histological studies of the thyroid gland of the rabbit

Histological Studies of the Thyroid Gland

of the Rabbit During Pregnancy

Yoshiteru Sawasaki

Department of Anatomy, Nagoya City University Medical School, Nagoya, Japan

(Director: Prof. Dr. S. Sugiyama)

Ten Figures

Introduction

Until recently, many papers have been published on the relation-ship between the human thyroid gland and pregnancy, but these have been chiefly confined to the gross anatomical, physiological and bio-

chemical (B. M. R., blood iodine concentration, thyroid iodine content, thyroid hormone and thyrotropic hormone secretion) and clinical aspects

(development of goiter). The literature on the histology of thyroid.

glands during pregnancy is relatively scarce and also much less complete than might be expected (see Ba rgmann, '39). Most histological studies of the thyroid gland during this period have included a generally

accepted finding represented by the terms " hyperplasia and hypertrophy," but not to the extent of obtaining more precise evidence. As suggested by Barg mann ('39), there seem to remain many gaps to be filled.

This paper presents the observations of histological changes of the

thyroid gland of rabbits made during pregnancy on quantitative histometrical grounds. New evidence concerning the changes has been brought foreward here and their possible significances are discussed in relation to times of ovulation, implantation of fertilized ova and

further differentiation of the embryonic thyroid gland (Toga r Sugi-

yama and Sawasaki, '52). It remains for future studies to answer many of the questions brought into focus.

1 This work was supported by a grant from the Department of Education.

2 Present adress: Department of Anatomy, Nagoya University School of Medicine,

Tsurumai-Cho, Nagoya.

270 Y. Sawasaki

Materials and Methods

A total of 110 healthy pregnant rabbits weighing about 2.5 to 3.5 kg, whose reproductive history and age were unknown, were used for this study. The days of gestation were determined partly by the lapse of time from observed coitus indicated as day 0 and partly estimated from the averaged crown-rump length of littermate embryos, according to A m m a's data ('38). To confirm the establishment of early pregnancy, during which period implantation swellings are not yet observed in the uterus, Pincus' ('36) and Rome i s' ('48) methods were used : the

presence of wandering fertilized ova was confirmed by washing out the lumina of the oviducts with warm physiological saline (39°C). By injection of air into the auricular veins, pregnant animals were sacri- ficed at regular intervals (immediately after mating : 0, 5, 10, 16.5 and

19 hours ; later : 24 hours exactly). Thyroid glands were immediately removed, with the trachea and

esophagus, from the neighboring organs and were divided into two

parts along a mid-sagittal plane. One half was fixed in Zenker's .fluid for general observation, sectioned at 6 to 8 ,u transversely and mounted serially. The sections were stained with Hansen's hematoxylin and eosin, and partly with Weigert's resorcin fuchsin for elastic fibers, and

partly impregnated with silver according to the method of Bielschowsky for argyrophilic fibers. Some of the sections were colored with azan stain. The other half was fixed in Kolster's fluid for detailed cytological

examination and sectioned at 6 p, transversely and mounted also serially. The sections were stained with Heidenhain's iron hematoxylin or Altmann-Schridde's method.

The histometrical measurements for the various components of the thyroid glands were made exclusively at the middle one-third of the lateral lobe. This will be used as a more reliable means of evaluating histologically the functional phases induced by pregnancy of the thyroid

glands. In the graphs (figs. 1 and 2) each point represents the obser-vation based upon one animal.

The frequency per square millimeter of occurrence of follicles over 50 p, in diameter was calculated as follows : The number contained within 20 sections composed of every 5th section was examined and the averaged frequency per square millimeter was determined by the ratio, frequency averaged per section/averaged section area (table 1

and fig. 1). The diameters of follicles were obtained by adding the

Thyroid of Pregnant Rabbit 271

length of the largest diameter and that of the diameter at right angles to it, and dividing the sum by 2. The frequency per square millimeter

of occurrence of irregular-shaped follicles was calculated in the same way (table 2).

In order to obtain some quantitative expression of the serial change

of follicular colloid, the following crude examination was made with Kolster-fixed preparations stained by Heidenhain's iron hematoxylin.

An unevenness or spottiness of staining between the extremes (blue-

black in the whole area within follicles and uniformly faintly gray) was for convenience reckoned as moderately stained colloid. In 20 fields of every 4th section, under low magnification (ocular x 8, objective x 40), follicles containing colloid of three staining orders, respectively,

were counted. The total for each animal at different stages was averaged and the percentages of the three orders were derived. Such a compromise with practicality led to the data as indicated in figure

2. In the graph, the percentages of follicles containing darkly stained colloid were plotted, and, to avoid congestion, the other two kinds were omitted. The percentages do not represent the exact rate of staining changes of colloid, but give an approximate indication of

changes, which are useful for a supplementary criterium to determine the functional phase.

Four female rabbits were used as control. They were supposed to be on heat from the observations of the presence of fully developed ovarian follicles and appearance of the external genitalia. The above

quantitative measurements were made on them. Pregnancy was divided into the following three periods subdivisible

into two stages, first and second halves, respectively : (1) early pregnancy—from the first to the 10th days of gestation ; (2) mid pregnancy—

from the 11th to the 20th days, and (3) late pregnancy—from the 21st day to full term.

Observations

Follicles. The majority of large follicles were always located in the peripheral zone of the glands and small follicles chiefly in the

central zone, as were recorded in rats (Jackson, '16; Su gi y a ma, '39)

and mice (Oh i d a, '54). Follicles exceeding 50 ,u, in diameter were regarded as large follicles, and, as shown in figure 1, their frequency

per square millimeter was plotted according to the progress of gesta-

272 Y. Sawasaki

tion. Based upon the examinations of figure 1, four follicular types of the thyroid gland were classified as follows : microfollicular type (below 50 per square millimeter), macrofollicular type (over 100) and intermediate type subdivisible into transitionally microfollicular (from 50 to below 75) and transitionally macrofollicular types (from 75 to below 100). The incidences of the various types found during different stages were shown as percentages in table 1.

The microfollicular type appeared to be characterized by an absolute predominance of follicles between 30 and 40 y in diameter, being ad-mixed with extremely small follicles between 20 to 25 ,u, (figs. 4 and 6). Detailed observations showed that, among these follicles there is a small number of large follicles between 50 and 70 p, chiefly at the periphery of the glands. The large follicles showed generally round to oval shapes, but during the second-half of early pregnancy most of them were very irregular in shape (fig. 6).

The macrofollicular type, in the majority of the cases examined, appeared to be characterized by an absolute prevalence of large follicles between 80 and 90p. This type sometimes contained a number of large follicles over 100 p, in diameter which ranged in number per square millimeter from 5 to 10, and sometimes included a large number of follicles measuring 60 to 70 p. The largest follicles found in this type were occasionally 220 the maximum value being 320 ,u. During early pregnancy, most of the large follicles indicated various degrees of irregularity in shape (fig. 7).

Immediately after mating, intermediate types were found, but thereafter between 5 to 19 hours microfollicular types began to be seen (figs. 1 and 4; table 1), and remained at almost the same incidence until the first day of gestation.

During the first half of early pregnancy, macrofollicular types including transitional types occurred at the beginning (fig. 5), while microfollicular types including transitional types appeared to be found toward the end. During the second half microfollicular types increased significantly (47.0%) and their transitional types also appeared (29.4%)

(fig. 6 and table 1). At the end of this stage or at the beginning of mid pregnancy, macrofollicular types including transitional types were found (figs. 1 and 7).

During the first half of mid pregnancy, various types were found almost evenly in rate as shown in table 1. However, further detailed examination of figure 1 reveals that, just at the beginning of this stage, transitionally macrofollicular types occurred, a continuation of the same


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274 Y. Sawasaki

category found at the end of the previous period ; at the middle, microfollicular types were found ; and again at the close, macrofollicular types including transitional types were observed together with transitionally microfollicular types which may be related to the same types found in the next stage. During the second half, microfollicular types

containing transitional types increased markedly (68.7%) with a smaller incidence of macrofollicular types including transitional types (31.3%). The latter types were chiefly found toward the end of this stage and

further at the beginning of late pregnancy (fig. 1).

During the first half of late pregnancy, both macrofollicular types including transitional types and microfollicular types including transi-tional types, were seen, in almost equal proportion (50.0% respectively).

The scatter diagram (fig. 1) indicates that it is much difficult to determine the trend of follicular types in any stage. During the second half, microfollicular types including transitional types (56.2%) were somewhat more predominant than transitionally macrofollicular types

(43.8%). No macrofollicular type was found. It is much more difficult to determine the trend of the follicular type.

Table 1. Percentages of occurrence of different follicular types in various stages of pregnancy, based upon the examination of frequency per square

millimeter of follicles over 50 i in diameter in representative sections per gland.

Note : 1: microfollicular type ; 2: transitionally microfolicular type ; 3: transitionally macrofollicular type ; 4: macrofollicular type ; F Frequency.

Follicles were generally round to oval in shape. Irregular-shaped follicles which have been interpreted as a sign of increased activity, especially for colloid release (A r on, '34, guinea pigs ; Sugiyam a, '39,


rats; Sugiyama and Yagizawa, '50, rabbits; Sugiyama and Sato, '54, guinea pigs) were found. Some of the irregular-shaped follicles indicated papillary projections extending into the follicular cavity due to collapse and folding of the follicular wall and in so doing became devoid of colloid (fig. 6). As described by WOW er (1880, human) and Sanderson-Damberg human), the papillae contained a slender core consisting of some connective tissue and capillaries and bore columnar follicle epithelium uniformly throughout their whole surfaces (fig. 6). Others showed elevated swellings of the follicular walls which were broader at their base and projected toward the follicular cavity, but did not resemble the " Proliferationsknospe " (Sand erson-Damberg,

'11, human; Biir kl e-de la Cam p, '24, human ; Ewe, '36, human). The development of such two formations led the follicles to assume various forms such as dumbells, star-fishes, arched or branching tubules, with gradual reduction of the follicular cavity (fig. 6). They varied in occurrence according to advancing stages of gestation.

Based upon the examination of the frequency per square millimeter of such irregular-shaped follicles, the thyroid glands were conveniently divided into five orders as shown in table 2. The majority of all the cases examined throughout pregnancy indicated a frequency below 20 per square millimeter, and the glands showed a prevalent occurrence of round to oval follicles. A small number of the cases (13.7%) showed values of over 30 per square millimeter, which imparted a much unstable appearance to the glands (fig. 6). The same sequence of quantitative observations as that for the frequency of large follicles of over 501a was repeated for investigation of serial changes of irregular-shaped follicles (table 2).

Immediately after mating, the majority of the cases examined showed a slight sign of irregularity in follicular shape (20Z.F.730 :

27.3%; 1OLFZ20 : 45.4%). During early pregnancy, the irregular changes in shape proceeded chiefly in large follicles, frequently with papillary projection of their walls. During the first half irregular-shaped follicles increased in number significantly per square millimeter toward the end, and cases showing more than 30 per square millimeter constituted 46.1% (30Z.FZ40 : 30.7% and 404F: 15.4%). During the second half, this was partly more emphasized (figs. 6 and 7). The extreme cases of over 40 per square millimeter increased to 35.3%. Around the 7th day of gestation this phenomenon was most exaggerated . The other cases showing numerous occurrence of regular-shaped follicles

(FZ.10 : 11.7% and 10/F/20: 412%) chiefly represented the cases

276 Y. Sawasaki

found at the end of this stage.

Table 2. Percentages of occurrence of different glandular types in various stages of pregnancy, based upon the examination of frequencies per square

millimeter of irregular-shaped follicles in representative sections per gland.

Note : F: Frequency.

During the first half of mid pregnancy, the cases of irregularity in follicular shape decreased in incidence, the glands being composed of almost regularly round to oval follicles (FL 10 : 70.6% and 10Z_F

L20: 23.5%). During the second half, irregular-shaped follicles increased slightly (20Z FL30 : 18.7% and 30Z.F40 : 6.3%).

During late pregnancy, irregular-shaped follicles were significantly reduced in the majority of the cases examined. Most follicles were again almost round to oval, irrespective of the follicular type of the

gland.. Follicle cells—were swollen cubical throughout the period of gesta-tion and averaged 9 to 10 p, in height. They were generally low in large follicles, while they were high and swollen in medium-sized to small follicles or in irregular-shaped follicles. Detailed examination, however, showed that their height and form are relatively variable according to the progress of pregnancy, and varied according to

glandular activity as well ; they collectively were columnar and attained heights of 16 to 18 th and their apical portion expanded to become dome-shaped in some of the cases examined, but they were low and measured 4 to 6 /J. in others.

The nuclei were generally round to oval in shape, and the


chromatin reticulum was moderately dense in Zenker-fixed preparations

stained with hematoxylin and eosin. They were located centrally or somewhat basally in the cell bodies and were increased in size in

proportion to swelling and elevation of the cell bodies. Mitotic figures were found in small numbers and difficult to find throughout pregnancy.

The cytoplasm was finely granular, containing a number of vacuoles.

It is of particular interest that the cytoplasm often showed accumula-tion of several secretory droplets (fig. 10). The droplets were found

generally in swollen cubical or columnar follicle cells which appeared clearly vacuolated, and rare in low cells. They were of various sizes and appeared chiefly in the supranuclear zone or lateral to the nuclei,

and were stained in various shades of black with iron hematoxylin or red to green by Altmann-Schridde's stain in combination with after-stain by light green. Large droplets appeared often in the apical

portion of the cells, and some of them were colored light gray by the former stain or green by the latter. The preparations fixed in Kolster's

fluid were very convenient in preserving the secretory droplets. During early pregnancy, follicle cells, collectively, were charac-

terized by swelling and clarification of the cell bodies and enlargement of the nuclei. Immediately after mating, follicle cells remained cubical and not yet swollen, but 5 hours later began to increase in height and to swell (fig. 4). Until the 5th day of gestation, they maintained almost the same condition, except in the cases of macrofollicular type or its transitional type (fig. 5). During this first half of early pregnancy, a wide-spread occurrence of intracellular secretory droplets was observed in the majority of the cases examined (figs. 1 and 2). On the 6th to

7th days of gestation, follicle cells became significantly swollen, cubical or columnar and clearly vacuolated throughout the whole area of the

glands (fig. 6). Colloid cells appeared relatively often, remaining in situ without receiving any visible factor or pressed by neighboring

swollen cells. Toward the 10th day of gestation (CRL : 4 mm) general follicle cells indicated a slight decrease in swelling and height (fig. 7).

During the first half of mid pregnancy (11th to 15th days of

gestation ; CRL : 6 to 16 mm) follicle cells, generally, showed still an appearance similar to that observed at the close of early pregnancy,

but contained numerous secretory droplets (fig. 10). During the second

half they indicated transiently some fluctuations in swelling and height, but finally showed a decrease in swelling at about the 20th day of

gestation (CRL : 37 mm). During late pregnancy, follicle cells began to swell again. During

278 Y. Sawasaki

the second half, they became more swollen throughout the entire area

of the glands, the cytoplasm becoming much vacuolated, frequently

with secretory droplets. Colloid cells appeared again to be considerably

numerous.

Parafollicular cells—throughout pregnancy, were found, as were described in normal histogenesis of thyroid glands of rodents (Su g yama, '39, '42, rats and mice ; Sugiyama, '50, rabbits ; Su giy ama, '54, guinea pigs). Dark parafollicular cells appeared more or less numerous in most of the cases examined (fig. 9), especially during the second half of early pregnancy and mid pregnancy.

Dark parafollicular cells were round to oval or polygonal, either when isolated or when aggregated as buds, and appeared usually like parietal cells of the gastric glands, without bordering the follicular cavity directly (fig. 9). Their distribution was found to be in a considerable number of follicles placed centrally in the lateral lobe. The nuclei were round to oval and as large as or smaller than those of ordinary follicle cells, and the chromatin reticulum was moderately dense. Some of the nuclei were darkly stained, and became finally pyknotic with progression of a cytoplasmic change as will be described later. Dark parafollicular cells contained numerous dust-like granules, partly admixed with granular or rod-shaped mitochondria and they give one an impression that they may arise through fragmentation of the mitochondria from some mitochondria-rich cells (fig. 9), and in ordinary preparations they stained more eosinophilic and highly granu-lar. Although their cell boundaries, when together, were not clearly visible in ordinary preparations, their approximate contours could be determined by the outermost extent of their granules in Kolster-fixed preparations. The sequence of the cytoplasmic change may be divided into two parts The first part included a change to maximum granu-lation ; the second part included a sequence from maximum granulation to dark hyalinization with cytoplasmic thinning (fig. 10). As this progressed, finally dark parafollicular cells became degenerative as suming an appearance similar in part to that of colloid cells.

Clear parafollicular cells appeared far less numerously throughout pregnancy. They became somewhat increased in number during early pregnancy and mid pregnancy. They contained only a few rod-shaped mitochondria which were often scarcely visible. Both vacuoles and secretory droplets were scarcely found in them.

Colloid—in Zenker-fixed preparations stained with hematoxylin and eosin, was as a rule not granulated, eosinophilic and rarely partly


eosinophilic and partly basophilic. The colloid was stained more intensely at the peripheral zones of the glands. With Heidenhain's iron hematoxylin, differences in staining were more clearly distinct,

ranging from blue black in the entire area within follicles, to faintly

gray. With azan stain, the colloid was red to blue in color. Iron hematoxylin stain was found to be superior to other stains for quanti-tative expression of serial changes of colloid, because of the relatively rare occurrence of moderately stained colloid which makes it impossible to determine its staining order. Intracolloidal vacuoles have the appearance of artifacts by fixation, but their numerous occurrence

may well bear a relation to colloid release because of their charac-teristic distribution induced under certain conditions (Krogh and

Okkels, '33, guinea pigs ; Severinghaus, '33, guinea pigs and mon- keys; Aron, '34, guinea pigs; Florentin, Fontaine and Hennequin, '36

, rabbits; Sugiyama, '39, rats; Sugiyama and Yagizawa, '50, rabbits; Ohida, '54, mice ; Sugiyama and Sato, '54, guinea pigs).

The marginal vacuoles (Randvakuolen) were found numerously in Kolster-fixed preparations and less numerously in Zenker-fixed prepa-

rations. Furthermore, they occurred to a greater extent in feebly stained colloid than in darkly stained ones, but their frequent occurrence was occasionally seen in large follicles containing dark colloid, which consisted of swollen cubical or columnar cells. Crevices which are obviously artifacts made by cutting with the microtome knife but are

related to dense colloid, were found in darkly stained colloid and infrequent in feebly stained colloid. Colloid contained desquamated follicle cells very rarely.

With Kolster-fixed preparations stained by He idenh a in's iron hematoxylin, examination of serial changes in staining of colloid during

pregnancy gave an interesting finding as shown in figure 2. This finding may be comparable to a certain extent with that observed concerning the follicular type of the gland. Although it is decreased in late pregnancy, the reliability of comparison between both is de-

pendent upon the expectation that a possible connection seems to exist and the glands present some color change of colloid consonant to a considerable degree with the trend of the follicular type. Dark staining

of colloid was generally found during tendency toward size-increase of follicles (figs. 5, 7 and 8), while poorer staining was seen in tendencies toward size-decrease of follicles or toward irregular change of general follicles (fig. 4 and 6). Some discrepancy of the relationship between both was often seen and difficult to explain, but it may be eliminated

280 Y. Sawasaki

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in considerable part by comparisons of the general trends of data indicated in figures 1 and 2.

During the first 24 hours just immediately after mating, colloid appeared to fluctuate promptly in staining with reduction in follicle size. Finally, it reached a minimum reduction in dark staining at about 24 hours after mating (first day of gestation) when the glands still remained as microfollicular type.

During early pregnancy excluding the period immediately after mating, varying degrees of prevalence of darkly stained colloid occurred alternately in peak and trough. From the second day of gestation the colloid became increased in staining with a trend toward size-increase of follicles (fig. 5) and it became reduced at about the 7th day of gestation, with a trend toward size- decrease of follicles (fig. 6). At the end of early pregnancy (9th to 10th days of gestation ; CRL : 3 to 4 mm) it increased again markedly in staining intensity concurrent with a trend toward size-increase of follicles (fig. 7).

Mid pregnancy : After the 10th day of gestation, colloid rapidly and transiently was reduced in staining and formed a trough (11th to 13th days ; CRL : 6 to 12 mm), irrespective of the follicular types. Some of the cases found during this phase showed microfollicular types. Immediately after this phase the colloid temporarily became enhanced in its staining (first peak) and the glands indicated a trend toward size-increase of follicles. During the second half, it indicated again an indistinct reduction in staining with a trend toward size-decrease of follicles and subsequently an increase with a trend toward size-increase of follicles at the end (second peak) (20th day ; CRL : 40 mm). Late pregnancy appeared to consist of alternate three peaks and

three troughs (fig. 2), and initiated with a distinct peak as the first one, a continuation of a peak which was already found at the end of mid pregnancy (CRL : 40 to 46 mm) (fig. 2). At the 21st to 23rd days

(CRL : approximately 45 to 55 mm), colloid showed a prevalence of feeble staining (first trough) ; at approximately the 23rd to 24th days

(CRL : 57 to 61 mm) a prevalence of dark staining (second peak) (fig. 8) ; and at the 67 to 72 mm stages a prevalence of feeble staining

(second trough). At approximately the 25th to 27th days (CRL : 73 to 81 mm), the third peak occurred with some fluctuations, and sub-sequently at the 27th to 28th days (CRL : 83 to 85 mm) the third trough appeared. At the end of late pregnancy (CRL : 90 to 100 mm) the cases examined were too small to permit evaluation of the staining

282 Y. Sawasaki

trend. Intracolloidal vacuoles were also variable in occurrence according

to the progress of gestation. They occurred in general markedly in each trough as indicated in figures 1 and 2 (figs. 4 and 6). Crevices in colloid were found in small numbers throughout pregnancy .

Interstitial Connective Tissue. Interlobular connective tissue origi-

nated from the glandular capsule and it divided the parenchym indistinctly into several areas ; the lobulation was relatively distinct

only in the medial peripheral zone. The interlobular connective tissue contained a considerable amount of collagenous fibers with a small amount of argyrophilic fibers and elastic fibers. Perifollicular connective

tissue was derived from both the interlobular and capsular tissues, and was small in amount, being composed of delicate argyrophilic fibers forming a reticulum around follicles and a few connective tissue cells . No elastic fibers were found here.

In Kolster-fixed preparations stained with iron hematoxylin, there was found often a colloid-like substance which stained lightly gray in interlobular tissue and sometimes in perifollicular tissue.

Blood Vessels. Arteries ran within or beneath the capsule and most of them entered the interior of the glands from the medial

peripheral zone along interlobular tissue to ramify into smaller branches. Large arteries had a distinct lamina elastica interna and somewhat

indistinct lamina elastica externa. At the branching points of large to medium-sized arteries, there were often found sphincters (S ugiyama and Yagiza w a, '50, rabbits). Veins were accompanied by arteries

and generally were composed of the intima and adventitia, without

having the media.

Capillaries formed a dense network around follicles, accompanying argyrophilic fibers. Capillary hyperemia varied in degree according to

the advance of gestation. Engorged capillaries became often sinusoidal and bulged into the follicle wall, indenting its basal surface. This

arrangement enhanced still further the intimacy of the capillaries with the follicle cells. The capillary hyperemia occurred markedly from soon after mating and continued to until the 7th day of gestation (fig. 6). During this stage, intrafollicular bleeding was found although very rare. During mid pregnancy as well as during late pregnancy,

capillary hyperemia appeared to be generally found in the glands of microfollicular type including transitional type.

In Kolster-fixed preparations stained with iron hematoxylin, there were often found colloid-like substances which stained lightly gray and


containing vacuoles, in some of the veins and capillaries. Whether or not the substances found here as well as in perifollicular or interlobular tissue are identical in biochemical property with follicular colloid remains problematical.

Control data on four Females. Thyroid glands showed microfol-licular or transitionally microfollicular types (frequency per square

millimeter of follicles over 50 13-69), follicles of which were generally round to oval (frequency per square millimeter of irregular-shaped

follicles : 3-8). Colloid was stained feebly (follicles containing dark colloid : 1-11% per field). Follicle cells were cubical and not markedly swollen, and contained no secretory droplets.

Discussion

Repeatedly presented data of thyroid enlargement found during

pregnancy have been generally interpreted as suggestive of the existence of a certain relation between the gonad and thyroid gland or an increased activity of the thyroid glands (Seitz, '26 ; Berberich and Fischer-Wasels, '32; Bargmann, '39; Selye, '49). Nevertheless,

concerning the character of the histological changes of the gland in women during pregnancy, no consistent description has been offered.

Engelhorn ('12) found that the glands indicate marked hypertrophy accompanied by the presence of numerous mitotic figures in follicle epithelium and a great amount of colloid. Further, he stated that

there occur a formation of numerous young follicles and marked release of colloid into lymph vessels. Wegelin ('26) observed that the glands during pregnancy indicate manifold manifestation of increased activity which consists of active colloid secretion, epithelial proliferation leading to formation of new follicles, and release of colloid into lymph vessels.

S t a nder ('36) believed that the hypertrophy of the glands which was found clinically in 65 to 90 per cent of pregnant women, is due to a

true hyperplasia with an increased blood supply and formation of new follicles. The results reviewed present variable pictures and an available

conclusion can scarcely be drawn. The principal impression gained, however, is that increased activity may occur during pregnancy.

As suggested by Bargm ann ('39), the reaction readiness (Reak- tionsbereitschaft) of the thyroid glands to stimuli which is based upon

the complicated mode of human life, may overshadow some of the characteristic changes induced by pregnancy. Investigations of thyroid

glands of laboratory mammals which are allowed to maintain almost

284 Y. Sawasaki

constantly their living conditions, might be expected to elucidate the

present problem to a certain extent. Despite this advantage in experi- ments, in previous publications there seem to remain many questions to be further investigated. Chief among the sources of diversity of the data appears to be due naturally to differences of animal species, but partly to paucity of the number of the cases examined in relation to the length of gestation period of the animals used, or partly to

failure of reasonable arrangement of the data according to the advance of gestation.

V. B o r z y s t o w s k i ('02) noted hypersecretion of the thyroid gland

of cats during the first half as well as during the second half of

pregnancy, which is characterized by the presence of distended follicles, strongly stained colloid and darkly stained low columnar epithelium,

together with a possible release of colloid into lymph vessels. Further he noted the formation of small follicles from undifferentiated epithelial masses and the diminution in number of large follicles. Another examination of the glands of pregnant cats (Lowe, '30) indicates that, although on the whole the glands show a greater degree of activity with capillary engorgement than those in non-pregnant females, yet they vary in accordance with the seasonal changes recorded for normal

animals ; the glands obtained during mid-winter contain less colloid

and more new secretion than those of non-pregnant females while those obtained during spring and summer contain large follicles and much colloid. Bernard ('27) observed the following changes in

pregnant dogs : During the early days of gestation the glands are transiently followed by slowing of function ; colloid is very slowly accumulated in the follicles. A period of such functional slowing

ceases at about the 32nd day of gestation, when resorption of colloid becomes manifest. After 43rd day of gestation there is seen considerable exaggeration of cell activity which attains its maximum at full term.

SpOttel ('29) suggested hyperactivity of the glands of sheep during

pregnancy, from observations of diminution in follicle size, irregular formation in follicle shape, elevation of follicle epithelium with pro-liferative activity, and increase in amount of diluted colloid. V er do zz i

('31) obtained the following findings with guinea pigs : The glands during the first half of pregnancy show marked activity evidenced by hyperemia of thyroid tissue and follicles are lined by cubical epithelium and filled with colloid. During the second half the colloid is hyaline and reduced in amount. Follicle epithelium is lower and interfollicular

cell islets are scarce. Herold ('33) stated that the histological change


of the glands of guinea pigs during pregnancy may be interpreted as an increased secretory activity similar to that of animals stimulated by thyrotropic hormone. Investigation of the glands of rats during

pregnancy reveals the following (A h a r a, '33) : During early pregnancy the glands are characterized by the presence of slightly enlarged, oval follicles, low cubical to cubical follicle cells and capillary dilatation. During mid pregnancy the glands are marked by the prevalence of enlarged irregular-shaped follicles, large vacuoles in colloid, swollen

cubical cells and capillary dilatation. During late pregnancy the same condition as that found in the previous period is more exaggerated for a while, but it soon later becomes quiescent. B en az z i ('34) stated that the glands of mice during pregnancy indicate various degrees of

glandular activity although no effective increase of it is confirmed. According to Charda r d-R a imba ult ('52), the gland of mice during

pregnancy shows two periods of activity, separated by a short stage of rest. From the llth to 14th days (first active period) an intense

resorption of colloid is shown and in the last days of pregnancy there is formation of small follicles which very soon show signs of functional activity (second active period). Zalesky ('35) pointed out hyperplasia in the glands of ground squirrels during pregnancy, which is followed by an exaggerated increase in parenchyma and active colloid resorption.

Histological observations based upon a series of 103 cows of similar. age at various stages of pregnancy, pointed to mild increased physiological activity, which is more pronounced in the earlier months of

pregnancy (Abbott and Prendergast, '36). A survey of the literature also indicates that the same situation as presented in human pregnancy holds true in pregnancy of lower mammals.

Although the limits of individual variation are large (Krogh and Okkels, '36), female rabbits appear to be suitable for investigations

of the progressive change occurring during pregnancy, and its signifi-cance, because of the adequate length of the gestation period and the accurate determination of the advance of gestation age following ovulation which is usually provoked 10 hours after mating (Hammond and Marshall, '25).

As concerns the histological changes of the thyroid glands of rabbits during pregnancy, the present data reveal on histometrical

grounds, that an orderly sequence of similar events may occur in follicles and colloid in accordance with the advance of gestation. In a comparison of figure 1 with figure 2, a fairly close confirmity will

be noted although it is decreased in late pregnancy. This seems to

286 Y. Sawasaki

provide a critical basis for correlating structure and function. Early pregnancy excluding immediately after mating appears to

consist of two trends toward increase of follicle size with prevalence of darkly stained colloid, as two peaks (transitory storage of colloid), and two trends toward decrease of follicle size with lesser staining of colloid, as two troughs (release of colloid). Presumably the thyroid

glands appear to go through recurring cycles of activity. The troughs presented are histologically characterized by still other changes such as general swelling of follicle cells, colloid vacuolation and capillary hyperemia. An almost similar situation may exist also in other troughs

found during mid pregnancy. According to Krjlow and Sternberg ('32), coition leads the

thyroid glands of female rabbits to colloid release which is indicated by reduction of the follicular cavity, poorer staining and vacuolation of colloid, marked swelling of follicle epithelium and further capillary

hyperemia. Their finding is partly in favor of the present result ; however, a short time fluctuation of colloid staining is observed in the

present result (figs. 1, 2 and 4). Further, one point remains doubtful in comparison with the observations of the control glands presented here. According to them control thyroid glands consist of large follicles containing dark colloid. This is in marked contrast with the

result of the present controls. Histological changes of thyroid glands in a transitional period from before coitus to immediately after coitus

remain undetermined at the present stages of investigation. An additional word should be said that a relationship may exist between thyroid hormone secretion and ovulation. The pigeon's thyroid glands appear histologically more active at time of ovulation (Marza and

Bl in ov, '36). According to Winchester ('39), thyroxin injection leads thyroidectomized fowls to produce eggs again.

The travel through the oviducts to the uterus of fertilized ova of rabbits has been reported to require approximately three to 4 or 5

days (Bischoff, 1842 ; van Beneden, 1875; Fraenkel, '03). Ac-cording to the present observations, fertilized ova in a two-cell stage

(24.4 hours post coitum) and in a morula stage (72 hours post coitum) are found in oviducts. Sax and Leit son ('38) demonstrated the indispensability of thyroxin for the development of fertilized ova in rabbits. The colloid storage phase found during the second to about 4th days of gestation, although it is transitory, may correspond with the time of development of fertilized ova (fig. 5).

Approximately 7 or 8 days of gestation have been estimated for


the time of establishment of implantation of fertilized ova in rabbits (Kossmann, 1898; Fraenkel, '03 ; Grosser, '19; Corner, '28). It is of particular interest in combination with these data, that the present study indicates that a colloid release phase appears to be initiated already from the 4th day of gestation and reaches a maximum on the 6th to 7th days (fig. 6). This evidence is also confirmed by the numerous occurrence of extremely irregular-shaped follicles, as indicated by epithelial projections of the follicular walls which lead the follicular lumina to reduction (table 2, fig. 6). It may be supposed that the thyroid glands at this stage play a synergic part in creating an optimal condition for implantation of fertilized ova. At the 10th day of gesta-tion (3 to 4 mm), an accumulation of colloid, which is indicated by dark staining of colloid with a trend toward increase of follicle size, is observed (fig. 7). After a period of further development of implanted ova, the thyroid glands appear to accumulate transiently.

Some of the relationships between pregnancy and thyroid glands, if not most of them, may be interpreted by the pituitary-thyroid axis. The stimulation of the thyroid glands during pregnancy is probably due to excessive production of thyrotropin by the pituitary although this has been not yet definitely proven (Selye, '49). Herold ('33) pointed out this possibility with guinea pigs in pregnancy. The administration of thyrotropic hormone of the pituitary has been recently reported to lead the gland to colloid release which is indicated by vacuolation and lesser staining of colloid, and diminution of follicle size and irregular change of follicle shape. Further studies established a relationship between the formation of intracellular secretory droplets and thyrotropic hormone. The droplets appear after presumably increased endogenous thyrotropic hormone secretion caused by partial thyroidectomy (Hiir thle, 1894 ; I s h im ar u, '26). In addition, special attention has been paid to the droplets as a basis for thyrotropic hormone assay (De Robertis, '42; De Robertis and Del Conte , '44, guinea pigs; Dvoskin, '47, '48, cockerels and rats). Ohida ('54) also suggested that the numerous occurrence of intracellular secretory droplets, in the thyroid glands of mice shortly after castration , represents part of a picture marked by increased thyrotropic hormone secretion by the pituitary. The majority of the cases examined show numerous occurrence of the droplets in follicle epithelium , especially from the second to 6th days of gestation, during which enlargement and subsequent reduction in size of follicles occur in consonance with change of colloid (figs. 1 and 2). The thyroid gland of rabbits during

288 Y. Sawasaki

early pregnancy appears to indicate a peculiar pattern affected by an

increased secretion of tropic hormone by the pituitary. Mid pregnancy appears to consist of two trends toward size-increase

of follicles with a prevalence of dark staining colloid, as two peaks, and two trends toward size-decrease of follicles with feeble staining of colloid as two troughs (figs. 1 and 2, table 1). Regularization of follicle shape proceeds during the first half and a slight irregularization is followed during the second half (table 2). K ri 1 o w and Sternberg

('32) reported in rabbits the restitution of colloid amount which has been decreased after coitus, to the original value toward the middle of pregnancy.

During mid pregnancy, secretory droplets in follicle epithelium occur also in the majority of the cases examined (figure 2). According to Dvoskin ('48), the secretory droplets present in the follicle epi-thelium or rats exposed to cold or fed thiouracil, promptly disappear following hypophysectomy, and the adrenal or its secretions are not responsible for droplet formation. Probably, the droplet-positivity due to the stimulation upon the thyroid glands by the pituitary may be

presented during this period also. There remains a problem concerning what physiological entities

have reflected the histological findings found during mid pregnancy. Starting approximately at the 16th day of gestation (CRL : 18 mm), embryonic thyroid glands of rabbits enter the preparatory differentiation stage subdivisible into the first part characterized by an increasing

prevalence of primitive follicles containing none and the second part marked by a prevalence of transitional follicles containing a little secretory substance stained feebly (Toga r i, S u g i yama and Saw as a k i, '52) (fig. 3). It is supposed that the colloid release observed during the second half of mid pregnancy may have occurred as a prelude

for giving support to the advanced differentiation of the embryonic thyroid glands, especially to a beginning accumulation of secretory substance (figs. 1 and 2). The evaluation of the present consideration must await additional data. There is some evidence which supports

this presumption. When radioiodine is given intraperitoneally into rabbit embryos, the embryonic thyroid glands at the 20th day of

gestation are able to concentrate it although little in amount (Jost, Morel and Marois, '49). Furthermore, that administered thyroid sub-

stance is capable of affecting embryonic thyroid glands, passing through

the placental barrier, is evident from a number of well-established observations (Gr umbrecht and Loeser, '38, Theresa, '39, guinea


pigs, rats and rabbits Peterson and Young, '52, guinea pigs). It is of particular interest that the administration to pregnant rabbit of thyroid substance induces an earlier development of definitive follicles in the experimental embryonic glands than in controls (A m m a, '38). These observations are important in as much as it must be considered later in the interpretation of the results found during late pregnancy.

Late pregnancy is characterized by so much variability in follicular type from case to case that it is difficult to find any definite trend, while follicle colloid indicates a change which alternates in its staining degree according to the advance of gestation (figs. 1 and 2). There are three peaks and three troughs. This suggests, presumably, that recurring cycles of activity may have occurred in the glands.

Considerable interest centers on the problem of what physiological entities are involved in the present findings. The author will concern himself here with some evidence that is of endocrine interest regarding the thyroid glands only. Despite the clinical recognition of a possible association, the obscurity surrounding histological observations of the embryonic-maternal thyroid relationship has persisted. In Hamblen's monograph ('39), that the functional reciprocity between fetal and maternal glands is supplement for their deficient thyroid function has been described.

The present histological comparison of the maternal and embryonic glands yields some new information. The corresponding maternal glands at the start (CRL : 46 mm) of the follicle stage of embryonic glands, at which time a complete status consisting of follicles containing typical colloid is achieved throughout the whole area of the glands

(Toga r i, Su g i yam a and S a was a k i, '52), represent a possible picture of high colloid release indicated by an abrupt reduction in colloid staining and partly in follicle size (figs. 1, 2 and 3). As a preliminary assumption, it is probable that this is suggestive of the existence of a relation to a final definitive differentiation of the embryonic thyroid gland.

Subsequent to the attainment to a definitive status of the embry-onic glands, fluctuations in the frequency of follicles over 50 ik occur as three peaks distinctly in the embryonic side, while the corresponding maternal glands appear to show two distinct peaks of dark staining of colloid (figs. 2 and 3). The first embryonic follicular peak may correspond in time relation with the second maternal colloid peak in late pregnancy, and the third embryonic follicular peak with the second peak appears to correspond with the third maternal colloid peak with

290 Y. Sawasaki

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a preliminary fluctuation. Such almost synchronous coincidence of

peaks in both sides appears to indicate a possible existence of some of the functional relationships between the maternal and embryonic glands. The data described bear a clue to this question.

The occurrence of secretory droplets in follicle epithelium is also found during late pregnancy (figs. 1 and 2), and is marked especially during the second half. The same presumption, as that for mid

pregnancy may be presented for this finding. Pregnancy alters metabolic balance or endocrine balance (Salter,

'40) . It has been reported by Marine, Cipr a and Hunt ('24), that the increased metabolism which occurs normally during pregnancy in rabbits depends on the presence of the thyroid gland. Despite past controversies, the present data enable the author to state that the

changes in the thyroid gland during pregnancy appear to fluctuate markedly in histological character and the structural modifications

reflect changes occurring in the secretory process, presumably passing through recurring cycles of activity.

Detailed examination of the quantitative data (figs. 1 and 2)

presents often some discrepancy in the relation between the follicular type and staining trend of colloid. Considerable difficulty was experi-enced in interpreting it. Some of the macrofollicular types including

transitional types indicate a lesser staining of colloid and some of the microfollicular types show a strong staining. These may be due to individual variation, difference of age and other unknown factors of rabbits. However, a considerable part of the discrepancy may be

eliminated by an accurate orderly arrangement of the data according to the progress of gestation and comparison of the trends of the data.

No apparent correlation between the histological data of the thyroid

glands during pregnancy and the number of littermate embryos or seasons during which materials were taken, was found at the present

stage of investigations. Parafollicular cells have been reported to increase during pregnancy

(Vicar i, '37, dogs). In the present observations, they, especially dark elements, are found abundantly during early and mid pregnancy, but in pregnancy, they appear to play no special role. Dark parafollicular

cells appear to be identical with Bensley's ovoid cells ('14) in the opossum and are destined to degenerate. S u g i y a ma ('54) has con-firmed also this fact in histogenesis of the thyroid gland of guinea

pigs.

292 Y. Sawasaki

Summary

A total of 110 pregnant rabbits were used in this study. Quan-

titative histometrical measurements (frequencies per square millimeter of occurrence of follicles over 50 /.4 and irregular-shaped follicles, and frequency per field of occurrence of follicles containing dark colloid) were made and it was revealed that an orderly sequence of similar

events concerning colloid and follicles may occur in the peak and trough with the advance of gestation. Based upon this, it was

presumed that the thyroid glands appear to pass through recurring cycles of activity. From this point of view, the significances of the histological changes observed were discussed in relation to times of ovulation, implantation of fertilized ova, and further differentiation of the embryonic thyroid gland.

Early pregnancy : Immediately after mating, the glands are composed predominantly of small follicles and colloid is feebly stained, with some fluctuation. During the passage through the oviducts of fertilized ova the glands have generally larger follicles and reveal a transitory storage phase in which dark colloid is abundantly contained. Shortly before and at the presumed time of implantation of fertilized . ova, the glands show again a trend toward microfollicular type, concurrent with irregular formation of large follicles, lesser sthining of colloid, marked swelling of follicle epithelium, and high capillary hyperemia. This is evidently a colloid release phase. After a while, the glands are composed predominantly of large follicles with an intense staining of colloid and reveal another transitory storage phase of colloid at this period.

Mid pregnancy : At the beginning, colloid is reduced in staining,

partly with a reduction of follicle size, and subsequently it is increased with an increase in size of follicles. Again, after this colloid storage

phase, another colloid release phase is followed. This may correspond in time relation with the preparatory differentiation stage of the

embryonic thyroid gland. At the end, a colloid storage phase is found with an increase of large follicles and dark colloid.

Late pregnancy : At the beginning the glands show an abrupt reduction in staining of colloid. This phase may correspond in time relation with the onset of wide-spread occurrence of definitive follicles containing typical colloid in the embryonic thyroid gland. Later they indicate some change of colloid in staining, which may be comparable


in time relation with some structural fluctuation in the fully developed

embryonic thyroid gland. The follicular types of the glands are highly

variable and it is difficult to determine their trend at any stages.

Parafollicular cells are found abundantly during early and mid

pregnancy.

Acknowledgement

The author wishes to acknowledge his sincere thanks to Dr. S.

Sugi yam a, presently, Professor of Anatomy, Nagoya University School

of Medicine, for his kind guidance throughout the course of this study.

Further, the author is indebted to Prof. Dr. Ch. T oga r i, Nagoya

University, School of Medicine, for his many valuable criticisms, and

to Prof. Dr. K. Mor i, Nagoya City University Medical School, for

kind encouragement and help.

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Abbott, A. C. and J. Prendergast: Canad. med. Assoc. J., 34: 609-614, 1936. Ahar a, M.: Mitt. med. Akad. Kioto, 8: 381-394, 1933 (Japanese).

Amma, Y.: Kaibo Zasshi, 12: 426-487, 1938 (Japanese). Aron, M : C. r. Ass. Anat., 29: 7-22, 1934.

Ba rg m an n, W.: Die Schilddriise. In: v. M011endorff's Handb. d. mikr. Anat. d. Mensch , VI/II TI/I: 2-136. Julius Springer, Berlin. 1939.

Benazzi, M.: Arch. ital. Anat. Embr., 32: 430-448. 1934 (cited by Anat. Ber., 33: 1936). Bensley, R. R.: Anat. Rec., 8: 431-440. 1914.

Berberich, J. und Be rnh. Fische r-Wasels: Schilddriise and innere Sekretion. In : M. Hirsch's Handb. d. inneren Sekretion, I: 368-371. Curt Kabitzsch, Leipzig. 1932.

Bernard, W.: Rev. franc. Endocrin., 5: 395-452. 1927. Bischoff : Entwickelungsgeschichte des Kanincheneies. Braunschweig. 1842 (cited by

Fraenkel '03). Blirkle-de la Camp, H.: Arch. klin. Chir., 130: 207-236. 1924.

Chardard-Raimbault, S.: Arch. Anat. microsc., 41: 227-236. 1952. Corner, G. W.: Amer. J. Physiol., 86: 74-81. 1928.

De Robertis, E.: Anat. Rec., 84: 125. 1942 (cited by Dvoskin '47). De Robe rtis, E. and E. Del Conte: Rev. Soc. argent. Biol., 20: 88. 1944 (cited by

Dvoskin '47). Dvoskin, S.: Endocrinology, 41: 220-229. 1947.

: Endocrinology, 43: 52-70. 1948 Engelhorn: Habil.-Schrift Erlangen, 1912 (cited by Bargmann '39, Berberich u. Fischer-

Wasels '32, Herold '33, and Wegelin '26). Ewe, H.: Beitr. pathol. Anat., 97: 195-204. 1936.

Florentin, P., Th. Fontaine et L. Hennequin: C. r. Soc. Biol., 121: 1410-1412. 1936.

Fraenkel, L.: Arch. Gynàk., 68: 438-545. 1903. Grosser, 0.: Arch. Gynak., 110: 297-327. 1919.

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Grumbrecht, P. und A. Loeser: Klin. Wschr., 17: 233-235. 1938. Ha mblen, E. C. : Endocrine Gynecology : 155-156. Charles C Thomas, Springfield. 1939.

Hammond, J. and F. H. A. Ma rsha I I: Reproduction in the Rabbit: Oliver and Boyd, Edinburgh & London. 1925.

Herold, L.: Arch. Gyn5k., 154: 256-264. 1933. Mir thle, K.: Pfliigers Arch., 56: 1. 1894 (cited by Dvoskin '48).

Ishimaru, S.: Fol. Anat. Jap., 4: 13-32. 1926. Jack son, C. M.: Amer. J. Anat., 19: 305-352. 1916.

Jost, A., F. F. Morel et M. Ma r o is: C. r. Soc. Biol., 143: 142-145. 1949. Kossmann, R.: Arch. Ginak., 57: 224-260. 1898.

Krjlow, L. N. und Ar. J. Sternberg: Endokrinologie, 10: 37-43. 1932. Krogh, M. et H. Okkels: Bull. Histol., 10: 105-112. 1933.

: Klin. Wschr., 15: 203-205. 1936. Lowe, E.: Quart. J. microsc. Sci., 73: 577-592. 1930.

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Ma rz a, V. D. et A. V. B Ii no v: C. r. Soc. Biol., 121: 1438-1440. 1936 Ohida, S.: Fol. Anat. Jap., 26: 347-370. 1954.

Peterson, R. R. and W. C. Young: Endocrinology, 50: 218-225. 1952. Pincus, G.: The Eggs of Mammals: Macmillan, New York. 1936.

Rome i s, B.: Mikroskopische Technik. 15 verbesserte Aufl. : 557. Leibniz, Miinchen. 1948.

Salter, W. T.: The Endocrine Function of Iodine : Harvard University Press, Cambridge. 1940.

Sanderson-Damberg: Frankf. Z. Path., 6: 312. 1911 (cited by Bargmann '39). Sax, M. G. and R. G. Leitso n: Bull. Biol. et Med., 4: 496. 1938 (cited by G. Ma nsf el d :

The Thyroid Hormones and their Action, Transl. by E. P u lay: 100. Frederick Muller, London. 1949).

Seitz, L.: Handb. d. norm. u. path. Physiol., 14(1: 472-473. Julius Springer, Berlin. 1926.

Se lye, H.: Textbook of Endocrinology, 2nd ed.: 710. Acta Endocrinologica Inc., Montreal. 1949.

Severingha us, A. E.: Z. Zellforsch., 19 : 653-680. 1933. SpOttel, W.: Z. Anat. u. Entw. gesch., 89: 606-671. 1929.

Stande r, H. J. In Williams, J. W.: "Obstetrics," 7th ed., ed. by H. J. Stan d e r. Appleton-Century Co., New York. 1936 (cited by Hamblen '39).

Sugiyama, S.: Nagoya Igakkai Zasshi, 50: 1171-1246. 1939 (Japanese). : Jap. J. Med. Sci., 1 Anat., 9: 18-19. 1942. : Fol. Anat. Jap., 23: 57-64. 1950. : Anat. Rec., 120: 363-377. 1954.

Sugiyama, S. and T. Sato: Anat. Rec., 120: 379-393. 1954. Sugi yam a, S. and T. Y agi z a w a: Fol. Anat. Jap., 23: 67-79. 1950.

Theresa, S.: Bull. Biol. et Med., 7: 544. 1939 (cited by Peterson and Young '52). Togari, Ch., S. Sugiyama and Y. Sawasaki: Anat. Rec., 114: 213-230. 1952.

van Beneden: Bulletins de l'academie royale de Belgique, T. 40, No. 12. 1875 (cited by Fraenkel '03).

v. Borzystowski: Inaug.-Diss. KOnigsberg, 1902 (cited by We gelin '26). Verdozzi, C.: Policlinico, sez. med., 38: 375-403. 1931 (cited by Abbott and Pren-

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Hist., VIII. Driisen mit innerer Sekretion : 57-62. Julius Springer, Berlin. 1926. Winchester, C. F.: Endocrinology, 24: 697-701. 1939.

WOlfler, A.: 'Ober die Entwicklung und den Bau der Schilddriise mit Rucksicht auf die Entwicklung der KrOpfe, Berlin. 1880 (cited by Wegelin '26).

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Explanation of Plate figures

All figures represent photomicrographs of thyroid glands fixed in Kolster's fluid and stained with Heidenhain's iron hematoxylin. Note: F: Frequency per square millimeter of follicles over 50 p.; f: Frequency per square millimeter of irregular-shaped follicles.

Fig. 4. Nineteen hours immediately after mating. Microfollicular type (F: 12.1; f: 3.8). Colloid is little in amount, stained feebly, and much vacuolated. Follicle cells are much swollen and clear in cytoplasm. x 170.

Fig. 5. Three days of gestation age. Transitionally macrofollicular type (F : 85.9 ; f: 17.8). Follicles are somewhat irregular in shape. Colloid is much in amount,

. stained darkly and not vacuolated. Follicle cells are cubical and not swollen x 170.

Fig. 6. Seven days of gestation age. Microfollicular type (F : 43.0 ; f: 42.5). Follicles are much irregular in shape. Colloid is little in amount, stained feebly, and

much vacuo:ated. Follicle cells are much swollen and clear in cytoplasm. Capillaries are engorged. x 170.

Fig. 7. Ten days of gestation age. Macrofollicular type (F : 104.8 ; f: 45.1). Follicles are much more irregular in shape. Colloid is much in amount, stained darkly,

and less vacuolated. Follicle cells are cubical but relatively swollen. x 170. Fig. 8. Twenty-four days of gestation age. Macrofollicular type (F : 142.8 ; f: 20.2).

Follicles are slightly irregular in shape. Colloid is much in amount, stained darkly and not vacuolated. Follicle cells are cubical. x 170.

Fig. 9. Fifteen days of gestation age. Dark parafollicular cells are seen. x 800. Fig. 10. Twelve days of gestation age. Numerous secretory droplets are distinctly seen

in follicle epithelium. x 800.

Y. Sawasaki

histological studies of the thyroid gland of the rabbit

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