Sandra W. Potter

Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy

Mouse uterine epithelium is a tissue that undergoes cyclic endocrine‐regulated cell dissociation and regeneration. It shows a dramatic cell loss following normal estrus. If pregnancy ensues, cell loss is averted during the first 2.5–3.5 days. However, this is followed by a precipitous loss of basal‐lateral cell adhesion and apoptosis in preparation for blastocyst invasion. By comparing epithelia isolated by protease treatment, we show that a reduction of lateral cell adhesion is a primary event in these instances of normal tissue loss. It was readily induced in ovariectomized adult and immature mice by injections of estradiol (E2), and to some extent also by progesterone (P4). The reduction of lateral adhesion induced by including ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA) in the isolation medium mimicked and was additive to the effect of E2 injection. However, the E2 effect was different in not being prevented by adding Ca2+. The E2 effect also was mimicked by the action on isolated epithelium of monoclonal antibody against the calcium‐dependent cell adhesion molecule, E‐cadherin, suggesting that inactivation of E‐cadherin was induced by E2. In detergent extracts of estrous and metestrous epithelium there was an increase in 80‐kDa extracellular domain of E‐cadherin relative to the intact 120‐kDa molecule. The loss of adhesion between 3.5 and 4.5 days of pregnancy was associated with a loss of both intact membrane‐associated 120‐kDa E‐cadherin and cleavage products. Cleavage of 80‐kDa E‐cadherin was uniquely induced by E2 in ovariectomized adult and immature mice; P4 was without effect. The cleavage of E‐cadherin correlated with increased basal accumulation of E‐cadherin antigen in estrous and E2‐injected mice and a loss of both basal and lateral antigen at 4.5 days of pregnancy. Only the E‐cadherin antigen within junctional complexes appeared unaffected. The data are consistent with the hypothesis that the cyclic and pregnancy‐dependent disruption of uterine epithelial integrity are promoted by E2‐dependent modification of E‐cadherin, including its extracellular cleavage.

Response ofNp mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts

TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned byNp, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10−4 weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callus-inducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constitutent(s) was stable and nonpolar. Thus, theNp allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil.

A comparison of developmental changes in surface charge in mouse blastocysts and uterine epithelium using DEAE beads and dextran sulfate in vitro

The ability of 3.5-day mouse blastocysts and vesicles prepared from maternal uterine epithelium to adhere to surfaces by charge interactions was compared by observing their adhesion to DEAE-Sephadex beads in the presence of increasing concentrations of dextran sulfate. The adhesion frequency for both the blastocysts and epithelium declined in a manner suggesting that predominantly ionic sites were being titrated, but differences between the two tissues in characteristics of the titration curve and susceptibility to neuraminidase digestion indicated that nonionic interactions were relatively more important for blastocysts. Because the threshold concentration of dextran sulfate required to initiate displacement of uterine epithelium from the DEAE beads was at least 4X that required to initiate the displacement of blastocysts, we argue that the uterine epithelium had at least 4X more interactive charged groups on its surface than the blastocysts. These differences were even more pronounced 4.5 days after mating, a time when attachment to the uterine epithelium is normally first seen in vivo. Blastocysts isolated at this time showed a marked increase in resistance to polyanion competition, but the epithelium showed a nearly 50% decline in surface negative charge that was not compensated by nonionic mechanisms. These observations support the conclusion that the initial adhesion of blastocysts in vivo is accompanied by a reduction in negativity of the uterine epithelial surface and by the formation of new trophoblast cell surfaces that adhere by nonelectrostatic interactions.

An In Vitro Model for Studying Interactions Between Mouse Trophoblast and Uterine Epithelial Cells

Even a cursory examination of the published attempts to devise an in vitro implantation system for mammalian embryos reveals that there have been two distinct approaches (Table I). One approach involves the use of complex tissues to mimic as closely as possible the natural conditions of the embryo or invasive cells. The other approach uses single cells in order to identify specific cellular and molecular events.

Specific stimulation of basal lamina heparan sulfate proteoglycan in mouse uterine epithelium by matrigel and by transforming growth factor-β1

SummaryThe basal lamina of differentiated epithelium normally turns over only slowly unless stimulated by tissue repair and growth. We show here that one mechanism of this stimulation, as modeled by basal lamina proteoglycan synthesis, may be the release of basal lamina-bound transforming growth factor (TGF-β). A large heparan sulfate proteoglycan (HSPG, 0.2Kav on Sepharose CL-4B) that was extractable from mouse uterine epithelium with 4M guanidine-HCl or 1M KCl was recognized by a specific monoclonal antibody to the basal lamina HSPG, perlecan. This HSPG was metabolically inactive with respect to [35S]-sulfate labeling in pieces of whole uterus during 4 h of culture, but it was labeled in isolated cells under the same conditions, provided that the cells had been cultured at least 6 to 12 h before labeling. The rate of labeling was then constant during at least 4 days in culture in serum-containing medium. Cultures on Matrigel showed an enhanced [35S]-sulfate labeling specifically in the 0.2Kav HSPG fraction. Partial stimulation was obtained with a serum-free medium extract of Matrigel, which fractionated on Sephadex G-50 in two components; a major one >30 kDa and the other at about 15 to 25 kDa. The specific stimulation was mimicked by the addition of 10 ng/ml of TGF-β1, but there was no specific stimulation by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulinlike growth factor-1 (IGF-1), or interleukin-1 (IL-1). TGF-β1 was identified as a 12.5 kDa monomer in thiol-reduced Matrigel and Matrigel extracts by polyclonal blocking antibodies on transblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Failure of excess amounts of these antibodies to block Matrigel-stimulated basal lamina HSPG synthesis indicates that TGF-β1 may be only one component of Matrigel that is important in stimulating basal lamina HSPG synthesis in culture. We suggest that in vivo TGF-β1 is bound to macromolecular components of mouse uterine epithelial basal lamina, where it may be sequestered until microenvironmental changes make it available to promote basal lamina HSPG synthesis.

In Vitro Analysis of Epithelial Surface Changes During Implantation

Blastocyst implantation involves interaction between two independently controlled yet highly interdependent systems, the embryo and its maternal environment. In rodents the preimplantation blastocyst becomes closely surrounded by uterine epithelium in an “implantation chamber” (Enders, 1975), which precedes the first irreversible interaction between the embryo and uterus — adhesion between the apical surfaces of the first embryonic epithelium (trophectoderm) and the uterine epithelium (“1” in Figure 1). In mice, adhesion occurs about 100 hours after the morning in which the coital vaginal plug is discovered (Potts, 1968). In rats this is brought about by a nidatory surge of estradiol, accompanying increasing levels of progesterone (Psychoyos, 1973). There is no clear separation in time between the appearance of apical adhesion and the initiation of basolateral changes, which occur between about 3.5 and 4.5 days after mating (“2” in Figure 1). These changes are characterized by a loosening of lateral cell associations, involving ultimately the junctional complexes (“3” in Figure 1) and a separation of the epithelium from its basal lamina (Schlafke et al., 1985), preparatory to its subsequent sloughing and cell death (Parr et al., 1987).