RESUMO
Rabbits aged 1, 4, 10, 15, and 20 days, and 4 months were anesthetized and perfused with 4% formaldehyde. One eye of each rabbit was processed for paraffin embedding, while the other eye was embedded intact in methacrylate. Rabbits aged 1 and 15 days and 4 months were perfused with 2.5% glutaraldehyde, and the eyes were processed for Epon embedding. The paraffin sections were immunostained to allow detection of a high molecular weight cartilage matrix glycoprotein (CMGP), which is synthesized by the ciliary body and found in the vitreous in adult animals, using a specific mouse monoclonal antibody. CMGP was identified in the vitreous and in the inner layer of the ciliary epithelium only after the fifteenth day of life in amounts comparable to those detected in adult rabbits. Before this time immunostaining with the monoclonal antibody was seen only in the apical region of the inner ciliary epithelial cells. However, electron microscopic observations revealed that the cytoplasmic organelles responsible for the secretion of glycoproteins, i.e. the rough endoplasmic reticulum, Golgi apparatus, and vesicles, were present in the inner layer of ciliary epithelial cells as early as the first day of life. Anteroposterior sections of whole eyes embedded in methacrylate revealed a relatively dense meshwork of vitreous fibrils on the first day of life. The blood vessels were concentrated at the posterior region of the lens, and isolated cells were visible. The blood vessels were not seen after the age of 15 days, and the fiber meshwork and cells were inconspicuous by then.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas da Matriz Extracelular , Corpo Vítreo/ultraestrutura , Animais , Vasos Sanguíneos/ultraestrutura , Corpo Ciliar/ultraestrutura , Retículo Endoplasmático/química , Glicoproteínas/análise , Complexo de Golgi/química , Proteínas Matrilinas , Microscopia Eletrônica , Coelhos , Fatores de Tempo , Corpo Vítreo/química , Corpo Vítreo/crescimento & desenvolvimentoRESUMO
L-[3H]fucose was injected either intravitreally or intra-aqueously into adult rabbits which were killed at several time points after injection. SDS-polyacrylamide gel electrophoresis and fluorography of iris extracts revealed that most of the proteins are glycoproteins containing fucose residues. Autoradiography of semi-thin histologic sections demonstrated that glycoprotein synthesis was most prominent in the epithelium of the iris, while little protein synthesis was evident in the stroma of the iris. The results of these experiments indicated that the glycoproteins of the iris undergo renewal. The protein band pattern of the iris extracts was very similar to that of extracts of the ciliary body. The high-molecular-weight cartilage matrix glycoprotein (CMGP), an intrinsic component of the ciliary body, vitreous, and aqueous humor, was detected by immunohistologic studies only in the stroma of the iris. The results of immunohistochemical analyses of the eyes of young rabbits (1-21 days old), in addition to the autoradiographic findings, strongly suggest that CMGP is not an intrinsic glycoprotein of the iris stroma, at least in this species.
Assuntos
Proteínas do Olho/biossíntese , Glicoproteínas/biossíntese , Iris/metabolismo , Animais , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Epitélio/metabolismo , Fucose/metabolismo , Imuno-Histoquímica , Masculino , Coelhos , Fatores de Tempo , TrítioRESUMO
A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1-22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic cross-reactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.
Assuntos
Proteínas do Olho/análise , Corpo Vítreo/análise , Animais , Antígenos/análise , Autorradiografia , Corpo Ciliar/análise , Epitélio/análise , Feminino , Técnicas Imunoenzimáticas , Masculino , CoelhosRESUMO
L-3H-fucose was injected into the lateral cerebral ventricle of vasopressin-deficient Brattleboro and control Long-Evans rats which were subsequently killed at several time intervals after the injection. The hypothalamus and the neurohypophysis were processed for light- and electronmicroscopic radioautography. Other complementary experiments using immunocytochemical and enzyme-histochemical techniques were also undertaken. L-3H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus of supraoptic and paraventricular neurons, and later on labelled glycoproteins migrated to lysosomes and the plasma membrane surrounding the perikaryon. The Golgi apparatus of the vasopressin-deficient neurons remained heavily labelled as long as 3 days after injection, in sharp contrast with the normal neurons in which there was a remarkable decrease of label in the Golgi region between 4 and 24 h after the isotope administration. Labelled glycoproteins also migrated to the neurohypophysis and were mainly found in the axonal plasma membrane, vesicles and axoplasm. The renewal of glycoproteins in the neurohypophysis of Brattleboro rats was faster than in the normal rats and this was attributed to the lack of formation of products which are normally packaged in secretory granules in the perikaryon and released at the axon terminal in the neurohypophysis. Colchicine caused a disturbance in the topography of the organelles of the perikaryon and the most striking features were the displacement of Golgi stacks to the periphery of the perikaryon and an accumulation of mitochondria in this neuronal region. No secretory granules were observed in the vasopressin-deficient neurons of untreated or colchicine-treated Brattleboro rats. By contrast, secretory granules (most of them labelled with 3H-fucose) were concentrated in the perikaryon of colchicine-treated Long-Evans rats. In these rats, colchicine caused a severe block in the migration of 3H-fucose-labelled glycoproteins to the neurohypophysis, but this did not occur in the Brattleboro rats. The results of the experiments were interpreted in the light of the genetic defect known to occur in Brattleboro rats which causes the inability to produce vasopressin and also remarkable morphological and physiological changes in the affected neurons.