Judy M. Strum

Harderian glands in mice: Fluorescence, peroxidase activity and fine structure

The Harderian glands of albino mice are composed of tubulo-alveoli which contain two secretory cell types. The most common cell (type A) displayed a natural red fluorescence due to the presence of porphyrins. Lipid droplets in this cell and along its apical border were often intensely fluorescent. The less common cell (type B) did not fluoresce. The type B cell contained unusual lipid droplets surrounded by concentric layers of membranes, and sometimes displayed cylindrical organelles believed to be associated with the formation of pigment. A dense red-brown pigment was observed in the lumens of a few tubulo-alveoli and it did not fluoresce, but areas where pigment formation was taking place fluoresced brightly. Myoepithelial cells, containing thick and thin filaments, were found underlying both secretory cell types. Fenestrated capillaries and adrenergic and cholinergic nerve endings were abundant in the adjacent connective tissue. Endogenous peroxidase activity was identified in both secretory cell types and was found localized only within tubules and vesicles of the smooth endoplasmic reticulum.

Constant light exposure induces damage and squamous metaplasia in Harderian glands of albino mice

Harderian glands from control albino mice kept in a cyclical light/dark environment had tubulo-alveoli comprised of lipid-filled glandular epithelial cells. The porphyrin content of the gland measured 122 microgram/100 mg gland. Constant light exposure for 24 hr caused exopthalmos grossly. Histologically most of the secretory cells were swollen and the lumens of many tubulo-alveoli were obliterated; a few areas of the gland showed damage. The porphyrin content had decreased to 116 microgram/100 mg gland. After 3 days of constant light exposure the tubulo-alveoli were markedly altered. Lipid and cellular debris filled the lumens, and lining cells were highly irregular, ranging in shape from columnar to squamous. The porphyrin content had decreased to 72 micro/100 mg gland and leukocytes and macrophages were abundant. Despite this extensive damage a number of tubulo-alveolar epithelial cells were observed under-going mitosis. After 7 days of constant exposure to light, some tubulo-alveolar epithelial cells had undergone squamous metaplasia, and the porphyrin content had dropped markedly to 50 microgram/100 gland. These pronounced cellular changes are believed to result from a direct effect of light on the gland.

Resting human female breast tissue produces iodinated proteins

Normal resting human breast tissue was obtained from immediate autopsies performed on six women who had died from head injuries sustained in accidents. Tissue samples containing epithelium were dissected asceptically and either fixed immediately or placed into culture. Samples in culture for 2 or 3 days were exposed to radioiodide for 4 hr in order to establish whether or not the isotope became incorporated into proteins. Light and electron microscope autoradiographs were prepared and evaluated. Radiolabeled secretory material was observed in both the terminal ductules and intralobular terminal ducts, but not within the larger ducts. Therefore the products in these separate compartments of the mammary epithelial tree differ in composition. Extensive gap junctions were discovered between adjacent myoepithelial cells in the terminal ductules and intralobular terminal ducts. These junctions probably serve to coordinate contractions which facilitate the movement of material from the most distal parts of the gland into larger ducts.

Modulation of glycogen stores in epithelial cells during airway development in Syrian golden hamsters: a histochemical study comparing concanavalin A binding with the periodic acid-Schiff reaction.

We studied glycogen storage in the developing airway epithelium of Syrian golden hamsters from gestational Day 11 to neonatal Day 2 using concanavalin A (ConA) staining as an adjunct approach to the periodic acid-Schiff (PAS) reaction. One hundred and fourteen fetuses and neonates were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol, embedded in paraffin, and stained with ConA-horseradish peroxidase conjugate as well as with PAS. ConA staining was abolished by alpha-glucosidase digestion or by pre-treatment with periodic acid, demonstrating that ConA bound to glycogen. In tissues fixed with mercury and/or aldehydes, ConA staining was greatly enhanced by pepsin digestion. Airway glycogen stores, revealed by ConA and PAS, fluctuated during development. At first all the undifferentiated epithelial cells contained abundant glycogen. Then, coincident with the appearance of the first endocrine cells, the glycogen stores were depleted. Thereafter, glycogen accumulated in pre-secretory and basal cells until birth, but by 2 days after birth the glycogen stores were again depleted. The initial depletion of glycogen followed by repletion was observed at all levels of the conducting airways; changes in the trachea preceded those in the bronchi and bronchioles by 1 and 2 days, respectively.

The regulated expression of mRNA for Clara cell protein in the developing airways of the rat, as revealed by tissue in situ hybridization

Tissue in situ hybridization has been used on sections of developing rat lung to follow the cellular sites of mRNA expression for a protein identified only in bronchiolar Clara cells. The mRNA for this Clara cell protein (CCP) was first detected on gestational day 16 in only one of the two types of tubules existing in the lung at this developmental stage. During the next 2 days CCP mRNA expression increased uniformly only in the epithelium lining the respiratory tubules. By gestational day 19, CCP mRNA expression became limited to secretory epithelial cells lining the bronchi, and terminal bronchioles. By neonatal day 1, an intense hybridization signal was observed along all of the conducting airways, but it was irregular due to the fact that expression of the CCP gene was limited to the secretory epithelial cells. In adult rats, CCP mRNA was expressed not only in secretory cells of the intrapulmonary airways at all anatomical levels, but also in secretory epithelial cells lining the trachea and its glands, as well as in specific alveolar cells thought to be type II pneumocytes. These findings demonstrate that the regulation of the CCP gene during lung development is a complicated process and that the expression of CCP mRNA does not parallel exactly the sequential development of the airways.

Vitamin A deprivation in hamsters. Correlations between tracheal epithelial morphology and serum/tissue levels of vitamin A.

SummaryThe effects of vitamin A deprivation on the tracheal epithelium of young hamsters were investigated. Colchicine was administered 6 h prior to death to induce metaphase arrest, thus making it possible to quantify the mitotic rates of basal cells and secretory (mucous) cells in the epithelium. Blood samples were taken from all hamsters, and liver samples from some, in order to measure serum and tissue levels of vitamin A. Age-matched controls were compared with the following groups of hamsters maintained on a vitamin A deficient diet: (1) pre weight plateau animals (those gaining weight), (2) weight plateau-early weight loss animals (those maintaining approximately the same weight for 3 or 4 days, followed in some cases by a loss of weight for 3 or 4 days), and (3) prolonged weight loss animals (those showing a loss of weight for 5 or more days). Four week old hamsters in a pre weight plateau had undetectable amounts of vitamin A in their livers and declining levels in their serum, whereas 41/2 week old hamsters still gaining weight had barely detectable levels of vitamin A in their serum. Nevertheless, the tracheal epithelium of these animals was not different from controls in appearance, proportions of different cell types, mitotic rates of secretory and basal cells, or in the number of cells per millimeter of basement membrane (cell density). Vitamin A was undetectable in the serum and livers of hamsters in the weight plateau-early weight loss stage. At this time the tracheal epithelium showed minimal morphological change, with small focal areas of epidermoid metaplasia in some animals. The tracheas of animals in early weight loss were smaller than tracheas in the control group, and there was a trend towards an increase in the number of epithelial cells per millimeter basement membrane. Cell types in the minimally changed epithelium appeared nearly normal, but there was an increase in the proportion of basal cells, and an absence (or near absence) of division in both basal and secretory cells. Tracheal rings from hamsters in the prolonged weight loss stage were lined by a cornifying metaplastic epidermoid epithelium.Our findings demonstrate that barely detectable levels of vitamin A in the serum are sufficient to maintain normal growth and differentiation of hamster tracheal epithelium (late pre weight plateau stage). When vitamin A serum levels fall below detectable limits the animals enter the weight plateau-early weight loss stage. This stage is accompanied by an inhibition of tracheal epithelial cell growth, although nearly normal cellular differentiation is maintained. As weight loss persists (prolonged weight loss stage), the epithelial cells fail to maintain normal differentiation and cornifying epidermoid metaplasia becomes widespread.

A method demonstrating motor endplates for light and electron microscopy

The cytochemical localization of acetylcholinesterase activity at motor endplates is commonly employed prior to electron microscopic examination. Methods which involve the use of thiocholine esters often lead to a loss of resolution due to the spread to surrounding tissues of an extremely dense reaction product. A method is presented in which esterase positive sites are revealed by incubation with hexazotized pararosaniline and indoxyl acetate. The osmiophilic property of the reaction product which remains localized to the synaptic cleft allows speedy identification and detailed examination of the endplate at the ultrastructural level. An application of the procedure is also described.

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase

SummaryThe purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormonedependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.

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

SummaryEighteen 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 (> 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.

Effect of iodide-deficiency on rat mammary gland

SummaryWhen rats are kept iodide-deficient, atrophy and necrosis takes place in the mammary gland and areas of dysplasia and atypia are seen. Administration of estradiol to iodide-deficient rats stimulates cell division in the gland and leads to the formation of alveoli. Continued stimulation by estradiol produces changes in the newly-formed alveolar cells. Their nucleoli are altered and show a separation of components. Ribosomes and lipid droplets increase and the cells synthesize large vacuoles containing protein. The secretion of great quantities of this material into areas of the tissue where regressive changes have occurred undoubtedly contributes to the formation of cysts within the gland. The present findings indicate that iodide-deficiency alters the structure and function of mammary gland alveolar cells and makes them highly sensitive to stimulation by estradiol.