Rapid atraumatic sex identification of developmental day 14–16 mice

Abstract

Embryonic mice have been used widely to study organ development. Days 14–16 are critical for sex organ development and differentiation in mice. Current methods for sex identification are limited. Even the simplest polymerase chain reaction method may injure the embryo. We determined that morphologic analysis of embryonic mammary anlagen could be used for rapid atraumatic sex identification of day 14–16 mice. The accuracy of our method was verified by molecular and anatomical approaches.     

Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.     

Enzymatic O-glycosylation of kappa-caseinomacropeptide by ovine mammary Golgi membranes

The results of subcellular fractionation of sheep mammary gland membranes indicate that N-acetylgalactosaminyl polypeptide transferase and galactosyl-N-acetylgalactosaminyl transferase, which are involved in the assembly of disaccharide units of kappa-casein, are localized chiefly in Golgi membranes. The glycosyltransferase activities incorporating N-acetyl [1-14C] galactosamine and [U-14C] galactose from uridine diphosphate N-acetyl [1-14C] galactosamine and uridine diphosphate [U-14C] galactose, respectively, were measured after membrane solubilization with Triton X-100 either with unglycosylated caseinomacropeptide, or with this polypeptide containing the N-acetylgalactosamine side chain residues (desialylated and degalactosylated caseinomacropeptide). Radioactive N-acetylgalactosamine was incorporated in the unglycosylated acceptor peptide, and the glycosidic bonds in the product were alkali labile, suggesting that they were linked to the hydroxyamino acid residues. In addition radioactive N-acetylgalactosamine was released after alpha N-acetyl-D-galactosaminidase treatment of labelled caseinomacropeptide. [U-14C] galactose was incorporated in the desialylated and degalactosylated acceptor peptide. Reductive alkaline treatment of [U-14C] galactose peptide resulted in the release of a major product, the chromatographic properties of which in TLC were identical with authentic galactosyl (1 leads to 3) N-acetylgalactosaminitol. The structure of the labelled disacchariditol determined after periodate oxidation (two equivalents) by gas liquid chromatography-mass spectrometry revealed that the [U-14C] galactose was linked to position C-3 on the N-acetylgalactosaminyl-residue. The anomery of the galactose, as determined by a chemical method, indicates unambiguously a beta configuration.     

Induction of malignant transformation in vitro and mammary tumors in rats by two new potent anthracycline antitumor antibiotics,morpholinodaunomycin and cyanomorpholinoadriamycin

The two new potent anthracycline antitumor antibiotics, morpholinodaunomycin and cyanomorpholinoadriamycin, are nonmutagenic or weakly mutagenic in Salmonella typhimurium or V79 Chinese hamster cells, but highly active inducing DNA repair in in cultured rat hepatocytes. Both agents were found to induce malignant transformation in vitro of C3H M2 mouse fibroblasts and mammary tumors in female Sprague-Dawley rats. The data indicate a) that these new anthracyclines, too, are highly oncogenic and b) in conjunction with previously published results, that the predictive value of in vitro short-term tests for in vivo carcinogenicity is dependent on the employment of a battery of such tests.Abbreviations ADM adriamycin - CNMoADM cyanomorpholinoadriamycin - DNM daunomycin - MNNG N-methyl-N-nitro-N-nitrosoguanidine Dedicated to Dr. J.H. Weisburger on his 65th birthday     

Temporal changes in tissue glutathione in response to chemical form,dose, and duration of selenium treatment

Selenium has been reported to affect glutathione (GSH) concentrations in short-term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L-selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds.     

Glucocorticoid effects on zinc transport into colostrum and milk of lactating cows

Colostrum Zn concentrations were measured in eight randomly selected Holstein dairy cows. Overall mean Zn concentrations were highest within 12 h postpartum (257 +/- 14 microM, mean +/- SEM), fell to 141 +/- 8 microM by 24 h, and then declined at a linear rate of 30 microM/d during the following 48 h. Zn concentrations at 3 d (82 +/- 5 microM) were not different from 150-d milk samples (72 +/- microM). In a second experiment, 32 early-gestation cows were blocked by stage of lactation into four groups in a randomized block design and injected with 0, 15, 30, or 45 mg of dexamethasone. Milk and blood samples were collected at 0, 12, and 24 h after injection and analyzed for Zn, and for fat, protein, and lactose in milk. Cows administered 0 and 15 mg of dexamethasone showed no difference in milk Zn concentrations compared to pretreatment measurements; however, milk Zn concentrations in cows administered 30- and 45-mg doses increased significantly. Plasma cortisol decreased in the dexamethasone-treated cows. Plasma Zn and milk fat, protein, and lactose did not change. These data indicate that glucocorticoids can mediate Zn uptake and transport by the mammary glands of lactating cows and suggest that the high Zn concentration in colostrum could be a result of the preparturient surge of cortisol.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

Elektronenmikroskopische Zytomorphologie der männlichen Brustdrüse

Zusammenfassung Im Vergleich zu lichtmikroskopischen Untersuchungen an der Mamma virilis wird anhand von 2 operativ entfernten Brustdrüsen eines 57 und 63 Jahre alten Mannes die elektronenmikroskopisch erfaßbare Zytomorphologie beschrieben. Die Befunde werden den physiologischen Wachstumsimpulsen dieses Organs gegenübergestellt und Fragen der Zelldifferenzierung, der Desquamation und apokrinen Sekretion beantwortet. Elektronenmikroskopisch werden am Drüsenepithel Basalzellen, größere Zellen der oberflächlichen Zellreihen und Myoepithelzellen unterschieden. Diese Zellen entsprechen den Gangepithelien der weiblichen Brustdrüse und besitzen intracytoplasmatische Filamente. Diese stellen ein häufiges Differenzierungsprodukt des Zytoplasmas dar. Mechanismen einer Sekretion waren nicht nachweisbar. In die Drüsenlichtung werden pseudopodienartig vorgewölbte Zytoplasmateile abgeschnürt (Extrasionsvorgang). — Superfiziale Zellen werden desquamiert, wobei die Zytolyse in den marginalen Zytoplasmaschichten erfolgt. Kern und Teile des Zytoplasmas gelangen in die Drüsenlichtung. — Die Befunde zeigen die von Lebensalter und Proliferationsreiz abhängigen Vorgänge eines permanenten Zellersatzes in der männlichen Brustdrüse an.

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Ultrastructure of the mammary gland of the human male

Summary The ultrastructure of two mammary glands obtained operatively from a 57-year old and a 63-year old man was compared to the structure observed in the light microscope, and related to stimuli controlling growth of the gland, cellular differentiation and desquamation, and apocrine secretion. The glandular epithelium, which is analogous to that of the female mammary gland, is differentiated into basic cells, large superficial cells, and myoepithelial cells. The cells have intracytoplasmic filaments, that may be a sign of differentiation. Mechanisms for secretion were not observed, although pseudopodia-like parts of the cytoplasm are extruded into the glandular lumen. Superficial cells are desquamated, followed by cytolysis of their margins. These findings illustrate the replacement of cells due to age and altered stimuli.

Frl. St. Walter, lt. Assistentin des elektronenmikroskopischen Labors, danken wir für Präparationen und Photoarbeiten.