Trypsin‐like hatching protease from mouse embryos: Evidence for the presence in culture medium and its enzymatic properties

Abstract

Enzymatic properties of a protease involved in hatching of mouse embryos were examined. A trypsin-like protease, which most efficiently hydrolyzed t-butoxycarbonyl-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide, was demonstrated in culture medium of mouse hatching embryos. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, N alpha-tosyl-L-lysyl-chloromethane, soybean trypsin inhibitor, and Trasylol, but not or weakly inhibited by p-chloromercuribenzoic acid, EDTA, E-64, pepstatin, chymostatin, and bestatin, suggesting a trypsin-like serine proteinase. The protease activity in the medium gradually elevated during the course of hatching, whereas the embryo-associated activity showed no significant change. Furthermore, pyroglutamyl-Leu-argininal, the strongest inhibitor for the enzyme among peptidyl argininals, all of which are potent trypsin inhibitors, showed the strongest inhibition toward hatching. Thus, a trypsin-like protease secreted from hatching embryos into the culture medium may participate in mouse hatching, probably as a hatching enzyme.

Effect of the site of assisted hatching on vitrified-warmed blastocyst transfer cycles: a prospective randomized study

“…Firstly, the hatching process in vivo differs from that of in vitro. In addition to the increased internal pressure, "protease" with zonalysis activity from the trophectoderm and (or) the uterus [19][20][21], repeated collapses and expansions of the blastocyst [22] and sphincter-like zona-breaker cells [23] are involved in the hatching process. Data from hamster and mouse showed that successful hatching in vitro was dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding.…”

Trypsin‐like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos6

“…Morulae (1 6-cell-stage) and early blastocysts (32-cell-stage) were collected by flushing the uterus obtained from mated female mice (ICR: ScSlc) as described previously (13,14,20). Unless stated, embryos were harvested 88 h after hCG injection.…”

“…The hatching enzyme, a proteinase responsible for digestion of the embryo coat, is also expressed at a certain stage of development in the course of the hatching process (9, 10). Although hatching enzymes from vertebrates (1,2,5,7,11,13,14,(19)(20)(21)(22)(23)(24) and invertebrates (3,6,9,10,17) have been studied enzymatically and molecular biologically, little is known about the mammalian hatching enzyme (4,11,13,14,20,21).…”

“…The blastocysts then expand and make a small hole in the zona pellucida, through which the embryos squeeze by embryonic movement from the unrestricted sites of embryos including mural and polar trophectoderms (20). Although the embryonic shedding appears to be simply mechanical, this process is substantially prevented by several naturally occurring and synthetic protease inhibitors (2, 11,13,14). Among protease inhibitors so far tested, benzyloxycarbonyl-Phe-Ser-argininal, one of the leupeptin analogs, was found to be the most potent inhibitor against the hatching of mouse embryos (14).…”

Chemical zona pellucida thinning with acidified Tyrode’s solution: comparison between partial and circumferential techniques

“…However, even with proper care that embryos of good quality are chosen for transfer to the uterus, the implantation rate remains low. As part of the natural hatching process in vivo, the zona pellucida (zona) is thinned and softened by a protease-like enzyme [1,2]. In contrast, embryo culture conditions in vitro are believed to induce abnormal hardening of the zona [3], which may impair the hatching process and thus contribute to low implantation rates after embryo transfer [4].…”