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 blastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocysts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with zona 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 cultured with zona-escaping blastocysts. However, zonae from mice, rats, sheep and humans were resistant to lysis under these conditions. Pronase treatment 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 +/- 8 s). When cysteine protease inhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were tested, they completely inhibited zona escape, while trypsin inhibitors (TLCK and SBTI) did not. Uterine zona lysin contribution in zona escape was discounted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae and (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopregnant recipients) were all zona-intact, while 71% of transferred blastocysts exhibited zona escape, following their recovery after 24 h. These observations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blastocysts in hamsters.