Histological Studies of the Prenatal Development of the Human Thyroid Gland By Akira Taki Department of Anatomy, Nagoya University School of Medicine (Directors : Profs. Drs. Chikataro Togari and Shooich Sugiyama) Introduction Regarding the histological differentiation of the human thyroid gland in prenatal life, there are a few detailed reports (N or r i s, '16 and '18 ; T a k a s h i ma and H a r a, '34), and the observations are concerned chiefly with the morphogenesis and histogenesis of the pri- mordium and follicles of relatively early life, and no attempt has been made to elucidate fully the histology of the pattern of the de- finitive parenchyma in later life. Recently, E. Hammer and H. L o e s c h c k e (36/37), by the use of the wax plate reconstruction method on the thyroid glands of human infants, demonstrated that acinus units are present in the lobules and that the units are com- parable in structural plan with those of the exocrine gland. To solve the problem whether such a pattern may exist in differentiation of the human thyroid gland in prenatal life or not is now indispensable for an understanding of the thyroid structure of the human adult. The primary purpose of this study is to clarify this point, and the second to determine cerfain important critical stages in differentia- tion of the human thyroid gland. The author thereby hopes that this paper may present fundamental data for the background of other investigations and for further consideration of this organ under pa- thological conditions. Materials and Methods A total of 165 japanese embryos and fetuses were examined and 144 of them were subjected to detailed histological observations (table 1). Intrauterine age of the embryos and fetuses was estimated by measuring the crown rump lengths, crown heal lengths and men- 65 66 Akira Taki Table 1. Numbers of human embryos and fetuses examined. ( ) : No. of cases, used for histological observations. * : Presumed values of CRL and CHL, derived from observed CHL and CRL b 2 CHL+3 3 CRL-3 y two formulas, CRL : 3 and CHL : 2(Fischeli '29) Prenatal Development of Human Thyroid 67 strual age (in weeks). Embryos and fetuses ranging from 3 mm in crown rump length to 90 mm in crown heal length were fixed in toto, chiefly in Zenker-formol fluid, and some in Car no y's fluid or 10% neutral formol, embedded in paraffin, and sectioned at 5 to 8// serial- ly, transversely or sometimes sagittally. Thyroid glands in fetuses measuring over 90 mm were removed from the neighboring organs and treated in the same way. The sections prepared were stained mainly by Ma y e r's hematoxylin and eosin, sometimes by Heide n- h a i n's iron hematoxylin, by W e i g e r t's resorcin fuchsin stain for elastic fibers, van G i e s o n's stain and azan stain for connective tissue fibers and Biel s c ho w s k y's silver impregnation for argyro- philic fibers. The silver impregnation method was employed for ex- amining the changing patterns of the thyroid differentiation. Some of the sections fixed in Zenker-formol fluid and Car n oy's fluid were stained by periodic acid-S c h i f f's method and B e s t's method for demonstrating glycogen and other substances, in combination with saliva digestion. Fig. I. Growth of the human thyroid gland in fetal life, estimated from the ventrodorsal, transverse, and cephalocaudal diameters of the lateral lobe and isthmus. Lateral labe— Q: ventrodorsal diameter ; • : transverse dia- meter ; (D: cephalocaudal diameter. Isthmus— A : ventrodorsal diameter ; A: transverse diameter ; A : cephalocaudal diameter. 68 Akira Taki Investigations of growth change of the thyroid primordium and gland were made by measuring the transverse, ventrodorsal and cephalocaudal diameters of the lateral lobes and isthmus by the usual method (fig. 1). To examine various forms of development of the primordia and follicles, wax plate reconstruction models were made by the use of serial transverse sections which were impregnated by silver according to B i el s c h o w s k y's method . Observations The Morphogenesis and Growth of the Thyroid Gland In the 3 to 5 mm CRL stages (5 weeks) the thyroid primordium appeared as a globular or elliptic body with a stalk arising from a shallow eva- gination of the pharyngeal floor, suggesting the later Foramen , caecum, and located in the mesenchymal tissue above and in front of the primitive heart and Truncus arteriosus (figs . 3 and 18,_2). The surface became rougher soon later. Some of the primordia were al- ready separated from the pharyngeal floor at the end of these stages . In the 6 to 9 mm CRL stages (6 to 7 weeks) the primordium was completely detached and free from the floor , and grew laterally to an initial bibbed structure, frequently with a remnant of the stalk (Ductus thyreoglossus) (figs. 183,9). The primordium was situated upon the Truncus arteriosus which moved caudad with the primitive heart . In these stages the primordium had one or a few completely closed cavities which sometimes contained a cloudy substance stained faint - ly with eosin (fig. 4). The cavities appeared to develop individually as independent spaces, which later partly communicated with each other, and finally opened to the outside. In the 10 to 13 mm CRL stages (7 weeks), the thyroid primordium grew further laterally and came to have the lateral lobes and isthmus, and was situated anter- ior to the primitive trachea (figs. 187 ,9 and 6). The primordium as a whole was sheet-like. The lateral lobes enclosed the carotid arteries (figs. 6 and 189). From the 14 mm to 31 mm CRL stages (8 weeks) , the lateral lobes underwent a marked increase in size becoming an ellipsoid , while the isthmus grew at a slower rate (fig. 1). Near full term , the cephalo- caudal diameter of the lateral lobes,became about 15-fold , the ventro- dorsal about 7-fold and the transverse about 12-fold in size that of the 15 mm stage (8 weeks). On the other hand , the ventrodorsal diameter of the isthmus became about 7-fold and the transverse about Prenatal Development of Human Thyroid 69 8-fold increase in size. Follicle Formation Until the 6 to 9 mm CRL stages (6 weeks) the primordium, containing a few intraprimordial cavities (fig . 4), consisted in great part of cell conglomerates . Somewhat later (10 to 13 mm stages, 7 weeks), cell plates appeared added to these conglo- merates. Some of the plates were produced by opening to the outside of the intraprimordial cavities (figs. 6 and 7) . The cell conglomerates and cell plates became increased and some of them became separated into cell cords and cell bands by ingrowth of mesenchymal elements . The cell plates and cell cords were usually two or three cells in thickness and 15 to 17a in width, while the cell conglomerates were several cells in thickness and 21 to 34g in width (figs . 6 to 9). These arrangements became maximum in number in the 31 mm CRL stage (10 weeks) and then gradually decreased although they were recognized up to 90 mm CHL stage (12 weeks) . Some of the epithelial cells in these different arrangements became arranged radial- ly, showing the most primitive type of follicle (figs . 8 and 9). In the center of these radial groups intercellular spaces appeared and were transformed into small cavities, which later were occupied by secre- tory substances-stained poorly with eosin. As development continued, the cell cords and cell plates exhibited varicosities and string-of-pearl appearances (fig. 10), with cavitation and differentiation of the epi- thelial cells, finally developing into the definitive follicles. Three types of follicle were distinguishable : namely, (1) the primitive type with radial cells and a central intercellular space, (2) the transitional type containing a distinct cavity, but whose cells were still immature and (3) the definitive follicle. The primitive follicles appeared for the first time in the 14 mm CRL stage (8 weeks) in • a very small number, and increased in number, reaching their maximum in the 32 to 48 mm CRL stages (10 to 11 weeks) (figs. 9 and 10). They were 21 to 25g in diameter. With some fluctuations after the 50 mm CRL stage (11 weeks), they decreased in number, but maintained a relatively low proliferative level until full term. They were produced also through budding processes from the walls of transitional and definitive follicles, without passing through the cell cords or cell plate formation. The epithelial cells of these primitive follicles varied from the primordial epithelium to the follicle epithelium, depending upon whether they were derived from budding of the follicle walls, in which case they assumed the same appearance as the follicle cells. 70 Akira Taki The transitional follicles began to appear from the 23 mm CRL stage (9 weeks) only in very small numbers , in the peripheral zone of the primordium , and were most numerous during the 32 to 48 mm CRL stages (10 to 11 weeks) . Despite possessing distinct cavities of various sizes , most of the transitional follicles contained none . Small numbers of them in the peripheral zone had only traces of secret ory substance, which was stained poorly with eosin or with periodic a cid-S c hif f's stain . From the 50 mm CRL stage (11 weeks) , a slow gradual conversion i nto almost definitive follicles took place . Some of the transitional follicles situated peripherally came to have half -mature colloid which was feebly stained with eosin , but red purple by periodic acid- S c h i f f's stain , and on the verge of developing into definitive follicles. The variously developing types of immature follicle were directly connected with each other without communication of their cavities and formed follicular conglomerates , bands, cords and net- works (figs. 11 and 19 „). From the 32 mm CRL stage (10 weeks) on, some of the transitional follicles preceded others in growth , and b ecame unproportionally large , often irregular-shaped, premature follicles. Such follicles had often several primitive follicles as buds . T heir epithelium was highly columnar . The cavity contained nothing or only small amounts of secretory substance . The secretory substance was periodic acid-S c h i f f positive , red purple, vacuolated and almost identical in appearance with the typical colloid found later . Follicles The definitive follicles made their first appearance at the 95 mm CHL stage (13 weeks) (fig . 14) and increased in number and size with age . They were, for the most part , round to oval in section, but some of them showed different degrees of irregularity . Large follicles were present more frequently at the periphery of the gland, and small follicles in the central part . The dimensions of the large follicles near full term reached about a three to 4 -fold increase over the 95 mm CHL stage (figs . 2 and 14). Follicles of different sizes , i ncluding primitive follicles as buds , were connected directly with each other. This fact was also confirmed by preparing wax plate reconstruction models (figs. 19 3_7). The connections were complicated and formed follicular cords or bands , conglomerates and networks. The cords and bands appeared as a kind of beaded chain with alternate swellings and constrictions (figs . 194_, and 17). In some of the folli- cular conglomerates , small follicles and primitive follicles were arranged as satellites around large follicles (figs . 17 and 19„6), and they formed Prenatal Development of Human Thyroid 71 an arboreal system (DrUsenbaumchen) or a follicle family. At full term, however, there was found a considerable number of dissociated follicles. Some of the large follicles showed an irregularity of shape and possessed a number of hollow out-projections of their wall, which appeared as independent follicles separated clearly by argyrophilic fibers in other consecutive serial sections. Their epithelium was highly columnar. - Epithelial Cells In early stages (3 to 13 mm CRL , 5 to 7 weeks), the epithelial cells were closely packed, without distinct cell bounda- ries. The nuclei were vesicular, with a weak chromatin reticulum and an eosinophilic nucleolus, and varied in size and contour. Gener- ally the cells having large nuclei were found in greater numbers than those with small nuclei. In sections fixed by Zenker-formol fluid and stained with hematoxylin and eosin, these nuclei were colored slightly reddish violet. The cytoplasm had a clear, almost hyaline appearance with fine scattered granules. Glycogen was found as granules in the cytoplasm but not in the nuclei (figs. 5 and 7). In the 14 to 31 mm CRL stages (8 to 10 weeks), the epithelial cells did not show any marked difference from those above, and formed plates, bands, cords and conglomerates. Some of the epithelial cells underwent changes to exhibit cell boundaries although indistinct, and became triangular or low cubical to form primitive and transitional follicles. Glycogen was found also in the same manner (fig. 8), and especially in abundance in the supranuclear area. In the 32 to 48 mm CRL stages (10 to 11 weeks) the majority of the epithelial cells formed primitive and transitional follicles. Some of them became eosinophilic or faintly red purple with periodic acid-S c h i f Is stain in their apical portion, suggesting the forerunning sign of secretory activity. Glycogen was found also in the same manner (fig. 9). From the .50 mm CRL to 90 mm CHL stages (11 to 12 weeks), the epithelial cells became cubical to columnar in some of the transitional follicles verging on maturity, and began to contain periodic acid- Sc hi f f positive secretory droplets in their apical portions. This positivity remained uninfluenced by saliva digestion. Glycogen was found (figs. 12 and 13). At the 95 mm CHL stage (13 weeks) the epithelial cells became completely definitive follicle cells. They exhibited relatively distinct cell boundaries and had somewhat smaller nuclei than before. The nuclei showed moderately dense chromatin networks. The cytoplasm 72 Akira Taki was finely granular and slightly eosinophilic , and sometime loaded with a few secretory droplets , which were stained red purple by periodic acid-S c h i f f's stain. The positivity was not changed by saliva digestion. Glycogen was still found in a very small amount at the 95 mm CHL stage, but soon later disappeared . The follicle cells were cubical to columnar , and were sometimes highly columnar in large follicles , chiefly in those placed peripherally, while they were generally cubical in small follicles in the central part of the gland. The colloid cells began to appear in the 95 mm CHL to 110 mm CHL stages (13 weeks) , and were red purple with periodic acid-S c h i f f's stain (figs. 15 and 16). Their positivity remained uninfluenced by saliva digestion . Mitoses of the epithelial cells were numerous during the early period. They continued to increase in number until the 50 mm CRL stage (11 weeks). Mitoses of the follicle cells were also numerous but gradually decreased in number towards full term. In proportion with the length of time elapsing after death , folli- cle cells appeared to be more or less extensively desquamated into the follicle cavity. Colloid Until the 31 mm CRL stage (10 weeks), secretory products failed to be found. From the 32 mm CRL stage (10 weeks) onwards , traces of secretory substance, which were poorly stained with eosin or periodic acid-S chi f f's stain, and believed to be the so-called precolloidal substance, began to appear in some of the transitional follicles placed peripherally, and increased at a very slow rate towards the 48 mm CRL stage (11 weeks). However, in the 32 to 48 mm CRL stages (10 to 11 weeks) transitional follicles containing none domi- nated the entire primordium. From the 50 mm CRL stage (11 weeks) on , half-mature colloid, which was stained red purple with periodic acid-S c h i f f's stain but feebly stained with eosin, occurred in transitional follicles maturing to definitive follicles, in the peripheral zone of the gland (fig . 13), and increased in amount slowly towards the 90 mm CHL stage (12 weeks). The positivity of periodic acid-S c h i f f reaction was not changed by saliva digestion. At the 95 mm CHL stage (13 weeks), typical colloid prevailed over the whole gland. The colloid was moderately eosinophilic and red purple with periodic acid-S chi f f's stain, and was partly vacuolated (figs. 14 to 16). The colloid found at the periphery was more intensely stained than in the central part of the gland, and was partly eosino- Prenatal Development of Human Thyroid 73 philic and partly basophilic. The positivity for periodic acid-S c h i f f's reaction remained unchanged by saliva digestion. Capsuk and Interstitial Connective Tissue Until the 10 mm CRL stage (7 weeks) mesenchymal tissue surrounded directly the thy- roid primordium. From the 14 mm CRL stage (8 weeks) onward, the mesenchymal tissue and primitive capillaries invaded the paren- chyma, preparatory to differentiation of the different cell arrange- ments. In the 14 mm stage the mesenchymal cells became possessed with slightly elongated nuclei, and exhibited frequent mitotic divi- sions. From the 16 mm CRL (8 weeks) on, argyrophilic fibers began to appear within the primordium and became increased towards later life, surrounding directly the different cell arrangements including primitive and transitional follicles (figs. 10 and 11). In the surround- ing zone of the primordium, the argyrophilic fibers were arranged in layers with mesenchymal cells and formed a primitive capsule (fig. 10). In the 50 mm CRL stage (11 weeks) the mesenchymal tissue was changed to young connective tissue which was arranged more densely than before. In the 95 mm CHL stage (13 weeks) the connective tissue was arranged densely and formed better developed interstitial tissue and a capsule. The interlobular connective tissue, as part of the inter- stitial tissue, began to be found as such at the 95 mm stage, but was indistinct. In the 200 mm CHL stage (15 weeks), the interlobular connective tissue was relatively distinctly formed in the dorsal, medial and lower peripheral zones of the lateral lobe. The tissue contained many collageneous and argyrophilic fibers but only a few elastic fibers. Some of the argyrophilic fibers were distributed in the follicles and associated follicle groups to form a fine reticular layer around the follicle epithelium (fig. 17). Other argyrophilic fibers surrounded circularly the capillaries. Blood Vessels Primitive ordinary capillaries appeared first in the interior of the primordium in the 14 mm CRL stage (8 weeks) and increased in number slowly, while sinusoidal capillaries occurred somewhat later (16 mm CRL, 8 weeks). Both types of capillary were increased markedly at about the 48 mm CRL stage (11 weeks) with frequent occurrence of mitotic divisions of endothelial cells. With the dominant occurrence of the definitive follicles (95 mm CHL, 13 weeks) the capillaries were increased further with repeated division of endothelial cells, and formed plexuses around the follicles and associated follicle groups. 74 Akira Taki Primitive arteries appeared for the first time at the periphery of the primordium in the 27 mm CRL stage (9 weeks) and in its interior in the 30 mm CRL stage (10 weeks). They gradually increased in number in the peripheral zone as well as in the central zone of the gland, and appeared complete as such in the 95 mm CHL stage (13 weeks). These arteries had a distinct lamina elastica interna. No arterial buds were found in the branching points of the arteries. Veins in the interior of the primordium were delayed in appearing, and were found first in the 30 mm CRL stage (10 weeks). They ap- peared to develop from some of the sinusoidal capillaries, and ran with the arteries early in the wide interstitial connective tissue and later in the interlobular connective tissue. In the 215 mm CHL stage (19 weeks), most of the veins appeared considerably developed and in the 300 mm CHL stage (24 weeks) some of them came to have a weak elastic layer. Most of the veins found in full term, however, were composed only of endothelium and adventitia, without muscle fibers. Discussion Biochemical, enzymological, radioautographic and radiochromato- graphic experiments have suggested that there are some stages of functional differentiation in thyroid glands of mammals in prenatal life. Rankin ('41) investigated some stages of change of the con- tent of iodine compounds in the thyroid gland of pigs in fetal life. Wolf f, C h a i k o f f and Nichols ('49) observed stages of accumu- lation of thyroxine-like and other iodine compounds in fetal bovine thyroid gland. McAlpine ('55) investigated different stages of change of alkaline glycerophosphatase in the developing thyroid gland of albino rats in embryonic life. Waterman and G or b man ('56) found some stages of functional differentiation in the fetal rabbit by the use of radioautography and radiochromatography. The histological observations also make it possible to divide the prenatal development of the human thyroid gland into the following three stages related to glandular activities : (1) early differentiation stage (3 to 13 mm CRL, 5 to 7 weeks) ; (2) preparatory differentiation stage subdivisible into three parts (14 mm CRL to approximately 90 mm CHL, 8 to 12 weeks) ; and (3) follicle stage (95 to 520 mm CHL, 13 weeks to full term). By definition these stages are clearly classi- fied, but it is true that the histological structure of the gland undergoes no corresponding sudden characteristic change at the beginning of