ABSTRACT
The teratogenic activity of the purified components of trypan blue was studied in pregnant mice. The blue component induced abnormalities similar in type and frequency to those caused by the whole dye, and the water-soluble purple fraction exerted a mild teratogenesis when the concentration was increased to 0 · 3 per cent. The sodium hydroxide-soluble purple and red fractions were without harmful effects.
The blue and water-soluble purple fractions are absorbed and retained by the endodermal cells of the visceral yolk-sac from the time they become differentiated until term. The dye granules are associated with proteinaceous materials in apical vacuoles in the cytoplasm of these cells.
Prior to the closure of the yolk-sac the dye has direct access to the embryo. Microscopic examination of unstained sections at this stage in development reveals a diffuse staining of the embryonic mass immediately adjacent to the visceral endoderm. The suggestion is offered that cessation of the teratogenic activity of the dye after closure of the yolk-sac is due to its retention and inactivation by the cells of the yolk-sac epithelium.
INTRODUCTION
Since the discovery of the teratogenicity of trypan blue by Gillman, Gilbert, Gillman and Spence (1948) the dye has offered a very concise and convenient method for studying teratogenesis in mammals. Dijkstra & Gillman (1960) fractionated the dye and found that the purple component stimulated the endothelial system in rats, thereby raising the speculation that the varied effects of the dye were caused by contamination. According to our previous experiments (1957, 1963) and those of Wilson, Beaudoin & Free (1959) one aspect of the problem has remained fairly constant, namely, the narrow time limit of the maximal effect of the dye and the stability of the pattern of malformations. Wilson and his associates suggest that the teratogenic effect is due to direct access of the dye to the embryo before the visceral yolk-sac is completely formed. Our current experiments in mice confirm their observation, and indicate that the absence of detrimental effects of the dye when administered after the 10th day is due to its inactivation by the epithelial cells of the yolk-sac. These cells show an affinity for trypan blue from the time the endoderm is differentiated until the end of gestation. Also the light microscope shows that both the blue and purple, but not the red, components of the dye are immobilized by the yolk-sac epithelial cells. The purpose of this paper is to report our observations on the isolation and association of the trypan blue in the cells of the visceral yolk-sac of the mouse and the effects of its components on fetal development.
MATERIALS AND METHODS
The animals used in this study were taken from a colony of DBA/2 Jax mice that have been kept inbred in this laboratory for 10 years. During this time no external abnormalities have been observed in the young of the colony. The mice have served as controls in many previous experiments, and periodic examinations of pregnancies have revealed normal young averaging eight embryos per litter and 0·12 per cent, fetal wastage. Only nullipara females were used and day one of gestation was regarded as the 24 hr. preceding the morning on which a vaginal plug was discovered. Injections were given on the 7th, 8th and 9th day of gestation unless otherwise specified. The whole dye was administered in 0·25 ml. subcutaneous injections of a 0·3 per cent, aqueous solution, and the components in the percentage of the ratio found in the whole dye; i.e., the red fraction, 0·03 per cent.; water-soluble purple, 0 ·03 per cent.; hydroxide-soluble purple, 0·01 per cent.; and blue, 0·23 per cent, (all in terms of weight per volume). The concentration of the components was increased to 0·3 per cent, in later experiments (Table 1).
In the experiments designed to reveal the effects of the dye on development, pregnancy was interrupted on the 15th day, 5 days prior to term, in order that all surviving fetuses might be recovered. After removal from the uterus the fetuses were examined under the dissecting microscope for external malformations, and selected specimens were prepared for serial sectioning. At the same time blood was collected for serum studies, and the yolk-sacs were dissected for light and electron microscope studies and for enzymatic digestion. In experiments planned for the study of the distribution of the dye in the maternal tissues, the females were killed at 24-hr. intervals after the last dye injection (Table 2).
Components and trypan blue
The dyes used in these experiments consisted of trypan blue, C.I. 447; an enriched red sample, B.C. 27775; and a low red sample, B.C. 27776, received from the National Aniline Division of Allied Chemical & Dye Corp., N.Y. These dyes were tested for their composition by two methods.
The first method was devised as a modification of Gregersen & Rawson’s (1943) technique of extracting a red component from trypan blue. We shall refer to this scheme as Scheme 1 (Text fig. 1).
Scheme showing a modification of Gregersen & Rawson’s (1943) technique for extracting a red component from trypan blue.
The enriched red sample was separated by Scheme 1 into a 570 mμ filtrate, 81·4 per cent., and a 559 mμ precipitate, 10·5 per cent. The low red sample was separated into a 603 mμ precipitate, 100 per cent., and a colorless filtrate.
Scheme 2 is Dijkstra & Gillman’s (1961) chromotographic method of separating trypan blue on an alumina column. Using whole trypan blue dye, we derived the same components as the authors. They are as follows in order of extraction with the solvents used for elution :
Red 559 mμ, 13 per cent., methyl-ethyl-ketone : water : ethanol (75:25:10)
Blue 603 mμ, 75 per cent., methyl-ethyl-ketone : water : ethanol (75:25:10)
Purple 570 mμ, 10· 8 per cent., water (1)
Purple 570 mμ, 1 · 2 per cent., 0· 1 N sodium hydroxide (2)
Fig. A. Section of mouse embryo at 7 days gestation stained with hematoxylin and eosin. The mother was injected with 0· 25 ml. of 0· 3 per cent, trypan blue 12 hr. previously. Cells of the visceral endoderm (arrows) contain dye granules.
Fig. B. Unstained paraffin section of gravid uterus on 8th gestation day. Mother received two injections of trypan blue. The maternal decidua is diffusely colored blue, the cells of the visceral endoderm contain masses of dye granules, and the embryonic mass is lightly colored blue.
Fig. C. Unstained paraffin section of mouse embryo in utero on 9th day of gestation. The mother received three injections of the dye. Pale blue coloration of the embryonic mass is visible inside the yolk-sac.
Fig. D. Unstained whole mount of the visceral yolk-sac at 13 days gestation. The mother received three injections of pure blue component. The distribution and localization of the blue fraction is similar to that of the whole dye.
Fig. A. Section of mouse embryo at 7 days gestation stained with hematoxylin and eosin. The mother was injected with 0· 25 ml. of 0· 3 per cent, trypan blue 12 hr. previously. Cells of the visceral endoderm (arrows) contain dye granules.
Fig. B. Unstained paraffin section of gravid uterus on 8th gestation day. Mother received two injections of trypan blue. The maternal decidua is diffusely colored blue, the cells of the visceral endoderm contain masses of dye granules, and the embryonic mass is lightly colored blue.
Fig. C. Unstained paraffin section of mouse embryo in utero on 9th day of gestation. The mother received three injections of the dye. Pale blue coloration of the embryonic mass is visible inside the yolk-sac.
Fig. D. Unstained whole mount of the visceral yolk-sac at 13 days gestation. The mother received three injections of pure blue component. The distribution and localization of the blue fraction is similar to that of the whole dye.
(Hereafter (1) is referred to as water-soluble purple, and (2) is referred to as sodium hydroxide-soluble purple.)
Using the enriched red sample, we derived 10· 5 per cent. 559 mμ with methyl-ethyl-ketone solvent, 9 · 1 per cent. 570 mμ with water, and 81· 4 per cent. 570 mμ with 0· 1 N sodium hydroxide. The low red sample contained only 603 mμ.
The aqueous components from Scheme 1 were substituted in Scheme 2 to test their purity by this method. The blue 603 mμ completely eluted as a single fraction with the alcoholic solvent, as did the red 559 mμ. However, the purple 570 mμ divided into 88 per cent. 570 m/x with water and 12 per cent. 570 m/x with 0· 1N sodium hydroxide.
Apparently the same three major components may be obtained from trypan blue by either method, but by Dijkstra & Gillman’s method the purple can be separated into a water-soluble and an alkali-soluble fraction. It proved more efficient to collect the purple component by Scheme 1, dialyze it against water, then separate it on the alumina column. Contamination was eliminated by exposure of the sodium hydroxide to CO2 and dialization to an aqueous solution.
In order to standardize the components, concentration versus optical density graphs were made with dry, uncontaminated weights of the pure components. Standard concentration curves using serum and veronal buffer were prepared for each component and the whole dye. The blue 603 mμ. was found to shift to 600 mμ in the buffered serum, and the correction factor for normal sera became larger as 559 mμ was approached. Concentration graphs were then made for the components contained in mouse sera during gestation, using three mice of approximately the same weight for each point on the curve. Care was taken in blood collection to prevent hemolysis.
RESULTS
Of the surviving embryos of mothers receiving the blue component in the same ratio as that found in the whole dye, 57· 1 per cent, were defective, whereas 55· 5 per cent, of the living offspring of mothers receiving a 0· 3 per cent, solution of the whole dye were affected. Fetal death was only slightly greater; 58 per cent, absorptions occurred following treatment with the blue component as compared with 54· 5 per cent, after treatment with the whole dye.
Treatment of pregnant mice with a 0· 3 per cent, solution of the water-soluble purple fraction resulted in a mild teratogenic action on the surviving young.
The sodium hydroxide-soluble purple and the red fraction caused no abnormalities in our mice nor any significant increase in the number of absorptions sites.
Serum studies
Since it was found to be impracticable to obtain enough blood from a single mouse each day throughout gestation to establish a continuous curve, three mice were used to confirm each point on the curve. The concentration of the dye in the blood was determined 6 and 24 hr. following each injection, thereafter on each succeeding day in gestation.
Text-fig. 2 shows that from the 6th to the 24th hr. following the first injection, the concentration of the whole dye falls only slightly, indicating that the body tissues become saturated before the 6th hr. During the same period following the second injection, a sharp rise occurs in the serum level, indicating that the dye is not being deposited in the tissues to the same extent as following the first injection. The sharper drop in the curve after 24 hr. also suggests that the dye is eliminated faster when the concentration in the serum is greater. This agrees with the findings of Dijkstra & Gillman (1961) who found that the rate of removal of the dye from the circulation is proportional to its concentration and that the dye is removed much faster at high than at low concentrations. After the third injection a rise of approximately the same degree as that following the second takes place. The slight increase is probably due to an accumulative effect. There is also the same rate of fall 24 hr. after the third injection.
During the remainder of gestation a gradual fall in the concentration of dye in the serum is noted. Non-pregnant animals show a higher concentration of the dye than pregnant animals, which is consistent with the normal increase in body tissue in the pregnant animal and with the development of physiological dilution factors. It was also noted that sera from animals treated with whole trypan blue (596 mμ) had a maximum absorption at 600 mμ, indicating elimination of the red component.
Text-fig. 3 depicts the concentration curve obtained following three injections of the blue component (603 mμ) from the 10th through the 18th day of gestation.
Concentration of blue component in blood of mice after injection.
The sera reads at a maximum absorption of 600 mμ. This in vivo shift is consistent with the in vitro shift of maximum absorption to the blue end of the spectrum. However, Rawson (1943) and Gregersen & Gibson (1937) reported that addition of plasma to aqueous solutions of the dye shifts the point of maximum absorption toward the red end of the spectrum. Rawson suggests that the alteration of the absorption curve is due mainly to the preferential binding qualities of the albumin fraction. We found the concentration curve to be very similar to that obtained from the whole dye.
Text-fig. 4 shows the fall in concentration of the water-soluble purple component, 570 mμ, following three injections. The maximum absorption of the dye in the sera is 570 mμ, The curve resembles those of the whole trypan blue and of the pure blue component, but reveals a faster rate of removal from the serum. The readings at the lower concentrations during the last few days of gestation were very difficult to obtain because normal serum absorbed at 570 mμ.
Concentration of water-soluble purple component in blood of mice after injection.
Another experiment was devised to study the appearance of the water-soluble purple component. First, the pure blue and water-soluble fractions were mixed in equal amounts, 0 · 25 ml. of a 0 · 3 per cent, solution of each, and administered in the usual three injections. Secondly, 0· 25 ml. of a 0 · 3 per cent, solution of the water-soluble purple was injected and followed 24 hr. later by the same amount of the pure blue. In each instance, the results were similar. Grossly the yolk-sac was colored blue and the axillary lymph nodes were deep lavender. The serum read 590 mμ, and the body tissues and urine reflected a bluish lavender coloration.
The concentration of the sodium hydroxide purple, 570 m/x, in the sera was barely perceptible after three injections. It could be read on the colorimeter only at the lowest point of our standard curve 24 hr. after the third injection, after which it could not be detected. This suggests either that none is absorbed into the circulation, or that it is eliminated as fast as it is absorbed.
Other observations in connection with the sodium hydroxide purple are of interest. The endodermal cells of the yolk-sac do not accumulate this fraction. Also there is no coloration of the urine visible on the 10th day as is the case with the water-soluble purple. However, the axillary lymph nodes show absorption of the dye. The gross appearance of the body tissues indicate that the dye migrates slowly and then only to those tissues in the immediate vicinity of the sites of injection. Thus it appears that the small amount of sodium-hydroxide purple absorbed by the animal accumulates mainly in the reticulo-endothelial system.
The red component cannot be detected in the sera nor in any of the body tissues 6 hr. after the third injection. It is grossly visible in the urine within 3 hr. following an injection, but cannot be detected after 24 hr. This is consistent with other reports in the literature (Von Möllendorf, 1915;Efskind, 1940; Kelly, 1958).
Time of teratogenic effect
Three 0 · 25 ml. injections of a 0 · 3 per cent, aqueous solution of the whole trypan blue were given on various days in gestation in order to determine the time at which the greatest damage occurred to the mouse embryos.
From Table 2 it can be seen that the greatest amount of damage to the embryos occurs when the injections are commenced on the 7th day of gestation. Injections begun prior to the 7th day usually result in complete absorption. Those begun after the 8th day have less teratogenic effect. Single injections given on any one of these days was without harmful effect, and injections given on two of the three days had little effect. Thus the greatest incidence of embryonic defects follows three injections during the susceptible period. Also the types of abnormalities and the percentage of fetal wastage caused by treatment with the blue and water-soluble purple components are similar to the results of the whole dye (Table 3).
Fig. E. Visceral yolk-sac at 14th gestation day showing trypan blue granules in relation to the spindle in a dividing cell. Arrows indicate dye granules. × 970.
Fig. F. Section of mouse embryo at 10th day of gestation. The mother received three injections of trypan blue. Dye is present in the cells of the yolk-sac epithelium to the margin of the gut epithelium (GE). A, amnion. R, Reichert’s membrane. Approx. × 170.
Fig. G. Visceral yolk-sac at 13th gestation day. The mother was injected intraperitoneally with 0· 25 ml. of a 0 · 3 per cent, solution of trypan blue on the 7th, 8th and 9th day. Masses of trypan blue (arrow) can be seen in vacuoles in the supranuclear cytoplasm. The contents of these vacuoles are PAS-positivc. × 970.
Fig. E. Visceral yolk-sac at 14th gestation day showing trypan blue granules in relation to the spindle in a dividing cell. Arrows indicate dye granules. × 970.
Fig. F. Section of mouse embryo at 10th day of gestation. The mother received three injections of trypan blue. Dye is present in the cells of the yolk-sac epithelium to the margin of the gut epithelium (GE). A, amnion. R, Reichert’s membrane. Approx. × 170.
Fig. G. Visceral yolk-sac at 13th gestation day. The mother was injected intraperitoneally with 0· 25 ml. of a 0 · 3 per cent, solution of trypan blue on the 7th, 8th and 9th day. Masses of trypan blue (arrow) can be seen in vacuoles in the supranuclear cytoplasm. The contents of these vacuoles are PAS-positivc. × 970.
Histology and cytology
Grossly the uterine wall and maternal decidua are diffusely stained within a few hours after a single injection of dye. Daily observations reveal that the coloration of the decidua decreases and is not conspicuous after the 11th day but the uterine wall remains colored throughout gestation.
In unstained sections of the gravid uterus on the 7th, 8th or 9th day, the decidua is diffusely colored pale blue. Intracellular dye in granular form is found only in an occasional giant cell or macrophage and in the endodermal cells of the visceral yolk-sac (Plate 1, A & B). Through the 10th day the dye in the yolk-sac cells appears as large, amorphous masses; but from the 11th day until term, the dye granules become progessively finer and more spherical in shape (Plate 1, C & D). From the 11th through the 14th day mitotic divisions are very rapid in the endodermal cells. The dye granules are always found in the peripheral cytoplasm beyond the spindle in dividing cells and are bequeathed to the daughter cells at random (Plate 2, E).
Embryos on the 7th, 8th or 9th day sectioned in situ show a faint blue coloration of the embryonic mass where it is in direct contact with the visceral endoderm (Plate 1, B). Late on the 9th day or early on the 10th day the yolk-sac encloses the embryo proper and the yolk-sac stalk begins to form (Plate 2, F). Dye granules are present in the endodermal cells at the margin of the stalk, and occasionally a few cells containing dye are incorporated in the stalk itself, but the dye never invades the cells of the embryonic gut epithelium.
Surgical injections of dye into the yolk-sac fluid resulted in diffuse coloration of the surface of the embryo and the mesodermal tissue associated with the yolk-sac splanchnopleur 6 hr. later. Paraffin sections revealed no dye in the endodermal cells of the yolk-sac, but the blue coloration had penetrated to the vitelline vessels and even the blood cells were colored.
Sections of the yolk-sac from the 10th through the 18th day stained with hematoxylin and eosin reveal pink-staining globules in the apical cytoplasm of the epithelial cells. When stained by the periodic acid-Schiff method, these globules, as well as droplets in the cytoplasm of the giant cells lying between Reichert’s membrane and the decidua, show a positive reaction. The embryonic viscera also reacts positively by this method. In yolk-sacs from animals treated with trypan blue, the dye granules appear to be associated with the PAS-positive material contained in vacuoles in the apical cytoplasm of the epithelial cells (Plate 2, G).
The PAS-positive material in the yolk-sac cells and the giant cells was not removed by digestion with saliva, malt diastase or hyaluronidase even when previously oxidized; but much of it was removed by trypsin after oxidation. The PAS-positive material in the embryo was removed by both malt diastatse and hyaluronidase.
When yolk-sacs were exposed to thorium dioxide and studied by the aid of the electron microscope, the colloid was found to be located in vacuoles in the supranuclear cytoplasm similar in location and appearance to those containing trypan blue (Plate 3, H & I).
DISCUSSION
Components of trypan blue
The components of trypan blue are: blue (603 mμ), 0·23 per cent.; watersoluble purple (570 mμ), 0·03 per cent.; sodium hydroxide-soluble purple (570 mμ), 0*01 per cent.; and red (559 mμ), 0·03 per cent. When given in the ratio found in the whole dye, only the blue component exerts a teratogenic action. However, when the concentration of the water-soluble purple is increased to 0·3 per cent., it also exerts a mild teratogenesis. Table 1 shows that each time the concentration of the water-soluble purple fraction was increased, a few defective young were produced and the fetal wastage was increased. The water-soluble purple migrates to the gravid uterus and is deposited in granular form in the cells of the vitelline membrane in the same manner as the blue fraction, but in smaller quantities. Also, as can be seen in Text-fig. 4, it is retained in the maternal circulation during the period of greatest susceptibility, and Table 3 shows the pattern of abnormalities to be the same as that induced by the blue fraction. Our experiments combining the blue and water-soluble purple components indicate that the pathway of the purple fraction is the same as that of the blue; and although a portion of the purple was immobilized in the lymph nodes, enough was present in the circulation to compete for binding sites and to shift the wave length from 600 mμ to 590 mμ.
Beck, Spencer & Baxter (1960) reported external abnormalities in the fetuses of rats following maternal injections of all fractions of trypan blue, including the red fraction. Later Beck & Lloyd (1963) treated pregnant rats with purified components of the dye and found that only the blue fraction induced abnormalities.
Fig. H. Visceral yolk-sac epithelium, 13 days gestation. The mother was injected with trypan blue on the 7th, 8th and 9th day of pregnancy. The surface plasma membrane is modified into microvilli that are more numerous at the lateral margins of the cell surface. There are vacuoles of varying density in the apical cytoplasm that are larger in the region of the nucleus. The membrane about the supranuclear vacuoles typically is incomplete. The Golgi apparatus is separate from the vacuoles. Trypan blue cannot be seen in the electron micrographs. Compare appearance with light micrograph Test-fig. 7. × 5900.
Fig. I. Visceral yolk-sac epithelium, 11 days gestation explanted into tissue culture and exposed to colloidal thorium dioxide for 12 hr. An aggregate of thorium particles (arrow) is seen in a vacuole similar in location and appearance to those containing trypan blue ×13,400.
Fig. H. Visceral yolk-sac epithelium, 13 days gestation. The mother was injected with trypan blue on the 7th, 8th and 9th day of pregnancy. The surface plasma membrane is modified into microvilli that are more numerous at the lateral margins of the cell surface. There are vacuoles of varying density in the apical cytoplasm that are larger in the region of the nucleus. The membrane about the supranuclear vacuoles typically is incomplete. The Golgi apparatus is separate from the vacuoles. Trypan blue cannot be seen in the electron micrographs. Compare appearance with light micrograph Test-fig. 7. × 5900.
Fig. I. Visceral yolk-sac epithelium, 11 days gestation explanted into tissue culture and exposed to colloidal thorium dioxide for 12 hr. An aggregate of thorium particles (arrow) is seen in a vacuole similar in location and appearance to those containing trypan blue ×13,400.
Relation of trypan blue to the embryo
Our experiments indicate that the dye has direct access to the mouse embryo on the 7th, 8th and 9th days of gestation. The dye reaches the uterus within 2 hr. after injection, diffusely colors the uterine wall, and gradually spreads to the maternal decidua. We have not observed gross coloration of the embryos at any time in gestation, only the uterine wall and maternal decidua are colored after subcutaneous dye injection. However, microscopic examination of unstained paraffin sections of embryos on the 7th, 8th and 9th days reveals a diffuse blue coloration of the embryonic mass similar to that seen grossly in the maternal decidua, and large dye granules are localized in the endodermal cells of the visceral yolk-sac (Plate 1, A, B & C). The proximal endoderm is differentiated on the 5th day (Snell, 1941) and we have observed trypan blue granules in these cells on the 6th day of gestation. At this stage the yolk-sac does not enclose the embryo, and fluids crossing Reichert’s membrane have direct access to the embryo. Ferm (1956) also demonstrated that trypan blue crossed the blastocyst wall and was present in the yolk-sac fluid, indicating that the dye has direct access to the rabbit embryo during the early stages of differentiation. According to Wilson et al. (1959) and Waddington & Carter (1953) the pattern of abnormalities, such as non-closure of the neural folds, enlargement of the pericardium, and defective development of the eyes, indicates that trypan blue acts within a very short span of time early in gestation. Waddington gave a single large injection (0·5 ml. of a 1 per cent, solution) which probably prevented survival of the more abnormal young.
Cessation of teratogenic activity of trypan blue
Our Table 2 shows the peak of teratogenic action of the dye is reached during the 7th, 8th and 9th days of gestation and that after the 9th day there is an abrupt cessation of activity. This functional change coincides with closure of the yolk-sac and the formation of the yolk-sac stalk (Plate 2, F). The yolk-sac epithelium, however, still contains masses of dye granules (Plate 1, D).
Other evidence indicates that the colloidal dye is absorbed into the cells of the yolk-sac epithelium and entrapped there in vacuoles in the cytoplasm. Trypan blue accumulates as particulate matter in close association with PAS-positive material in the endodermal cells, and after enzymatic digestion the dye granules remain in the empty vacuoles. Bridgman (1948) felt that the supranuclear proteinaceous granules visible before the 8th day held the vitally injected trypan blue particles; but after the 8th day the dye appeared to be inside pre-formed apical vacuoles.
Electron microscope studies show that colloidal thorium dioxide enters the yolk-sac cells between the microvilli at the free surface, and aggregates of the particles can be found in vacuoles in the supranuclear cytoplasm similar in location and appearance to those containing trypan blue (Plate 2, H & I). The findings in the yolk-sac epithelium are similar to those reported by Trump (1961) in a study of the uptake and transport of thorium and trypan blue in the renal tubular epithelium of frogs and rats. The supranuclear vacuoles in the yolk-sac epithelium may represent lysosomes, but histo-chemical studies are needed to establish this point.
Yolk-sac epithelium as maternal-fetal barrier to trypan blue
Our experiment in which trypan blue was injected into the yolk-sac fluid was terminated before the dye reached the epithelium, but it had penetrated through the associated mesodermal tissue. Beaudoin & Ferm (1960) studied explants of rat yolk-sac epithelium grown on the chorio-allantoic membrane of the chick. Trypan blue accumulated in the endodermal cells in granular form after being injected into the chick yolk-sac or applied directly to the allantoic membrane, showing that the dye passed through the coelomic mesothelium and reached the explanted yolk-sac epithelium through the basal portion of the cell. Thus it would appear that the endodermal cells are capable of immobilizing the dye in granular form whether it reached them through the apical or basal end of the cell. This suggests that the yolk-sac epithelium is the essential maternal-fetal barrier to trypan blue after closure of the yolk-sac.
RÉSUMÉ
Recherches sur les propriétés tératogènes du bleu trypan et de ses constituants, chez la souris
L’activité tératogène des constituants purifiés du bleu trypan a été étudiée sur des souris gestantes. Le constituant bleu a induit des anomalies semblables, par leur nature et leur fréquence, à celles que provoque le colorant complet, et la fraction pourpre hydrosoluble a exercé un léger effet tératogène quand sa concentration s’est élevée à 0,3 %. Les fractions pourpre, soluble dans la soude, et rouge n’ont pas eu d’effets nocifs.
Les fractions bleu et pourpre hydrosoluble sont absorbées et retenues par les cellules endodermiques du sac vitellin viscéral depuis le moment où elles deviennent différenciées jusqu’à terme. Les granules de colorant sont associés à des substances protéinacées dans des vacuoles apicales du cytoplasme de ces cellules.
Avant la fermeture du sac vitellin, le colorant a directement accès à l’embryon. L’examen microscopique de coupes non-colorées à ce stade du développement révèle une coloration diffuse de la masse embryonnaire immédiatement adjacente à l’endoderme viscéral. On suggère que la cessation de l’activité tératogène du colorant après la fermeture du sac vitellin est due à sa rétention er a son inactivation par les cellules de l’épithélium du sac vitellin.
ACKNOWLEDGEMENTS
This investigation was supported by research grants GM 08266-01, NB 03481 and HE-2549 from the Institutes of Health, United States Public Health Service; and Research Career Development Award GM-K3-15, 333, from the Division of General Medicine, National Institutes of Health, USPHS. (J. C. Geer, M.D.)
We would like to acknowledge the very competent technical assistance of Mrs Sydney Simmons and Miss Catherine Catsulis.
All dyes used in this investigation were received from National Aniline Division, Allied Chemical & Dye Corp.