GH is known to increase the formation of bone and hard tissues of the tooth (dentine, cementum, and enamel), as do bone morphogenetic proteins. GH receptors are expressed in these tissues and could mediate local growth responses. Here we report that both GH and insulin-like growth factor I (IGF-I) are able to increase expression of bone morphogenetic protein-2 and -4 messenger RNAs 4- to 5-fold in human dental pulp fibroblasts in vitro. Induction was seen at physiological concentrations of hormone (25-100 ng/ml GH; 50-200 ng/ml IGF-I) and reached a maximum at 4-8 h. Immunoblot analysis demonstrated that the increase in messenger RNAs resulted in an increase in expressed protein. Anti-IGF-I inhibition experiments indicate that GH is able to induce the response without a requirement for local IGF-I production. These results raise the possibility that bone morphogenetic proteins mediate the local osteogenic actions of GH and IGF-I, and lend support to the view that GH can act through the mediation of factors other than IGF-I. These factors may combine with IGF-I in different tissues to enhance GH action and specificity.

To document the effect of hypophysectomy and growth hormone replacement on the ultrastructure of cementogenesis in the developing rat third molar, 12 female Wistar rats were randomly allocated to normal control, hypophysectomized or hypophysectomized plus human growth hormone (for 10 days) treatment groups. The results of this study by electron and light microscopy and morphometry have shown that qualitative and quantitative changes occur in the organelles of cementoblasts forming cellular cementum as a result of hypophysectomy and growth hormone replacement. After hypophysectomy, the changes of less prominent nucleoli and nuclear pores, less prominent Golgi apparatuses and decreased endoplasmic reticulum can be interpreted as diminished cementum matrix biosynthesis--an interpretation that can be confirmed morphometrically by less cellular cementum formation. Growth hormone replacement for 10 days reactivates protein synthesis and cementogenesis as evidenced by ultrastructural changes in cementoblasts and a greater production of cementum.

Dental organs of incisors from normal, dwarf and growth hormone-treated dwarf rats were analysed histochemically using a panel of lectins. A distinctive pattern of differential staining was obtained with Helix pomatia agglutinin, a lectin specific for N-acetylgalactosamine. In Bouin's perfused and paraffin-embedded undecalcified tissues from normal rats, reaction product for N-acetylgalactosamine was visible in the odontogenic cells and some extracellular matrices. In the growth hormone-deficient dwarf rats, the N-acetylgalactosamine reaction was consistently minimal in the odontoblasts, predentin, cementoblasts, cementoid, osteoblasts and osteoid matrices, although the staining of ameloblasts and osteoclasts was similar to normal. Administration of growth hormone to dwarf rats for six days (66 micrograms/100 g rat b.i.d.) restored the reaction for N-acetylgalactosamine in the affected matrices. Thus, an N-acetylgalactosamine rich matric component is differentially expressed during odontogensis. Growth hormone may regulate this component in these matrices, which may be a proteoglycan or a glycoprotein, essential for normal growth of the teeth.

Nucleolar organizers are major sites of ribosomal RNA synthesis and provide an index of transcriptional activity. In order to further define growth hormone actions on nucleolar organizer regions in tooth forming cells, hypophysectomized rats treated with growth hormone for 4 and 24 h, hypophysectomized and sham-operated animals were used. After demineralization and standard paraffin embedding, longitudinal sections of maxillary incisors were stained by a silver stain technique to reveal nucleolar organizer regions. The area of these regions per nucleus was measured using a modified microdensitometer. Analyses of variance of the resulting data showed that preameloblasts and preodontoblasts have greater silver stained nucleolar organizer region values than ameloblasts and odontoblasts. Hypophysectomy reduced and growth hormone partly restored the level of nucleolar organizer regions in preameloblasts and preodontoblasts, but not in mature ameloblasts or odontoblasts. In the case of the younger preameloblasts and preodontoblasts, the effect of growth hormone was seen within 4 h of growth hormone injection. In conclusion, rRNA synthesis, as revealed by the specific silver staining of nucleolar organizer regions in tooth forming cells, appears to be regulated by growth hormone over a relatively short time frame.

KEY W O R D S . Microdensitometry, microspectrophoto-microscopy, nucleolar organizer regions. S U M M A R YNucleolar organizer regions (NORs) are major sites of ribosomal RNA synthesis, providing an index of transcriptional activity and possibly determining the malignant status of cells. Difficulties lie in quantifying them. This study reports a methodology to assist in the standardization of the assessment of interphase NORs. Regenerating hepatocytes, which have increased rRNA synthesis, were chosen as a model to test automated microdensitometry for silver-stained NORs. Quantification employed a microspectrophoto-microscope as a microdensitometer. Significant differences in silver-stained NORs in hepatocytes were recorded among treatment/fixative groups. As the quantitative method avoids subjective observer error and thus improves the accuracy of measurement, it would potentially have routine application to diagnostic pathology.