Metabolic changes in the poly(ADP-ribosyl)ation pathway of differentiatingrat germinal cells

Endogenous levels of poly(ADP-ribose) and beta NAD(+) have been determined in rat male germinal cells at different stages of differentiation. The leve ls of both metabolites decreased progressively from primary spermatocytes t o secondary spermatocytes and especially in spermatids, We have also determ ined the size and complexity of the ADP-ribose polymers synthesized in perm eabilized germ cells. Polymers of different chain length and complexity wer e observed in cells incubated with different concentrations of [P-32]beta N AD(+); short polymers characterized primary spermatocytes incubated with lo w beta NAD(+) concentration. In all cell fractions, polymers of over 20 res idues in size were observed at high beta NAD(+) levels. Long polymers were associated with the sulfuric acid-insoluble proteins (nonhistone proteins s uch as PARP itself). By contrast, oligomers of 20 ADP-ribose units or less were found in the sulfuric acid-soluble proteins (histone proteins). We hav e also identified the main ADP-ribose protein accepters formed in each cell type. In all cells examined, PARP appears to be extensively automodified, However, by far, the H1t variant of histone H1 appeared to be the preferred ADP-ribose target among the acid-soluble proteins separated by reverse-pha se HPLC, Therefore, we conclude that an active protein-poly(ADP-ribosyl)ati on system is concentrated in primary spermatocytes, based on a high level o f PARP automodification accompanied by the preferential heteromodification of the histone H1 variant specifically expressed in the cells undergoing th e pachytene phase of the meiotic division. (C) 2000 Academic Press.