200 DETECTION OF mRNA ENCODING HEPARANASE IN PORCINE OVIDUCT

Polyspermy is an important anomaly of fertilization and is responsible for the low efficiency of IVF in the porcine species. It was reported that the exposure of oocytes to oviducal fluid increases monospermy after IVF in pigs. This fact has been related with the binding of OVGP1 to the ZP and is affected by heparin glycosaminoglycan. The OVGP1 is removed from the ZP during the embryo transit towards the uterus; however, the mechanism responsible for this process is unknown. Taking into account the contribution of the heparin in this process, the presence of heparanase in the oviduct milieu was postulated. This enzyme could degrade the heparin and unstabilised the binding of the OVGP1 to ZP. The aim of this study was to describe the presence of mRNA codifying for heparanase in the porcine oviduct. Oviducts from 4 animals in preovulatory phase were obtained. Epithelial cells from the oviducal mucosa (ampulla) was obtained by scraping, and total RNA was isolated using the RNAqueous® kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Complementary DNA was synthesised with an oligo dT as primer. This cDNA was used as the template in RT-PCR amplifications using specific primers designed based on GenBank sequence for Sus scrofa heparanase (NM_001146130.2). The amplification by RT-PCR resulted in a 199-bp amplicon. This PCR product was automatically sequenced and showed a high similarity with heparanase of different mammalian species. In conclusion, the heparanase mRNA is present in the porcine oviduct. This glycosidase could participate in the regulation of heparin content in the oviducal milieu. More studies are necessary to clarify its effect in the oviducal fluid and fertilization. This study was supported by MICINN (AGL2009-12512-C02-01-02).