Evidence for the involvement of a species-specific embryonic protease in zona escape of hamster blastocysts

The source and nature of zona lytic factors during zona escape of hamster b lastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocy sts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with tons escaping-blastocysts (at a ratio of 1:10), exhibited zona lysis. Various other embryos at the 1-8-cell stages also showed zona lysis when c ultured with zona-escaping blastocysts, However, zonae from mice, rats, she ep and humans were resistant to lysis under these conditions. Pronase treat ment resulted in rapid zona lysis in hamsters (7 +/- 1 s), whereas in other species zona lysis was much slower: mouse (662 +/- 27 s), rat (532 +/- 16 s), sheep (120 +/- 12 s) and human (104 +/- 13 s). When cysteine protease i nhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were test ed, they completely inhibited zona escape, while trypsin inhibitors (TLCK a nd SBTI) did not. Uterine zona lysin contribution in zona escape was discou nted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae an d (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopr egnant recipients) were all zona-intact, while 71% of transferred blastocys ts exhibited zona escape, following their recovery after 24 h. These observ ations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blasto cysts in hamsters.