Gamma crystallins in Wolffian lens regeneration demonstrated by immunofluorescence

Abstract

A fraction containing γ crystallins was isolated from the newt lens, and an antiserum against the fraction was prepared. Tests with immunoelectrophoresis and immunodiffusion indicated that the fraction was not contaminated with other crystallins and that the antiserum reacted with γ crystallins, but not with other crystallins. An immunofluorescent reagent was prepared with this antiserum and used to detect γ crystallins. In the normal lens the reaction was positive in the fiber area but not in the lens epithelium. With this immunofluorescent reagent, appearance and distribution of γ crystallins were studied in Wolffian lens regeneration of adult Triturus viridescens. The γ-crystallin reaction was negative in the regenerates of stages I–IV, as well as in the normal iris epithelium. The reaction became positive in a small number of cells located in the internal wall of lens vesicle at stage V. Subsequently, increase was observed in the number of positive cells and in intensity of immunofluorescence in each cell. All cells which could be recognized as primary lens fiber cells were positive. Later than stage IX, when the secondary lens fiber cells were formed, they all become positive. In all fiber cells, γ crystallins were detected in the nucleus as well as in the cytoplasm. No immunofluorescence was observed in the external layer of lens vesicle or in the lens epithelium of the growing lens. It was concluded that in Wolffian lens regeneration γ crystallins became detectable after the cells entered the terminal cell cycle and were accumulated in the cells during lens-fiber differentiation.