Isolation and characterization of a plasma membrane fraction derived from the luminal surface of the pig uterus during the estrous cycle and early pregnancy.
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abstract
A plasma membrane-enriched fraction was isolated from the uterine epithelium of the pig at different times during the estrous cycle and early pregnancy as well as at Day 60 of pseudopregnancy. This preparation was 10-50-fold enriched in certain plasma membrane marker enzymes relative to the homogenate, including the brush border enzymes 5'-nucleotidase, leucine aminopeptidase and alkaline phosphatase. There was also a 27-fold increase in specific 125I-radioactivity associated with the membrane preparation after introduction of [125I]-concanavalin A into the uterine lumen to bind to exposed glycoproteins prior to homogenization, and a 10-26-fold enrichment of 125I or 3H after the covalent attachment of 125I or 3H to surface-exposed components by the lactoperoxidase or periodate-[3H]-KBH4 techniques, respectively. The membrane polypeptides of this fraction were then solubilized in alkaline urea solution and analyzed by two-dimensional polyacrylamide gel electrophoresis. A large number of polypeptides were detected by Coomassie blue staining. Glycoprotein composition was followed by staining such gels with several [125I]-lectins with different carbohydrate group specificities, such as concanavalin A (-mannosyl units) and ricin I (-galactosyl units). In each case the patterns were shown to be highly complex. However neither the glycoprotein nor polypeptide make up of the membranes showed marked changes during either the estrous cycle or early pregnancy, even after the spread of the chorioallantois over the uterine surface. Lactoperoxidase-catalyzed iodination of the surface membranes was inefficient and failed to incorporate isotope into any of the major polypeptides or glycoproteins. Labeling was confined to a few minor components. It appears that the surface epithelium is efficiently protected from direct exposure to reagents introduced into the uterine lumen and shows little change in composition either in response to alterations in steroid hormone levels or during pregnancy.
