Patterns of ovarian cell proliferation in rats during the embryonic period and the first three weeks postpartum.

The purpose of this study was to characterize the patterns of ovarian cell proliferation during the earliest stages of folliculogenesis, which occur in the embryonic period and the first weeks postpartum in rats. Rats were given continuous infusions of [3H]thymidine (3H-TdR) or bromodeoxyuridine (BrdU), and cells that were synthesizing DNA were visualized by autoradiography or immunohistochemistry. There were dramatic changes in the patterns of cell proliferation during the period studied. Mesenchymal cells proliferated extensively in the embryonic and neonatal ovary, but their growth fraction declined precipitously as follicles formed. Epithelial cells in the medulla of the ovary left the cell cycle at about embryonic Day 12, then resumed proliferation as soon as they were incorporated into follicles just after birth. Epithelial cells towards the cortex of the organ continued to proliferate until late in the embryonic period; they apparently became quiescent around the time of birth, and incorporation into follicles did not release them from their quiescent state. After the follicles had formed, patterns of cell proliferation continued to change. At 5 days postpartum, approximately 36% of the smallest follicles (1-8 granulosa cells in cross section) had at least 1 granulosa cell that was labeled following a 24-h infusion of 3H-TdR; by Day 20 only 14% of these follicles were labeled, and by Day 30 only 4.4% were labeled.

Patterns of cellular proliferation and airway branching in cultured fetal hamster lung explants.

Lungs were removed from hamsters at gestational day 12 and cultured in BGJb medium under the following conditions: (1) alone, (2) with 5% fetal bovine serum (FBS), (3) with defined additives (with and without vitamin A), and (4) with defined additives but lacking both epidermal growth factor (EGF) and vitamin A. Patterns of cell proliferation were determined by immunochemical labeling for bromodeoxyuridine (BrdU) and airway branching was evaluated in each explant. After 4 days in BGJb alone, an orderly but limited branching pattern occurred, and labeling was greater in the epithelium than in the connective tissue. With FBS a relatively normal branching pattern occurred, and labeling was nearly equivalent in connective tissue and epithelium. With defined additives the connective tissue was heavily labeled in the compact region supporting the distorted bronchioles and in the loose flange at the periphery; removing only vitamin A did not alter these patterns. Removing both vitamin A and EGF caused an extensive but distorted epithelial branching pattern that extended to the periphery of the explant (a flange of loose connective tissue did not form); BrdU labeling was sparse. These results indicate that EGF played a major role in contributing to alterations in the growth and development of fetal hamster lung.

Airway branching patterns and cytodifferentiation in cultured fetal hamster lung.

Intact fetal hamster lungs were taken for culture on gestational day 12, when only lobar bronchi and primary bronchioles are established and the epithelial cells are undifferentiated. Explants were maintained on Transwell collagen membranes for 2 and 4 days in BGJb medium alone, with 5% FBS, or with the following additives: insulin, transferrin, hydrocortisone, cholera toxin, EGF, and vitamin A. Development of the respiratory tree was affected differently by each medium formulation. BGJb medium with 5% FBS permitted near normal branching of airways and presumptive alveoli. In contrast, BGJb medium alone permitted only limited branching of these structures. BGJb medium with additives permitted branching but markedly altered normal development. The differentiation of endocrine and secretory cells was monitored by immunolabeling for serotonin and calcitonin gene-related peptide, and Clara cell protein, respectively. Ciliated cells were identified by morphology. All medium formulations supported the timely differentiation of endocrine, secretory, and ciliated cells. The ultimate goal of our studies is to characterize factors that influence airway branching and cytodifferentiation during fetal lung development. This study showed that near normal airway branching and cytodifferentiation were supported in vitro by BGJb medium with 5% FBS. Although cytodifferentiation occurred with the two other formulations, airway development was impaired.

The immunocytochemical detection of cytochrome P-450 monooxygenase in the lungs of fetal, neonatal, and adult hamsters.

Antibodies against rabbit cytochrome P-450 reductase (reductase), cytochrome P-450 isozyme 2 (P-450 IIB), and cytochrome P-450 isozyme 5 (P-450 IVB) were used to detect homologous enzymes in the developing lung of the Syrian golden hamster. No immunocytochemical labeling was observed on gestational days 11, 12, and 13. On gestational day 14, light immunoperoxidase labeling for reductase and P-450 IIB was observed over cells lining the trachea and cranial portions of lobar bronchi. On gestational day 15, these enzymes were detected in conducting airways at all anatomic levels, and in the media of the pulmonary vein and its branches. Light labeling for P-450 IVB was first observed over cells lining the trachea and lobar bronchi on gestational day 15, but the smallest bronchioles and the media and endothelium of the pulmonary vein did not label for this enzyme until gestational day 16 (neonatal day 1). Type II pneumocytes and the pleural mesothelium first labeled for each of the three enzymes on neonatal day 1. Although the mesothelium no longer labeled for reductase or P-450 IIB in hamsters 3.5 wk old, the other labeling sites persisted in adult hamsters. Because cytochrome P-450 enzymes are associated with the endoplasmic reticulum, an ultrastructural examination of differentiating secretory cells was made to detect its appearance. At each conducting airway level, smooth endoplasmic reticulum was present in the cells 2 d before cytochrome P-450 enzymes could be detected immunocytochemically. The appearance of these enzymes paralleled the development of the hamster lung; they were first present in the trachea and lobar bronchi, then in the bronchioles, and finally in the alveoli.

Localization of healing tracheal wounds using bromodeoxyuridine immunohistochemistry.

Immunohistochemical demonstration of the thymidine analogue bromodeoxyuridine (BrdU) in regenerating cells was useful in determining the size and location of the wounded areas (epithelial and submucosal) during regeneration of the hamster tracheal epithelium, at times late in the healing process (72-144 hr postinjury) when the wound sites and their boundaries were not recognized with certainty in conventionally stained paraffin sections. Cells distant from the wound sites remained unlabelled. The success of this method resulted from prolonged exposure to BrdU released over several hours from a 25mg tablet implanted subcutaneously at 24 hr postwounding at the time when DNA synthesis and cell proliferation are maximal. This simple technique promises to be useful in determining the size and location of wound sites with application to a wide variety of organs and tissues in studies of repair and healing.

Effects of vitamin A-deficiency and inflammation on the conducting airway epithelium of Syrian golden hamsters.

The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48-55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6-7 fold in the epithelium lining the lobar bronchus (p less than 0.0002) and the bronchioles (p less than 0.0001), and the proportions of ciliated cells were decreased (p less than 0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that was not reversed when vitamin A was restored to the diet.

Correlations between blood glucose levels and bromodeoxyuridine labelling indices of pancreatic islet cells following streptozotocin administration to pregnant Syrian golden hamsters.

Eighteen timed-pregnant Syrian golden hamsters were injected subcutaneously with streptozotocin (STZ, 60 mg/kg bw) early on gestational day 10. The response varied widely, and based on changes in blood glucose levels during gestational days 11 to 15, the hamsters were categorized into four groups: 1) no change; 2) mild diabetes (200-250 mg/dl), which reverted; 3) moderate diabetes (greater than 300 mg/dl), which reverted; and 4) moderate to severe diabetes (300-500 mg/dl), which was sustained. Two hours before sacrifice, a 25 mg tablet of bromodeoxyuridine (BrdU) was implanted subcutaneously into each experimental hamster and into 17 control pregnant hamsters that had not received STZ. BrdU-labelling was demonstrated immunochemically in the pancreatic islet cells. In control hamsters, the mean labelling index (LI) of the islet cells was 0.07% and did not exceed 0.2% in any hamster. Following injection of STZ, islet cell LI's remained low (0.13%) if the blood glucose levels were not altered by the diabetogenic drug. However, LI's were increased in islet cells of hamsters which showed a mild to moderate diabetes which rapidly reverted; the highest LI's (5% +/- 2.1) occurred in four hamsters that were killed 2 days after receiving STZ. The LI's were moderately increased (1.4% +/- 0.42) in two hamsters with moderate diabetes killed 2 days after STZ, but LI's were low (0.12% +/- 0.04) in six hamsters with moderate to severe diabetes killed 3, 4, and 5 days after STZ. Reversion of hyperglycemia to normoglycemia correlated closely with increased DNA synthesis in the islet cells of the pregnant hamsters. These observations strongly suggest that following mild cytotoxic injury induced by STZ, the B cells regenerated and insulin production was restored sufficiently to maintain normoglycemia.