Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis

The objectives of this study were to identify, characterize and examine dif ferences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippie nsis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state. After capture, alligators from lakes in north cent ral Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of t he tract dissected out and partitioned for explant culture. Analysis of the biosynthetic activity indicated regional variations within the tract, diff erences among reproductive groups and region by status interactions. When o viductal regions were considered regardless of reproductive status, the gre atest incorporation of [H-3]Leu into secreted nondialysable macromolecules was by the anterior and posterior infundibulum and oviductal tube compared with the transition zone and the uterus. When status was included, the bios ynthetic activity of the anterior and posterior portion of the tract in non -reproductive alligators was not different, whereas that of the posterior r egion of the reproductive group (vitellogenic, gravid, post-clutch) was sig nificantly lower than that of the anterior region. This finding indicates t hat regulation of protein synthesis and secretion by the non-reproductive a lligator tract is different from that in the tract of the reproductive grou p. Explant-conditioned media were analysed by one-dimensional and two-dimen sional SDS-PAGE and fluorography. Sixteen major proteins in culture media w ere identified as de novo synthesized, by relative molecular weight, by iso electric point and by differences in distribution determined for reproducti ve status and oviductal region. Six proteins were examined by N-terminal am ino acid microsequence analysis. On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M-r 55000, basic), f ound in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen. Four proteins (a P4-aP7) were sequenced and shown to be significantly related to immunoglobu lin heavy chains from several species. This study demonstrated that a large number of proteins are synthesized de novo and released by the female alli gator reproductive tract and that there are biosynthetic activity differenc es by reproductive status and region. Six proteins have been identified, se veral of which may be incorporated into alligator egg albumen and some of w hich appear to be different from proteins found in the egg albumens of othe r species.