The ultrastructure of the developing neural lobe

Summary The ultrastructure of the developing neural lobe of rats was studied. The results revealed three periods in its development. The first period lasts till the 17th day of fetal life. At its beginning the anlage of the neural lobe is formed as a mass of cells very similar in appearance and in connection with the subependymal cells of the future median eminence. During the first period the cells of the anlage differentiate into pituicytes, and the penetration of the first nerve fibres and blood vessels among them is seen. The second period is from the 18th day of fetal life till one month after birth. At its beginning the first signs of neurosecretory activity were detected. During the period increasing numbers of neurosecretory fibres penetrate into the neural lobe, and the pituicytes show morphological signs characteristic of active cells. An increase in the functional activity of the neural lobe is also detected. The third period is from the end of the 1st till the end of the third month. During this period the development of the neural lobe proceeds and at the end it has the appearance of the adult gland. During this period the pituicytes gradually lose the signs of activity and at the end of the period they look like those observed in adult animals. Considering the results from the study, together with some data from previous investigations it is suggested that the pituicytes exert some stimulating and regulative influences on the process of neurosecretion in the neural lobe.Research fellow of the Alexander von Humboldt Foundation.     

Morphogenesis of structural elements of the cervical spine in the white rat in prenatal ontogeny

Embryonal development of the spinal column cervical part has been studied in 100 series of sagittal, transversal, frontal sections; time of the main structural elements anlagen (vertebral bodies, arches, joints, ligaments) is noted. The prenatal development of the spinal column cervical part is divided into 3 stages--mesenchymal, cartilagenous, osseous. The first stage lasts up to 16 days of development; during this period anlagen of vertebral bodies, arches, joints, ligaments are formed. The second stage--cartilagenous; mesenchyma is substituted for cartilagenous tissue, cartilagenous cells are differentiated. This stage lasts from the 16th up to the 18th day of embryogenesis. The third stage--osseous--lasts from the 18th up to the 21st day of embryogenesis. During this period structures of the spinal column cervical part acquire a definitive form, the cartilagenous tissue is substituted for the osseous one.     

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.     

Early secretory activity in the hypothalamic nuclei and neurohypophysis in the rat; determined by selective staining

Visualization of stainable material in the neural lobe of the rat provided the most reliable index of the age at which secretory activity can first be recognized, though preceded by both hypothalamic synthesis and axonal transportation. A problem of interpretation was encountered in the neural lobes of fetal and infant animals, due to different staining responses obtained during this age period, to the two methods of staining employed; chrome alum hematoxylin-phloxin and aldehyde fuchsin after oxidation by either acidified potassium permanganate or performic acid. With aldehyde fuchsin the material of the neural lobe is stainable selectively from the eighteenth day of fetal life to adulthood. With hematoxylin phloxine the first staining response also occurred in the posterior lobe but much later, at the end of the first postnatal week. The staining situation in the pars neuralis has its counterpart in the differentiating hypothalamic nuclei; complicated by the differentiation of the supraoptic nuclei some days in advance of the paraventricular nuclei. After aldehyde fuchsin staining, evidences of neurosecretory activity were present in the perikarya of the supraoptic nuclei at birth, but mature neurons were rarely seen in the paraventricularis until at least 24 hours later. Nuclei of fetal hypothalami were not studied, but the demonstration of stainable material in the fetal neural lobes constitutes circumstantial evidence of functional competence of some neurons of either one or both types of nucleus, most likely the supraoptic.     

Cyto- and synaptogenesis in the arcuate nucleus of the rat hypothalamus during fetal and early postnatal life

Summary Morphogenesis of the arcuate nucleus of the rat from the 15th fetal day to the 6th postnatal day was investigated light and electron microscopically. The arcuate neurons exhibit a gradual development after the 15th fetal day. All cytoplasmic constituents are present in these nerve cells already during the last days of gestation. Nevertheless, they are not fully differentiated at birth. The first synapse-like structures (presynapses) were observed in 17 day-old, the first synapses in 18 day-old fetuses. During the early postnatal period the number of presynapses decreases, but at the same time there is a gradual increase in the number of the relatively mature synapses. This process starts already during the last days of prenatal life. Although all structural elements of the arcuate nucleus of the adult rat appear to be present at birth, the extent of the neuropil area and the number of the presynapses indicate that the arcuate nucleus is still in a fairly undeveloped stage during the first postnatal days.     

Regulation of fetal Na+/K+-ATPase in rat kidney by corticosteroids

The enzymatic differentiation of various tissues is under hormonal control in the perinatal period. Since the regulation of Na+/K+-ATPase has not been explored prenatally, the aim of this study was to determine the corticosteroid sensitivity of sodium pump maturation in the fetal period. Na+/K+-ATPase activity was both measured in kidney homogenates of fetal rats and localized by in-situ histochemistry. Sodium pump activity was first quantifiable on day 18 of fetal development as 1.4 +/- 0.17 mumol Pi/h per mg protein, and was increased 3.4-times by day 22 of gestation. While the Na+/K+-ATPase activity was the most intense in cortical tubules at an earlier fetal age (18th and 19th day), the reaction product in the medullary tubules increased with fetal age, becoming highly intense on the 21st and 22nd day of gestation. From the 18th to 21st day of fetal development homogenate Na+/K+-ATPase activity increased as a function of chronologic age. While mineralocorticoids were without any effect on Na+/K+-ATPase activity, on the last day of the fetal development, the glucocorticoid dexamethasone proved to be successful in stimulating enzyme activity in corticosteroid-suppressed animals. According to our results, glucocorticoid hormones seem to be operating as an endogenous driving force for sodium pump maturation at the end of fetal development.     

Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats

During the perinatal period, calcium metabolism is stressed. As intestinal Ca-binding protein is considered as a molecular expression of the hormonal effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), Ca-binding protin measurements may document the vitamin D roles during this period. We describe the variations of Ca-binding protein concentrations in the rat during the last 5 days of gestation, in the maternal duodenum, placentas, fetal membranes and fetal intestines. We also report intestinal Ca-binding protein changes from birth until weaning. The evolution of the maternal intestinal Ca-binding protein, which increases on day 19.5 of gestation, is consistent with that of calcium intestinal absorption and may be explained by increased 1,25(OH)2D3 production. Placental Ca-binding protein rises from day 17.5 until the end of gestation, and may be related to the profile of calcium transfer from mother to fetuses. It is noteworthy that the placental Ca-binding protein is predominantly found in the fetal part of the organ where materno-fetal exchanges occur. The yolk sac synthesizes substantial amounts of Ca-binding protein. In the fetal membranes, Ca-binding protein plateaus from day 17.5 until day 20.5 and decreases on day 21.5. The Ca-binding protein presence in the fetal placenta and in the yolk sac may suggest that these tissues are also targets for vitamin D. In the fetus the intestinal Ca-binding protein s is detected as early as day 17.5 of gestation and increases markedly during the last day of gestation. From birth and during the first 3 weeks of postnatal life, the intestinal Ca-binding protein concentration does not change. It undergoes a sharp rise just at the time of weaning. We have also shown that the specific distribution of Ca-binding protein along the intestine is acquired during intrauterine life and does not change with sucking or weaning. The two main changes of intestinal Ca-binding protein, observed just before birth and at weaning, may reflect the intestinal maturation and/or variations in vitamin D metabolism.     

Ascorbic Acid in Fetal Rat Brain

Ascorbic acid in fetal rat brain increases from 374 mg/g on the 15th day of gestation to 710 mg/g by the 20th day and remains at that level until birth. There is an 18% drop from this plateau after birth.     

Neuronal maturation in an experimental model of brain tissue heterotopia in the lung

Neural maturation involves diverse interaction and signaling mechanisms that are essential to the development of the nervous system. However, little is known about the development of neurons in heterotopic brain tissue in the lung, a rare abnormality observed in malformed babies and fetuses. The aim of this study was to identify the neurons and to investigate their maturation in experimental brain tissue heterotopia during fetal and neonatal periods. The fetuses from 24 pregnant female Swiss mice were used to induce brain tissue heterotopia on the 15th gestational day. Briefly, the brain of one fetus of each dam was extracted, disaggregated, and injected into the right hemithorax of siblings. Six of these fetuses with pulmonary brain tissue implantation were collected on the 18th gestational day (group E18), and six others were collected on the 8th postnatal day (group P8). The brain of each fetus from dams not submitted to any experimental procedure was collected on the 18th gestational day (group CE18) and on the 8th postnatal day (group CP8) to serve as a control for neuronal quantitation and maturation. Immunohistochemical staining of NeuN was used to assess neuron quantity and maturation. The NeuN labeling index was greater in the postnatal period than in the fetal period for the experimental and control groups (P8 > E18 and CP8 > CE18), although there were fewer neurons in experimental than in control groups (P8 < CP8 and E18 < CE18) (P < 0.005). These results indicate that fetal neuroblasts/neurons not only survive a dramatic event such as mechanical disaggregation, in the same way as it happens in human cases, but also they retain their development in heterotopia, irrespective of local tissue influences.     

Ontogeny of Tyrosine Hydroxylase Concentration in Locus Coeruleus of Newborn Rats: Long-Term Effects of RU24722

The ontogenetic variations of tyrosine hydroxylase (TH) have been studied in locus coeruleus of developing rats. During the first 2 weeks after birth, a large increase in TH content (6.04-23.99 TH units) in the noradrenergic structure was observed, followed by a period of progressive increase of the protein concentration (42 TH units in adult rats). The expression of TH was studied in the same ontogenetic period after treatment by RU24722 (20 mg/kg, i.p.). The long-term increase in TH concentration produced by the drug was found to follow ontogenetic variations. It becomes significant around the middle of the second week after birth and gradually increases until the 24th day of postnatal development, indicating a maturation of the mechanisms involved in the inducing effect.     

Changes in endonuclease activity and in chromatin structure of rat hepatocytes during fetal and neonatal life

A developmental study of rat hepatic endonuclease has been performed. Nuclei, from different stages of hepatocyte maturation, were analyzed for endogenous endonuclease activity. The chromatin extracted from these nuclei does not show any fragmentation during the first 17 days of fetal development. On the 18th day of fetal life there is a massive increase in specific endonuclease activity. At birth this activity reaches a maximum level (3.5 units/mg DNA); thereafter it undergoes a gradual decrease. The size of the basic DNA repeats produced by the endonuclease action is 218.9 +/- 1.6 in 18-day-old fetuses and decreases to 204.9 +/- 2.5 in 19-day-old fetuses, a value which remains constant in the following fetal and postnatal life. This difference in monomer size is due to changes in the chromatin structure. Micrococcal nuclease digests show that the "nucleosome core" does not change during hepatocyte development. Therefore, the difference in size of the endonuclease DNA fragments must be due to the linker regions.     

Morphometric analysis of the developing mouse soleus muscle

The pattern of organogenesis of the soleus muscle of the 129 ReJ mouse was evaluated quantitatively using spaced, serial, ultrathin sections and computer-assisted morphometric analysis. Muscles from 14-, 16-, and 18-day in utero mice and muscles of 1- and 5-day-old mice were analyzed to determine age-related alterations in the maximal girth and length of the muscle, number of myotubes, cluster frequency, and the lengths and diameters of myotubes. Primary myotubes are found in the muscle at 14 days in utero. There is little de novo myotube formation between 14 and 16 days in utero, this interval being principally one of primary myotube growth and maturation. The interval between 16 and 18 days in utero is marked by extensive secondary myotube formation, with more myotubes being formed during this period than in any period studied. Morphometric data support the hypothesis that secondary generation myotubes use primary myotubes as a scaffold on which they are formed. Morphometric data also confirm the hypothesis that cluster formation and cluster dispersal occur concurrently during the prenatal period. Secondary myotubes continue to form until birth. At birth, the soleus muscle contains the adult number of myofibers. The first 5 days postnatally are marked by myofiber growth and maturation.     

Cytochemical and biochemical studies on the differentiation of microperoxisomes in the small intestine of the fetal mouse

The localization of catalase activity during the morphogenesis of duodenum and ileum has been studied in Swiss ICR mouse embryos from the 16th day of fetal life until birth. Catalase activity was also measured by a spectrophotometric method. Few diaminobenzidine-positive microperoxisomes are present at 15 days of gestation in undifferentiated cells of the stratified epithelium lining the lumen of duodenum and ileum. The number of microperoxisomes increases considerably in the duodenal enterocytes at 17 days; the highest concentration of microperoxisomes is attained at 18 days, after which time their number becomes stable until 4 weeks after birth. Biochemically, catalase activity is barely detected at 15 days in the first half of the small intestine, but afterwards it increases steadily up to 1 day after birth. In the ileum, the increase in microperoxisome number is far less important than in the duodenal enterocytes and reaches a maximum at 19 days of gestation, that is, immediately at birth. The level of catalase activity in the second half of the small intestine is also much lower than that measured in the first half. These results are discussed in relation to the biogenesis of microperoxisimes in the small intestine before birth.     

The development of the peripheral autonomic nervous system in relation to the gastro-intestinal tract

In this study the development of vagus nerves and the development of sympathetic nerves related to the development of the upper gastro-intestinal tract was studied in the rat from 12.5 days p.c. until birth by means of enzyme histochemical methods applied to sections and toto preparations. A striking time relation between the ingrowth of the vagus nerves as well as the sympathetic nerves and the appearance of acetylcholinesterase (AChE)-positive cells in the wall of the upper gastro-intestinal tract is established. The maturation of the upper enteric ganglion cells is dependent on the presence of a vagal influence. The vagal ingrowth of the stomach starts at about day 12.5 p.c. In AChE toto preparations it is established that the basic distribution pattern as described in the adult rat is complete at day 15 p.c. However, in contrast with the adult state, gastric branches can be traced up to the pylorus and the greater curvature until day 18 p.c. During the 18th day p.c. there is a tremendous increase in the surface area of the stomach. This increase in surface area of the gastric wall and relative decrease in vagal outgrowth leads to the adult configuration of vagal ramifications in relation to the wall of the stomach which can already be observed on the 19th day p.c.     

The hypothalamic magnocellular neurosecretory system of the guinea pig. III. Ultrastructure of the fetal neural lobe.

The ultrastructure of the fetal guinea pig neural lobe was studied from day 40 to day 60 of gestation. Pituitaries were taken every five days and at least four glands from each gestational period were examined. Bundles of nerve fibers had invaded the periphery of the gland on day 40. By day 50 axon profiles were distributed throughout the entire posterior pituitary though pituicyte processes continued to act as a barrier between axons and the perivascular space of capillaries. Neural processes established contact with the capillaries between days 55 and 60. Neurosecretory granules (NSG) were present within a few axons on day 40. The number of axons with NSG and the total quantity of granules increased gradually throughout fetal development. Electron-lucent granules (microvesicles) were observed infrequently until the day of birth. A population of dense-cored vesicles, 70-80 mmu in diameter, was present from day 50 onward; a second population with larger diameters was also present throughout the developmental sequence and these increased from 90-130 mmu in diameter to 170-220 mmu in diameter between days 40 to 60. The presence of neurosecretory granules is discussed in relation to the onset of synthesis and storage of neurohypophysial hormones.     

The effects of prenatal protein deprivation on thyrotroph development

Maternal dietary protein restriction produced by feeding a diet containing 4% casein throughout gestation adversely affects body size and retards development of various organs in the progeny. For the most part, alterations are present in structural or functional entities which evolve during the last trimester of gestation. Fetal thyroid follicle formation and iodine concentrating capacity, which increase rapidly between the 17th gestational day and birth in pups of dams fed the control (24% casein) ration, are retarded in age-matched pups of protein-deficient females. The first immunoreactive thyrotrophs appear in the fetal pituitary on day 17 in both control and prenatally protein-deprived (PPD) young. The total number of thyrotrophs per pituitary was unaffected by maternal protein deficiency, except on day 21 when there were significantly more thyrotrophs per pituitary in fetal control rats. Although pituitary volume was significantly reduced in 18-, 19- and 21-day old fetal PPD rats, as compared with controls, pituitary volume:body weight ratios differed between young in the two dietary groups only on day 21, when the ratio was significantly higher in PPD as compared to control young. Maternal protein deprivation does not affect the morphological maturation of the thyrotrophs of the anterior pituitary of the fetal rat.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

Postnatal maturation of the parenchymal cell types in the rabbit pineal gland.

An ultrastructural study on the maturation of the parenchymal rabbit pineal cell types from the first postnatal day up to 120 days is presented. Two main cell types are distinguished from the first 24h of postnatal life. Pinealocytes of the types I and II display different developmental degrees. Both immature cell types are arranged in groups. In addition, type II pinealocytes form rosette-like structures. Both cell types progressively become isolated and display cell processes. The nucleus and the cytoplasm of type I pinealocytes are barely electrondense. During the postnatal period, the number of cytoplasmic organelles, cell processes and terminal clubs increase progressively. Terminal clubs are frequently seen near blood vessels. After 30 days, type I pinealocytes show characteristics of adult pinealocytes. However, the maturation of most type I pinealocytes does not complete until the 90th postnatal day. Type II pinealocytes present a fairly electrondense nucleus and cytoplasm. Mature forms can be seen after the 5th postnatal day. During the postnatal period, a close relationship is determined among type II pinealocytes and cell processes and terminal clubs of type I pinealocytes.     

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.     

Development of pancreatic enzymes in fetal and suckling rats with emphasis on lipase and colipase

Pancreatic amylase, chymotrypsinogen, lipase and colipase were assayed, at intervals, in rats from day 16 of fetal life until weaning. In the fetus, amylase and chymotrypsinogen accumulated regularly, in parallel, until birth. Lipase and colipase accumulation slowed down between day 20 and birth. The ratio of colipase to lipase was extremely high (9.5) and decreased until weaning towards adult values. Enzyme contents of the pancreas were depleted after birth and remained low until day 14. Intestinal concentrations were equally low, showing that pancreatic depletion was not due to hypersecretion. Protein synthesis was very active, intermediate between that of the fetus and of the adult. It is concluded that in the early suckling phase the proteins synthesized are mainly constitutive and not enzymatic. Starvation followed by refeeding showed that secretion sensitivity to nutritional stimulation only appears at 14 days. During the suckling period amylase concentrations decreased, evidencing a degree of nutritional sensitivity to the low level of carbohydrate in the diet. The productive capacity for lipase underwent a slow maturation which was not even complete at weaning, since concentrations had not yet reached adult level despite the high fat content of milk. This was in part compensated for by the high proportion of colipase but shows that lipase was not adaptative during this phase and that pancreatic lipase can hardly account for lipid digestion before weaning.