Structural Changes in the Suprarenal Gland of the Mouse During Pregnancy

GUIEYSSE (1889) observed that the suprarenal gland of the guinea-pig underwent enlargement during pregnancy, this increase chiefly affecting the zona fasciculata. This observation was confirmed by Kolmer (1912) and extended to the case of the rabbit by Kolde (1913) and others. According to Guieysse the hypertrophy of the zona fasciculata in the guinea-pig is due to an enlargement of the cells themselves and not to an increase in their number. He also states that there is no enlargement of the zona glomerulosa or of the medulla.

An increase in the weight of the suprarenal during pregnancy was noted in the rat by Herring (1920) and in the guinea-pig by Verdozzi (1917) and Castaldi (1922). Donaldson (1924) states that there is no change in the weight of the adrenal in the healthy albino rat during the phase of pregnancy lactation. Masui and Tamura (1924) have shown that the suprarenal of the mouse increases in size at the beginning of pregnancy, but that at the same time degeneration of the zona reticularis sets in and proceeds until this zone has completely disappeared so that towards the end of pregnancy the gland as a whole is greatly reduced in size, although the zona fasciculata itself has increased slightly.

The present work was undertaken in order to examine the effect of unilateral ovariotomy upon the suprarenal. Observations were made on unilaterally ovario-tomised mice during pregnancy. The results obtained show that unilateral ovariotomy is not followed by any change in the histological structure of the adrenal (apart from such variation which is also found in normal non-pregnant females), apart that during pregnancy in the unilaterally ovariotomised female both adrenals undergo similar modifications in structure identical with those that occur in the normal pregnant female.

Ordinary mixed tame mice were used as material. Each experimental group was composed of individuals from one and the same litter, and virgin females from a few litters were kept as controls. Unilateral ovariotomy was performed under ether anaesthesia at three, five, seven and eight weeks after birth. The operated and control animals from each experimental group were kept in the same box. The ovariotomised animals were mated at nine weeks and killed with chloroform at the first, second or third pregnancy. They were weighed and measured immediately after killing; the ovaries and other endocrine organs were removed and fixed.

Allen-Bouin, Regaud, and Chura’s solutions were used as fixative agents. The last solution gave good results for the purposes of examination of mitochondria, granules and chromosomes. The thickness was 6μ for structure and 3 or 4μ for granules examination. The stains used were Delafield and Heidenhain’s haema-toxylin for structure, Champy-Kulle’s acid-fuchsin-toloidin-aurantia for mitochondria and granules, and osmic acid for lipoid and fatty substances. The uterus and its contents were weighed; then all foetuses were weighed and their measurements taken. It is impossible by examination to determine with exactitude the stage which pregnancy has reached in the mouse. If the length of the foetus is used as a standard for classification, the whole period of pregnancy may be conveniently divided into the following five stages :

Stage I. Before the formation of the placenta, i.e. the stage of fixation of the fertilised egg, or the coelomic stage.

Stage II. From the formation of the first traces of placenta to the stage when the length of the foetus is 5 mm.

Stage III. The length of foetus from 6 to 12 mm.

Stage IV. The length of foetus from 13 to 20 mm.

Stage V. The length of foetus from 21 mm. and over.

In order to determine the range of variation in the length of the foetus during Stage V, measurements were taken of young at birth and lengths from 21 mm. to 27 mm. were obtained. To determine the relative sizes of the different areas of the adrenal a drawing was made of the largest section (fixed in Allen-Bouin solution for as short a time as possible in order to prevent shrinkage, and stained with ordinary Delafield haematoxylin) and the outline of each zone was traced with the aid of a planimeter.

My thanks are due to Dr F. A. E. Crew for providing accommodation and experimental material and also for his valuable help and constructive criticism during the course of this study.

Masui and Tamura (1924) have described in detail both histologically and cytologically the structure of the suprarenal cortex; it is not necessary there fore in this paper to deal specially with this. The changes in the histological structure of the adrenal during pregnancy will be described with reference to the five stages which have been defined.

Stage I (Pl. VII, figs, 1, 2 and 3). During this stage no change in the relative sizes of the areas of all zones either in the cortex or in the medulla is to be observed, as is shown in Tables I and II. The histological structure of the zones, however, shows modification, that of the zona reticularis most and that of the zona fasciculata least.

The cells of the zona glomerulosa have become active, some showing mitosis, others becoming enlarged in size whilst minute granules are present as in the gland of the normal non-pregnant female. In the zona fasciculata two types of cells appear. One is the typical vacuolated fasciculata cells (Pl. VIII, figs. 11 and 21) and these are present in large numbers throughout the zone; the others are much smaller, clear, non-vacuolated cells which lie at the border line between this zone and the zona reticularis. These cells are similar in structure to those of the zona reticularis. Signs of mitosis are visible in their nuclei. The region of these cells will be termed zona gestationis. The zona reticularis shows a remarkable and change due to a rapid degeneration of the tissues (Pl. VIII, fig. 10). This degeneration begins along the whole of the border-line of the zona fasciculata and proceeds centripetally ; at an early stage of degeneration a layer composed of contracted cells, packed closely together, can be seen between the two zones (Pl. VII, figs. 2 and 3). In some cases this process is far more advanced that in others; the zona reticularis has, for the most part, degenerated into large vacuoles or connective tissue and is represented by a thin layer of cells adjacent to the medulla, which still retain their original character. The cells of this layer are reduced in size and have irregularly-shaped nuclei with one or more very clear nucleoli and a small quantity of chromatin granules. Cells containing secretory granules are, when present, very few in number and the size of the granules is smaller than that of those in the non-pregnant female. Thus, although at this period the zona reticularis is present, the characteristic reticularis cells are almost absent, this finding being in agreement with that of Masui and Tamura.

In the medulla a few mitotic figures are present, but no other changes can be seen.

Stage II (Pl. VII, fig. 4). The total area of the section is reduced by about 18 per cent, and a remarkable change in the relative sizes of the zonae fasciculata and reticularis has occurred ; the latter zone is much reduced in size owing to the absorption of the tissue, which is the principal cause of the shrinkage of the section as a whole. In the zona glomerulosa no change is to be observed as is shown in Tables II and III. The area of the zona fasciculata shows an absolute increase by about 20 per cent., i.e. a relative increase (estimated in relation to the total area of the cortex) of about 56 per cent. On the other hand, the area of the zona reticularis has decreased by 84 per cent, in absolute, and 78 per cent, in relative value.

Notwithstanding the remarkable change in the relative size of the area, the histological structure of the zonae glomerulosa and fasciculata is almost the same as in the former stage. The smaller, clear and non-vacuolated cells (zona gestationis) of the zona fasciculata have increased in number towards the periphery; among them small cells having large clear nuclei and a relatively scanty protoplasm are also visible. The zona reticularis has either almost or entirely disappeared and is replaced by connective tissue. When the zona reticularis has degenerated, it does not surround the medulla as in the normal gland, but becomes compressed along the abscissa of the section and accumulates in elongated processes at each side of the medulla along its ordinate. No noticeable change has occurred in the medulla although its area has increased slightly. Several mitotic figures are observed as in the former stage.

Stage III (Pl. VII, fig. 5). There is no observable change in the relative sizes of the different zones when compared with those of the previous stage (see Tables III and IV) ; the zona reticularis has increased by 37 per cent. ; this is caused by the presence of large vacuoles (the so-called zona spongiosa). On histological examination the zona glomerulosa is seen to be still developing and mitotic figures in considerable numbers are visible, mostly in cases when the length of the foetus is 8 mm. and over. Granules, which were described in the previous stage are again found, though they are now of varying sizes.

The structure of the zona fasciculata in this stage may be of two different types; in one type almost the whole of the area is occupied by typical vacuolated fasciculata cells (as in the former stages) and the vacuoles near the centre are smaller than those in the peripheral region. In the second type the central portion is occupied by the clear cells (Pl. VIII, fig. 16) described in the previous stage, which are now more numerous. A few mitotic figures at varying stages are found.

It is noteworthy that when these latter cells are present the vacuolated cells in the peripheral portion are polyhedral in shape and the nuclei in some cases show degeneration. The relative size of the area of the medulla in this stage shows a decrease which, being but slight, may be due to fluctuation. Mitotic figures are more numerous, beginning from about the 8 mm. stage.

Stage IV (Pl. VII, figs. 6, 7 and 8). There is no observable change in the relative size of the zona fasciculata, while that of the zona glomerulosa has increased by about 26 per cent, as compared with that of former stages, and that of the zona reticularis by about 46 per cent.

On microscopical examination it is seen that the zona glomerulosa is remarkably changed. During the previous stage the minute granules were seen to be increasing in size; at this stage, mostly at from about 16 mm., they are seen to be typical secretory granules (Pl. VIII, figs. 14 and 15) having a structure identical with that described by Masui and Tamura (1924) in the cells of the zona reticularis. The maximal secretory activity is found at about 17 mm. As observed in the zona reticularis of the normal non-pregnant female, very big vacuoles or a network of connective tissue (Pl. VIII, fig. 13) in the cells are also found; the nuclei in these cases are similar to those of the secretory cells. As shown in Table IV, the area of this zone takes up on an average about 21 per cent, of the cortex and at its maximal 36 per cent.

In the zona fasciculata at this stage the clear cells (Pl. VIII, fig. 12), mentioned above, appear in all cases, although their number varies. In some cases they occupy the whole area of this zone; in others they press the vacuolated cells towards the periphery, so that the latter become diminished in size and rectangular or elliptical in shape, and their nuclei are irregularly shaped.

Some of the clear cells, mostly those situated in the peripheral region, show (Pl. VIII, figs. 18 and 19) “siderophile bodies” (secretory granules) of various sizes, while those situated in the central portion do not contain any granules or mitochondria.

At this stage the size of the zona reticularis varies very considerably ; in one case it was equal to that of the zona fasciculata (Pl. VII, fig. 8), though it consisted of nothing but big vacuoles and a network of connective tissue. In another case it occupied but 1 per cent, of the cortex (Pl. VII, fig. 7) and had the form of a thin line of connective tissue.

No marked change is found in the medulla as regards its microscopical structure ; its area is found to be increased by about 6 per cent. The mitotic figures are still increasing in number.

Stage V (Pl. VII, fig. 9). At this stage the relative size of the areas of the zonae glomerulosa, fasciculata, and reticularis have decreased by 17, 5 and 56 per cent, respectively. On histological examination the secretory activity in the zona glomerulosa is not visible, although in a few cases secretory granules are still observed. Likewise, in the zona fasciculata a remarkable change has occurred. In cases where the typical fasciculata vacuolated cells which have been pushed towards the periphery remain, they become enlarged. In other cases, where no fasciculata cells were present, they appear and the zona gestationis is seen to undergo degeneration (Pl. VII, fig. 9). Its cells become greatly vacuolated, in some instances the entire cell protoplasm being reduced to a network. The degeneration proceeds from the periphery. In some cases the zona disappears entirely and typical fasciculata cells occupy the whole area. The proportion of the area of the medulla increases by 14 per cent, in absolute, and by 30 per cent, in relative, value to the entire area of the gland.

Guieysse stated that in the guinea-pig the hypertrophy of the zona fasciculata is the result of an enlargement of the cells themselves and not of an increase in their number. However, in the mouse two types of hypertrophy may be observed. One is similar to that described by Guieysse in the guinea-pig. It takes place at a very early period of pregnancy and the cells increase in diameter by about 38 per cent., the largest cell increasing from 16μ to 22μ. Guieysse maintained that hypertrophy implies hyperfunctioning of the zona fasciculata, even though he was unable to demonstrate how this occurred. However, it is reasonable to assume that in the mouse this hypertrophy is only compensatory following a shrinkage caused by rapid degeneration and complete disappearance of the zona reticularis.

An examination of Tables II, III, V and VI will show that the average area of the zona fasciculata in Stage II shows a very great increase as compared with Stage I ; and that in Stage V a decrease, as compared with Stage IV. However, the initial increase is much greater than the subsequent decrease. This is probably due to the fact that the increase in size of the zona fasciculata is permitted by a very rapid degeneration of the zona reticularis and is thus a process of compensatory hypertrophy. In the later stage (V), when the zona gestationis has ceased to develop and begins to degenerate, this process is slow and therefore the increase in the area of the typical vacuolated fasciculata cell is very gradual.

Another type of the hypertrophy of the zona fasciculata is that caused by the development of the zona gestationis within the zona fasciculata. The new zone increases gradually in size and at Stage IV occupies almost the whole area of the zona fasciculata ; at Stage V it ceases to develop and begins to degenerate and consequently the area of the zona fasciculata becomes reduced. As shown in Table V, the area of the zona fasciculata is greatest at Stage IV.

Origin. As stated above, when at an early period of pregnancy the zona reticularis begins to degenerate, there appears on the border line between it and the zona fasciculata a layer of degenerated cells, which clearly separates these two zones. At the same time one can identify at the inner margin of the zona fasciculata clear cells which increase gradually in number and in the third quarter of pregnancy (i.e. 17 mm. in length) occupy almost the whole area of the zona fasciculata, while during the last quarter they become reduced in number. As stated above, the vacuoles of the cells in the inner portion of the zona fasciculata are smaller in size than those of the outer one. Mitotic figures are present in the cells up to the middle of pregnancy.

From these facts it is seen that the zona gestationis arises on the border line between the zona fasciculata and the zona reticularis. In a non-pregnant female the cells of this zone cannot be distinguished from those of the zona reticularis, but the occurrence of pregnancy stimulates their activity and produces structural changes.

At the early period of pregnancy the cells do not show any signs of secretory activity and have a very clear protoplasm and nucleus (Pl. VIII, fig. 16). Some of them show mitotic figures which increase gradually in number up to the beginning of Stage IV, when minute granules appear which are arranged irregularly around the nucleus (Pl. VIII, fig. 17); at the 17 mm. stage some cells show typical secretory granules (Pl. VIII, figs. 18 and 19), while at Stage V (21 mm. and over) the granules change into vacuoles (Pl. VIII, fig. 20) which are similar to those of typical fasciculata cells. It is noteworthy that the change begins along the whole outer margin of the zone and proceeds inwards.

Kolmer (1912) states that “Alle Rinderelemente aus der Keimschicht der glomerulosa und zwar erst durch Amitose, dann durch Mitose hervorgehen.” However, the course of the development and degeneration of the zona gestationis is not in agreement with this hypothesis, and it is more reasonable to assume that every zone has an independent origin, as well as developmental and functional activity.

The degeneration of this zone was noted by Kolmer (1912), and Masui and Tamura (1924); and its complete disappearance by the latter workers. The results of the present investigation confirm the observation that it disappears entirely and ranidly at a very early period of pregnancy. During Stage IV large vacuoles take rapidly site of this zone (Pl. VII, fig. 8). This “zona spongiosa,” according to Guieysse and others, is a characteristic feature of the suprarenal gland during pregnancy in the guinea-pig. In the mouse it is present only in the earlier part of Stage I and in Stage IV, and is not characteristic. The vacuoles are filled with a fatty substance which stains black with osmic acid.

According to Guieysse, no hypertrophy occurs in this zone. However, in the course of this investigation, it was observed at certain periods that an enlargement of this zone occurred resulting from an increase in number of the constituent cells and a heightened activity. During Stage IV there is an average hypertrophy of about 29 per cent, with a maximum of 80 per cent.

The presence of mitotic figures was noted by Kolmer (1912). In the course of the present investigation these were observed at an early period of pregnancy, mostly at the beginning of Stage IV, while no mitotic figures were visible at Stage V. As stated previously, cells of the zona glomerulosa are most active during the closing period of Stage IV.

According to Guieysse there is no hypertrophy in the medulla of the guinea-pig during pregnancy. Masui and Tamura reported that it atrophied in the mouse at the closing period of pregnancy. In the present investigation a marked increase of the area of the medulla was observed; this process is slow in certain stages, but towards the end of pregnancy there is a sudden acceleration so that in certain cases the medulla occupies as much as 26 per cent, of the total area, thus representing an increase of 46 per cent, over that which obtains at the beginning of pregnancy when the medulla is but 16 per cent, of the whole area of the gland. A few mitotic figures were observed during Stage II, and a considerably larger number in Stage IV. This suggests that there occurs a hypertrophy of the medulla during Stages IV and V.

In a recent paper, Donaldson (1924) comes to the conclusion that “there is no change in the weight of the adrenal of the healthy albino rat during the pregnancy lactation period,” although he observed a slight increase in weight in the first quarter, an observation also made by Verdozzi and Castaldi in the case of the guinea-pig. Similarly, Herring (1920) observed in the rat an increase in the weight of the adrenal at the close of pregnancy. It is not reasonable to hold that there must be a correlation between the size of the gland and the degree of its functional activity, as suggested by Riddle (1923) in the pigeon, especially as in the case of the suprarenal gland of the mammal there are several distinct zones.

There can be no doubt that the zona reticularis during pregnancy in the mouse is composed of nothing but connective tissue or big vacuoles in which fatty substances are present. Thus the increase in the weight of a gland cannot be considered as indicative of an increase in its functional activity. A gland may have a very large total area, yet if the area taken up by the degenerated zona reticularis is deducted from it, it will be seen that the area occupied by functional tissues is actually smaller than in the normal gland.

Guieysse, who observed vacuoles and siderophile bodies in the adrenal of the guinea-pig, regards them as signs of secretory activity of the gland. He suggests that in the guinea-pig the secretory activity is cyclic, increasing till the middle of pregnancy, and followed by a resting stage until parturition.

But in the present investigation vacuoles are present in great numbers in Stage I ; in Stage II they are reduced in number and are replaced by the cells of the zona gestationis which at this stage show no sign of secretory activity. As stated above, this process is very gradual. During Stage IV the vacuoles are almost absent. The cells of the zona gestationis as well as those of the zona glomerulosa exhibit the maximal degree of secretory activity, containing large numbers of siderophile bodies (referred to by Masui and Tamura as secretory granules).

Mahnert (1920) assumed that during pregnancy the suprarenal is in an exhausted condition. In explanation of this assumption he says, “Geht aus der Lipoidverar-mung dieser Organe bei den Schwangerschaftstoxikosen.” On histological examination there can be no doubt that the lipoids are scanty throughout pregnancy except at a very early stage, so that the assumption of Mahnert is probably true. If the functional activity during pregnancy is noted, it will be seen to increase slowly until the third quarter and to decrease during the last quarter.

Watrin (1914, 1919) attempted to discover the cause of the hypertrophy of the suprarenal in the rabbit and after numerous experiments concluded that hypertrophy is due to the presence of the fertilised ovum before fixation and of the foetal placenta. The results of the present investigation are such as tend to corroborate this suggestion.

Mahnert considers that during pregnancy the suprarenal is functioning abnormally and that this disfunctioning (or malfunctioning) of the endocrine organs during pregnancy is caused by the toxins of the foetal elements of the placenta. Further, he agrees with others that the disfunctioning of the ovary is primary and that of the adrenal secondary.

Current opinion concerning the relationship of the suprarenal ovary can be stated as follows :

1. The zona reticularis is hyperfunctioning at the period of “heat.” (Masui and Tamura.)

2. During pregnancy, when the ovary and adrenals are in the disfunctioning stage, the zona reticularis degenerates and disappears. (Tamura in this paper.)

3. The zona reticularis reappears when the ovary regains its normal functioning. (Masui and Tamura.)

4. In the blood of the gonadectomised female there is present an enzyme which stimulates the development of the ovary. (Long and Evans, and Goto.)

5. Complete gonadectomy in the female has no effect on the structure of the suprarenal. (Masui and Tamura.)

The results of the present investigation are such as tend to confirm Mahnert’s first suggestion, but in them there is no evidence which permits a decision as to whether the disfunctioning of the suprarenal in relation to that of the ovary is primary or secondary.