STUDIES ON THE ONTOGENY OF FETAL HEPATOCYTE (FH) PROLIFERATION DURING LATE GESTATION IN THE RAT. † 341

Abstract

We have found that late gestation rat FHs can proliferate in vitro in the absence of added mitogens or serum. However, this capacity varies with the gestational age at which the FHs are isolated: A gradual decline occurs as term approaches such that term FHs are non-proliferative in culture under these mitogen-free conditions. The present studies were undertaken to characterize the FH growth arrest which occurs at term. Primary cultures of FHs from day 19 of gestation (E19) showed a gradual decline in DNA synthesis (3H-thymidine incorporation) from very high levels during the first 24 hr in culture to low levels by 72 hr. In contrast, term (E21) FHs synthesized little DNA over the first 24 hr and showed a marked increase by 48 to 72 hr. In addition, E21 FH DNA synthesis was potentiated by Transforming Growth Factor α (TGFα) whereas E19 FH DNA synthesis was not. These findings correlated with immunocytochemical staining for Proliferating Cell Nuclear Antigen (PCNA). E19 FHs were PCNA positive immediately after plating and this persisted over 48 hr. E21 FHs showed a synchronized pattern of PCNA staining which was cytoplasmic at 12 hr and nucleolar at 24 to 48 hr. Intense, diffuse nuclear expression appeared for a brief period at 60 hr. Additional studies showed that: (a) E21 fetal liver has a high level of c-myc expression even though the cells are not proliferating; (b) The mitogenic Jun kinase signaling pathway is equally active at E19 and E21; (c) The cell cycle kinase, cdc2, is expressed at term but its inactive forms predominate. These results indicate that E21 FHs are growth arrested in vivo and that culturing releases them from inhibitory influences, resulting in synchronized proliferation in vitro.