\alpha$-D-mannosidase의 처리시 유의적인 차이를 보였다 (P<0.05). 본 연구의 결과, $\beta$-GlcNAc'ase가 주로 돼지정자의 원형질막내에 존재하는 것으로 추측되며, 배양된 정자에 의한 투명대 접착정도와 침입율이 낮았음에도 불구하고 glycosidase activity가 증가하는 것으로 나타났다.
