Smooth muscle development during postnatal growth of distal bronchioles in infant rhesus monkeys.

Development of smooth muscle in conducting airways begins early in fetal life. Whereas the pattern and regulation of smooth muscle differentiation are well-defined, the impact of airway growth on the process is not. To evaluate the transformations in organization during postnatal growth, smooth muscle bundle organization (size, abundance, and orientation) was mapped in five generations of distal airways of infant rhesus monkeys (5 days and 1, 2, 3, and 6 mo old). On the basis of direct measurement of the bronchiole proximal to the terminal bronchiole, length increased by 2-fold, diameter by 1.35-fold, and surface area by 2.8-fold between 5 days and 6 mo of age. Smooth muscle bundle size was greater in proximal bronchioles than in respiratory bronchioles and did not change with age. However, relative bundle size decreased in proportion to airway size as the airways grew. Relative bundle abundance was constant regardless of airway generation or age. The distribution of smooth muscle bundle orientation changed with age in each airway generation, and there were significant changes in the terminal and respiratory bronchioles. We conclude that smooth muscle undergoes marked organizational changes as airways grow during postnatal development.     

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.     

Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.     

Alterations in alveolar basement membranes during postnatal lung growth

We studied the ultrastructural characteristics of alveolar basement membranes (ABM) and capillary basement membranes (CBM) in rat lungs at birth, at 8-10 d of age, during alveolar formation, and at 6-10 wk of age, after most alveoli have formed. We also measured in vitro lung proteoglycan and heparan sulfate synthesis at each age. We noted three major age-related changes in pulmonary basement membranes. (a) Discontinuities in the ABM through which basilar cytoplasmic foot processes extend are present beneath alveolar type-2 cells but not alveolar type-1 cells. These discontinuities are most prevalent at birth but also exist in the adult. (b) Discontinuities are also present in CBM at the two earliest time points but are maximal at 8 d of age rather than at birth. Fusions between ABM and CBM are often absent at 8 d of age, but CBM and CBM/ABM fusions were complete in the adult. (c) Heparan sulfate proteoglycans identified with ruthenium red and selective enzyme degradation are distributed equally on epithelial and interstitial sides of the ABM lamina densa at birth, but decrease on the interstitial side with age. In vitro proteoglycan and heparan sulfate accumulation at birth was two times that at 8 d and five times that in the adult. Discontinuities in ABM allow epithelial-mesenchymal interactions that may influence type-2 cells cytodifferentiation. Discontinuities in CBM suggest that capillary proliferation and neovascularization are associated with alveolar formation at 8 d. When CBM becomes complete and forms junctions with ABM, lung neovascularization likely ends as does the ability to form new alveoli.     

Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).     

Differential effects of N-methyl-D-aspartate receptor stimulation on growth hormone secretion at specific stages of postnatal development of the male rhesus monkey

The present study attempts to examine the role of N-methyl-D-aspartate (NMDA) receptor in the central regulation of growth hormone (GH) secretion during specific stages of pubertal development of the male rhesus monkey (Macaca mulatta). Infantile (n=4), prepubertal (n=5), peripubertal (n=5) and adult (n=5) intact male rhesus monkeys were given an agonist of NMDA receptor, N-methyl-D,L-aspartate (NMA) (15 mg/kg BW) through a teflon cannula implanted in the saphenous vein. Blood samples were collected 20-60 min before and 40-80 min after the injection of the drug at 10-20 min intervals. NMA was dissolved in normal saline immediately before use and passed through a 0.22 microm filter at the time of injection. All bleedings were carried out under ketamine hydrochloride anesthesia (initial dose 5 mg/kg BW, im followed by 2.5 mg/kg at 30 min intervals). The plasma levels of GH and testosterone (T) were determined by using specific assay systems. The hypothalamic-somatotrope activity under basal conditions was studied by averaging all the GH concentrations obtained before NMA injection, whereas the sensitivity of NMDA receptor to NMA stimulation was determined by comparing basal GH levels immediately before NMA injection at 0 min and GH concentrations obtained 10 min after the injection. The mean basal plasma concentrations of GH in the four groups of animals showed marked age-related differences. The levels of GH were found to be higher in infantile and peripubertal monkeys as compared to those of prepubertal and adult animals. A single iv injection of NMA produced differential effects on GH secretion during specific stages of postnatal development depending upon the level of GH secretion under basal conditions. Whereas NMA had no demonstrable effect on GH secretion in infantile and peripubertal animals in which the basal GH levels were high, it produced pronounced effects on GH secretion in prepubertal and adult monkeys wherein baseline GH concentrations were low. In conclusion, the present study suggests that the glutamatergic component of the control system that governs GH secretion by utilizing NMDA receptor may participate in regulation of age-related changes in the secretion of GH in the male rhesus monkey.     

Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth

Circulating erythropoietin (EPO) stimulates erythrocytosis, whereas organ-specific local EPO receptor (EPOR) expression has been linked to angiogenesis, tissue growth, and development. On the basis of the observation of concurrent enhancement of lung growth and erythrocyte production during exposure to chronic hypoxia, we hypothesized that a paracrine EPO system is involved in mediating lung growth. We analyzed EPOR protein expression in normal dog lung tissue during postnatal maturation and during compensatory lung growth after right pneumonectomy (PNX). Membrane-bound EPOR was significantly more abundant in the immature lung compared with mature lung and in the remaining lung 3 wk after PNX compared with matched sham controls. COOH-terminal cytosolic EPOR peptides, which were even more abundant than membrane-bound EPOR, were also upregulated in immature lung but differentially processed after PNX. Apoptosis was enhanced during both types of lung growth in direct relationship to cellular proliferation and EPOR expression. We conclude that both developmental and compensatory lung growth involve paracrine EPO signaling with parallel upregulation but differential processing of EPOR.     

Pigment cell development in rhesus monkey eyes: an electron microscopic and histochemical study

The ontogeny of pigment cells in the eyes of rhesus monkeys was studied by electron microscopy and histochemistry.In 60- to 80-day-old fetuses, the pigment epithelium of the iris and retina has already differentiated whereas stromal melanocytes of the uveal tract differentiate much later. The morphological and histochemical difference between melanocytes of the iris stroma and the choroid suggests that during embryonic development melanocytes migrate from the iris toward the ciliary body and choroid.Similarly, melanosomes of pigmented epithelial cells may have their origin in the epithelium of the anterior layer of the iris, which is metabolically more active than both the posterior layer and the pigment epithelium of the ciliary body and retina.     

Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey Macaca mulatta

Of the eight categories of epithelial cells identified in pulmonary conducting airways, four are found in the trachea of adult primates: basal, mucous goblet, intermediate, and ciliated cells. While their ultrastructure is well characterized, little is understood about their origin or differentiation. This study describes the pattern of differentiation of the tracheal luminal epithelium in a species of nonhuman primate, the rhesus monkey, Macaca mulatta. Tracheas of 57 fetal and postnatal rhesus were fixed with glutaraldehyde/paraformaldehyde: ten at 29-54 days gestational age (GA), ten at 59-80 days GA (pseudoglandular stage), sixteen at 82-130 days GA (canalicular stage), ten at 141-168 days GA (saccular stage), eight at 1-134 days postnatal, and three adults (2 yr 11 months to 11 yr 11 months). Slices taken proximal to the carina were processed for electron microscopy by a selective embedding procedure. In the youngest fetuses, essentially one population of cells lined the tracheal epithelial surface. These cells were columnar in shape with a central nucleus, few organelles, and large amounts of cytoplasmic glycogen. At 46 days GA, ciliated cells were observed on the membranous side of the trachea. Some nonciliated cells had concentrations of organelles in the most apical portion of their cytoplasm. At 59 days GA, membrane-bound cored granules were intermixed with organelles in the apices of some glycogen-filled cells. They were observed first on the cartilaginous side. Between 59 and 100 days GA, a large number of cell forms which appeared to be transitional between ciliated, secretory, basal, and undifferentiated cells were present. These included ciliated cells with electron-lucent inclusions resembling mucous granules. Mucous secretory cells were more numerous and had more granules and less glycogen in older fetuses. By 105 days GA, few of the secretory cells had significant amounts of glycogen and the cytoplasm was condensed. Secretory granules were very abundant in some cells and minimal in others. The Golgi apparatus was prominent. In animals 120 days GA and older, small mucous granule cells and basal cells resembling these cells in adults were present. By 134 days postnatal age, the epithelium resembled that in adults. We conclude that most of the differentiation of tracheal epithelium in the rhesus monkey occurs prior to birth; the cells differentiate in the following sequences: ciliated, mucous goblet, small mucous granule, basal; and basal and small mucous granule cells do not play a role in ciliated and mucous cell formation in the fetus.     

Alterations in lung basement membrane during fetal growth and type 2 cell development

We studied basement membrane development in the late fetal and in the neonatal rat lung, from the 18th day of gestation (term = 22 days) through the 8th postnatal day, with particular emphasis on the gas-exchange region of the lung. In the periphery of the lung, as type 2 cells differentiate, the continuous basement membrane develops openings beneath these cells. Basal cytoplasmic foot processes extend through these discontinuities into the underlying interstitium, often approaching interstitial cells closely. These discontinuities and extensive foot processes are associated only with type 2 epithelial cells and not with either differentiated airway cells or with the type 1 alveolar lining cells derived from type 2 cells. The type 2 cell basement membrane discontinuities and penetrating foot processes are maximal in the perinatal period and decrease in the week after birth. The appearance of openings in type 2 cell basement membrane and changes in distribution, linear density, and ruthenium red staining of anionic sites suggest that the epithelial basement membrane undergoes continuous remodeling throughout development, particularly in association with type 2 cell differentiation and growth of lung surface area. Epithelial cell foot processes may interact with underlying interstitial cells and affect the coordination of lung surface growth with the development of its connective tissue framework.     

Ontogenetic and lung development in Tupaia belangeri during the early postnatal period

The Belanger's tree shrew (Tupaia belangeri) has an unusual reproductive strategy. The animals are born in altricial condition and remain in the nest for the first four weeks of life, nursed only once in 48 h. This is highly demanding for the constitution of the neonates. Despite their immaturity in the external appearance at birth, newborn tree shrews have to deal with the absence of the mother. We asked if the lung structure of the neonates match the high physiological requirements of this “absentee system”. To examine the lung development of nest young tree shrews, histological and ultrastructural investigations were performed. Newborn tree shrews are at the transition stage between the saccular and the alveolar stage of lung development. In addition to small saccules, the lung has alveoli and associated structures already at birth and thus appears more mature compared with typical altricial species. The results of the present study reveal that despite their immaturity in the external appearance newborn tree shrews are relatively mature in terms of lung development. This can be interpreted as a prerequisite for thermoregulatory abilities, necessary in neonate tree shrews to cope with the restricted nature of maternal care.     

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.     

Epidermal growth factor family in rhesus monkey uterus during the menstrual cycle and early pregnancy

This study examines immunohistochemically the presence of EGF, TGFalpha, HB-EGF, AR, and EGFR, members of the EGF family in the monkey uterus during the menstrual cycle and early pregnancy. EGF, TGFalpha, HB-EGF, AR, and EGFR were mainly localized in glandular and luminal epithelium. TGFalpha, HB-EGF, and AR staining were stronger in the glandular epithelium closer to the myometrium than in that closer to the luminal epithelium. The level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining was low on days 1 and 6, and began to increase on day 9 of the menstrual cycle. A high level of EGF, and EGFR staining was maintained on days 16, 20, and 25 of the menstrual cycle. The highest levels of TGFalpha, AR, and HB-EGF staining were seen on days 16 and 20 of the menstrual cycle. In early pregnancy, a low level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining appeared on days 1 and 2 of pregnancy, and then gradually increased from day 3 of pregnancy. The highest levels of EGF, TGFalpha, HB-EGF, and EGFR were detected on days 9, and 11 of pregnancy. Our data suggest that the EGF family may play a role in monkey implantation. Mol. Reprod. Dev. 55:164-174, 2000.     

Dynamics of muscle fibre growth during postnatal mouse development

Background  

Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.