Changes of hippocampal protein levels during postnatal brain development in the rat

Information on postnatal brain protein expression is very limited, and we therefore compared hippocampal protein levels in rat hippocampus at different developmental time points using two-dimensional gel electrophoresis followed by mass spectrometrical protein identification and specific software for quantification. Proteins from several cascades as e.g., antioxidant, metabolic, cytoskeleton, proteasomal, and chaperone pathways were developmentally regulated, which is relevant for design and interpretation of protein chemical studies in the mammalian brain.     

Response of the epididymis, ductus deferens & accessory glands of the castrated prepubertal rhesus monkey to exogenous administration of testosterone or 5alpha-dihydrotestosterone.

The response of the epididymis, ductus deferens, and accessory glands of the castrated prepubertal rhesus monkey to exogenous administration of testosterone or 5alpha-dihydrotestosterone (DHT) was investigated. 200 or 800 mcg of either steroid/day were administered for 60 days beginning on the day after castration. Castration caused a marked regression of the weight of and secretory function of the reproductive organs; testosterone/DHT stimulated their growth and secretory activity which were maintained at the level of the controls. The weight of the caput epididymides however, was unaffected by testosterone but was stimulated by DHT. DHT caused a greater stimulation of the growth and secretory activity of the reproductive organs than testosterone and also caused a hyperstimulation of secretion by the seminal vesicles. The data, analyzed statistically, show that the accessory organs of the prepubertal rhesus monkey are affected by castration and vary in their response to stimulation by exogenous androgens.     

Subcortical projections of the visual cortex in the adult albino rat and during postnatal development

We have investigated the subcortical projections of the rat striate cortex by using the silver-degeneration method and the HRP-technique too. Cortical lesions were made in 60 young animals (1, 4, 5, 6, 7, 10 and 14 days old) and in 6 adult rats. The terminal regions of projection occurred only ipsilateral to the lesions. After passing the internal capsule the degenerating pathway divides into 2 bundles. In the dorsal thalamus one of them runs in caudal direction. The other bundle turns ventrally, reaches the cerebral peduncle and terminates in the pons. The first fibre bundle terminates in the following structures: Nc. reticularis thalami, Nc. lateralis thalami, Nc. lateralis posterior thalami, Corpus geniculatum laterale, pars dorsalis (Cgld), Corpus geniculatum laterale, pars ventralis (Cglv), Nc. praetectalis anterior et posterior and Colliculus superior. The fibers of the second bundle innervate the Nc. lateralis pontis. Fibers from this bundle terminate probably in the Cglv and in the Zona incerta too. By using the HRP-technique it could be demonstrated that the axons terminating in the Cgld originate in layer VI of the area 17. In contrast, the projection to Cglv, Nc. lateralis posterior, Colliculus superior and Nc. lateralis pontis originates from pyramidal cells in layer V. The development of the projection in young animals indicates: Like in adults rats, terminal degeneration is present in all subcortical projection regions at postnatal day (PD) 10. At PD 4-7 we can observe heavily degenerating axons but the terminal degeneration is different. It is remarkable in the "visual" part of the reticular nucleus and iln the Cgld (decreasing from inside to outside). Only a weak terminal degeneration is visible in the pretectal region and in the superior colliculus. At PD 1 the trajectory of degenerating fibres is clearly visible. Signs of terminal degeneration can only be found in the reticular nucleus. It is discussed whether the date of generation of the cortical neurons and the time of the differentiation of the cortical layers is of importance for the time of innervation of the subcortical projection fields. The question when the axons arrive at their terminal region and form there synaptic contacts has not yet been exactly answered. To solve this problem electronmicroscopic investigations are necessary.     

Histological and histochemical study of rat salivary glands during their postnatal development

The postnatal development of the three major salivary glands (parotid, submaxillary and sublingual) was comparatively followed up from the histological viewpoint and in relation with some histochemical reactions. The sublingual gland presented a well developed cytomorphological structure at birth, whereas the parotid and the submaxillary one, immature at birth, gradually reached the overall appearance of adult glands, the former at 5 - 6 weeks, the latter at 8 weeks. In relation with the product secreted, it is already from birth that the parotid and the submaxillary glands presented negative reactions for mucosubstances and positive ones for revealing the protein-bound groups. The sublingual gland exhibited from the first postnatal 24 hrs positive reactions for revealing mucosubstances at the level of glandular secretory glands.     

Insulin-regulated protein kinases during postnatal development of rat heart

The control of glucose uptake and glycogen metabolism by insulin in target organs is in part mediated through the regulation of protein-serine/ threonine kinases. In this study, the expression and phosphotransferase activity levels of some of these kinases in rat heart ventricle were measured to investigate whether they might mediate the shift in the energy dependency of the developing heart from glycogen to fatty acids. Following tail-vein injection of overnight fasted adult rats with 2 U of insulin per kg body weight, protein kinase B (PKB), the 70-kDa ribosomal S6 kinase (S6K), and casein kinase 2 (CK2) were activated (30–600%), whereas the MAP/ extracellular regulated kinases (ERK)1 and ERK2 were not stimulated under these conditions. When the expression levels of the insulin-activated kinases were probed with specific antibodies in ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats, phosphatidylinositol 3-kinase (PI3K), PKB, S6K, and CK2 were downregulated (40–60%) with age. By contrast, ventricular glycogen synthase kinase-3β (GSK3β) protein levels were maintained during postnatal development. Similar findings were obtained when the expression of these kinases was investigated in freshly isolated ventricular myocytes, where they were detected predominantly in the cytosolic fraction of the myocytes. Compared to other adult rat tissues such as brain and liver, the levels of PI3K, PKB, S6K, and GSK3β were relatively low in the heart. Even though CK2 protein and activity levels were reduced by ∼60% in 365 day as compared to 1-day-old rats, expression of CK2 in the adult heart was as high as detected in any of the other rat tissues. The high basal activities of CK2 in early neonatal heart may be associated with the proliferating state of myocytes. J. Cell. Biochem. 71:328–339, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of estramustine-binding protein during postnatal development of rat brain

Estramustine-binding protein (EMBP) is expressed in several types of brain tumors, such as astrocytoma, ependymoma, and meningioma. It binds the cytotoxic drug estramustine with high affinity and is suggested to cause accumulation of the drug in EMBP-expressing tumor cells. In this study, the spatial distribution of EMBP in normal rat brain was studied with immunohistochemistry. Brains from male and female rats of different ages were used. EMBP was found in the cytoplasm of ependymal cells, in the leptomeninges, mainly the arachnoid, and in scattered neurons. Moreover, staining was seen in nuclei of choroid plexus cells, in the granular cell layer in the cerebellum, and in a few scattered endothelial cells. The nuclear staining was more frequent in younger animals. No obvious difference in EMBP expression between male and female rats was observed. The expression of EMBP in rat brain was confirmed with nested RT-PCR. Future studies are justified to elucidate the role of EMBP-like proteins in CNS and in brain tumors.     

Photoperiodic influence on the innervation of the ductus epididymidis and ductus deferens of the Djungarian hamster,Phodopus sungorus: electron-microscopic and biochemical results

Summary The influence of long (light: dark 168) and short (light:dark 816) photoperiods on the autonomic innervation of the ductus epididymidis and the ductus deferens of Phodopus sungorus was studied using electron microscopy with morphometric analyses, and biochemical methods. At short photoperiods, only the large smooth muscle cells in the ductus deferens became atrophic, the number of mainly adrenergic varicosities in the smooth muscle layer decreased, and the mean distance between varicosity and smooth muscle cells increased. The content of noradrenaline was determined by high-performance liquid chromatography with electrochemical detection. For the ductus deferens, the noradrenaline content was reduced at LD 816 to less than 10% of the initial value. Short photoperiods are proposed to influence only the adrenergic innervation of the large smooth muscle cells of the ductus deferens. These cells are believed to exert a trophic influence on their nerves.     

Metabolism of 3H-testosterone in epididymis & accessory glands of reproduction in the castrate hamster Mesocricetus auratus

The metabolism of tritiated-testosterone in epididymis and accessory glands of reproduction was studied in the castrate hamster, Mesocricetus auratus. The amount of tritium accumulating in the epididymis was similar to that in the accessory glands 60 minutes postinjection but was significantly higher (p .02-p .001) at 150 minutes. In all accessory organs the rate of tritiated testosterone was slow; marked quantities of testosterone and androstanediol were present but the accumulation of dihydrotestosterone (DHT) was relatively minimal at both time intervals studied. These results may indicate a simultaneous requirement of testosterone and androstanediol to mediate the action of androgens in the hamster while DHT may be of secondary importance.     

NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.     

Nucleolar and nucleoplasmic RNA polymerase activity in different regions of rat brain during postnatal development.

RNA polymerase activities of whole nuclei, of isolated and purified nucleoli and of the nucleoplasmic fractions obtained from cerebral hemispheres, cerebellum and brain stem of rat at different days of postnatal development have been determined. In the whole nuclei the fraction of RNA polymerase which is sensitive to alpha-amanitin, is strongly affected by salt concentration; at low ionic strength most of the activity is resistant to the drug while at high ionic strength the enzymatic activity shows a greater sensitivity to the drug. In isolated nucleoli RNA synthesis is not inhibited at all by alpha-amanitin. The biosynthesis of RNA, at low ionic strength, is inhibited by low doses of actinomycin D, whereas at high ionic strength it is remarkably inhibited only by higher doses of the drug. The sensitivity of the reaction to alpha-amanitin and actinomycin D provide good evidence that UTP or GTP incorporation into RNA in purified nuclei and nucleoli, is dependent on RNA polymerases acting on DNA template and is not dependent on homopolymer formation. These results show that in the whole brain nuclei at low ionic strength there is a preferential synthesis of rRNA, whereas at high ionic strength the synthesis of heterogenous RNA predominates. In isolated nucleoli the synthesis of RNA is restricted to rRNA.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Morphological study on the hypoplastic testis with aplasia of the epididymis, ductus deferens, and gland of the ductus deferens in the TW inbred rat

In the TW inbred rat, about 50% of the males show bilateral or unilateral testicular hypoplasia with aplasia of the ipsilateral epididymis, ductus deferens and gland of the ductus deferens. To investigate the pathogenesis of the testicular abnormality in the TW rats, the weight and morphology of the testes on the aplastic and normal side were studied between one week and one year of age. The weight of the testes on the affected side was greater than those on the normal side at four and five weeks. However, it rose to a plateau at six weeks and then remained at about one half to one third the weight of a normal testis. As for the testicular histology, there were no obvious changes from one day to three weeks of age. The diameter of the seminiferous tubules became larger and the number of germ cells decreased at four and five weeks. At six weeks, degeneration and loss of germ cells were observed and many multinucleated giant cells appeared. Thereafter, the loss of germ cells became more severe, and they eventually disappeared with increasing age, but Sertoli's cells continued to exist. In the interstitial area, edematous changes and proliferation of Leydig's cells were observed. The efferent duct of another strain, with normal testes, was ligated at three weeks of age, and changes of the testis after the operation were examined to investigate whether or not these anomalies of the TW strain were due to the absence of the accessories, which may block the excretion of the testicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)