Germ cell-sertoli cell interactions: Analysis of the biosynthesis and secretion of cyclic protein-2

Abstract

Cyclic protein-2 (CP-2) is secreted in vitro in substantial amounts by mature rat Sertoli cells in intact Stage VI and Stage VII seminiferous tubules. This stage-dependent secretion has led us to postulate that the biosynthesis of this molecule is stimulated by germ cells at a specific state of development. In order to explore this hypothesis and to examine the steps in CP-2's biosynthesis, we generated a polyclonal antiserum against this protein and used it to analyze the biosynthesis and secretion of CP-2. Analysis of the steps in the biosynthesis of CP-2 indicated that its polypeptide core represented most if not all of the translation product of the CP-2 mRNA and that a single asparginelinked oligosaccharide became attached to this core. Analysis of the rate of biosynthesis of CP-2 at specific stages of the cycle of the seminiferous epithelium was also conducted. Two-millimeter segments of tubules at Stages II, VI, VIIa, b, VIII, and XII were cultured for 1 hr in the presence of [ 35S]methionine and radiolabeled CP-2 immunoprecipitated from the tubules. Data ( 35S-CP-2 synthesized per hour) demonstrated that the rate of CP-2's biosynthesis increased 9-fold from Stage II to Stages VI and VIIa, b and then decreased 13-fold by Stage XII. To determine whether these rates of biosynthesis were identical to the rates of secretion, tubules were cultured for 17 hr with [ 35S]methionine, CP-2 was immunoprecipitated from the culture medium and data were expressed as 35S-CP-2 secreted per hour. This analysis demonstrated that the rate of secretion of CP-2 varied in the same stage-specific manner as its rate of synthesis. However, at each stage, the apparent rate of biosynthesis of the molecule exceeded its apparent rate of secretion. In order to explain this observation, we analyzed the rate of export of newly synthesized CP-2 out of the tubules. This demonstrated that quantitative export of the protein into culture medium required at least 17 hr. This period of time was most likely due to the retention of the protein within the tubular lumen, since primary cultures of Sertoli cells were shown to rapidly secrete newly synthesized CP-2. We, therefore, concluded that CP-2 was biosynthesized in a stage-dependent manner and that all CP-2 was secreted.