ABSTRACT The implantation of the mouse embryo requires the controlled invasion of the uterine stroma by the embryonic trophoblast. This event is dependent, in part, on the secretion of matrix metalloproteinases and serine proteinases for the extracellular degradation of the uterine matrix. Proteinase activity is controlled by stromal decidualization and specific proteinase inhibitors. This work adds to our understanding of implantation and placentation by reporting the expression and function of another class of proteinases/inhibitors closely related to invasive cell behavior. We focused on the cysteine proteinases, cathepsins B and L, and their inhibitor cystatin C. Northern blots showed that trophoblast expressed cathepsin B throughout the invasive period (days 5.5-10.5). Both cathepsin B message and cathepsin L protein were localized to the mature, invasive trophoblast giant cells. Substrate gel electrophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that cystatin C, the main inhibitor of cathepsins, was a major product of the decidualizing stroma. Message levels first increased in peripheral decidualizing cells, with the protein localizing close to the embryo during implantation (days 5.5-8.5). With the regression of the decidua beginning on day 9.5, a coordinated upregulation of both cathepsin B and cystatin C was observed implying a role for controlled cathepsin expression during apoptosis. E-64, a synthetic inhibitor of cathepsins B and L, was injected into pregnant females at the stage of blastocyst attachment (days 4.5-5.5). High doses resulted in the complete failure of implantation while lower doses resulted in stunted embryos and a reduced decidual reaction. These results suggested that cathepsins B and L are necessary for normal embryo development and uterine decidualization, and that decidua contributes to their control by a coordinated expression of cystatin C within the implantation site.
Cuckoldry was studied in New Jersey population of European Starlings (Sturnus vulgaris). Biopsy of both blood and pectoral muscle was done on 550 wild birds, including both adults and all chicks at 95 nests between 1983-1985. Vertical thin-layer polyacrylamide gel electrophoresis (PAGE) was employed rather than starch gel electro- phoresis because we found through using both that PAGE has greater resolution for the enzymes we examined. Thirty-three loci were screened, but only three were both resolvable and found to have bona fide polymorphism. Two unambiguous cases of were discovered, each involving two chicks. Another six cases may have been either or intraspecific brood parasitism; they involved only one chick each. If only the two un- ambiguous cases are counted, the frequency of was 2. 1%. If the six ambiguous cases are included, the frequency was 8.4%. Low measured frequencies do not necessarily imply low risk of because they may reflect the conservatism of electrophoresis and the effectiveness of anticuckoldry behaviors. Cuckoldry may be serious, but contained, risk in our population. Received 18 Mar. 1987, accepted 15 June 1987. Cuckoldry is defined as a male's involuntary rearing of another male's offspring as result of the latter male (the cuckolder) having insemi- nated the mate of the former male (the cuckold) (Power et al. 1981). This can occur as result of extrapair copulation(s) by female when she is unguarded by her mate, with or without her cooperation. Although there has been some disagreement regarding its usage (Gowaty 1982, 1984; Power 1984), the term cuckoldry seems to be the best term to describe the above defined phenomenon. Cuckoldry specifically refers to the sit- uation in which males are the victims of an act of mate infidelity, and distinguishes between that phenomenon and brood parasitism. In this paper we use the term cuckoldry as it is this specific phenomenon that we wish to address. Cuckoldry is significant from an evolutionary standpoint, because it negatively affects the reproductive success of the cuckold. Thus, males should evolve anticuckoldry behavior, and this behavior should evolve in tandem with the evolution of male parental investment. Without cuck- oldry avoidance behavior, should impede the evolution of male parental investment, because males who do not provide parental invest- ment will be rewarded genetically while males who do provide it will be
Implantation of the mouse embryo depends on the interaction of the trophoblast and the uterine environment, which includes an extracellular matrix with abundant amounts of laminin. The experiments reported here analyzed the interaction of trophoblast with laminin in vitro. Secondary trophoblast attachment and spreading on laminin matrices was competitively inhibited in the presence of the laminin peptides YIGSR (Tyr-Ile-Gly-Ser-Arg), RGD (Arg-Gly-Asp), and IKVAV (Ile-Lys-Val-Ala-Val). Substrates of multiple, but not single, peptides supported attachment and spreading of trophoblast. The presence of laminin-binding proteins on trophoblast cell surfaces were detected by immunofluorescence using antiserum directed against a fusion protein encoded by the full-length clone for the 32-kDa laminin-binding protein. Immunoblotting with the same antiserum identified immunoreactive proteins of 33, 41, 45, 62, and 76 kDa in trophoblast lysates. The antiserum also inhibited the spreading of trophoblast on laminin matrices.
Decidualization results in the remodeling of the extracellular matrix with the loss of collagen type I and the appearance of basement membrane matrix components. We have developed an in vitro assay system to study matrix metalloproteases during mouse decidualization. Uterine stroma, or decidua isolated from day 7.5 pregnant mice, were grown on a three-dimensional collagen type I matrix (Vitrogen). Gelatin zymography of conditioned media from these cultures showed constitutive secretion of processed forms of gelatinase A at 65, 62, and 59 kDa with 62 kDa predominating. Similar patterns of gelatinase A expression were obtained from tissue lysates of decidualizing uteri from days 5.5 to 7.5 of development. Cells cultured on Vitrogen, but not on plastic or matrix-coated dishes, were able to process the proenzyme to the 59 kDa form as observed in vivo. Only stroma cells cultured on a coating of collagen type I displayed the same increase in the 59 kDa zymogen. Decidua cells grown on Vitrogen attached and then migrated into aggregates that eventually penetrated the gel and spread as differentiated decidua on the underlying plastic. These preliminary results suggested that the in vitro assay system can be used to study the role of metalloproteases in matrix remodeling during decidualization.Key words: matrix metalloproteases, uterine stroma, decidua, gelatinase A, Vitrogen.
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