Electron microscopy of cultured mammalian lenses. II. Changes preceding and accompanying insulin-induced mitosis

Abstract

Morphological changes preceding insulin-induced mitosis in cultured rabbit lenses were characterized. The fine structure of these lenses was compared with that of lenses exposed to culture conditions in which a mitotic response failed to materialize. Lenses were cultured in: (1) medium KEI-4, i.e., a completely defined medium, which retains the central epithelium in a nonproliferating state, (2) KEI-4 plus insulin, and (3) KEI-4 plus insulin, theophylline, and dibutyryl adenosine 3':5'-cyclic monophosphate (I-DBcAMP-T). The insulin-induced mitotic response was inhibited by dibutyryl cAMP and theophylline or papaverine or 8-bromoadenosine cAMP. At one hour, lenses cultured in KEI-4 alone or in KEI-4-I-DBcAMP-T showed a moderate increase in free ribosomes relative to zero hour controls. In contrast, lenses exposed to KEI-4-insulin exhibited a pronounced increase in the number of free ribosomes and had prominent nucleoli. An increase in the degree of folding of the cell membrane was detected in all lenses at three hours but was decidedly more prominent in lenses cultured in KEI-4-insulin. At seven hours, lenses exposed to KEI-4-insulin showed an increase in the size and apparent number of mitochondria. At all intervals, ribosomes were more numerous in lenses cultured in KEI-4-insulin. Lenses cultured in medium KEI-4 alone for 24 and 52 hours displayed a morphology ostensibly similar to that of zero hour controls. At 52 hours the epithelial cells of lenses exposed to insulin, DBcAMP and theophylline were multilayered and markedly elongated. The increase in ribosomes (one hour), changes in the cell membrane (three hours), and the heightened mitochondria profile all precede the initiation of DNA synthesis and mitosis which commenced at 24 and 40 hours, respectively.