Influence of chromatographic fractions of extracts derived from bovine neural retina on newt ( Notophthalmus viridescens ) lens regeneration in vitro

Abstract

Removal of the ocular lens in adult newts ( Notophthalmus viridescens ) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2 :247–283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2 :247–283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131 :151–172, 1958; Reyer, Dev. Biol. 14 :214–225, 1966) and in vitro (Yamada et al., Differentiation 1 :65–82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240 :343–351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67 :265–269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19 :923, 1979; Connelly, Differentiation 16 :85–91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230 :485–490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.