1. The uptake of radiosulphate in rat embryos 7–22 days old was investigated with autoradiography and compared with the occurrence of metachromasia after toluidine blue staining and a positive PAS reaction.

  2. The youngest embryos retained rather small amounts of radiosulphate, which was evenly distributed. In the older embryos the amount of retained radiosulphate was considerably higher and regionally well differentiated.

  3. Marked changes in the autoradiographic pattern of certain organs occurred with growth.

  4. The uptake of radiosulphate was as a rule paralleled by metachromasia with toluidine blue. No direct relationship was found between fixation of radiosulphate and PAS reactivity of the tissues.

  5. Radiosulphate was found to be incorporated into cells and tissues containing inorganic sulphate (mineral part of bony tissues) and sulpho-mucopoly-saccharides (mesenchyme, mast-cells, &c.).

In the last few years various groups of workers have studied extensively the fate of radiosulphate in the animal body. Accounts of the research performed have recently been given by Bostrom (1953) and Bostrom & Jorpes (1954). If a small amount of radiosulphate is given to an animal most of it is rapidly eliminated in the urine and faeces. In the adult rat approximately 95 per cent, is excreted in this way in 5 days (Dziewiatkowski, 1949). Some of the retained radiosulphate is present as inorganic sulphate, and is probably incorporated as such, in the mineral part of bone (Engfeldt et al. 1954b). Labelled sulphate can be recovered in large amounts from the sulpho-mucopolysaccharides, and autoradiographic studies have disclosed that radiosulphate is retained to the greatest extent by cells and tissues known—or supposed—to contain these substances. Recently it has also been demonstrated (Holmgård, 1955) that a certain amount of radiosulphate enters the sulpho-cerebrosides. On the other hand, little sulphate is utilized in the synthesis of taurine, methionine, and cystine (Tarver & Schmidt, 1939; Bostrom & Åqvist, 1952) or, consequently, keratin. The information available is still incomplete, however; for instance, little is known about the possible intervening steps between inorganic sulphate and sulpho-mucopolysaccharides or sulpho-lipids.

In connexion with the incorporation of labelled sulphate into the sulpho-mucopolysaccharides, the uptake by the embryo is of considerable interest. It has been shown that metachromatic substances—presumably sulpho-mucopoly saccharides—occur in large quantities in embryonic tissues as well as in other rapidly growing tissues (Holmgren, 1940, 1949). A transfer of radiosulphate from the maternal rat to the foetus was first reported by Layton et al. (1950). Bostrom & Odeblad (1953) briefly reported the incorporation of radiosulphate by rabbit embryos as revealed by autoradiography. The uptake of S35 varied markedly in different tissues, being highest in those presumably containing sulpho-mucopolysaccharides. Applying chemical and to some extent autoradio graphic methods as well, Dziewiatkowski (1953) demonstrated that the amount of radiosulphate retained by the embryo was directly related to its degree of development, increasing with its age. He also found that the radiosulphate frac-tion insoluble in trichloroacetic acid increased from 40 per cent, at 10 days to 90 per cent, at 20 days. Some tissues of the embryo showed higher activity than their maternal equivalents. Embryonal cartilage, for example, contained 30 times the amount in maternal cartilage 24 hours after the injection of radiosulphate. Lately Amprino (1955) has investigated the uptake of S35-labelled sulphate in early chick embryos during cartilage and bone formation.

The aim of the present investigation was a systematic autoradiographic study on the incorporation of radiosulphate by the rat embryo and a comparison of the results with those obtained by histochemical staining reactions.

Some forty female albino rats representing various stages of pregnancy were given an intraperitoneal injection of 3 me./kg. of carrier-free radiosulphate (obtained from A.E.R.E., Harwell, England) in 0·9 per cent, saline with 0·02 per cent, sodium sulphate. The animals were killed 48 hours later and the embryos were withdrawn. Their weights and C.R.-lengths served as a control of their age as estimated from copulation dates. Some forty embryos 7–22 days old and five new-born from different litters were used for autoradiography. The new-born rats were born at the end of the 48 hours that were allowed to elapse between injection and killing, the radiosulphate having been injected into their mothers. Specimens were also taken from the mothers (and non-pregnant females) for comparison.

The majority of the preparations were fixed in absolute methanol and the remainder were fixed in 10 per cent, formaldehyde containing 4 per cent, basic lead acetate. After dehydration in ethanol and clearing in xylene they were embedded in paraffin. Serial sections were cut 8 μ thick, and were alternately placed on glass slides (for staining) and on methacrylate slides (for autoradiography).

Autoradiography was performed with Gevaert Dentus Rapid film. The methacrylate slides were passed through xylene and ethanol and dried. They were then placed in contact with the X-ray film between cardboard strips in iron screw presses for 45 days. The films were developed in Kodak DK-20. The resolution was 15–20 μ. For orientation one-third of the slides were stained1 with haematoxylin and eosin or van Gieson connective tissue stain. One-third of the sections were stained in a 0·1 per cent, solution of Toluidine Blue O, C.I. 925, in 30 per cent. ethanol for 30 minutes. The remainder was stained with periodic acid-leucofuchsin (periodic acid-Schiff, PAS) in a modification of the original Hotchkiss’s method (1948) previously elaborated (Friberg et al., 1953). All solutions were made in 70 per cent, ethanol and the reducing rinse was omitted. The Schiffreagent contained 0·1 per cent. Basic Fuchsin, C.I. 677, decolorized by SO2 from a cylinder. The sulphite wash consisted of 70 per cent, ethanol saturated with SO2. The slides were left for 30 minutes in the periodic acid-solution as well as in the leucofuchsin reagent.

No significant differences between embryos fixed in methanol and embryos fixed in formaldehyde-basic lead acetate could be observed with regard to content of radiosulphate or histochemical staining reactions. As judged from the autoradiographs the youngest embryos retained only small quantities of radiosulphate which were evenly distributed. There occurred a steady increase and differentiation in the uptake of radiosulphate towards birth. The ratios between the uptake by different organs within the same embryo varied with its age. Thus no general statement valid for all organs can be formulated. At 11 days there was a distinct differentiation of the uptake of radiosulphate—areas of mesenchyme containing more than the other tissues of the embryos. During the second half of the gestation period the highest uptake of radiosulphate occurred in areas of mesenchyme or its derivatives, e.g. cartilage, tendons, heart-val ves. It also appeared that the autoradiographic picture of the internal organs was greatly influenced by their content of mesenchyme. Uptake of radiosulphate, however, was also found without relation to mesenchyme, for instance, in the mucous glands of the digestive tract and in some parts of the central nervous system.

Mesenchyme

The first areas of mesenchyme to show an uptake of radiosulphate higher than other tissues of the embryo were those around the notochord and the neural tube and in the heart-valves. In embryos 16–22 days old, cartilage, tendons, fasciae, connective tissue septa, dermis, and tooth papilla showed a high uptake of radiosulphate in addition to the structures already mentioned. As a rule, there was a close relationship between the amount of metachromatic ground substance and sulphate retention. The tissue mast-cells which showed high incorporation of radioisotope were not visible in the autoradiographs before 17–18 days of age. Their presence could only be safely established in full-term embryos when they occurred in great numbers (Plate 3, figs. K, L, M; Plate 4, fig. O). Their granules exhibited a more or less distinct metachromasia and sometimes a PAS reaction.

Where cartilage was formed, a progressive increase of radiosulphate incorporation was recorded which ran almost parallel to the occurrence of metachromatic ground substance, but because of the limited resolution it could not be excluded that radiosulphate was present also in the cells. The ground substance of cartilage showed metachromasia and weak to moderate PAS reaction. The chondrocytes showed little metachromasia but often contained granules which stained heavily with PAS.

During enchondral bone formation the uptake of radiosulphate diminished as cartilage was replaced by bone. Calcified bone thus showed a lower incorporation of radiosulphate (Plate 4, figs. O, P), but displayed a stronger PAS reaction. Intramembranous ossification was preceded by an accumulation of great amounts of mesenchymal ground substance showing intense metachromasia and moderate PAS reactivity. Simultaneously there was a high uptake of radiosulphate (Plate 1, fig. B, brain capsule).

Cardiovascular system

The uptake of radiosulphate in the heart-muscle was low during embryonic development and distinct metachromasia could not be observed. The PAS reaction, however, was markedly positive already in embryos 10 days old. The mesenchymal components of the heart, i.e. annulus fibrosus and heart-valves, showed a high uptake of radiosulphate in embryos of 14 days and older (Plate 1, figs. A, B). These structures were metachromatic. In the wall of the aorta moderate to large amounts of radiosulphate were retained from about 15 days onwards (Plate 2, fig. D). The uptake of the main arteries exceeded that of the main veins. Blood-cells were not found to incorporate any significant amounts of radiosulphate.

Alimentary system

Increased uptake of radiosulphate occurred in connexion with the downgrowth of the lip furrow band in the surrounding mesenchyme, which showed metachromasia and PAS reactivity (Plate 1, fig. B). In the teeth of full-term embryos and new-born rats a high uptake of radiosulphate was noted at the pulpodentinal border and in the tooth papilla (Plate 3, fig. L). The predentine was weakly metachromatic whereas a stronger metachromasia could be seen in the tooth papilla. The PAS reaction occurred in a similar way. In the tongue a moderate uptake of radiosulphate was found in the superficial layers, especially in the basal parts (Plate 1, figs. A, B). It appeared to be housed mainly within the subepithelial mesenchyme. A moderate uptake was found in salivary glands of full-term embryos and new-born rats. In the oesophagus a distinct uptake of radiosulphate occurred from 15 days onwards. The uptake was marked at 18 days, but much less prominent in new-born rats (Plate 1, figs. A, B; Plate 2, fig. D). This uptake appeared to have occurred mainly within the mesenchymal parts of the wall, which showed metachromasia and a moderate PAS reaction.

The stomach was found to take up moderate amounts of radiosulphate at 17–19 days. The retention of radiosulphate within the walls of the intestine was found to vary during different stages of development. In embryos 18 days old the highest uptake of radiosulphate was found in the metachromatic subepithelial mesenchyme (Plate 1, fig. B; Plate 2, fig. G). A lower uptake was found in the epithelium. At birth the uptake of the mesenchymal parts of the intestine was less prominent. The intestinal epithelium was at birth producing a mucous substance that was intensely metachromatic and strongly PAS reactive. Considerable amounts of radiosulphate were found in the epithelium and intestinal contents (Plate 2, fig. H). Mucus with the same staining properties and high uptake of radiosulphate was also found in the intestinal lumen of some embryos 10 days old. It was not possible, however, to demonstrate any such mucus or uptake of radiosulphate at 14–19 days.

The uptake of radiosulphate in the liver and the pancreas was low during the complete gestation period. In the bile ducts a moderate uptake was found in the later stages.

Respiratory system

A great uptake of radiosulphate was found in the cartilages of the nose (Plate figs. A, B), the trachea and the bronchi (Plate 2, fig. D). Otherwise the uptake of radiosulphate was insignificant in the respiratory passages. In the lung, the uptake was low during embryonic development.

Urogenital system

In the metanephros, a considerable uptake of radiosulphate combined with metachromasia occurred in the mesenchymal stroma around the pelvis (Plate 2, fig. F). The uptake of radiosulphate in the blastemal areas was low. Towards the end of the gestation period the uptake of sulphate within the pelvic stroma was less striking. The differentiation of the bladder, genital papilla, and vagina was associated with an accumulation of metachromatic substances and an increased uptake of radiosulphate in the mesenchyme (Plate 2, fig. G). The uptake of radiosulphate was low in areas rich in differentiated muscle-cells.

Nervous system

The uptake of radiosulphate in the peripheral nervous system was low during all stages of development. The peripheral nerves and ganglia exhibited little metachromasia and reacted weakly to PAS. In the central nervous system the uptake of radiosulphate was also low as a rule, but a higher uptake occurred in some parts of the brain at certain stages of development. In the CNS of embryos about 10 days old the uptake of radiosulphate was low and evenly distributed. At 15 days, however, a differentiated uptake could be observed (Plate 1, fig. A). At 17–18 days, there was a fairly high uptake in the corpus striatum and certain layers of the cerebral cortex (Plate 1, fig. B). On the other hand, the ependymal cells surrounding the lateral ventricles did not retain much radiosulphate (Plate 2, fig. E). No significant difference between grey and white matter was observed in the cord. Those parts of the brain retaining more sulphate displayed a stronger PAS reaction than did parts with lower uptake. The metachromatic reaction was somewhat variable. The chorioid plexus reacted strongly to PAS and showed a fairly high uptake of the radioisotope.

Sensory organs

Uptake of radiosulphate in the eye was observed in embryos from 15 days. The uptake in the optic cup and the corpus vitreum was low. The peripheral but not the central part of the lens contained moderate amounts of radiosulphate. The cornea and sclera and the eyelids showed a moderate uptake of radiosulphate and metachromasia in embryos 17 days old (Plate 3, fig. I). In the inner ear maximal incorporation of radiosulphate occurred in the cartilaginous labyrinth. The uptake in the membranous labyrinth was low. A moderate uptake was seen in the membrana tectoria, the gelatinous mass of the cupula, and the otolithic membrane. These structures showed little or no metachromasia, whereas the PAS reaction was quite strong (Plate 3, fig. K).

Skeleto-muscular system

In the autoradiographs the picture is dominated by the intense uptake of the cartilaginous skeleton. A decrease in the amount of retained radiosulphate was registered in areas of calcification as cartilage was replaced by bone, the uptake of calcified bone being lower. The uptake of radiosulphate within cartilage was not uniform. In cartilage undergoing calcification, the highest concentration of radiosulphate was found at the level of the proliferating hypertrophic cells. The periosteum was metachromatic and retained moderate to large amounts of radiosulphate. The bone-marrow presented a low retention of radiosulphate. In the skeletal muscles the uptake of radiosulphate was low, except for mesenchymal septa, which often contained mast-cells. Tendons retained large amounts of radiosulphate (Plate 4, fig. P). The articular capsules showed a moderate sulphate fixation (Plate 4, fig. O).

Skin

In the embryos at 10–15 days, a moderate uptake of labelled sulphate was present in the skin. At full term a low uptake was found in the epidermis, but the corium contained a moderate amount. The tactile hair follicles showed a high uptake especially in the outer layers, whereas hair-shafts did not absorb any sulphate. The corium contained numerous mast-cells with a high incorporation of the isotope (Plate 3, fig. M).

Extra-embryonic structures

In the pregnant uterus at 10 days, a high uptake of radiosulphate was found in the decidua and especially its basal parts, whereas the myometrium showed a considerably lower uptake (Plate 1, fig. C; Plate 3, fig. N). The uptake in these areas was paralleled by the occurrence of the PAS and metachromatic staining reactions. Similar relationships were seen in the later stages of gestation. The occurrence of a very high uptake was found to correspond to the presence of cells with metachromatic granules in the decidua beneath the placental insertion. These cells are probably identical with the ‘specific cells’ of Asplund et al. (1940).

More cells, however, were seen in the sections than on the autoradiographs. The granules of these cells were found to be PAS positive. The cells were larger than average tissue mast-cells and lay close to the vessels. They were present in large numbers at 10 days, but seemed to decrease in number towards birth.

The placenta showed low uptake of S35 before 11 days, but moderate amounts were incorporated in the later stages of gestation. The uptake was evenly distributed and only the walls of larger vessels stood out clearly. In the umbilical cord a high uptake of radiosulphate was often found both in or around the walls of the vessels. The rest of the cord showed a diffuse, weak metachromasia and moderate uptake of radiosulphate.

The indirect mode of administration of the isotope to the embryos makes certain precautions necessary in the interpretation of the autoradiographs. The uptake of radiosulphate will depend on both extra- and intra-embryonal factors. We found a steady increase in the fixation of radiosulphate towards birth. This may be due in part to a facilitation of passage through the placental barrier and in part to increased sulphate-retaining activity of the embryonal tissues. It is not possible to state exactly when the main uptake of radiosulphate occurred, but only that it took place during the 48 hours of intra-uterine life following the injection. This interval is indeed long in relation to the rapidity of embryological development, and during it the individual embryo may have undergone considerable changes.

The amount of inorganic radiosulphate still present after the interval between the injection of the isotope and the withdrawal of the specimens can be considered as low. The autoradiographs thus mainly visualize the distribution of the incorporated radiosulphate. The incorporation can be expected to have occurred into the mineral part of bone (in older embryos, as inorganic sulphate), into the sulpho-mucopolysaccharides and perhaps the sulpho-lipids (as ester sulphate). There was a striking parallelism between the occurence of metachromasia and retention of radiosulphate. Metachromasia in vitro with toluidine blue may be caused by a fairly large number of substances, containing acid radicals (Walton & Ricketts, 1954, and others), such as sulphate groups, phosphate groups, and carboxyl groups. It appears that the sulpho-mucopolysaccharides and to a lesser extent the sulpho-lipids, hyaluronic acid, and nucleic acids can be expected to have occurred in such amounts as to have caused metachromasia in the sections. This points in favour of an incorporation of the sulphate into the sulphomucopolysaccharides. Certain discrepancies, however, occurred between the distribution of metachromasia and uptake of radiosulphate. Thus the membranes of the inner ear were found to incorporate radiosulphate and to exhibit a very strong PAS reaction but very little or no metachromasia. Sulphate uptake in the membranes of the inner ear of new-born rats has previously been reported by Bélanger (1953). Our staining reactions seem to be in accordance with those reported by Wislocki & Ladman (1955). It must be remembered that metachromasia is influenced by a large number of factors, e.g. temperature and presence of other compounds such as basic protein (French & Benditt, 1953, and others). Thus the lack of metachromasia in this case does not exclude the presence of sulpho-mucopolysaccharides.

The retention of radioisotope was not directly related to the presence of substances giving the PAS reaction, although structures taking up radiosulphate were as a rule PAS positive. Mast-cells, for example, showed a high uptake of S35 but a weak PAS reaction, whereas muscle-tissue had a low uptake of radiosulphate but gave a strong colour with PAS in many cases. PAS is stated to stain any compound containing the 1,2-glycol group —CHOH—CHOH— (hydroxyl groups may be replaced by amino or alkylamino groups) or its oxidation product —CHOH—CO. It is probable, however, that a substantial colour is produced in the sections only by high molecular substances of polysaccharide nature (Hotchkiss, 1948). Thus glycogen, heparin-mono-sulphuric acid and hyaluronic acid will be stained. On the other hand, heparin-trisulphuric acid and chondroitin sulphuric acid cannot be expected to react (Jorpes et al., 1948). The absence of any direct relationship between fixation of S35 and PAS reactivity is due to the fact that the PAS reaction is given by a wide range of compounds, many of which do not contain sulphates. Besides, certain sulpho-mucopolysaccharides do not react.

The resolution of the autoradiographs did not always permit the exact localization of the isotope, whether intraor extra-cellular or in a special cell layer. In most cases an extra-cellular retention is probable, as the incorporation of radiosulphate was parallel to the amount of ground substance present. The uptake seen in connexion with, for example, mast-cells and ‘specific cells’ of the decidua, however, appears to be intra-cellular. Certain authors have found an incorporation of S35 into the nuclei of cells (Odeblad & Bostrom, 1953). Our material did not permit observations of that refinement.

As a rule the highest uptake occurred in connexion with the mesenchyme and its derivatives. This uptake most probably occurred into the sulpho-mucopolysaccharides. The formation of cartilage involved an accumulation of metachromatic material. These areas also incorporated great amounts of radiosulphate. From the histochemical findings it seems probable that a large portion of the metachromatic and radiosulphate-containing material would be identical with chondroitin-sulphuric acid.

Enchondral and intramembranous bone formation as well as the formation of dentine commenced with an accumulation of metachromatic, PAS reactive ground substance exhibiting a high uptake of radiosulphate. Thus these three calcification processes seem to take place in a similar way from a histochemical standpoint. Similar findings have recently been reported by Bélanger (1954) and Engfeldt et al. (1954 a, b).

The most conspicuous cells of the connective tissue are the mast-cells. According to Holmgren (1946–7) mast-cells are not present in 14-mm. rat embryos but have appeared at the 20-mm. stage. In our material they could be safely identified in the autoradiographs only in the oldest embryos, i.e. older than 17–18 days. At this time, they incorporated large amounts of radiosulphate, as do mast-cells of adult tissues (Jorpes et al., 1953). Metachromatic ground substance, incorporating radiosulphate, is undoubtedly produced long before any typical mastcells can be seen in autoradiographs or sections.

In some way related to the mast-cells are the ‘specific cells’ of the uterine wall. Asplund et al. (1940) stated that the metachromasia of these cells was less alcohol-resistant than that of the mast-cells. They assumed that the ‘specific cells’ contain sulpho-mucopolysaccharides which are not as highly esterified with sulphate as heparin. Similar cells have been described in the human decidua by Wislocki & Dempsey (1948). The metachromasia of these cells was, however, removed with ribonuclease. We found cells probably identical with the ‘specific cells’ which, besides being metachromatic, also gave a positive PAS reaction and showed a high incorporation of radiosulphate. These cells thus showed some of the more important characteristics of the mast-cells. Possibly they contain heparinoid substances and are merely variants of mast-cells.

In the digestive tract of the embryo, uptake of radiosulphate occurred in connexion with the development of the mesenchyme concomitant with the production of mucous substances presumably of epithelial origin.

In some parts of the central nervous system an increased uptake of radiosulphate was found at certain stages of development. The correlation with metachromasia, however, was somewhat inconsistent, but a better correlation was found to PAS reactivity. This incorporation of radiosulphate may be due to sulpho-mucopolysaccharides. The presence within the central nervous system of a ground substance containing mucopolysaccharides has recently been claimed by Hess (1953) and Bairati (1953). It is thus tempting to assume that some radiosulphate might have been incorporated into sulpho-mucopolysaccharides. On the other hand, an incorporation into sulpho-lipids is plausible and would possibly be associated with the process of myelination. It is, however, not known to what extent sulpho-lipids occur in embryonic nervous tissues. Besides, it is probable that the sulpho-lipids are removed by the present histological methods (xylene-alcohols). No certain conclusions can thus be drawn as to the nature of the sulphate-incorporating compounds of the embryonic brain.

Evidence in favour of the incorporation of radiosulphate into myelin has recently been given by Ringertz (1956).

The authors are indebted to Dr. L. Gyllensten for stimulating interest during the work and for criticism of the manuscript, and to Mrs. M. Bjorkman and Mrs. M. Bohlin for technical assistance. AB Acierex Ltd. kindly supplied the iron presses. This investigation was supported by grants from the ‘Reservations-anslaget’ of Karolinksa Institutet and from the Foundation ‘Konung Gustaf V:s 80-ârsfond’.

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The pieces of film exposed to the radioactive slides were used as negatives from which prints were taken. The photographs in the following plates are such prints, and in them, therefore, the lighter the area the more intense is the radioactivity represented.

PLATE 1

Fig. A. Autoradiograph of a sagittal section of a 15-day embryo. A high uptake of radio-sulphate is seen in the following cartilaginous skeleton parts: base of skull, nasal cartilage, hyoid bone, and vertebral column. A high uptake is also seen in the heart-valves. A moderate incorporation is present in the meninges, base of tongue, skin, aorta, oesophagus, and intestines. The uptake in the liver and muscular tissue, including myocardium, is low and even. A low but somewhat uneven uptake is presented by the brain. Magnification ×10.

Fig. B. Autoradiograph of a sagittal section of an 18-day embryo. A high uptake of radiosulphate is seen in the cartilaginous skeleton, heart-valves, oesophagus, and brain capsule. A moderate incorporation is found in the aorta, intestinal submucosa, brain, skin, and fasciae, and a low uptake in the lung, myocardium, liver, and muscle-tissue. Magnification × 10.

Fig. C. Autoradiograph of pregnant uterus at 10 days showing high uptake in the decidua, and low uptake in the placenta and the embryo. Note numerous white dots in the decidua below the placental insertion, probably corresponding to the ‘specific cells’. Cf. Plate 3, fig. N. Magnification × 10.

PLATE 1

Fig. A. Autoradiograph of a sagittal section of a 15-day embryo. A high uptake of radio-sulphate is seen in the following cartilaginous skeleton parts: base of skull, nasal cartilage, hyoid bone, and vertebral column. A high uptake is also seen in the heart-valves. A moderate incorporation is present in the meninges, base of tongue, skin, aorta, oesophagus, and intestines. The uptake in the liver and muscular tissue, including myocardium, is low and even. A low but somewhat uneven uptake is presented by the brain. Magnification ×10.

Fig. B. Autoradiograph of a sagittal section of an 18-day embryo. A high uptake of radiosulphate is seen in the cartilaginous skeleton, heart-valves, oesophagus, and brain capsule. A moderate incorporation is found in the aorta, intestinal submucosa, brain, skin, and fasciae, and a low uptake in the lung, myocardium, liver, and muscle-tissue. Magnification × 10.

Fig. C. Autoradiograph of pregnant uterus at 10 days showing high uptake in the decidua, and low uptake in the placenta and the embryo. Note numerous white dots in the decidua below the placental insertion, probably corresponding to the ‘specific cells’. Cf. Plate 3, fig. N. Magnification × 10.

PLATE 2

Fig. D. Autoradiograph of a sagittal section of the thorax of an 18-day embryo. The heart is seen in the centre of the picture. The myocardium and the blood-filled cavity have not retained significant amounts of radiosulphate. A high uptake is seen in the skin, the aorta (upper centre and lower left), and the tracheal cartilages. Maximal uptake in the oesophagus (upper left) and the ribs. Lung (lower left), liver (lower right), and thymus (upper centre) contain little radiosulphate. Magnification × 25.

Fig. E. Autoradiograph of a horizontal section through the basal parts of the skull of a 17-day embryo. Maximal uptake is seen in the cartilaginous skeleton. A moderate uptake of radiosulphate is seen in the meninges. The brain has retained moderate to large amounts of radiosulphate. A marked spatial differentiation of the uptake is seen. The lateral ventricles are seen in the upper central part of the picture. Magnification × 25.

Fig. F. Autoradiograph of kidney of an 18-day embryo. The pelvis, to the left, and the ureter, to the right, have taken up large amounts of radiosulphate. The uptake in the metanephric blastema and the lobulated liver is low. Magnification × 25.

FIG. G. Autoradiograph of a sagittal section from the lower abdominal region of an 18-day embryo. Maximal uptake is seen in the vertebral column in the lower corners. A high uptake is found in the genital papilla and bladder. In the intestines the retention is low in the mucosa, but high in the mesenchymal layer; no radioisotope is contained within the lumen. Magnification × 25.

Fig. H. Autoradiograph of intestines from new-born rat. In the left coil mucus-producing cells with a high S35-incorporation are seen. Radioactive mucus in the lumen of the right coil. Magnification × 25.

PLATE 2

Fig. D. Autoradiograph of a sagittal section of the thorax of an 18-day embryo. The heart is seen in the centre of the picture. The myocardium and the blood-filled cavity have not retained significant amounts of radiosulphate. A high uptake is seen in the skin, the aorta (upper centre and lower left), and the tracheal cartilages. Maximal uptake in the oesophagus (upper left) and the ribs. Lung (lower left), liver (lower right), and thymus (upper centre) contain little radiosulphate. Magnification × 25.

Fig. E. Autoradiograph of a horizontal section through the basal parts of the skull of a 17-day embryo. Maximal uptake is seen in the cartilaginous skeleton. A moderate uptake of radiosulphate is seen in the meninges. The brain has retained moderate to large amounts of radiosulphate. A marked spatial differentiation of the uptake is seen. The lateral ventricles are seen in the upper central part of the picture. Magnification × 25.

Fig. F. Autoradiograph of kidney of an 18-day embryo. The pelvis, to the left, and the ureter, to the right, have taken up large amounts of radiosulphate. The uptake in the metanephric blastema and the lobulated liver is low. Magnification × 25.

FIG. G. Autoradiograph of a sagittal section from the lower abdominal region of an 18-day embryo. Maximal uptake is seen in the vertebral column in the lower corners. A high uptake is found in the genital papilla and bladder. In the intestines the retention is low in the mucosa, but high in the mesenchymal layer; no radioisotope is contained within the lumen. Magnification × 25.

Fig. H. Autoradiograph of intestines from new-born rat. In the left coil mucus-producing cells with a high S35-incorporation are seen. Radioactive mucus in the lumen of the right coil. Magnification × 25.

PLATE 3

Fig. I. Autoradiograph of a horizontal section of the eye of a 17-day embryo. Maximal uptake in the cartilaginous skeleton. Moderate uptake in the skin (lower right), sclera, cornea, lens capsule, and mesenchymal tissue around the eye. Low uptake in the lens, corpus vitreum, retina, optic nerve, and eye-muscles. Magnification × 28.

Fig. K. Autoradiograph of inner ear of a 21-day embryo. Maximal uptake in the cartilaginous temporale anlage. A high uptake is seen in the mast-cells in the mesenchyme areas. A fairly high uptake is noted in the otolithic membrane and the gelatinous mass of the cupula. Low uptake in the membranous labyrinth. Magnification ×28.

Fig. L. Autoradiograph of a transverse section through a tooth from a 21-day embryo. A maximal uptake is seen in the cartilaginous upper jaw. A high uptake is seen in the dental pulp, at the pulpodentinal border, and in mast-cells of the mesenchyme (above). Magnification ×28.

Fig. M. Autoradiograph from the nose region of a 21-day embryo. Low uptake of radiosulphate in the epidermis and moderate uptake in the corium. Numerous mast-cells can be seen as white dots. The tactile hair follicles show a high uptake, especially in the outer layers, whereas the hair shafts have not taken up any radiosulphate. Magnification × 28.

FIG. N. Autoradiograph of pregnant uterus at 10 days. Very low uptake in the uterine lumen, placenta (lower end of section) and in scattered vascular spaces. Low uptake in the myometrium (upper end). A moderate to high uptake of the decidua and the decidual-myometrial border. In the centre of the picture adjacent to dark vascular spaces, white dots are seen—most of which probably correspond to the ‘specific cells’. Magnification × 28.

PLATE 3

Fig. I. Autoradiograph of a horizontal section of the eye of a 17-day embryo. Maximal uptake in the cartilaginous skeleton. Moderate uptake in the skin (lower right), sclera, cornea, lens capsule, and mesenchymal tissue around the eye. Low uptake in the lens, corpus vitreum, retina, optic nerve, and eye-muscles. Magnification × 28.

Fig. K. Autoradiograph of inner ear of a 21-day embryo. Maximal uptake in the cartilaginous temporale anlage. A high uptake is seen in the mast-cells in the mesenchyme areas. A fairly high uptake is noted in the otolithic membrane and the gelatinous mass of the cupula. Low uptake in the membranous labyrinth. Magnification ×28.

Fig. L. Autoradiograph of a transverse section through a tooth from a 21-day embryo. A maximal uptake is seen in the cartilaginous upper jaw. A high uptake is seen in the dental pulp, at the pulpodentinal border, and in mast-cells of the mesenchyme (above). Magnification ×28.

Fig. M. Autoradiograph from the nose region of a 21-day embryo. Low uptake of radiosulphate in the epidermis and moderate uptake in the corium. Numerous mast-cells can be seen as white dots. The tactile hair follicles show a high uptake, especially in the outer layers, whereas the hair shafts have not taken up any radiosulphate. Magnification × 28.

FIG. N. Autoradiograph of pregnant uterus at 10 days. Very low uptake in the uterine lumen, placenta (lower end of section) and in scattered vascular spaces. Low uptake in the myometrium (upper end). A moderate to high uptake of the decidua and the decidual-myometrial border. In the centre of the picture adjacent to dark vascular spaces, white dots are seen—most of which probably correspond to the ‘specific cells’. Magnification × 28.

PLATE 4

Fig. O. Autoradiograph of a section from the hind leg of a 21-day embryo. Note maximal uptake of radiosulphate in the epiphyseal cartilages. A fairly high uptake is seen in the calcified bone. Low uptake in the bone-marrow. The skeletal muscles show a low uptake. Numerous mastcells in the skin appear as white dots. The articular capsule and some vessels show a moderate uptake. Magnification × 28.

Fig. P. Autoradiograph of a section from the hind leg of a new-born rat (22 days from conception). Maximal uptake is seen in the cartilage at the right end, whereas a considerably lower uptake is found in the calcified bone at the left end. The uptake in the muscular portion of the leg appears to be confined to mesenchymal septa. Such strands are seen to converge on a tendon, which shows a fairly high uptake. Magnification × 28.

PLATE 4

Fig. O. Autoradiograph of a section from the hind leg of a 21-day embryo. Note maximal uptake of radiosulphate in the epiphyseal cartilages. A fairly high uptake is seen in the calcified bone. Low uptake in the bone-marrow. The skeletal muscles show a low uptake. Numerous mastcells in the skin appear as white dots. The articular capsule and some vessels show a moderate uptake. Magnification × 28.

Fig. P. Autoradiograph of a section from the hind leg of a new-born rat (22 days from conception). Maximal uptake is seen in the cartilage at the right end, whereas a considerably lower uptake is found in the calcified bone at the left end. The uptake in the muscular portion of the leg appears to be confined to mesenchymal septa. Such strands are seen to converge on a tendon, which shows a fairly high uptake. Magnification × 28.

1

Certified stains from the National Aniline Division, Allied Chemical & Dye Corp., New York, U.S.A., were used.