Impact of Sperm Hyaluronidase and VLMWHA on Sheep Blastocyst Formation In Vitro, Viability after Cryopreservationand Pregnancy Rate after Embryo Transfer

Background: Recent research in our laboratory has reportedthe presence of members of the hyaluronan (HA) system includingHA synthases and receptors and hyaluronidase (hyals)in reproductive system and embryos and the critical role ofhyal۲ in early stage embryo development. We hypothesised thatvery low molecular size HA fragments (VLMWHA; <۱۰kDa)produced during degradation by sperm hyaluronidase (PH۲۰),function as a survival factor and growth stimulator during preimplantationembryo development.Materials and Methods: Sheep oocytes were collected fromslaughterhouse derived ovaries and matured and fertilized invitro. Experiment ۱: Cleaved embryos were cultured in theabsence (control) or presence of ۱۰ng/ml PH۲۰, or ۱۰۰μg/mlVLMWHA or anti HA cell membrane receptors CD۴۴ andRHAMM for ۶ days when development to blastocyst was recordedand the number of hatched blastocysts counted. Experiment۲ assessed quality of the blastocysts based on survivalafter cryopreservation by vitrification of early blastocyst stageembryos. Experiment ۳ analysed pregnancy and live birth ratesafter embryo transfer to oestrus synchronised recipient ewes.Pregnancy was assessed by ultrasound scanning on day ۳۵, andnumber and normality of lambs were recorded.Results: Significantly higher percentage of blastocysts wereproduced in PH۲۰ (۵۶.۸ ± ۶.۹) or VLMWHA (۶۳.۶ ± ۴.۰) versuscontrol (۳۲.۴ ± ۳.۴ %). Similarly higher proportion of theseblastocysts were hatched (PH۲۰; ۲۱.۶ ± ۳.۱, VLMWHA; ۲۲.۶ ±۴.۴, Control; ۷.۲ ± ۱.۲) P<۰.۰۵). These effects were abrogatedin the presence anti-CD۴۴ and RHAMM. Higher percentage ofthe blastocysts cultured in PH۲۰ or VLMWHA survived aftervitrification as observed by re-expansion and hatching after reculture(۷۶.۲% and ۸۰% v. ۵۲.۲%, P<۰.۰۵). Higher number ofpregnancies and live birth was observed in the ewes receivingblastocysts developed in the presence of PH۲۰ (۸/۱۱; ۷۳%) orVLMWHA (۹/۱۲; ۷۵%) versus control (۶/۱۱; ۵۵%). No abnormalitywas observed in the lambs weight, behaviour and survival.Conclusion: These studies have defined a new a new role forsperm in supporting early stage embryos and provided evidencefor a receptor-mediated role of sperm hyaluronidases or VLMWHAin enhancing embryo development and quality during thepreimplantation period.

کلیدواژه ها:

Hyaluronan ، Hyaluronidase ، Sperm ، Blastocyst ، Pregnancy and Live Birth