Ivan Damjanov

Teratocarcinogenesis as related to the age of embryos grafted under the kidney capsule.

SummaryMouse egg-cylinders of C3H/H strain with two and three germ layers were transplanted under the kidney capsule. They developed into well differentiated teratomas and teratocarcinomas. Older embryos developed invariably into teratomas composed of adult tissues only. We consider the period of germ layer inversion as a critical stage when embryonic cells loose their potentiality for uncontrolled growth.

Ultrastructure of mouse egg-cylinder.

SummaryThe mouse egg-cylinder prior to and after mesoderm formation was studied by means of electron microscopy. The ultrastructural appearance of the proximal entoderm of both embryonic and extraembryonic segments suggests an intensive absorptive and nutritional activity. Numerous pinocytotic vacuoles, microvilli, primary and secondary lysosomes and fair amounts of rough endoplasmic reticulum and free ribosomes were the most important characteristics of these cells. After mesoderm formation, the extraembryonic entoderm showed the aforementioned characteristics even more prominently, while the cells of embryonic entoderm became flattened and depleted of microvilli and of almost all organelles. The cells of the extraembryonic and embryonic ectoderm prior to and after mesoderm formation had the same ultrastructural appearance as mesodermal cells. The cytoplasm of these cells was replete with free ribosomes, but other organelles such as mitochondria and rough endoplasmic reticulum were few in number. The architecture of all cells of the egg-cylinder except those of the extraembryonic entoderm suggested a very low level of differentiation. The criteria and possibilities for the determination of the degree of differentiation on the ultrastructural level and possible differences in protein synthesis in extraembryonic entoderm as compared with other parts of the embryo are considered.

Enzyme histochemistry of experimental embryo-derived teratocarcinomas.

Teratocarcinomas were obtained by transplantation of 7 day old mouse egg-cylinders under the kidney capsule of adult isogeneic animals in order to visualize the histogenesis in the teratocarcinomas. The sequence of histochemical changes roughly corresponds to the events taking place in the developing organism. The origin of differentiated tissues could not be traced histochemically because of the changes which occur in the enzyme pattern during differentiation. No “marker” enzymes were found to enable us to link the differentiated tissues with the undifferentiated embryonal carcinoma cells. The latter cells displayed a low activity of oxydative enzymes and virtually no activity of acid hydrolases, but were rich in alkaline phosphatase. Histochemically they are similar to ecto-mesodermal cells of the egg-cylinder from which the teratocarcinomas were derived. In conjunction with previous ultrastructural findings these data indicate that embryonal carcinoma cells are cells which did not differentiate, but proliferate, retaining morphologic and probably some functional characteristics of undifferentiated ecto-mesodermal cells from the early postimplantation stages of development. Teratocarcinome wurden durch Transplantation von Eicylindern 7 Tage alter Mäuse unter die Nierenkapsel von erwachsenen isogenen Tieren erzeugt, um die Histogenese in den Teratocarcinomen zu verfolgen. Der Ablauf der histochemischen Veränderungen entspricht etwa dem des sich entwickelnden Organismus. Die Herkunft der differenzierten Gewebe konnte wegen der während der Differenzierung sich abspielenden Veränderung des Enzymmusters histochemisch nicht verfolgt werden. „Marker“-Enzyme, die es erlauben würden, die differenzierten Gewebe mit undifferenziertem embryonalen Gewebe in Verbindung zu bringen, konnten nicht gefunden werden. Die embryonalen Zellen zeigten eine geringe Aktivität der oxydativen Enzyme und praktisch keine Aktivität saurer Hydrolasen, waren aber reich an alkalischer Phosphatase. Histochemisch sind sie ähnlich den ecto-mesodermalen Zellen des Eicylinders, von dem das Teratocarcinom abstammte. Zusammen mit früheren elektronenmikroskopischen Feststellungen deuten diese Befunde darauf hin, daß embryonale Carcinomzellen Elemente darstellen, welche sich nicht differenzieren, sondern bei ihrem Wachstum morphologisch und wahrscheinlich auch manche funktionellen Charakteristika undifferenzierter ectodermaler Zellen aus den ersten Entwicklungsstadien beibehalten haben.

Distribution of hydrolytic enzymes in early rat and mouse embryos--a reappraisal.

SummaryThe time of appearance and the distribution of alkaline and acid phosphatase and nonspecific esterase was investigated in cleavage and early postimplantation stages of mouse and rat embryos.Alkaline and acid phosphatase appeared for the first time in 8-cell embryos. Activity of both enzymes grew progressively stronger to blastocyst stage. Acid phosphatase activity was revealed in the form of fine and coarse granules distributed evenly in the cytoplasm. Alkaline phosphatase was predominantly localized in plasma membranes. There was no difference in intensity of reaction between trophoblastic cells and the inner cell mass.After implantation acid phosphatase was localized in coarse granules in the apical portion of entodermal cells. With the appearance of mesoderm, the cells of embryonal entoderm became flattened and devoid of acid phosphatase activity which was restricted to cells of extraembryonic entoderm. The activity of nonspecific esterase was not detected in preimplantation stages. In postimplantation embryos it roughly corresponded to the activity of acid phosphatase. Alkaline phosphatase was localized in cell membranes of ectodermal cells. The mesodermal cells of mouse embryo displayed a somewhat weaker activity than ectodermal cells, while in the rat embryo the same layer remained completely nonreactive.Our findings on the distribution of the enzymes mentioned did not reveal any kind of polarity or bilateral symmetry in preimplantation stages. In postimplantation stages acid phosphatase and nonspecific esterase are probably bound to lysosomes and play an important role in embryonic nutrition. The absence of alkaline phosphatase from entodermal cells is somewhat puzzling and suggests that the process of molecular transport in those cells is most probably restricted to endocytosis. Our results suggest that all blastomeres are identical with respect to enzyme distribution and that the first signs of differentiation of enzyme content appear with the formation of germ layers.

Host-related factors determine the outgrowth of teratocarcinomas from mouse egg-cylinders.

7 day old mouse egg cylinders transplanted under the renal capsule give rise to both teratomas and teratocarcinomas in mice of C3H/H, CBA and A strains. The proportion of benign to malignant tumors is approximately the same in the three strains. In C57BL mice, only an occasional graft examined histologically 2 months after transplantation of the embryo contains undifferentiated embryonal cells and could thus be considered a teratocarcinoma. None of these “teratocarcinomas” in C57BL mice could be retransplanted and thus unequivocally proven to be malignant. C57BL egg cylinders grafted to C57BL × C3H/H F1 hybrids gave rise to numerous teratocarcinomas. It was possible to obtain teratocarcinomas from C57BL egg cylinders grafted to lethally irradiated isogenic hosts salvaged with the bone marrow cells of C57BL × C3H/HF1 hybrids. These tumors were retransplantable in hybrids but not in inbred C57BL recipients. It was concluded that some host related factors, possibly of an immunologic nature, govern and regulate the outgrowth of tumors from mouse egg cylinders and determine whether the grafted embryo will give rise to benign teratomas or teratocarcinomas. Aus 7 Tage alten Mäuse-Eicylindern, die unter die Nierenkapsel transplantiert wurden, wachsen in C3 H/H, CBA und A-Mäusen Teratome und Teratocarcinome aus. Das Verhältnis zwischen gut- und bösartigen Tumoren ist etwa das gleiche bei diesen 3 Mäusestämmen. Bei C57BL-Mäusen wurde nur ein einziges Transplantat nach 2 Monaten untersucht; es enthielt undifferenzierte embryonale Zellen und konnte somit als Teratocarcinom angesehen werden. Aus C57BL-Eicylindern, die C57BL × CSH/F1 Hybriden implantiert waren, entwickelten sich zahlreiche Teratocarcinome, doch konnte keines dieser Teratocarcinome in C57BL-Mäusen transplantiert und somit als zweifellos bösartig bestätigt werden. Es war auch möglich, Teratocarcinome aus C57BL-Eicylindern zu erhalten, die in mit letalen Dosen bestrahlte und durch Knochenmarkzellen von C57BL × C3H/H F1 — Hybriden am Leben erhaltene Wirtstiere transplantiert worden waren. Diese Tumoren waren in Hybride retransplantierbar, nicht aber in die C57BL-Tiere. Daraus wird geschlossen, daß gewisse, möglicherweise immunologische Wirtsfaktoren das Auswachsen von Tumoren aus den Eicylindern leiten und regulieren, sowie bestimmen, ob die implantierten Embryonalzellen zur Bildung von gutartigen Teratomen oder Teratocarcinomen führen.

(Na+ −K+) dependent ATPase in mouse submaxillary gland

SummaryThe influences of castration and of testosterone administration on (NaK)-ATPase in mouse submaxillary gland has been studied. Electron microscopical and histochemical data showing a profound change in the structure of the granular tubules after castration are also presented. Whereas testosterone administration is followed by a proliferation of the rough and smooth endoplasmic reticulum in the cells of the granular tubules, castration results in an opposite change. After castration, alkaline phosphatase, which is primarily localized in the basal membranes of the granular tubules, is drastically reduced.The tissue was fractionated, by the procedure of Katz and Epstein [15], and microsomal membranes were isolated by a modification of the procedure described by Schwartzet al. [29]. Plasma membranes were isolated by the method of Henninget al. [9]. As regards MgNaK-ATPase activity in plasma membranes, castration produced a slight decrease inVmax values. In the same membrane preparation, a completely opposite results was obtained for NaK-ATPase. In microsomal membranes a tremendous increase inVmax with a change inKm occured when potassium chloride was varied. When sodium chloride was constant and KCl concentration varied, the same high increase inVmax was recorded, but inKm the decrease was not so strongly pronounced. The conclusion was reached that the high specific activity of NaK-ATPase in castrated mouse submaxillary gland may be a consequence of a different amount of membrane protein per unit of tissue weight.

Cytology of experimental teratomas and teratocarcinomas.

Von bei Mäusen erzeugten Teratomen und Teratocarcinomen entstehen in der Hälfte der Fälle ungefähr gleich häufig bösartige wie gutartige Mischtumoren. Bei der Punktion und der nachfolgenden cytologischen Untersuchung war es jedoch nicht möglich, aus den Abstrichen maligne und benigne Tumoren zu trennen.

Demonstrability of some oxidative enzymes in early rodent embryos with and without fixation.

Abstract Several oxidative enzymes [NADH-TR (reduced nicotinamide-adenine dinucleotide-tetrazolium reductase), NADPH-TR (reduced nicotinamide-adenine dinucleotide phosphate-tetrazolium reductase), SDH (succinic dehydrogenase) and LDH (lactate dehydrogenase)] were studied by histochemical means during early development of rat and mouse. All investigated enzymes could be easily demonstrated in zygote and also to some extent in somitic stages without any pretreatment. However, in cleavage and early postimplantation stages enzyme activity could be revealed only after the embryos were pretreated in some way. This pretreatment can be fixation with formalin or acetone, freezing and thawing, slight mechanical damage or very prolonged incubation time. The formazan granules as a sign of enzymatic activity were present in all stages of embryonic development and were more abundant in reactions for NADH-TR and LDH than in reactions for NADPH-TR and SDH. Our results suggest that the investigated enzymes are present in all embryonic cells during early development. It seems that the permeability of embryonic cells for histochemical media must be increased otherwise the histochemical reactions cannot be accomplished.

Aspiration Cytological and Cytochemical Study of Changes Induced in Rat Liver Cells by Carbon Tetrachloride (CCl4)

Summary The effect of a single and several repeated exposures to CCl 4 on the rat liver was studied in aspiration smears. The cytological and cytochemical findings were correlated with the histological changes in order to establish the reliability of the cytological examination of aspirates for the diagnosis of acute and chronic lesions of the liver. It was found that there is an almost complete correlation between the data obtained from aspiration smears and histological findings but only after an acute single exposure to CCk. In animals exposed repeatedly to the vapors of CCl 4 , the liver changes showed histologically a grudual transition to postnecrotic cirrhosis. Cytologically this course of the hepatic lesions could not be followed as exactly as it was in histological sections. It was nevertheless possible to establish the diagnostic criteria for differentiating cirrhotic lesions from the less adcanced ones as well as from the acute toxic insult. This study has shown that aspiration cytology can give a valuable and reliable insight into the liver changes induced by CCl 4 .