Interaction oi Proteinase Inhibitors with Blastocyst Proteinases involved in Implantation

RABBIT blastocysts exhibit a remarkable proteinase activity during the first (abembryonic) phase of implantation in the uterus. The activity becomes detectable around 6 days post-coitum (d p.c.) and rises rapidly until 7-7'/3 d p.c. when syncytial trophoblastic "knobs" penetrate the extracellular blastocyst coverings and establish contact to the uterine epithelium; thereafter blast~cyst proteinase activity declines rapidly until 8-8% d p.c.(4,6) Blastocyst coverings are c,?mposed of both protein and sialic acidand sulfate ester-rieh glycosaminoglycan components.(5) Presumably a number of enzymes including glycosidases and proteinases are involved in the breakdown of this "barrier" between trophoblast and uterine epithelium, (5,8) blastocyst proteinase activity apparently playing a major role as suggested by the following observations. This enzyme activity is always found maximal at the abembryonic pole where lysis of blastocyst coverings starts, whieh holds true also in the case of abnormal orientation of the blastocyst in the uterus. Cl0) Blastocysts that show signs of beginning lysis of their coverings have never been found to lack high proteinase activity. Furthermore, hormonal regulation of blastocyst proteinase activity is reflected by hormonal regulation of lysis of the coverings: in ovariectomy and progesterone replacement therapy experiments both respond in a parallel way.(7) So far the histologic site of synthesis of the proteinase molecules is unknown (trophoblast? endometrium?),(1l,16) but there is strong evidence that the trophoblast provides at least an essential cofactor (activator) if not the enzyme (proenzyme) molecules.ClO-12) According to the apparent physiological importance of this proteinase, biochemical characterization seems to be very desirable but has still not been achieved yet duc to the small quantities of enzyme present in an embryo. So far only gelatin membranes have been successfully used as a substrate.( 4,6-12,16) Therefore, until purified preparations are obtained and/or a specific substrate has been found, we decided to study the action of a number of purified and well-characterized inhibitors on this proteinase using a highly sensitive histochemical gelatin substrate fIlm test(9) (Fig. 1).