Differential growth in the ovaries and genital tract near the time of ovulation in rats treated with colchicine

DIFFERENTIAL GROWTH I N THE OVARIES AND GENITAL TRACT NEAR THE TIME O F OVULATION

I N RATS TREATED WITH COLCHICINE1

EDGAR ALLEN, THURLO B. THOMAS, JAMES G . WILSON AND DOROTHY A. HESSION

Department of Anatomy, Yale University School of Medicine, Xew Haven, Connecticut

FIVE TEXT FIGURES AND FIVE PLATES (EIGHTEEN FIGURES)

This study has the advantages of two approaches which justify further study of the reproductive organs of the rat at and soon after ovulation. I n the first place, the animals were carefully timed from the first indication of mating responses, a method which has proved very effective in studies of the rabbit, cat and guinea pig. Secondly, the colchicine technique was used to accumulate mitotic figures and so accentuate evidence of cell division. By means of this technique it is possible to tell at a glance how rapidly various tissues are growing; for instance, whether an ovarian follicle is growing or resting, and if it is growing, whether the growth occurs evenly or unevenly.

Much evidence has accumulated from experiments involving growth reactions of genital tissues in response to injected hormones. It is interesting now to restudy normal animals and compare the rates of growth in these tissues responding to autogenous ovarian hormones and in the ovarian structures which are responsible for the production of these hormones.

This study includes observations of the follicles of a current ovulation and the follicles which would require 4 days of

'This investigation was aided by grants from the Committee for Research in Problems of Sex, National Research Council, and the Fluid Research Fund of Yale University School of Medicine.

291

292 EDGAR ALLEN AND OTHERS

growth to attain ovulation at the next estrous period. It also includes a description of the high incidence of mitosis among the follicle cells which escape with the eggs at ovulation and surround them as they pass down the proximal part of the tubes. The early stages of transformation of ruptured follicles into corpora lutea are also studied with this technique and several interesting points stressed. Differential growth of the tissues of the vagina, uterus and tubes are correlated with stages of development of ovarian structures.

At the outset some clarification of the term growth is necessary. Under a broad definition any increase in size or weight might be included. Such increases are usually determined by comparison of two or more successive measurements.

There are at least four different factors which might be included as contributing to growth :

(1) increase in size of individual cells without cell division, (2) cell multiplication by mitosis, (3) the accumulation of fluid either in intercellular spaces

or in lumina. (This type of physiological increase in intercellular fluid is especially important in studies of growth of the ovaries and uterus. The retention of fluid in the uterine lumina at estrus (in rodents) and the accumulation of liquor folliculi in the follicle may be cited as examples) and

(4) in any study of growth, the relative vascularity; especially the engorgement of the vessels with blood and lymph, must be considered.

I n this study, the second point, cell multiplication by mitosis, is given especial attention because of the action of colchicine in arresting mitoses. \

ANIMALS A N D METHODS

The rats used in this study were timed from the onset of estrus as determined by the first mating response to manual stimulation. As other students of mating behavior have emphasized, an animal shows an abrupt change in behavior at the onset of estrus. When not in estrus, she is evasive or

GROWTH I N OVARIES AND GENITAL TRACT 293

resists an approach strongly. Suddenly her behavior changes and she becomes quite responsive. The change is sharp and definite. Several investigators have shown that if the rat is not mated, she experiences a remarkable spurt of spontaneous activity at this time, which has been recorded by hundreds of revolutions of a revolving cage or rotating drum. Since rats are nocturnal animals, this timing necessitated testing at hourly intervals, sometimes well into the night.

Immediately after the first signs of receptivity, the first pair of animals were injected subcutaneously with colchicine and killed 8 hours later. Others were injected at 8,16 and 24 hours and killed at 16, 24 and 32 hours respectively. These stages were chosen because 8 hours after one injection of colchicine, one finds a maximum accumulation of arrested mitoses. This gave us four different stages for study, 8, 16, 24 and 32 hours after onset of ipating reactions. Obviously the number of mitotic figures observed represented the cells dividing during the last 8 hours of life. Ten hours should be subtracted in computing the approximate time of ovulation, i.e., 2 hours before and 6,14 and 22 hours after ovulation (Boling, Blandau, Soderwall and Young, '41, p. 324).

The dosage of colchicine used was 0.1mg. per 100gni. of body weight. At this level colchicine accumulates mitoses for 8 hours or more. Further comment on the colchicine technique is included under discussion.

One ovary and the vagina, uterus and one tube were studied from nine animals, two in each stage, with the exception of the 16-hour stage in which three animals were available. I n addition, the rectum adjacent to the vagina was included, because mitotic division in the rectal glands is a reliable indication of the effectiveness of the action of this drug. Tissues were fixed in Bouin's solution, cut at 10 p and stained with haema- toxylin and eosin.

The main effort of the study was concerned with a detailed analysis of the ovaries. .As studied with the colchicine technique, several phases of follicular growth are remarkably clear, while without this technique thev are much less apparent.


For instance, an apparently normal follicle when studied with the colchicine method, may show spotty or uneven growth. Also, one is seldom impressed with cell division in the theca, even in growing follicles, without the colchicine technique. With it, the theca is revealed as a rapidly growing outer zone.

A careful census was taken of the follicles which were classified in three groups :

Stage I11 > 700p, the large preovulation follicles during estrus.

Stage I1 - 500-700 p, the largest follicles after ovulation of the previous groups, probably aberrant and destined for atresia. These were present in the 24- and 32-hour animals. < 500 p, follicles of medium-size or smaller from which follicles would have been chosen f o r ovulation at the next estrus, 4 days later. Stage I was further divided into three sub-classes (table 1).

Many studies of growth have utilized the mitotic index ; i.e., the proportion of dividing to resting cells. When the colchicine technique is used, mitotic figures in rapidly growing tissues are so numerous that detailed counts seem unnecessary. Con- sequently, in this study the frequency of mitotic figures were rated on a scale of + to + + + +. Occasionally counts were made as supplementary evidence, but reference to the illustra- tions will show how tedious detailed counts would have been. During the course of this study it was found that independent estimates by the observers participating usually coincided.

Stage I

OBSERVATIONS

Eight hours after the o m e t of mating respomses

The ovary studied from animal no. 15 had one ovulation point and the egg was found in the ovarian capsule right beside the ostium of the tube. The egg was surrounded by many follicle cells and a considerable number of these cells were in mitotic division (fig. 8). The ovum was cut into nine sections.

GROWTH IN OVARIES A N D G E N I T A L TRACT 295

Counts were made of mitotic figures among the follicle cells surrounding the ovum, and in ten sections there was an average of twelve mitoses per section (fig. 1). These will be compared later with the number of mitoses in the cumuli of ova in preovulation follicles and also around tubal ova in the the 16-hour animals (fig. 2) .

2

Fig. 1 An outline drawing to show the incidence of mitosis among the follicle cells around a very recently ovulated egg located in the ovarian capsule near the ostium of the tube in ra t no. 15 8 hours a f te r first mating response. A count of the cells of the former cumulus in this section showed twenty-two mitoses among a total of 246 cells surrounding the ovum, a ratio of 1: 11. It is probable tha t most of these mitoses occurred in the follicle before ovulation. See also figure 8.

A drawing illustrating the many mitoses around one of the tubal ova of a 16-hour animal about 6 hours a f te r ovulation. There were eighteen mitoses in this section. These ova had passed through the fimbriated end of the tube and were located in the second loop. Note that a t this time the follicle cells are not widely dispersed as they were immediately a f te r ovulation. Compare with figure 1.

Fig. 2

It is unusual to find eggs in the ovarian capsule. They apparently pass very quickly into the tube and rather quickly through its fimbriated end. Thi% fact, and the finding of many former follicle cells around the eggs, show that this single ovulation had occurred very recently. It is probable that many of these mitoses had occurred before ovulation.


There were six large preovulation follicles in this ovary. Death of this animal had caught them on the verge of ovulation. The ova of these follicles had all entered the first maturation spindle stage (fig. 10). There was evidence of partial formation of the corona radiata and some dispersion of the cells of the cumulus. In all of these follicles there was some secondary liquor folliculi, the clear secretion formed rapidly a few hours before ovulation which disperses the cumuli and frees the ova from their attachment to the follicular walls (Robinson, '18). The walls of the follicles of the preovulation set were somewhat folded, perhaps indicating partial release of intrafollicular pressure, or possibly an artefact.

I n the cumuli of these preovulation follicles many mitotic figures were counted (rated as + + in table 1). The mitoses per section of cumulus varied from eleven to twenty-two, and averaged sixteen for six follicles. The total number of follicle cells surrounding the ova varied from 200 to 350 cells per section.

The mitoses were in most cases evenly distributed in the granulosa and theca of these large preovulation follicles. In a few of the follicles, however, there was a higher incidence of mitosis in the granulosa near the ovum; i.e., at the base of the cumulus (fig. l o ) , while distal parts of the granulosa were quiescent (fig. 11). In the theca, mitoses were abundant and evenly distributed. Actual counts of these mitotic figures ranged from 80 to 105 per section in the granulosa (average 90). The counts in the theca ranged from 24 to 28 per section (average 27). The total number of cells involved in the theca was much less than in the granulosa. The figures cited are actual counts of single sections.

These counts of mitoses show that continued cell division is one of the outstanding features of the transformation of a follicle immediately preceding ovulation. The fact that cell division also continues so rapidly in the theca is worth empha- sis, as it is frequently overlooked when the colchicine technique is not used.

GROWTH IN OVARIES AND GENITAL TRACT 297

I n addition to the six preovulation follicles, which might have ovulated, there were two other follicles strikingly different. The ovum of one of these had the chromatin in the first maturation spindle; in the other the nucleus was intact. The point of greatest difference between these two and the six “probable ovulating” follicles was the complete absence of cell division in both cumulus and granulosa. However, both follicles had many mitoses in the theca (f +). The follicle cells, especially of the cumulus, of the two atypical follicles were tightly packed. There was no corona formation. Some secretion of secondary liquor folliculi was apparent in the follicle containing the ovum with the maturation spindle, but not in the one in which the nucleus of the ovum was intact. The uneven distribution of mitoses is clearly an indication of uneven growth. This will be considered later as evidence for an interpretation of the hormonal stimuli responsible for cell division at this time (see discussion).

At this point a comparison of the recent corpus luteum from which the ovum in the ovarian capsule had just been ovulated with the walls of the preovulation follicles is interesting, for both belong to the same generation of ova. The walls of the corpus were completely collapsed (fig. 12) and the rupture point was clearly visible. The former granulosa contained an average of 100 mitoses per section, and the former theca about thirty-six (fig. 13). This compares quite favorably with ninety and twenty-seven, the averages of the granulosa and the theca of the unruptured follicles (figs. 10 and 11). These counts represent cell divisions during the 8 hours previous to death. Therefore much difference in the preovulation follicles and the recent single corpus luteum is not to be expected, since in the recent corpus many of the arrested colchicine mitoses may have been accumulated before ovulation.

After study of the “follicles of ovulation,” including the one which had just ovulated, the six “probables” and the two “atypicals ’’ which obviously had no chance of ovulating, studies of the next largest follicles were begun. The search

298 EDGAR A L L E N A N D OTHERS

now centered upon the follicles which would have provided the eggs for ovulation at the next estrous period 4 days later.

The next largest follicles (listed as stage I) showed rapid growth, and in many the mitoses were fairly evenly distributed in both granulosa and theca. They were characterized by medium size (250-500cr) and by eight to fifteen layers of follicle cells. Some had small lakes of primary liquor folliculi but others had none. Nuclei of the ova were vesicular. From this description the smallest of the stage I follicles will be recognized as the most advanced stage possible after hypophysectomy (Smith and Engle, ’27; Lane and Qreep, ’35). I t should be recalled at this point that further development of these follicles requires gonadotropic stimulus from the pituitary.

One ovary of rat no. 15 contained eighteen follicles which might be included in this class, and which might be designated “4-day follicles”; i.e., some of them (4 to 8) would have developed to ovulation size at the next estrous period 4 days later. Closer study of this group of follicles, however, showed clearly quite a difference in development, which might justify dividing this group into several sub-classes (table 1).

Two follicles, somewhat larger than the others (400-500 p), were designated as stage I, (fig. 2 2 ) . They were characterized by approximately fifteen layers of follicle cells and one or two lakes of primary liquor folliculi, but secretion of liquor had not progressed to the point where the cumuli could be clearly distinguished from the granulosa. These follicles were growing a t a + rate in the granulosa and + + + in the theca.

There were six follicles of the stage I, group (350-400 p).

These follicles (figs. 19 and 20) with ten or more layers of follicle cells showed a higher rate of growth in the granulosa (+ +) and even more active growth in the theca, in some

a In considering the measurement of follicles, it should be noted that when the colchicine technique is used, mitosis does not proceed to the formation of two daughter cells. In normal animals these mitoses would be completed and tlierc would be at least as many more cells in the follicle as there are colchicine mitoses. Consequently, follicles of this study would be smaller than in animals in which the colchicine technique had not been employed.

u, 0 1

I--

a

300 EDGAR ALLEN AND OTHER8

cases as high as + + +. Small lakes of primary liquor folliculi were present.

Ten smaller stage I, follicles (250-350 p ) were counted (fig. 17). I n a majority, mitoses were evenly’ distributed in follicle cells and theca. ’ There was little accumulation of intercellular secretion. Five other follicles of approximately stage I size were in early phases of atresia.

Follicles smaller than stage I were not counted, but it was frequently noted that many of these smaller follicles contained high enough numbers of mitotic figures to indicate that some of them were growing rapidly (both follicle cells and theca). Others were entirely quiescent. The colchicine technique shows this clearly. This is emphasized because several descriptions of rat ovaries by other investigators give the impression that these small follicles are resting or “in storage.” Further discussion of this phase of follicular development appears in a later section.

Thorough search of many areas of interstitial tissue showed the absence of mitotic figures, indicating that cell division does not occur in this tissue, at least at these stages of the estrous cycle. This is in marked contrast to the high incidence of mitosis in the majority of follicles, including the theca.

Sections of the vagina showed the cornified layer still intact. Therefore, rat no. 15 was in estrus. Mitotic figures were scarce in the vaginal epithelium (characterized as +). It is clear that cell multiplication in the vaginal epithelium at the beginning of ovulation was at low ebb.

The uterus was still considerably distended with secretion (table 1). There were no mitotic figures in the uterine glands at this stage, indicating that the first wave of cell division of the next cycle had not yet begun in these structures. There was extensive infiltration of the uterine stroma by leucocytes. The surface epithelium, however, was still in good condition, neither vacuolated nor invaded with leucocytes.

Rat no. 1, the second animal sacrificed 8 hours after the onset of mating responses, was in most instances quite similar to no. 15, with the exception that no follicles had yet ovulated.


There were four preovulation follicles present. Differences in the growth rate of the various follicles can be seen in table 1.

Sixteen hours after the olzset of mating responses Rat no. 13 had four eggs in the fimbriated end of the tube.

They were still surrounded by many former follicle cells in which mitoses were listed as + +. Examples are shown in figures 2 and 9.

It was surprising to find a large number of mitotic figures among the follicle cells around these tubal ova. Supposedly these eggs had been in the tube at least 6 hours, since ovulation occurs at an average of 10 hours after the onset of mating responses (Boling, Blandau, Soderwall and Young, '41). It is well known that these follicle cells, ovulated with the eggs, usually disappear during the second day after ovulation. Counts were made of the mitotic figures around these four ova. Forty-five sections contained a total of 600 mitotic figures, which would mean an average of 150 mitoses around each egg. To put the count on a basis comparable to that of rat no. 15, the average per section of egg was thirteen, as compared with sixteen per section of preovulation cumulus and twelve around the very recently ovulated single egg in the ovarian capsule of rat no. 15.

The continued high rate of cell division immediately after ovulation among the follicle cells adjacent to the tubal ova is remarkable. Although some of these mitoses may have occurred in the follicle, cell division must have continued among cells of the former cumulus during passage through the proximal part of the tube. The ova were not fertilized, for the animals had not been mated.

There were four recent corpora lutea corresponding to the tubal ova. These corpora were completely collapsed and the inner layers of their walls were composed of darkly staining, tightly packed cells which were quite different from those of the outer layers (fig. 14). Collapsed walls would indicate that the rupture points of ovulation had not yet healed. The inner zone of tight-packed, dark-staining cells might be open to two


interpretations which will be considered further in the discussion, page 318.

Colchicine mitoses were unevenly distributed throughout the walls of these collapsed corpora. They were numerous among the cells of the former theca, where the incidence was listed as + +, approximately the same as in the theca of the preovulation follicles. The decrease in the number of mitoses in the inner layers of luteal cells immediately after ovulation is noteworthy (fig. 3). A surmise as to its significance will be found under discussion.

The ovary of rat no. 13 was remarkable in that it contained forty-two follicles of stage I, which would supply the four or five follicles of ovulation at the next estrus 4 days hence. Of these, three were classified as I2 follicles, having a growth of T i n the granulosa and + in the theca. There were thirty- nine I, follicles showing somewhat more rapid growth, + in the granulosa and + + in the theca. I n addition, five follicles did not appear normal, and small, hyperchromatic cells char- acteristic of early atresia were present. It is possible that these five follicles were members of the present ovulation group and had failed to obtain sufficient stimulus for ovulation. If it were not for the + growth in the theca they would have been classed as clearly atretic.

The vaginal wall of rat no. 13 was in an early metestrous condition ; i.e., the cornified layer had been completely sloughed into the lumen, but there was no leukocytic invasion of the cells in the vaginal lumen. Mitoses in the vaginal epithelium were listed as +.

The uterus was considerably distended with secretion. There were as yet no mitotic figures in the uterine glands nor in the surface epithelium. Leukocytic infiltration of the stroma was well advanced.

Animals no. 11 and no. 14, also l 6 - 1 1 0 ~ stages, were quite comparable to no. 13. Four eggs had been ovulated from one ovary of rat no. 11, and eight eggs from one ovary of rat no. 14. With one or two possible exceptions there were no follicles which might be considered as left over from the

G R O W T H I N OVARIES AND GENITAL TRACT 303

“ovulation class” in the ovgries of any of these 16-hour animals. There were no clearly atretic large follicles. Other data are listed in table 1.

Twenty- f our hours a f ter onset of mat ing responses Six eggs had been ovulated from one ovary of rat no. 5.

They were in the second loop of the tube. There were still a few follicle cells around the tubal ova, but no mitoses could be identified among them. These cells were pycnotic, darkly staining and degenerate. Therefore cell division ceases among the follicle cells around tubal ova in the 24-hour stage, during the interval from 6 to 14 hours after ovulation. This is in contrast to the condition described for the 16-hour animals (6 hours after ovulation) where dividing cells were still present.

There were six recent corpora lutea in this ovary. All were redistended (fig. 16), which would mean a healing of the rupture points and secretion of a considerable amount of tertiary liquor folliculi. This term was proposed by Robinson f o r the fluid which accumulates in the recent corpora after healing of the rupture points. With the redistention of the corpora. lutea, the inner zone of darkly staining luteal cells mentioned in the 16-hour stage, had almost completely disappeared. The cells of the inner zone, however, still seemed more follicular than luteal in type.

In the inner half of the walls of the corpora some mitotic figures could be identified (characterized as +). In the outer part of the walls there were greater numbers in mitosis (+ +). The adjacent connective tissue, the remains of the theca of the preovulation follicle, still contained many mil otic fiyures (+ +). I t should be stressed that at the 24-hour stage, blood vessels were actively growing into the outer layers and that some of the recorded colchicine mitoses occurred in their endothelium. This reservation is necessary when comparing the incidence of mitosis in the granulosa of preovulation follicles which is avascular, with that in the walls of early corpora lutea.

304 EDGAR ALLEN A N D OTHERS

There were fifteen follicles in this ovary from which the follicles of the next ovulation might be chosen. Thirteen of these were classified as stage I. The remaining two follicles were larger than the others and had well differentiated cumuli, indicating a more advanced stage in the secretion of primary liquor folliculi. Growth in the theca of both was listed as + + +, in the granulosa as +, and in the cumulus i. These follicles required a more advanced classification and were therefore designated as stage 11. It will be noted in table 1 that the number of these follicles increases in the ovaries of the 24- and 32-hour animals. Of the thirteen stage I follicles, two were classified as I,, five as I,, and six as I,. The smallest were growing most rapidly (granulosa + +, theca + + +).

The vaginal wall a t the 24-hour stage was characterized as in early metestrum. Some of the cornified layer had sloughed into the lumen, but in other places it was still intact. There was an increase over the 8-hour stage in the number of mitoses in the basal layers, characterized as + + (fig. 6). There were no leukocytes in the vaginal wall or in the lumen.

The uterus was collapsed, the lumen was slit-shaped in cross section and contained little secretion. The uterine glands a t this stage showed a high incidence of mitosis (fig. 7). They were crowded with mitotic figures (in this animal listed as + + +). Attention is called to this feature as the first indication of rapid growth of the uterus in preparation f o r a new cycle. The tips of the glands had previously been described as the first tissues to respond to injected estrogenic hormone in ovariectomized mice (Allen, Smith and Gardner, '37). I t was surprising, however, to find mitoses here 14 hours after ovulation in the normal rat. The stroma was heavily infiltrated with leukocytes (fig. 7 ) , and the surface epithelium was vacuolated.

Rat no. 6, the second 24-hour animal, almost completely duplicated the conditions in animal no. 5 ; slight differences in rate of growth of ovarian structures will be noted in table 1,

Thirty-two hours after onset of nzatiizg resporzses Rat no. 8 had four ova which had passed through the ciliated

portion of the tube and were located in the second loop. These


ova were completely naked; the follicle cells having disappeared.

The corresponding corpora lutea were redistended (fig. 16). Tlie walls had thickened somewhat beyond those of the 24-hour stage, but there were still large lakes of tertiary liquor folliculi in the centers. Ingrowth of capillaries had reached the inner part of the wall, as had also some connective tissue cells. Even at the 32-hour stage, 22 hours after ovulation, some of the inner cells of the corpora appeared small and closely packed, but the majority had been transformed into luteal cells. Mitotic figures were quite plentiful and evenly distributed throughout the inner and outer zones of the wall, and the surrounding connective tissue (+ +). As previously described, many of these mitoses were in the endothelium of vascular sprouts. It was noted that in this stage the external limiting membrane, if present, was very difficult to identify. There were many cells of the surrounding stroma, however, which were still dividing rapidly.

Six stage I1 follicles were present in this ovary. Growth of the theca was much more rapid than in the cumulus and granulosa (fig. 23).

A total of fifteen stage I follicles were found in the ovary of animal no. 8. There were ten follicles of stage I,, having very little growth in the granulosa (+), and somewhat more in the theca (+ + +). In addition to these there were two stage I, follicles growing almost as rapidly. There were three stage I, follicles having + + growth or more in both granulosa and theca.

The vaginal wall of rat no. 8 was in the early diestrum. Mitoses were listed as + + in the basal layers of the vaginal epithelium of this animal.

The uterus was completely collapsed, showing in cross section a slit-like lumen. The uterine glands were growing with extreme rapidity, more than half of their cells being in mitosis (listed as + + + +).

Rat no. 7, the second animal of the 32-hour stage, was similar in most respects to no. 8. Four eggs had been ovulated recently but the follicle cells had completely disappeared. The


corpora were at similar stages of development. There were also two stage 11 follicles. Slight variations in growt,h are listed in table 1.

The preouulation. follicles

The continued mitosis among the follicle cells of the preovulation follicles as revealed by the colchicine technique, is one important feature of late follicular growth. Occasionally a preovulation follicle is found in which mitotic figures are more abundant around the ovum or at the base of the cumulus, instead of being distributed evenly throughout the wall of the follicle (figs. 10 and 11). An interpretation of such uneven growth as a first sign of decline leading to atresia will be discussed later.

The theca of growing follicles has a high incidence of mitosis. Before the use of the colchicine technique in studies of the ovary, the theca had seemed a relatively unimportant adjunct to the follicle. If follicles were actively growing, the cells of the theca seemed flattened, or in section, elongated. The number of mitoses, without the use of colchicine, was not prominent enough to be easily e ~ i d e n t . ~ When colchicine is used to accumulate the mitotic figures, the theca is revealed as a rapidly growing outer zone of the follicle, probably of considerable importance in the preovulation growth.

The time of ovulation as correlated with onset of mating responses is of major import in studies of the estrous cycle. I n animals subject to multiple ovulations, the intervals between successive ovulations have only recently been timed (Walton and Hammond, '39, and Boling and collaborators, '41). I t is interesting in our series to find the ovulation of one follicle as early as 8 hours after tlie first mating response. Ovulation had occurred in all the animals killed at the l 6 - h o ~ r stage and after. Finding continued cell division of the former cumulus cells while the eggs were passing through the fimbriated ends of the tubes was surprising. In the 16-hour stage

3 A n exception to this would be the extreme development of the theca in the pocket gopher as described by Mossman ( '37).

GROWTH IN OVARIES A N D G E N I T A L TRACT 307

the follicle cells around the tuba1 ova were tightly packed, in contrast to their partial dispersion around the, egg in the ovarian capsule of the 8-hour stage and in the cumuli of preovulation follicles. This seemed to coincide with the dis- appearance of the corona phenomena.

The recent corpora lutea at the 16-hour stage, 6 hours after ovulation, were characterized by complete collapse of their walls, indicating that the rupture points had not yet healed and that if liquor folliculi was being secreted it was not being retained. I n addition, the cells of the whole inner half of the walls of these corpora were abnormal. They were darkly stained and closely packed (fig. 14). This condition so shortly following removal of the egg at ovulation might suggest either trauma or anoxia. Another point clearly shown in the 16-hour animals was the rapid decline in mitotic activity in the inner layers of the corpora. Some cell division still continued in the outer layers and in the transforming theca.

The corpora 14 hours after ovulation showed complete redistention in all cases, indicating a,relatively brief period for blockage and healing of the rupture points.

In the 32-hour stage, 22 hours after ovulation, the corpora were completely redistended. The luteal walls were thickened but large lakes of tertiary liquor folliculi were still present in the centers. The vascular sprouts had, in most cases, extended into the inner layers. In some areas of the inner wall small patches of cells still follicular in type had not yet been transformed into luteal cells (fig. 15).

Mitotic division continued in the connective tissue around the corpora lutea for some time after the external limiting membrane of the follicle had completely disappeared. This may indicate that the former theca had not been completely obliterated, or carried into the corpus with the ingrowth of the vascular sprouts, as described for “thecal-lutein” cells. Ex- amples of the distribution of mitotic figures in preovulation follicles and early corpora lutea at several stages of early development are shown in figure 3.

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The former follicle cells surrounding the tuba1 ova were still subject to a high rate of cell division, especially in those of the 16-hour animals, 6 hours after ovulation. These cells had disappeared almost completely 22 hours after ovulation, but still the rate of cell division among them during the first 6 or 8 hours remained almost as high as it was in the cumuli of the preovulation follicles. These were unfertilized ova, for the rats in this series had not been mated.

After studying the preovulation follicles in ovaries of the 8-hour animals and the recent corpora of later stages, a search was begun for follicles of the next generation. These would have had to grow sufficiently to become “follicles of ovulation” a t the next estrus within 4 days. These follicles, designated stage 11, ranged from 500-700 microns in diameter. They were advanced enough in secretion of primary liquor f olliculi so that the cumuli could be distinguished from the granulosa. They were definitely larger than stage I follicles (250-500 p in diameter), in which one or two small lakes of liquor folliculi might have appeared, but in which secretion had not continued to the point where a cumulus had been differentiated.

A study of table 1 shows the complete absence of stage I1 follicles in the 8- and 16-hour animals (with the exception of one in rat no. 14). It is not until the 24- and 32-hour stages that these follicles make their first appearance. There were fewer stage I1 follicles than had been expected; i.e., too few to supply the average number of follicles of ovulation for the next estrous period. There was also very little, if any, mitotic division in the cumuli, and cell division of the granulosa was seldom more than +. This rate of growth seems inadequate if these follicles were destined to grow to ovulation size in 3 or 34 days. The high incidence of mitosis5n the theca (+ + or more) of the stage 11 follicles indicated clearly that they had not yet begun atresia, and that they should still be classed as normal follicles. The waning growth rate in the inner layers might presage atresia.


Stage I follicles

As the study progressed it became more and more certain that the follicles of the next ovulation must be derived from follicles smaller than those of stage 11. These were designated stage I follicles; they ranged from 250 to 500 microns in diameter. As the time following ovulation increased, such differences appeared within this group that it had to be sub- divided into stages I,, I,, and I,.

Immediately after ovulation, follicles of the next group to ovulate 4 days hence usually have very little liquor folliculi, or at most one or two small lakes, but not enough to differentiate cumulus from granulosa (figs. 17-22). Many of these stage I follicles were usually growing rapidly and evenly.

The smallest stage I follicles, as studied in the 8-hour animals are clearly at the stage of development which follicles reach in hypophysectomized rats and mice; i.e., they are autonomous structures as far as cell division is concerned in that they do not require pituitary gonadotropic stimulus for growth. It might be noted here that in the ovaries of hypophysectomized animals the follicles which we have designated stage I,, grow quite evenly. As studied by the colchicine technique both follicle cells and theca of a majority of these follicles show rapid growth.

I n the ovaries of the 24- and 32-hour stages, many of the stage I follicles were subject to very rapid growth and ac- celerated secretion of liquor folliculi.

As has been noted, there are numerous stage I, follicles in the ovaries of animals no. 7 and no. 8. It is remarkable to find such an increase in the number of follicles of this stage as early as 32 hours after the onset of mating reactions, which would be 22 hours post-ovulation (table 1).

Intermediate between stage I, and stage I,, a stage I, was defined. This group was somewhat more circumscribed than either stage I, or stage I, in that the size of the follicles ranged from 350 to 400 microns. These follicles had a maximum of ten layers of cells and some primary liquor folliculi.


A further attempt was made to differentiate follicles of stage I by counts of mitoses in granulosa and theca. These were expressed in proportions as a possible means of indicating the sources of stimuli for mitosis. This was done with the idea that high incidence of mitosis in the granulosa might represent a growth stimulus coming from the egg and a high incidence in the theca, a follicle stimulating influence from the pituitary. It is clear from the counts that many of the stage I

TABLE 2

Counts of mitoses in granulosa and theca, showing different rates of growth in stage I follicles. (Slide 3, rat no. 15, at the 8-hour stage.)

___ PROPORTION

(QRANULOSA TO TKECA) GRANU W S A TIIECA

10 20 30 75 25 30 40 13 14 15 13

30 32 30 24 27

9 15 11 12 13 11

1 : 3 2 : 3 1: 1 3: 1 1: 1 3: 1 3: 1 1: 1 1: 1 1 : 1 1 : 1

Summary 6, 1 : 1 even growth

3, 3 : 1 1 1, 1 : 3 uneven growth 1, 2 : 3 J

- _ _ - ~

follicles must be eliminated during the course of development of the “follicles of ovulation. ” The retardation of growth, as shown by the colchicine technique, may be the first evidence to indicate which of these follicles are due for elimination.

For instance, in one slide containing fifty-five sections of an ovary of animal no. 15 killed at the 8-hour stage, eleven stage I follicles were studied in this way. These counts are included in table’2. Six of eleven follicles had a fairly even distribution of cell division in granulosa and theca, a proportion of 1: 1 (fig. 4). Three follicles had a greater number of mitoses in


the granulosa than in the theca, a proportion of 3: 1, and two follicles had reverse proportions, indicating more rapid growth in the theca than in the granulosa (fig. 5). This uneven growth in the former instance might indicate either an inadequate amount of pituitary gonadotropic stimulus which is necessary for continued growth of follicles past the stage characterized as stage I,, or in the later instance, a failure of

*

. . Fig. 4 “Spot ta rge t” to show the even distribution of mitoses among follicle

cells and theca of rapidly growing stage I, follicles. Follicles of this group require 4 days of development for ovulation a t the next estrous period.

“Spot ta rge t” to show uneven growth; many mitoses in the theca but very few in the granulosa. This follicle could not yet be considered atretic but the uneven growth is considered as presaging atresia.

Fig. 5

internal stimulus from the ovum. It is realized that a more detailed study would be necessary to establish this point con- clusively. It is felt, however, that this differential growth is significant.

Many smaller follicles, with two to six layers of follicle cells, may have several mitotic figures. Therefore, although some follicles of this size are undoubtedly atrophic or growing slowly, considerable numbers of them are growing rapidly.


This observation raises serious doubts that such small follicles can exist long “in storage” as a source of follicles of ovulation at long intervals in the future.

Numerous atretic follicles of various sizes were found in all ovaries. I n the 8- and 16-hour animals a few large atretic follicles were present. Clear signs of atresia were the pres- ence of small hyperchromatic cells and the close packing of the follicle cells. The colchicine technique emphasized the complete absence of growth in such follicles.

The interstitial tissue was carefully studied for mitotic figures. Mitoses are absent from this tissue even when the colchicine technique is used. This is one striking difference from the action of pituitary gonadotropin upon the follicles. If there is a pituitary gonadotropin specific for interstitial cells, it does not stimulate them to divide mitotically at least in the 8- to 32-hour stages included in this study.

Attempts were made to coordinate growth in the vagina, uterus and uterine tubes. The 8-hour animals, those which contained the preovulation follicles, had the outer cornified layers of the vagina still in place, therefore, by this criterion also they were in estrus.

Ovulation had occurred in all of the animals killed 16 hours or later after the first mating responses. By this time most of the cornified layers had been sloughed into the vaginal lumen. I n animals no. 11 and no. 14, remnants of the cornified layer were still in place in the upper vagina and in the cervical canal. I n the 24- and 32-hour stages the cornified cells had disappeared, the vaginal wall being composed of six to eight or nine layers of stratified epithelium.

In the five animals killed at 8 and 16 hours there were small numbers of mitoses in the basal layers in the vaginal epithelium, listed as + in the scale of + + + + as maximum. Animal no. 15, which had the large preovulation follicles in the ovary, was listed as +. a t the 24- and 32-hour stages, three of the four animals showed an increased number of mitotic fig-ures in the basal layers, indicating an increased growth rate at this time.


There was still considerable distention of the uterus, more marked in the 8- than in the 16-hour animals. I n some cases the glands were distended also, and it was noted that in these animals there was no mitotic division in the glandular epithelium. I n the 24- and 32-hour animals the uterus was completely collapsed. As seen in cross section the lumen was slit-like. I n all cases at the 24- and 32-hour stages the uterine glands were crowded with colchicine mitoses, listed as + + to + + + in the 24-hour animals arid as + + + + in both of the 32-hour animals. In some sections of glands nearly all of the epithelial cells seemed to be in division, an extremely high incidence of mitosis! This wave of intense mitosis in the uterine glands had previously been noted as the first response to the injection of estrogenic hormones in castrated animals (Allen, Smith and Gardner, '37). It was surprising to see it in normal animals as early as 24 hours after the beginning of estrus, which would be 14 hours after ovulation.

In a few of the animals in this series occasional mitoses were noted in the fimbriated ends of the uterine tubes, but they were by no means as numerous here as in th.2 uterine tubes of mice. I t is very difficult to find them in the tubes at other times in the estrous cycle. The non-ciliated epithelium which pre- dominates in the lower tube very seldom shows evidence of cell division. No mitoses were found here in the stages of the cycle studied.

DISCUSSION

Cell division, one of the most important factors in growth, occurs in several genital tissues as they respond to hormonal stimulation. This is true of responses of the genital tract to ovarian hormones and of responses of the ovaries to pituitary follicle-stimulatinq hormone. Various tissues show differential responses. When studied with the aid of the colchicine technique, the extent of cell division can be estimated and distinguished from other important factors in growth, such as cell hypertrophy, accumulations of tissue fluids or secretions, and rascular changes.


The timing of the stages studied from the onset of mating responses, which has proved so helpful in studies of ovulation in the rabbit, ferret and cat where mating is necessary for ovulation, has proved of value also when applied to studies in spontaneously ovulating animals. The vaginal smear technique was used to establish regularity of several successive cycles, but the more precise behavioral mating responses was the final criterion used in timing the series.

Colchicine “freezes” mitoses for at least 8 hours and thus accumulates evidence of hyperplasia, making tedious counts and computations of mitotic indices unnecessary. However, the colchicine technique has several limitations. One drawback is that the effective dose of this drug is quite close to the lethal dose. In the second place, its action on dividing cells is not clearly understood. As seen in tissue cultures, it seems to gel the cytoplasm during mitosis, especially in the metaphase, preventing completion of mitotic division (Ludford, ’36). It apparently does not prevent cells from entering mitosis, but merely holds them in that condition after mitosis has begun. Since mitotic division of cells in small rodents may be completed in 10 to 20 minutes, it is obvious that the moment of death will catch relatively few cells in division. One injection of colchicine in small rodents accumulates an optimum number of mitoses in 83 hours (Lits, ’34). These colchicine mitoses may be of two kinds. First, the chromosomes may be clumped in the center in a darkly staining mass surrounded by a wide, clear zone of cytoplasm, or second, the chromosomes may be dispersed throughout large cells. In the former condition, the mitoses are easy to count and estimate even with low power magnification. In our work the former type of clumped chromosomes were common.

One major point emphasized in this study is the continua- tion of mitotic division of the follicle cells up to the time of ovulation, and for some hours afterward in the cells of the former cumulus which are ovulated with the egg. For instance, from ten to thirty cells in mitosis in one section of the cumulus through the ovum is a not uncommon finding. The incidence is

I


almost as high around the unfertilized eggs in the tube while they are passing through the fimbriated and ciliated portion. Therefore, continued cell division may be emphasized as an important factor at these times. Several recent studies of the preovulation follicle describe a sharp decline in mitotic activity as the follicle enlarges preparatory to ovulation (Walsh, '17 ; Lane and Davis, '39). There may be some decline in cell division during the late stages of follicular development as compared with the medium-sized follicles which are literally packed with mitoses. However, the colchicine technique shows that mitotic division continues at a high level in the preovulation follicle.

Not only the number of mitoses but their distribution in the follicle may have important implications. These two factors may provide indicators of the source and strength of the hir- mones known to be necessary for growth of the follicles. For instance, in some of the preovulation follicles it was noted that the majority of mitoses occurred in the cells surrounding the eggs or at the bases of the cumuli, while in the more distal granulosa cells, mitoses were scarce or absent. This happened in unruptured follicles still present after ovulation of other follicles of the same group. We interpret this as a continued but waning stimulus from the egg for division of the follicle cells, which has become inadequate to reach the distal parts of the follicle.

A second type of uneven distribution of mitoses is to be found in some other moderately large follicles. I n these, the incidence of mitosis in the cumulus and granulosa is low, while it is consistently high in the theca. A possible interpretation of this distribution is that a stimulus for mitosis from the egg has stopped, while the follicle-stimulating hormone from the pituitary, known to be necessary at this time, may still be influencing the cells of the theca.

This uneven growth, revealed clearly by the colchicine technique, may be the first sign of local weaknesses in these follicles before other signs of atresia are clear. When follicles


are definitely atretic they invariably show absence of mitosis or an extremely low incidence.

It has been shown in the rabbit that the pituitary stimulus, acting for 1 hour after mating, is sufficient to carry through ovulation of a group of the follicles at the tenth hour after mating (Fee and Parkes, ’29). It is now well established that late growth of the follicles, ovulation and formation of the corpora lutea are all dependent upon anterior pituitary stimulation. Both follicle stimulating and luteinizing factors from the pituitary must be present simultaneously in animals like the rat with short estrous cycles. At the time that the mating responses are first apparent, a change occurs in the nature of the liquor folliculi. It is secreted more rapidly and has a different coagulation reaction (Robinson, ’18). Two of its functions are to disperse the cumulus and liberate the ovum from its attachment to the wall of the follicle and to distend the follicle immediately preceding rupture. It is also known that this secondary liquor folliculi is more fluid than the more slowly produced, viscous primary liquor. I n the moving picture on “Ovulation in the Rabbit’’ (Hill, Allen and Kramer, ’35) this fluid secondary liquor is seen leaking through the rupture point before the actual rupture occurs. This change in the nature of the liquor folliculi secreted in the preovulation follicle may be responsible for the mating behavior a t estrus. For instance, it might mark the beginning of secretion of progesterone by the preovulation follicle. Young and collaborators (’38) have shown that while estrous behavior can be induced in some guinea-pigs by injection of estrogen alone, the response can be induced in practically all by injection of progesterone after priming with estrogen. The time interval between injection of progesterone and the response falls within the 10-hour range. These results coincide with those of Hisaw and Foster ( ’35), where ovulation was induced by injecting a luteinizing fraction after treatment with follicle-stimulating hormone.

Therefore the large follicles just before ovulation may secrete progesterone. Also, after ovulation, the newly forming


corpora lutea continue to secrete estrogen. I n the rat, where successive generations of follicles and corpora lutea are super- imposed every 4 days, it is probable that the ovary secretes considerable amounts of both estrogen and progesterone at the same time. There may be a fluctuation in amount, but there can hardly be any period of extensive scarcity of either hormone.

Sixteen hours after the onset of the first mating responses and about 6 hours after ovulation the collapsed corpora lutea showed an abnormal inner zone of cells, tightly-packed and hyperchromatic. The number of mitoses among these cells was uniformly low, but in the outer zone of the wall it continued high. Also the incidence of mitosis around these early corpora remained equally as high as in the theca of the preovulation follicle.

Boling, Blandau, Soderwall and Young ( ’41) illustrate the collapsed stage of recent corpora shown in figure 14, but do not comment upon the possible significance of this inner zone. It might be interpreted either as due to trauma incident to the redistribution of these cells upon the collapse of the follicular walls after ovulation, or more probably to a transient anoxia due to loss of liquor folliculi. Since blood vessels have not as yet grown into these corpora, it seems probable that after removal of the ovum the inner cells are without adequate metabolic contacts.

Long and Evans (’22) sacrificed thirty-seven rats in estrus as determined by recognition of the cornified cells in the vaginal smear, without finding any corpora in the collapsed stage. They comment (p. 30) “the closure of the pore of rupture must be very rapid since we have failed to detect it in this relatively large series of cases.” This merely emphasizes the advantage of timing ovulation from the onset of mating responses. The corpora lutea were completely redistended at the 24-hour stage, which means that the rupture point of ovulation had been closed 14 hours after ovulation. The fluid redistending the early corpora has been called the


tertiary liquor folliculi (Robinson, ’18). This contains estrogen and may contain some progesterone.

During the immediate preovulation enlargement of follicles at estrus, the emergence of follicles of the next ovulation from the medium sized group indicates that in the rat with a short 4-day cycle, some of the follicles of the next ovulation respond to the gonadotropic stimulus which results in the ovulation of their predecessors.

Studies of ovaries after hypophysectomy show that without pituitary gonadotropin the follicles cannot grow beyond medium size (Smith, ’30). These follicles (I,) are characterized by eight to ten layers of follicle cells and only the beginning of secretion of primary liquor folliculi. In such animals, and also in mice hypophysectomized at mid-pregnancy, mitotic figures are distributed evenly among follicle cells and theca. The number of such follicles in the ovaries of the present series averaged twenty-two per ovary, from which four to six per ovary might be selected for ovulation at the next estrous period. Could the distribution of mitoses in the follicles indicate at this time which are to achieve ovulation and the source of stimuli for cell division during the remaining four days of their development?

The idea that the ovum may supply the stimulus for proliferation of the follicular epithelium is not new. Loeb ( ’11) called attention to the probability and Walsh (’17) made detailed counts of mitoses in search of further evidence. In rapidly growing follicles beyond medium size he found a graduated distribution of mitotic figures from the ovum as the center. In larger follicles in ferret ovaries Robinson calls attention to the “rapidity of the multiplication of the nuclei of follicular epithelium” with a statement that this increases and is most rapid in the region of the cumulus as the follicular antrum develops. This work was done before the denionstra- tion that pituitary gonadotropin was necessary for late f ollicular development.

Use of the colchicine technique in a study of the ovary of the rat confirms these conclusions for some of the follicles,

320 EDQAR ALLEN A N D OTHERS

but shows in others that the distribution is greater at the periphery, or even in the theca, than in the cumulus. .This distribution is also found in some follicles where the ova may shorn7 signs of early atresia. We suggest that this preponder- ance of mitoses a t the periphery indicates adequate pituitary gonadotropic stimulation, combined with inadequate stimulus from the ovum.

The rapid rate of cell division in the theca of growing follicles is eniphasized by the colchicine technique. Mitoses are seldom found in the them of clearly atretic follicles. The high incidence of mitosis in the theca shows this tissue to be truly a rapidly growing outer zone of the follicle. In animals where the theca is usually not well developed, the high incidence of mitosis is frequently overlooked. It was not even mentioned by Walsh in his study of the guinea-pig. In the pocket gopher, however, where the theca develops to extreme proportions and assumes the appearance of a “thecal gland,” very extensive proliferation must occur (Mossman, ’37). Similar bat much less developed theca has been described also for the preovulation follicle in the cat (Dawson and Friedgood, ’40). I n the rat the thecal cells of rapidly growing follicles do not assume secretory characteristics. Instead they are flattened by intrafollicular pressure and in section appear elongated.

Attempted analysis of the pituitary stimulus upon the follicles during the 10 hours immediately preceding ovulation emphasizes the following point. Where multiple ovulation is the rule, all the eggs do not ovulate at the same time. Tlicre is an interval of 10 or 15 minutes between successive ovulations. This has been shown definitely in the rabbit both by observation (Walton and Hammond, ’28), by moving picture photography of ovulation (Hill, Allen and Kramer, ’35) aiid by timing ovulations by changes in the electric potential (Burr, Hill and Allen, ’ 3 5 ) . But the pituitary stimulus for ovulation reaches an effective level in the rabbit 1 hour after mating. Slight differences in development of follicles may determine the sequence in which they ovulate. Several follicles of a par-


ticular generation might possibly fail to ovulate, even at this late time, because of weakness in the ovum (failure of an internal stimulus). Once follicles have reached a preovulation stage it is.clear that they could not exist as such until the next ovulation 4 days hence. Loeb ('ll), in his study of guinea-pig ovaries, recognized that large follicles which fail to ovulate do not persist but undergo atresia in the next day or two. Similar conclusions have been reached in this study of the rat.

That the follicles secrete estrogenic hormone is clearly shown by its isolation from follicular contents. This hormone must reach the theca to be picked up by the blood vessels and carried as a hormone to the genital tract, because blood vessels do not penetrate the growing follicle. I ts action on other genital tissues induces mitotic division. Why should it not act locally in the same way on the theca? Such local action, similar to the action of an embryonic inductor, seems the most logical reason for the rapid proliferation of the theca cells. Then this same mitosis-stimulating substance, picked up by the blood stream, acts as a hormone to induce mitosis in more distant genital tissues. Is it possible that this ovarian hormone is responsible for the wave of mitosis in the anterior pituitary recently described by Hunt ( ' 42 ) ?

After the follicles grow to medium size autonomously, their further development is impossible without gonadotropic stimulation from the pituitary. There is much experimental evidence to show that there is a limited amount of this pituitary gonadotropin, for if the amount is increased, many more follicles than would otherwise ovulate, develop and achieve ovulation (Smith and Engle, '27; Engle, '29). This would mean that if there were enough pituitary gonadotropin available, all stage I follicles might be brought to ovulation. The counts of stage I follicles in the ovaries of this series after ovulation range from thirteen to forty-two, the average is twenty-two per ovary, or forty-four for the two ovaries of one animal. This approaches the number of follicles ovulated in experimental animals by increasing the amount of pituitary hormone.


The distribution of mitoses as studied by the colchicine technique should show which of these follicles are beginning to be eliminated. The data listed in table 2 show local weaknesses in some of the follicles of this group. Uneven distribution of mitoses may indicate failure of either the stimulus from the ovum or from the pituitary. Although no signs of atresia are yet present, this uneven growth probably presages elimination of these follicles.

With the colchicine technique we find in the rat that a considerable number of small follicles are undergoing rapid growth. For instance, a follicle with only two or three layers of follicle cells may have from three to eight mitoses in a single section, and several mitoses in the newly forming theca. This finding is important in balancing the evidence for the two concepts concerning life span of ova; either (1) relatively short life span, with either active growth or death, or (2) storage of many follicles for varying and indefinite periods of time. For instance, Robinson (’18) was unable to find mitotic division among the follicle cells of small follicles in the ferret ovaries. A quotation emphasizes this point - “It is difficult to say whether the increase of the number of follicle cells, in the early stages, is due to mitotic or to amitotic division, and, as a matter of fact, although I have examined many thousands of primitive follicles, I have never seen any clear indications of mitotic division of the young follicle cells, although mitotic divisions were numerous in larger follicles of the same ovary.” Lane and Davis (’39) as their third point under summary and conclusions, report “Follicles less than 200 p in diameter are inactive mitotically and are thought to be physiologically quiescent. ”4 Use of the colchicine technique would have clarified this point.

In studies of ovogenesis in the rat, Evans and Swezy ( ’31) and Swezy (’33), and in studies of the ovary of the bat, Guthrie and Jeffers (’38) clearly recognized mitotic division of the cells in small follicles.

‘This conclusion seems questionable on the basis of the data given in their tables 2 and 3 fo r follicles of this size.

GHOTVTH I N OVARIES AND GEXITAL TRACT 323

Recent studies of ra t ovaries timed in relation to mating responses hare been contributed by Boling, Blandau, Soder- wall and Young ('41) and by Boling ('42). Attention is centered chiefly upon the size changes in the follicles and corpora lutea and accurate growth curves are plotted. Con- cerning mitotic division in the follicles, brief statements are made under each stage to the effect that mitoses were either numerous, frequent or less numerous in the granulosa. No mention is made of mitoses in the theca. Myers, Young and Dempsey ( '36) had previously contributed a similar study of the ovaries of the guinea-pig. Their attention also was focused particularly on the changes in size of the follicles. They clearly recognize elimination of follicles from the inedium-sized groups, from which the few follicles of ovulation are chosen.

Pincus ( '36) and Dawson and Friedgood ( '42) have studied closely spaced stages near the time of ovulation in the rabbit and cat. Their observations stress particularly the secretory activity and maturation changes of the preovulation follicles. Their studies do not include analyses of the part played by mitotic activitj- a t this time.

Schmidt ( '42)' studying mitotic proliferation of the guinea- pig ovary with the colchicine technique, coiicurs with Loeb and TValsli that mitosis in the granulosa declines rapidly and ceases in mature follicles. She also records a decrease in mitosis in the theca in progressively larger follicles. To the contrary we find in the rat that although the rate may decline, many mitotic figures are still present in the follicles of ovulation, and also in the former theca during the early transformation of ruptured follicles into corpora lutea.

The interstitial cells of the ovary respond to certain frac- tions of gonadotropic extracts. Evans and his co-workers have called this f raction interstitial-cell stimulating hormone (ICSH). A siniilar fraction isolated b\- Greep, van Dyke and Chow ('42) has been called metakentrin. Similar effects are produced by low concentrations of the luteinizing fraction of pituitary extracts. The action on the interstitial cells is one

324 EDGAIt ALLEN AND OTHERS

of maintaining them in functional condition or inducing hypertrophy after hypophysectomy. We have searched for evidence that such a fraction might induce mitotic division in interstitial cells. Careful study of this question, using the colchicine technique, has failed to disclose mitoses in ovarian interstitial tissue. I n contrast to the mitosis-stimulating effect on the follicles, including the theca, we find no evidence for mitosis in interstitial tissues in our series of ra ts near the time of estrus.

Studies of estrogenic hormones have shown that their major effect is to induce cell division of the tissues of the genital tract. By using the colchicine technique it has been shown that in ovariectomizeci animals within 8 hours after a single injection of hormone, the epithelial tissue of the vagina begins to undergo mitotic division. This growth wave reaches its peak a t about 36 hours (Allen, Smith and Gardner, ' 37 ) . It lias also been shown that after removal of the ovaries the tips of the uterine glands are the first tissues from the uterus to undergo mitotic division under the influence of injected estrogen. Therefore the findings in the 24- and 32- hour animals that (1) the tips of the uterine glands are growing rapidly and (2) there is an increase in mitotic division in the vagina, a re probably the first indications of growth for the next estrus resulting from autogenous estrogenic stimulation. From a study of table 1, however, it will be noted that coincident with this rapid growth of the uterine glands comes a redistention of the early corpora lutea with tertiary liquor folliculi. This might be a supplementary source of mitosis-inducing stimulus for the uterine glands. But the fact that growth occurs in the uterine glands as the immature animals come into the first estrous period, a t which time there are no corpora present, would indicate that the secretion from the corpora lutea is not nece s sa r y .

SUMMARY

1. Studies were made of differential growth (cell division) in the genital organs of rats timed from the first appearance


of mating responses. Evidence of cell division was accentuated by the use of the colchicine technique. The incidence of mitosis described represents dividing cells accumulated over a period of 8 honm before the animals were sacrificed.

2. The large follicles just before ovulation continued to be suliject to moderate mitotic division in cumulus, granulosa and theca. Soriie of them showed higher incidence of mitosis near tlie ovum, in the cumulus and adjacent follicular wall. Mitoses were especially numerous and evenly distributed in the theca.

3. Division continued in the cells of the former cumuli, extruded with the ova at ovulation, as they were passing through the proximal part of the uterine tubes. The rate of cell division at the 16-hour stage after the onset of mating responses was as rapid as 8 hours earlier in the unruptured follicles. These ova had not been fertilized. Some mitoses were found at this time in the ciliated tuba1 epithelium.

4. After ovulation (16-hour stage) the walls of the corpora were collapsed and cells of the inner layers showed signs of transient abnormality and a lowered rate of mitosis. I n contrast, cell division continued rapidly in the outer luteal wall and in the surrounding connective tissue (former theca).

5. After ovulation the follicles which would have provided the four or five ova per ovary to be ovulated at the next estrous period are 250 to 500 microns in diameter. There was an average of twenty-two such follicles per ovary from which four or five might be selected for the next ovulation. A t this time they contained very little liquor folliculi. The majority of these follicles was growing rapidly. Use of the colchicine technique showed that other follicles of this stage were subject to uneven rates of growth or that the growth had stopped. This may be the first indication of imminent atresia.

6. At the 24- and 32- hour stares some larger follicles for the next ovulation were emerging from the medium-sized group. At the same time the ruptured follicles (early corpora lutea) had become redistended with tertiary liquor folliculi. Distribution of colchicine mitoses in recent corpora showed moderate cell division of inner luteal cells, rapid cell division


in endothelial cells of ingrowing vascular sprouts, and a continued high rate in the outer zone of the wall and surrounding connective tissue.

7. Coincident with these events, a wave of mitosis appeared in the uterine glands. Previously they had been secreting, and mitotic figures had been absent. This cell division a t 14 hours after ovulation is the first sign of growth in the uterus in preparation for the next estrous period.

8. The incidence of mitosis in the basal layers of the vagina, a t low ebb near the time of ovulation, is doubled a t the 24- and 32-hour stages, 14 and 22 hours after ovulation. However, the most rapid phase of vaginal proliferation comes later.

9. These differential rates of growth, revealed by the distribution of colchicine mitoses, have been correlated in the discussion with hormonal stimuli from the ovaries and anterior pituitary gland known to he necessary and operating a t these times. Uneven growth in follicles of the next ovulation group, clearly revealed by the colchicine technique, is considered the first sign of decline leading to atresia and eventual elimination of a considerable number of this group.

LITERATURE CITED

ALLEN, E., G . M. SMITH AND W. U. GARDNER 1937 Accentuation of the growth effect of thcelin on genital tissues of the ovariectomized mouse by arrest of mitosis with colchicine. Am. J. Anat., vol. 61, pp. 321-341.

Growth and regression of corpora lutea during the normal estrous cycle of the rat. Anat. Rec., vol. 82, pp. 131-145.

Growth of the Graafian follicle and the time of ovulatioii in the albino ra t . Anat. Rec., vol. 79, pp. 313-331.

rabbit. Proe. Soe. Exp. B i d . and Med., vol. 33, pp. 109-111.

BOLING, .J. L. 1942

BOLING, J. L., R. J. BLANDAU, A. L. SODERTVALL AND W. C . YOUNG 1941

BURR, H. S., R. T. HILL AND E. ALLEN

DAWSON, A. B., AND H. B. FRIEDGOOD

1935 Detcctioii of ovulatioii in the intact

The time and sequence of pre- ovulatory changes in the cat ovary after mating or mechanical stimulation of the cervix uteri. Anat. Ree., rol . 76, pp. 4 1 1 4 2 9 .

The effect of daily transplants of the anterior lobe from gonadectomized rats on immature test animala. Am. J. Physiol., vol. 88, pp. 101-106.

Ovogenesis and the norinal follicular cycle in adult mammalia. Mem. Univ. Calif., rol. 9.

1940

ENGLE, E. T. 1929

EVANS, H. M., AND 0. SWEZY 1931

G R O W T H I N OVARIES AND GEKITAL TRACT 32i

FEE, A. R., AND A. S. PARKES 1929 Studies on ovulation. 1. The relation of the anterior pituitary body to ovulation in the rabbit. J. Physiol., vol. 67, pp. 383-389.

1935 Experimental ovulation and the resulting pseudopregnancy in anoestrous cats. Anat. Rec., vol. 62, pp. 75-93.

1942 Gonadotropins of the swine pituitary. 1. Various biological effects of purified thylakentrin (FSH) and pure mctakeiitrin (ICSH) . Endocrinology, vol. 30, pp. 635-649.

GUTHRIE, M. J., AND I(. R. JEFFERS Growth of follicles i n the ovaries of the bat, Myotis lucifugus lucifugus. Anat. Rec., vol. 71, pp. 477-496.

HILL, R. T., E. ALLEN AND T. C. KRAMER 1935 Cinemicrographic studies of rabbit ovulation. Anat. Rcc., vol. 63, pp. 239-245.

HUNT, T. E. Mitotic activity in the anterior hypophysis of female rats. Anat. Rec., vol. 82, pp. 263-276.

LANE, C. E., AND F. R. DAVIS 1939 The ovary of the adult rat. 1. Changes in growth of the follicle and in volume and mitotic activity of the granulosa and theca during the estrous cycle. Anat. Rec., vol. 73, pp. 429-442.

The follicular apparatus of the ovary of tlic hypophysectomized immature rat and the effects of hypophyseal gonadotropic hormones on it. Anat. Rec., vol. 63, pp. 139-146.

LITS, F. 1934 Contribution b 1 'Btude des reactions cellulaires provoquees par la colchicine. Compt. rend. SOC. de biol., rol . 115, pp. 1421-1423.

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FOSTER, M. A., A N D F. L. HISAW

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1938

1942

LANE, C. E., AND R. 0. GREEP 1935

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1942

1927


SWEZY, 0. 1933 Ovogeiiesis and its relation t o the hypophysis: (the effects of pregnancy, hypophysectomy, tliyroidectorny and hormone adininistra- tion on the ovary of the ra t ) . The Science Press, Lancaster.

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WALSH, L. S. N. 1917

~ V \ I , T O S , A., AND J. HAXMOND

YOTNO, W. C., E. W. DEMPSEY, C . W. HAGQUIST AND J. L. BOLING

1928

pp. 300-303.

PLATE 1

EXPLANATION OF FIGURES

6 Section of vaginal wall of rat no. 5, 24 hours after the onset of mating reactions. The numher of colcliicine mitoses in the epithelium was rated as + 4-, an approximate doubling of cell division when compared to the 8- and lG-hour animals. At this stage (MI), the cornified layer had been sloughed into the lumen, lint there was no leukocytic infiltration of the epithelium. X 225.

Section of the wall of the uterus from ra t no. 7, 32 hours after the onset of mating responses. The high incideiicr of mitosis was restricted to the tips of the uterine glands. This is typical of the 24- and 32-hour animals, and is in marked contrast to the complete absence of mitoses i n the 8- and If-hour animals (table 1, columns 5 and 6) . At the later stages the nterus was small and collapsed and there mas very little evidence of secretion. This is the first sign of increase in estrogenic stimulation leading to the estrous period 3 t o 4 days later. Notr lieav:~ leukocytic infiltration of the stroma. X 225.

An ovum found in the ovarian capsule ndjnpent t o tlie ostium of tlir tnhc in ra t no. 15, 8 hours a f te r the onset of mating reactions. This was the only egg ovulated frnrr, this ovar j . (Therr were six laige follicles on the verge of ovula tion.) X 225.

A section through two tuba1 orit from rat no. 13, 16 hours after the onset of mating reactions. There were 600 niitoscs arouild four ova in this tulw, an average of 150 mitoses aniong the follicle cells around each ovum. These eggs nere probably ovulated about 6 hours previous to death and a considerable numher of these cell divisious must have occurred as the eggs were passing through the fimbriated end of the tube. The cells of the former cumulus surrounding the orum were less dispersed in the 16-hour animal than those in the 8-hour animal. (Corn- paro ivith figure 8.) The chromatin of tlie ova is in maturation divisinii (at 0 o’clock in tlic npprr orum). X 225.

7

8

9

GROWTH I N OVBRIES AND GENITAL TRACT EDGAW ALLEN AND OTHERS

PLATE 1

PLATE 2

ESPLANATIOPT OF FIGURES

10 Section tlilough the ovuni and base of the cumulus of a preovulation follicle from aninial no. 1:. An average of sixteen mitoses per section were counted amoug the cells immediately surrounding the ovum. Granulosa cells a t the base of the cumulus continued to shorn a high rate of cell division, as did also tlic' thecs. maturation spindle can be seen a t 9 o 'clock. Note the corona phe~~nnienou. I n this particular field therc ~verc twenty mitoses immediately arouiid the ovuni, thirty-four in the section of granulosa shown, and four in the tlieca. X 2 2 5 .

Section throughout the wall of the same follicle opposite the cunlulus. Note the high incidence of mitosis in the thcca but almost complete cessation of cell division in the granulosa distal from the ovum. X 225.

A collapsed corpus lnteuni from animal no. 15, corresponding to t.he single egg (shown in fig. 3 ) ovulated 8 hours after onset of mating responses. X 110.

A section of the wall of the same corpus shows the high incidence of mitosis among cells of the former theca ( f + + ) and some mitoses in the former granulosa (+ +). This is tlir earliest corpus luteum of the series, proh:ihly repre- scwti i ig :I w r y recent ovulation.

11

12

13

x 225.

330

PLATE 2

331

PLATE 3

EXPLANATION OF F I G U R E S

14 A sect,ioii of the wall of a corpus luteum from animal 110. 14, 1 6 liours after the onset of mating reactions and about 6 hours after ovulation. Therc were still many mitoses in the former theca, a few in the outer layers of tlic ~r-all of the corpus, but practically none in the inner layers of the wall. (Compare the 16- and 8-hour animals in table 1, coluuins 9 to 13.) The dark staining, tiglit,ly packed cells a t the right of the photograph show the inner zone of the ivall typical of all tlic corpora of the l6-liour stage. X 225.

A section of the wall of a redistcnded corpus lnteum of rat no. 5, 24 hours after onset of mating reactions. There was still a fairly high incidence of mitosis, especially in the outer part of the wall, hut many of tliese divisions were jn a d o - thelial tissuo of vascular sprouts which were groiriiig into the wall a t this stagc. Occasional mitoses were found among the former granulosa cells which were lieginning to take on luteal characteristics. Occasionally small masses were found on the inner wall composed of cells more vascular than luteal in type.

16 Section of wall of corpus from animal no. 8, 32 hours after onset of mating rcactions. The early coqxis had hren redistended by accumualted tertiary liquor folliculi. There secmed to be rapid recorcry of the cells of the inner zone shown in figure 9. X 110.

1.7

X 225.

332

GROWTH I N OVARIES AND GENITAL TRACT EDGAB ALLEN A N D OTHERS

PLATE 3

333

PLATE 4

EXPLAKATION O F FIGURES

1 7

18

Section of a follicle of stage I, from rat no. 15 showing the high incidence of mitosis and even distribution in both follicle cells and theca. X 225.

A stage I, follicle from rat no. 11 a t 16 hours showing the high incidence of mitosis in the theca, only occasioiial cell divisions in the outer follicle cells, but conipleto cessation of mitosis among the follicle cells around the ovum. This un equal growth is interpreted as failure of stimulus for mitosis from the ovum, but continued adequate pituitary-gonadotropic stimulus for mitosis. X 225.

Follicle in stage I, from ra t no. 15 a t 8 hours with moderate numhcr of mitoses, evenly distributed in both follicular epithelium and theca. x 225.

A similar follicle from the same ovary in which mitotic activity in thr follicular epithelium had practically ceased, while the theca was still subject t o rapid cell division. There was no indication of atresia other than the more tightly paekrd appearance of the follicle cells. X 225.

19

20

334

GROWTH IN OVARIES AND GENITAL TRACT EDGAR ALLEN AND OTHERS

PLATE 4

335

PLATE 5

EXPLANATION OF FIGURES

2 1 Follicle of stage I, from rat 110. 11 a t 1 6 hours showing a moderate number of mitoses evenly distributed among follicle eeih and tliecn.

22 Follicle of stage I, from ra t no. 8 a t 32 hours showing unevcii distributioii of mitoses, a greater number in the theca and very few near the ovum. X 225.

23 A section through the cumulus of a stage I1 follicle from rat no. 5 : i t

01 hours. A few follicles cf this stage mere present in the 24 and 32-hour animals. They usually had a few initoses in the granulosa and many in the theca. The number of mitoses in the cumulus varied from i- to + +. The uneven distribution of mitoses might indicate eliiiiinatioii of these follicles before the nest estrus. x 22.5.

X 225.

336

GROWTH I N OVARIES *4ND G E N I T S L TRACT EDGAB ALLEN AND OT'HERS

PLATE 5

337

Documents

Differential growth in the ovaries and genital tract near the time of ovulation in rats treated with colchicine