Nikola Škreb

Separation of germ layers in presomite rat embryos

Les feuillets du cylindre-Æuf du rat ont été disjoints par une solution composée de deux enzymes: 0,5% de trypsine et 2,5% de pancréatine. La séparation a été achevée par une aiguille de tungstène. Les feuillets ainsi détachés retiennent leur pouvoir de croissance et de différenciation comme homogreffes, sous la capsule rénale.

Cell cycle analysis in the mouse egg-cylinder

Abstract The cell cycle and its phases in the ectoderm and mesoderm of a seven-day-old mouse embryo was investigated by means of autoradiography. It was found that the duration of the total cell cycle for ectoderm is 6.25 h, G1 phase lasted 0.25 h, S phase 4 h, G2 0.5 h and mitosis 1.5 h. The corresponding values for mesoderm were: cell cycle 7.5 h, G1 phase 0.7 h, S phase 5 h, G2 0.5 h and mitosis 1.3 h. Comparison of the duration of the cell cycle and its phases, particularly the duration of S phase, points to the difference existing between the ectoderm and mesoderm at the moment of mesoderm formation.

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.

Histogenetic capacity of rat and mouse embryonic shields cultivatedin vitro

SummaryThe organ culture technique was used for the study of early cytodifferentiation in explanted rat and mouse embryonic shields. After 15 daysin vitro the main tissues were differentiated in explants. The full differentiation depended on the presence of homologous serum in the culture medium. 95% oxygen in the atmosphere was either deleterious or without measurable effect if introduced from the beginning or toward the end of the cultivation period, respectively. Some chemically defined media supported the development for only a limited time span during the initial period of cultivation.

Partial differentiation of rat egg cylinders in serum-free and protein-free medium☆

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagles MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.

Effect of dibutyryl cAMP and theophylline on cultured rat embryonic shields.

Effects of N6, O2 dibutyryl adenosine 3′,5′-cyclic monophosphate (db-cAMP) and theophylline (Th) on cultured rat embryonic shields were studied. After addition of db-cAMP to the culture medium, an increase of the weight of explants and of the incidence of the skeletal muscle was observed. Theophylline seems to be ineffective.

Protein patterns during final differentiation of some rat organs

Summary1.Protein composition of rat brain, and of heart and skeletal muscle was analysed by polyacrylamide gel electrophoresis in their late fetal and postnatal development.2.Adult organs show specificity of their protein patterns as judged from the relative quantitative proportions and distribution of separated protein bands.3.Protein composition of morphologically already well defined organs in the late fetal stage of development is not changed by the event of birth and is preserved unmodified until the early neonatal period.4.Significant developmental changes in protein composition leading to formation of adult pattern take place between the 3rd and 30th postnatal day.5.Results and limitations of the technique applied are evaluated from the point of applicability to developmental studies.Zusammenfassung1.Das Protein-Muster von Gehirn, Herz und Skelettmuskel in spätembryonalen und postnatalen Entwicklungsstadien wurde durch Polyacrylamid-Gel-Elektrophorese untersucht.2.Adultorgane zeigen Spezifität ihrer Eiweißmuster mit Bezug auf relative Anteile und Verteilung der getrennten Eiweißbanden.3.Die Eiweiß-Zusammensetzung von morphologisch bereits gut definierten Organen im spätembryonalen Stadium wird nicht verändert durch den Vorgang der Geburt und bleibt erhalten bis in die frühe neonatale Periode.4.Signifikante Entwicklungsänderungen in der Eiweiß-Zusammensetzung, die zur Bildung des adulten Musters führen, finden zwischen dem 3. und 30. postnatalen Tag statt.5.Die Ergebnisse und die Limitationen der Methode werden beurteilt mit Bezug auf Anwendbarkeit auf entwicklungsbiologische Studien.