The effect of macromolecular rat serum fractions on conceptuses cultured in human serum: role of transferrin.

We describe the results of experiments to study the effects on rat conceptuses cultured in human serum supplemented with rat serum which was separated into high (greater than 30 kDa) and low (less than 30 kDa) molecular weight fractions by ultrafiltration. Ultrafiltered rat serum was found to lack certain growth-promoting substances which are necessary for embryonic growth and differentiation during the culture period. Culture in whole human serum or human serum supplemented with low molecular weight fraction (filtrate) results in conceptuses which grow reasonably well but are anaemic, whilst anaemia is relieved by the high molecular weight fraction. Addition of human or rat transferrin (MW 80 kD approx.) to whole human serum alleviates anaemia, an effect observed more distinctly with rat transferrin.

Growth of rat embryos in the serum of alcohol drinkers.

Seven healthy male volunteers who had fasted overnight consumed Scotch whisky (70-85 g absolute alcohol) in a period of 15 minutes after venesection at 9.30 a.m. An hour later a further quantity of blood was collected. Rat embryos (9.5 days of gestation) grown for four hours in 'post-drink' serum (115 mg alcohol/100 ml serum) followed by 44 hours in 'pre-drink' serum were compared to controls cultured in normal human serum for 48 hours. All cultures contained 90% human serum and 10% rat serum. The embryos were examined morphologically and their protein content was measured to assess in vitro growth and differentiation. The results demonstrated the teratogenic and growth-retarding effects of alcohol ingestion. Addition of ethanol (120 mg/100 ml) to the culture medium produced similar results. Culture of 9.5-day rat embryos for 24 hours in 'post-drink' serum (115 mg/100 ml alcohol) containing 10 or 20 micrograms acetaldehyde/ml or in pre-drink serum containing similar amounts of acetaldehyde showed a toxic effect of acetaldehyde only at concentrations of 20 micrograms/ml, in the absence of alcohol.

Morphological, total nucleic acid and total protein analyses of rat embryos cultured in supplemented and unsupplemented human serum.

Culture of explanted 9 1/2 day rat conceptuses for 48 hours (i.e. until 11 1/2 days) in 100% human serum supplemented only with glucose does not result in growth and development comparable to those conceptuses cultured for the same period in 100% rat serum. Significant differences were observed in the yolk sac diameter, crown-rump length, morphological score and total embryonic nucleic acid content. No difference was observed in the total embryonic protein content. A large proportion of the embryos cultured in glucose-supplemented human serum (100%) exhibited abnormalities of the neural tube. Supplementation of the human serum with glucose and 10% rat serum resulted in embryos showing higher indices of embryonic growth and differentiation (in all criteria of assessment) than those cultured in pure rat serum; no abnormalities were observed. The significance of better growth and differentiation of the conceptuses in human serum supplemented with glucose and 10% rat serum is discussed.

Normal and abnormal appearances of the 35-day ferret fetus studied by freehand (razor blade) sections and gross examination.

This communication reports the sectioning and subsequent examination of 1-mm freehand razor blade sections of the normal 35-day ferret fetus; pregnancy in the ferret lasts for 42 days. Skeletal and other abnormalities obtained after maternal treatment with teratogens, such as trypan blue, mustine hydrochloride, and sodium salicylate are included for comparison with the normal.

Trypan blue teratogenesis in the rat: further observations in vitro.

Explanted 9 1/2- and 10 1/2-day rat conceptuses were cultured for 24 or 48 hours and 24 hours respectively in immediately centrifuged, heat-inactivated rat serum to which trypan blue was added at concentration of 150 micrograms/ml or 300 micrograms/ml or 450 micrograms/ml. The embryos were assessed for normal growth and differentiation using the following criteria: heart beat, vitelline circulation, fusion of the allantois with the chorion, normal turning, normally closed neural tube, presence of optic vesicles, presence of forelimb buds, tail, somites, somite number, yolk sac diameter, crown-rump length, and protein content. The results indicated that trypan blue is more teratogenic when 9 1/2-day conceptuses are cultured for the first 24 hours, and that its teratogenicity decreases after 10 1/2 days. Morphological abnormalities produced included neural tube defects, turning defects, and tail defects, the tail abnormalities being manifest as fluid-filled blebs. Ten and one-half-day conceptuses cultured for 24 hours in treated serum produced predominantly tail defects. The conceptuses were cultured at 11 days, when the embryo lies completely within the yolk sac. Culture of such conceptuses in serum containing 450 micrograms/ml trypan blue produced mainly tail defects. Injection of 0.5 microliters of 0.5% dye solution into the yolk sac cavity also produced tail abnormalities; sham treatment, or the injection of sterile water or a nonteratogenic dye, azo blue, did not affect embryonic development.

Morphometric analysis of the visceral yolk sac endoderm in the rat in vivo and in vitro.

Rat embryos together with their visceral yolk sac were grown in vitro during the early period of organogenesis from days 9.5 to 11.5 and the ultrastructural morphology od the visceral yolk sac cells was compared with that in vivo at the beginning and at the end of the culture period. Identical areas of the visceral yolk sac endodermal cells were analysed morphometrically. A measure of the functional activity was obtained by comparison of the volume density of surface density of the vacoular system. At 9.5 and 11.5 days, the values obtained were virtually identical for yolk sacs in vivo and in vitro. At 9.5 days, the volume and surface density of the endocytotic vacuoles in the embryonic endoderm was significantly lower than in the visceral yolk sac endoderm. It is concluded that the digestive function of the yolk sac is almost certainly identical in culture and in vivo and that the cells of the embryonic endoderm do not take a significant part in embryonic nutrition.

The developing ferret palate--a scanning electron microscope study: I. Primary palate and secondary palatal shelves.

Palatogenesis was studied in the ferret (Mustela putorius) from day 27 to day 29 of gestation. At day 27 the primary palate is present as a flattened shield area, and the two secondary palatal shelves are directed at 45 degrees to the horizontal plane, passing downward on the lateral sides of the tongue, which occupies the space between them. By day 28 the primary palate is bulging convexly in the oral cavity, and the secondary palatal shelves have elevated and are in contact in the midline. Finally, at day 29 the primary palate and nasal septum are in contact with the two secondary palatal shelves. During fusion of the secondary palatal shelves, no structures (leading edges or filopedia) associated with cell movement were apparent. It is suggested there are two stages of secondary palate formation in the ferret; an initial contacting of the secondary palatal shelves with the production of cell debris, and further midline movement of the shelves and cell migration out of the epithelial seam.

Development of ferret limb buds in organ culture.

The forelimb bud explants from ferret embryos at days 20, 21, 22, 23 and 24 of gestation were cultured in a Trowell organ culture system. Satisfactory differentiation of the limb bud skeleton was achieved with limbs from embryos explanted at 22 days of gestation (up to 50 somites). Limb buds explanted at 20 and 21 days had poorly differentiated distal segments while those explanted at 23 and 24 days of pregnancy provided less information because chondrogenesis was already under way at the beginning of culture. Thus we conclude that the optimal time for culture of ferret limb buds is at 22 days of gestation. The level of differentiation corresponded to that obtained with mouse limb buds from 11 to 12 day embryos (40-43 somites). The time taken to achieve this level of differentiation in the ferret was 18 days compared with 6 days in the mouse.

Development and structure of the extra-embryonic membranes of the ferret. A light microscopic and ultrastructural study.

Light and electron microscopic observations on the development and structure of the extra-embryonic membranes of the ferret are described. By day 16 of pregnancy trophoblastic villi consisting of a cytotrophoblastic core with a peripherally located phagocytic syncytiotrophoblast have penetrated deeply into the endometrium. During its invasion the syncytiotrophoblast removes endometrial glandular cells and some of the adjacent stromal tissue. The maternal blood vessels remain intact and eventually become surrounded by the syncytiotrophoblast. A marked hypertrophy of the maternal capillary endothelium then begine. By day 28 the maternal capillary endothelial cells attain their maximum height. The cytoplasm of these cells contains numerous cisternae of rough endoplasmic reticulum and a well-developed Golgi apparatus, features which are typical of protein-secreting cells. The maternal blood vessels are separated from the syncytiotrophoblast by a thick layer of amorphous material which stains positively with PAS. Alcian blue staining in the presence of critical concentrations of magnesium chloride indicates the presence of both sulphated and carboxylated acid mucopolysaccharides. It may be that the amorphous material contributes significantly to the nutritional requirements of the developing fetus. Invaginations of the syncytiotrophoblast plasma membrane facing this amorphous layer are often seen. Coated vesicles and substances in contact with the outer surface of the membrane are therefore likely to be endocytozed. The acid phosphatase in the syncytiotrophoblast is consistent with the presence of lysosomes and catabolic function. Regions of paraplacental cellular trophoblast which lie in close association with the endometrium, but do not penetrate it, appear to specialize in endocytosis. The haemophagus organ, which is located at the antimesometrial pole, is the site of rupture of maternal blood vessels, and the extravasated blood lies in close association with the chorio-allantoic membrane. The maternal blood cells ingested by the trophoblast in this area are presumably an important source of iron for the embryo. The cellular trophoblast in the paraplacental regions is well equipped with hydrolytic enzymes, as shown by histochemical tests for acid phosphatase. It seems likely that these regions are concerned with embryotrophic endocytotic nutrition supplementing endothelio-chorial nutrition.