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Anti-Hyaluronidase Oligosaccharide Derived from Chondroitin Sulfate A Effectively Reduces Polyspermy During In Vitro Fertilization of Porcine Oocytes¹

Department of Bioproduction,³ Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan School of Bioresources,⁴ Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 µg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1–100 µg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 µg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10–100 µg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25–30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 µg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 µg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

¹ Supported by a Grant-in-Aid for Young Scientists (14760181 to H.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Correspondence. FAX: 81 98 895 8757; hidettmt{at}agr.uryukyu.ac.jp