In vivo modulation of proliferating cell nuclear antigen in growth plate chondrocytes from normal, hypophysectomized, growth hormone-treated hypophysectomized rats: a comparative immunohistochemical study with image analysis.

Growth hormone (GH) regulates the proliferation and maturation of chondrocytes in the epiphyseal growth plate, in which a temporal dimension is superimposed on the septal organization of the tissue. In this study we investigated the in vivo effects of hypophysectomy (Hypox) and injection of GH into Hypox animals (Hypox + GH) on the proliferative activity of the growth plate chondrocytes. We assessed the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in paraffin-embedded tissues, using monoclonal antibody PC 10 against PCNA combined with immunogold-silver staining. We subjected the immunostained sections to computer-based image analysis by ACAS 570 interactive laser cytometry employing a conventional microscopic light source. Hypox was carried out on 20 rats at 8 weeks of age, half of which received a hypodermic injection of human GH at a dose of 1 IU/kg twice a day for 1 week after the operation. Another group of five rats of the same age were used as normal controls. In normal rats, a distinct PCNA immunoreaction was observed in the proliferative zone, whereas a remarkable diminution of the number of immunoreactive cells in this zone was apparent in Hypox animals. On the other hand, marked hyperplasia of PCNA-positive cells was seen in the proliferative zone of the Hypox + GH rat growth plate. The immunoreactive cells of this zone in Hypox + GH animals exhibited increased nuclear size and staining intensity of PCNA compared with those in normal and Hypox rats. These findings were further confirmed by computer-based image analysis of the specimens in terms of cell integrated value, area, perimeter, and shape factor. These different patterns of PCNA expression may imply that GH specifically promotes the proliferation phase of the chondrocytes in the proliferative zone. The data also suggest that GH influences not only cell replication activity but also cell kinetics of chondrocytes in the growth plate during their lifespan.