Glucocorticoid effects on rabbit fetal lung maturation in vivo: An ultrastructural morphometric study

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animal were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body crosssectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissué cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell defferentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

An ultrastructural, morphometric analysis of rabbit fetal lung type II cell differentiation in vivo

Rabbit lung type II cell differentiation was evaluated by use of ultrastructural, morphometric techniques. Fetal lung epithelial cells decreased in size dramatically from day 19 to day 21 of gestation. Thereafter, the cell and cytoplasmic cross-sectional area declined gradually until the neonatal time point. The tall columnar cell shape characteristic of fetal lung epithelial cells at early stages of development became cuboidal by day 24 of gestation. The number of mitochondria per micron2 cytoplasmic area in presumptive alveolar epithelial cells and the mitochondrial volume density increased toward the end of gestation. The volume density of glycogen pools within fetal lung epithelial cells reached a plateau on day 21 of gestation and then declined sharply on day 26 of gestation in lamellar body-containing, type II epithelial cells. Lamellar bodies increased in number and volume density in epithelial cells starting on day 26 of gestation and peaked with respect to these parameters in the neonatal lung tissue. Multivesicular bodies, which are thought to be a precursor to the lamellar body, became more prominent in differentiated type II cells on day 26 of gestation and increased in volume density from day 28 of gestation to the adult time point. The distance between mesenchymal and epithelial cells in fetal lung tissue declined sharply between days 24 and 26 of gestation but remained relatively constant thereafter. Foot processes extending from connective tissue cells contiguous to the epithelium were generally more numerous than those extending from the basal plasma membrane of epithelial cells at every stage of development examined. These data quantitate for the first time key ultrastructural events that occur during the differentiation of fetal lung epithelial cells in vivo.     

Vagal nerve maturation in the fetal lamb: An ultrastructural and morphometric study

The maturation of the left vagal nerve was studied in the fetal lamb by transmission electron microscopy and by computer-assisted morphometry of sections of the entire nerve at seven gestational ages between 79 and 145 days (term is 147 days) and in the adult ewe. The number of unmyelinated axons per Schwann cell progressively decreased from 25 to 55 at 79 days to 1 to 5 at near-term. Unmyelinated axons of various sizes were enclosed within a single Schwann cell at all ages, but the mean axonal diameter increased in inverse relation to the number of unmyelinated axons. A few Schwann cells enclosed two myelinated axons, but in most instances myelination did not begin until a 1:1 ratio was achieved; some single axons with a Schwann cell remained unmyelinated in the adult. Myelinated fibers were rare at 79 days but myelination progressed rapidly thereafter until the adult ratio of myelinated: unmyelinated fibers was reached at about 100 days; myelinated axons were not uniformly distributed. The myelin sheaths and axons of small fibers progressively increased in diameter in late gestation, but new large fibers were not added. Early myelinating fibers and immature unmyelinated axons contained more microtubules than neurofilaments; neurofilaments predominated in mature axons with or without myelin. Cross-linkages between neurofilaments were already evident by 79 days. Maturation of the vagal nerve thus occurs first by an increase in number of myelinated fibers and then by an increase in the size of each fiber in this fixed population. The bimodal distribution in the size histogram of myelinated fibers is not achieved until 134 days gestation and correlates well with physiological maturation of respiratory patterns. © 1993 Wiley-Liss, Inc.     

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, ～twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, ～twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. © 1993 Wiley-Liss, Inc.     

The effects of ACTH on lung maturation in fetal lambs.

The ability of adrenocorticotrophic hormone (ACTH) to induce lung maturation was evaluated in fetal lambs. One member of 14 twin pairs between 114 and 138 days of gestation was infused intravenously with 0.5 mg ACTH over 5 days. The lungs of treated versus control lambs were judged more mature by morphologic criteria by the use of light and electron microscopy and by biochemical criteria by the use of lamellar-body-rich cell fractions. None of 5 premature lambs treated with ACTH and allowed to breathe showed evidence of hyaline membrane disease, while 3 untreated control lambs showed typical hyaline membranes.     

Parathyroid glands in primary hyperparathyroidism: an ultrastructural morphometric study of 25 cases.

In parathyroid glands removed from patients with primary hyperparathyroidism (pHPT), hyperplasias and adenomas cannot be distinguished from one another by light microscopy when only one gland is available for examination. When a second gland is available, it is necessary to establish whether it is normal, suppressed, or hyperplastic. This distinction may be difficult, and the main criterion is the amount of cytoplasmic lipid in the parenchymal cells. If the lipid is abundant, the gland is considered normal or suppressed, and if it is scanty, the gland is interpreted as hyperplastic. We have performed a morphometric ultrastructural study to test the reliability of this criterion. Twenty-five adenomatous glands removed from patients with pHPT, when compared with glands of normal size from euparathyroid patients, showed a significant increase in the parameters indicative of metabolic activity, namely the size of the Golgi apparatus, the amount of rough endoplasmic reticulum, and the length of plasmalemmas. In addition, the amount of cytoplasmic lipid was significantly reduced. Furthermore, 25 glands of normal size removed from the same patients with pHPT showed an amount of lipid similar to that of normal glands from euparathyroid patients. However, all the parameters indicative of metabolic activity were significantly higher than those in glands from euparathyroid patients and comparable to those found in adenomatous glands. These results suggest that in pHPT, normal-size glands are as active as adenomatous glands, regardless of a higher lipid content.     

An enzyme-histochemical, ultrastructural and morphometric study on fetal and neonatal rat diaphragms

The organogenesis of the rat diaphragm was studied with enzyme-histochemical, ultrastructural and morphometrical methods. 18 to 20 and 22 day in utero diaphragms were examined as well as 2-8-14 and 25 day old ones, in order to determine the age related changes in the diameter and the percentage of muscle fibers and mitochondria. In all examined ages, morphometric data showed type II fiber predominance. Type I and II fibers were equivalent in diameter. During fetal life, mitochondrial size increases appreciably with age while the mitochondrial percentage does not. Instead in postnatal life, the mitochondrial diameter is almost constant both for the small type I fibers and for the large ones (type II fibers). The mitochondrial percentage gradually increases particularly in the small fibers.     

A freeze-fracture study of tubular myelin in fetal rabbit lung.

A freeze-fracture replication study was undertaken to clarify the relationship between intracellular lamellar bodies of type II alveolar epithelial cells and intra-alveolar contents in late gestation rabbit fetuses. The interior of the inclusion bodies was composed of multiple stacks and/or whorls of membranes devoid of membrane-associated particles, while the limiting membrane of intracellular lamellar inclusion bodies was studied with membrane-associated particles of 150A diameter. The intra-alveolar contents were comprised of two components; spherical bodies, and tubular elements. Spherical bodies were identical to the internal contents of the lamellar bodies and also were devoid of membrane-associated particles. Tubular elements mostly appeared rectangular on cross-fractured faces, but triangular and hexagonal forms could also be seen. These tubules rested on the surfaces of the spherical bodies and appeared to be formed of the outer lipid monolayer of spherical bodies. The present observations suggest that the tubular element of the alveolar contents is formed through the interaction between the discharged lamellar body content and the alveolar fluid.     

Androgen insensitivity syndrome: an immunohistochemical, ultrastructural, and morphometric study

OBJECTIVE: To evaluate the morphometric, immunohistochemical, and ultrastructural lesions of the testes in prepubertal and adult patients with androgen insensitivity syndrome. METHODS: We examined the testicular biopsy using immunohistochemistry for vimentin, smooth muscle actin, and collagen IV antigens. Quantification of seminiferous tubules and testicular interstitium was performed in prepubertal and adult patients with androgen insensitivity syndrome and results were compared with normal testes from both infants and adults. RESULTS: The adult testes presented nodular and diffuse lesions that consisted of Sertoli-cell-only seminiferous tubules. Two types of Sertoli cells could be distinguished, namely, immature vimentin-positive Sertoli cells and nearly mature Sertoli cells. In the nodules, the lamina propria was thin and contained a scant number of actin-positive peritubular cells. Leydig cells were hyperplastic. The prepubertal patients showed only diffuse lesions characterized by Sertoli cell hyperplasia, decreased germ cell numbers, and a discontinuous immunoreaction to collagen IV. CONCLUSIONS: The testicular lesions in androgen insensitivity syndrome are probably caused by primary alterations that begin during gestation. These lesions become progressively more pronounced at puberty, when the nodular lesion pattern (adenomas) is completely developed.     

The maturation of the rabbit fetal lung following maternal administration of pilocarpine

Pilocarpine HCl, a parasympathomimetic drug, was administered to pregnant white rabbits in a daily subcutaneous injection of 5 mg/kg on days 24 through 27 of gestation. Fetuses from these animals and from salineinjected controls were obtained by caesarean section at day 28 of gestation. Light microscopic examination revealed thinner alveolar septa in the lungs of pilocarpine-treated fetuses and, morphometrically, a significant increase in the number of mature type II cells, both per unit area and per 1,000 lung cells of any kind. Examination by electron microscopy revealed that the alveolar epithelium of pilocarpine-treated fetuses demonstrated morphologic correlates of increased maturation. These included thinning of type I cells to form blood-air barriers and substantial reductions in the glycogen content of both epithelial cell types. Type II cells of pilocarpine-treated fetuses contained (as indicated by morphometric analysis) more and larger lamellar inclusion bodies, as well as more multivesicular bodies than those of controls. Biochemical determination indicated that the glycogen content of fetal lung, but not liver, was reduced significantly in the pilocarpine-treated group. The findings of this study indicate that maternal administration of pilocarpine results in increased maturation of the fetal alveolar epithelium, thus providing a basis for the autonomic manipulation of fetal lung maturation.     

The effects of red light with different intensity on retinal photoreceptor cells in rabbit: an ultrastructural study

The purpose of this research was to investigate the fine structure of the photoreceptor layer of male rabbits exposed to intermittent red light with different intensities. Thirty adult male rabbits were divided into three groups: control group, experimental group 1 (exposed to 3?h/3?h 60?W red light) and experimental group 2 (exposed to 3?h/3?h 100?W red light). The retina of the eye was removed and studied by transmission electron microscope. Results showed that the experimental group 1 exhibited slight loss of mitochondrial cristae and vacuolization in inner segment, but the other layer of photoreceptor appeared normal. Disorganization and loss of outer segment showed in experimental group 2. Vacuolization, loss of mitochondrial cristae, cell swelling, and pyknotic nuclei were observed in this group. These findings indicate that intermittent red light cause damage in the photoreceptor layer of the rabbit retina; also, higher light intensity increased these damages.     

Neuroendocrine tumors of the nasal cavity: an ultrastructural and morphometric study of 24 cases.

A series of 24 small cell nasal tumors with neuroendocrine differentiation was studied by electron microscopy, and the numbers of secretory granules and cell junctions were assessed. To investigate the relationship between the extent of dendrite formation and the behavior of the tumors, the size of the tumor cells and the area occupied by dendritic processes were determined for each tumor by morphometric analysis performed on low-magnification electron micrographs. A positive correlation was demonstrated between the dendritic area index and the survival of the patients (P = 0.017). Neither the number of secretory granules nor the frequency of cell junctions was prognostically significant.     

Cellular relationship impairment in maturation arrest of human spermatogenesis: an ultrastructural study

Ultrastructural examination of testicular biopsies from cases of maturation arrest showed that there were characteristic abnormalities of the Sertoli cell junctional connections. These abnormalities together with the meiotic failure afford an explanation for the severe oligospermia or azoospermia noted in patients with maturation arrest. Premature setting up of ectoplasmic specializations in front of early spermatids and/or spermatocytes were also observed.     

Total cortisol in amniotic fluid and fetal lung maturation.

Total cortisol was determined by radioimmunoassay in 48 samples of amniotic fluid obtained at various stages of normal pregnancy. Before the 34th week, all cortisol levels were less than 40 ng per milliliter. At 35 to 40 weeks the mean value was 2.4 times higher than that at 20 to 34 weeks. In pregnancies greater than 40 weeks, a further increase in total amniotic-fluid cortisol was observed, and values greater than 120 ng per milliliter were found only in patients of this group. A good rank correlation between cortisol and lecithin/sphingomyelin ratio was found in 43 samples (r = 0.83, p less than 0.001). No respiratory-distress syndrome of the newborn occurred when total amniotic-fluid cortisol was greater than 60 ng per milliliter (16 patients). Total amniotic-fluid cortisol may reflect initiation of fetal lung maturation, and may help identify pregnancies with a gestation period of over 40 weeks.     

The development of alveolar septa in fetal sheep lung. An ultrastructural and immunohistochemical study

The morphogenesis of pulmonary alveolar septa in the sheep was studied by light microscopy, transmission electron microscopy and light microscopic immunohistochemistry for the detection of elastin. The primordia of alveolar septa developed in the glandular stage in areas subjacent to the epithelium, and formed alveolar septa by protruding into the glandular lumina. In their earliest stage, the primordia consisted of groups of fibroblasts, which were associated with elastic fibers and unit collagen fibrils and were surrounded by epithelial basement membrane and by more immature fibroblasts. The fibroblasts in the primordia subsequently became myofibroblasts or smooth muscle cells. In the alveolar zone of the glands, elastic fibers were exclusively found in the primordia of alveolar septa in early developing lung. In early developing lung, wavy, thickened epithelial basement membranes were found in the regions of the glands, which eventually underwent considerable expansion of their surface areas, especially in the primordia of alveolar septa and the bifurcations in the alveolar zones. Areas of fusion of the basement membranes of capillary endothelial cells and epithelial cells in the alveolar zone were found after the formation of the primordia of alveolar septa was accomplished. These areas of fusion were not found in the primordia themselves, but in regions between the primordia. Epithelial cell flattening and differentiation occurred after the formation of the primordia of alveolar septa, and flattening was first observed in the areas of the primordia and the bifurcations of the alveolar zones.     

Carcinomas of the lung: an ultrastructural and immunocytochemical study

Fifty-two primary carcinomas of the lung were studied by electron microscopy and by an immunoperoxidase method, using an anti-human keratin antiserum. The results were compared with light microscopic observations. One-third of the carcinomas of the lung showed ultrastructural evidence of both glandular and squamous differentiation. The group of small cell carcinomas was found to be particularly heterogenous ultrastructurally with only three out of eight tumors showing neurosecretory-type granules. Indirect immunoperoxidase staining revealed presence of a keratin-type protein in the vast majority of carcinomas, including foci of small cell carcinomas. Our studies emphasize the heterogeneity and frequent intermixing of the four major categories of lung carcinomas: squamous cell carcinomas, adenocarcinomas, small cell carcinomas, and large cell carcinomas. It is suggested that all these tumors might be derived from pluripotential "reserve" bronchial or bronchioalveolar cells. The segregation of small cell carcinomas from other groups continues to be justified on pragmatic grounds because these carcinomas constitute a group of predominantly small fast-replicating cells amenable to chemotherapy.     

Development of ciliated cells in the human fetal oviduct: an ultrastructural study

Prenatal development of ciliated cells in the human fallopian tube was studied by light and electron microscopy in specimens obtained from 12 fetuses, aged 12-40 weeks. On light microscopy, transverse sections of the ampullary portion of the tube revealed a slit-like lumen at 12 weeks. The lumen began to fold by 15 weeks, and formed the typical villous structures by 31 weeks. On electron microscopy, the epithelial cells contained a large number of sub- and supranuclear glycogen particles until 18 weeks and an occasional solitary cilium. At 20-22 weeks, instead of glycogen particles, cytoplasmic organelles such as mitochondria, rough endoplasmic reticula and Golgi apparatus were well developed, and some cells possessed cilia with a 9 + 2 microtubular structure. Between 22 and 31 weeks, ciliated cells were sporadically observed. At 31 weeks, the epithelial cells accumulated a large number of sub- and supranuclear glycogen particles. Afterwards, numerous ciliated cells with well-developed cytoplasmic organelles were observed by 40 weeks.