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

Abstract

The objectives of this study were to identify, characterize and examine differences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippiensis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state. After capture, alligators from lakes in north central Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of the tract dissected out and partitioned for explant culture. Analysis of the biosynthetic activity indicated regional variations within the tract, differences among reproductive groups and region by status interactions. When oviductal regions were considered regardless of reproductive status, the greatest incorporation of [3H]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 biosynthetic activity of the anterior and posterior portion of the tract in non-reproductive alligators was not different, whereas that of the posterior region of the reproductive group (vitellogenic, gravid, post-clutch) was significantly lower than that of the anterior region. This finding indicates that regulation of protein synthesis and secretion by the non-reproductive alligator tract is different from that in the tract of the reproductive group. Explant-conditioned media were analysed by one-dimensional and two-dimensional SDS-PAGE and fluorography. Sixteen major proteins in culture media were identified as de novo synthesized, by relative molecular weight, by isoelectric point and by differences in distribution determined for reproductive status and oviductal region. Six proteins were examined by N-terminal amino acid microsequence analysis. On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M(r) 55,000, basic), found in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen. Four proteins (aP4-aP7) were sequenced and shown to be significantly related to immunoglobulin heavy chains from several species. This study demonstrated that a large number of proteins are synthesized de novo and released by the female alligator reproductive tract and that there are biosynthetic activity differences by reproductive status and region. Six proteins have been identified, several of which may be incorporated into alligator egg albumen and some of which appear to be different from proteins found in the egg albumens of other species.