In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts.
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abstract
The objective of this study was to identify, characterize, and examine oviductal secretory proteins (OSP) synthesized de novo by whole oviduct (WO), ampulla (A), and isthmic (I) tissue from ovariectomized (OVX), corn oil (CO)-, estrogen (E)-, progesterone (P)-, and E + P-treated gilts. Oviducts were collected from OVX gilts after CO, E, P, or E + P treatment for 11 consecutive days and tissue was incubated with 3H-leucine (3H-leu). Rates of 3H-leu incorporation into nondialyzable macromolecules by WO explants were greater (P less than 0.01) with E- compared to CO-, P-, or E + P-treated gilts and greater (P less than 0.05) by A explants with E- compared to CO-, P-, or E + P-treated gilts. An effect of location was noted, with A having a greater (P less than 0.01) rate of incorporation than WO or I. Conditioned culture medium was analyzed by one (1D)- and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Analyses by 1D-SDS-PAGE revealed three major E-dependent bands (335,000, 100,000, and 80,000 M(r)) in WO and A, and one (335,000 M(r)) in the I. A 20,000 M(r) band found in A was inhibited by E, while a 60,000 M(r) band found in the A was induced by P. Analyses by 2D-SDS-PAGE resolved major E-dependent bands 2 (100,000 M(r)) and 3 (80,000 M(r)) into basic and acidic 100,000 M(r) proteins and a 75,000-85,000 M(r) protein (pI less than 4), respectively, found in WO and A, but not in I. A basic 20,000 M(r) protein and an acidic 45,000 M(r) complex, both found in A, were inhibited by E. Gel filtration of culture medium revealed a high M(r) fraction (greater than 2 x 10(6)) that was induced by E and was 6.8-fold greater in medium from A than from I. This study clearly demonstrates that 1) WO and A tissue from E-treated gilts de novo synthesize and secrete three major proteins (basic 100,000, acidic 100,000, and 75,000-85,000 M(r)); 2) these E-dependent proteins are not found in I or with other treatment; 3) several protein complexes synthesized by A are inhibited by E treatment; and 4) a high M(r) fraction, produced primarily in the A, is induced or amplified by E.
