Paper - On the origin of the corpus luteum of the sow from both granulosa and theca interna (1919)
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|pig corpus luteum from both granulosa and theca interna.
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On the Origin of the Corpus Luteum of the Sow from both Granulosa and Theca Interna
From the Anatomical Laboratory, University of California
The history of the discussion, now of more than seventy years' standing, as to the origin of the corpus luteum, has been repeated so many times that it has become traditional, and the names of von Baer and Bischoff have been passed down to us as the original proponents of the two chief doctrines in question. It is said that the former, in his monograph "De ovi mammalium genesi" ('27) first stated that the corpus luteum is derived from the theca interna of the Graafian follicle, and that Bischoff first discarded this view in favor of the membrana granulosa as the site of origin. I have not been able to see von Baer's work, but judging at least from Bischoff's account of the early embryology of the rabbit ('42), there was no such clear-cut opposition of view as tradition declares, for Bischoff considered himself, rather, as an upholder of von Baer (and was so quoted by contemporary investigators).
It must be remembered that the first of these monographs appeared a decade before Schleiden and Schwann's enunciation of the cell theory, and the other not five years after; histology was studied with pincettes and the needle rather than by sections, and the first nuclear stain was not discovered. The layers of the follicle were as yet imperfectly differentiated, and the early descriptions are so vague that it is difficult to interpret them in present-day terms. New steps toward the solution of this problem have always followed fast upon the development of histological technique, and thus it is in the writings of Wilhelm His ('65) and Waldeyer (70) that we first find opinions and descriptions approaching those of recent years.
The studies of His led to the complete formulation of the view that the corpus luteum is derived from the theca interna of the Graafian follicle, which in the next two decades was supported by a number of investigators and still holds a place in the field against strong opposition. The chief arguments in favor of this view are that, first, the membrana granulosa of large follicles is often degenerated, and is believed to be cast off at the time of rupture; second, as the Graafian follicle ripens, the cells of the theca interna show marked changes — they swell in volume, become rounded, in some species they acquire granules of a yellowish pigment, and in short come to present a striking resemblance to the large cells of the corpus luteum; third, this resemblance is enhanced by the fact that not only are the large cells of the theca interna folliculi and the corpus luteum similar, but the presence of many blood-capillaries and connective-tissue cells causes a resemblance as well in the general structure of the two tissues; and, fourth, such follicles as do not rupture lose their granulosa by degeneration, become obliterated by proliferation of the theca interna, and in this process of atresia attain also a resemblance to the corpus luteum. .
None of the contributions disagreeing with this view in favor of the granulosa origin of the lutein cells were at all convincing, until the appearance in 1895 and 1896 of Sobotta's first researches, which mark the beginning of modern work upon the question. Here again the chief contribution was one of method. Sobotta pointed out that the arguments quoted above are based merely upon analogies between the layers of the follicle and the fully formed corpus luteum, and that from the writings of his predecessors it is apparent that few had actually seen corpora lutea in process of formation. Even when descriptions are given of mature follicles or supposed early corpora lutea, there is usually no proof that the structures in question actually represent the results of normal follicular development or recent ovulation. The problem should be worked out from a series of specimens gathered at known periods after rupture of the follicle; and in order to avoid confusion with atresia or other irrelevant processes, each follicle or corpus luteum studied should be certified as to its normal condition and stage of development by comparison with the fertilized ovum or embryos proceeding therefrom. To fulfill these high requirements calls for long and tedious labors — the investigator must spend hours and days in observation of his animals ; the reproductive cycle of the species used must be known well enough to acquaint him with the time of ovulation, the animals must be killed at definite times thereafter, and the ova must then be sought in the ovary, the oviducts, or the uterus. Sobotta himself chose the mouse, in which he had found that an ovulation takes place about twenty-one days after the birth of a litter, and in which the small size of the animal permits serial sectioning of the entire ovaries and Fallopian tubes. It must be admitted that his own postulates could not be followed to the full; the individual corpus luteum corresponding to a given ovum cannot be identified, because many ova are extruded at one ovulation in this species; the exact time of ovulation may vary by hours, and again there is so much variation of the interval between ovulation and the entrance of the spermatozoon into the egg that the condition of the ovum cannot be used as an exact measure of the age of the corpus luteum. Study of the ova merely provides assurance that the corpora lutea are normal and gives a rough means of determining their ages. The judgment of the investigator must finally be used to rank the corpora lutea in an orderly series. It is beyond denial, however, that Sobotta possessed such a series, collected from about 200 mice and based upon the study of nearly 1500 ova during the stages of maturation, fertilization, and the segmentation of the blastomeres. He found no degeneration of the membrana granulosa; instead the cells of this layer remain and undergo hypertrophy (without division), finally becoming the characteristic large cells of the corpus luteum. Meanwhile the cells of the theca interna undergo mitotic division, are converted into spindle-cells and invade the granulosa to form the connective- tissue reticulum of the corpus. In this process all the theca interna cells are used up, and the layer therefore disappears. Capillary blood-vessels grow in from the vessels of the theca interna, ultimately providing the corpus luteum with a rich circulation.
There will be no need to enter here upon a detailed account of the debate which began immediately upon the publication of these epoch-making studies. A full analysis of the literature upon the origin of the corpus luteum up to 1901 will be found in the papers just quoted and in the two reviews of Sobotta in the Ergebnisse der Anatomie ('99a, '02). In 1897 Sobotta himself studied corpus luteum formation in the rabbit, and found the process in all important points exactly as in the mouse. A year later, Stratz ('98) printed a research upon which he had been engaged before the publication of Sobotta's work, in which he had followed the early stages of the corpus luteum on ovaries of an insectivore, Tupaia javanica, the lemuroid ape Tarsius spectrum (the specimens being those of Hubrecht's well-known Javanese collections,) and of the 'Spitzmaus' or shrew, Sorex vulgaris. Although he did not have large numbers of cases, all were checked by the study of the ova or embryos. As far as the persistence of the granulosa cells and their direct conversion into the lutein cells was concerned, Stratz agreed fully with Sobotta, but with regard to the fate of the theca interna there is a minor difference. If I understand Stratz correctly, he considers the theca interna of the mature follicle merely as a zone of blood-vessels, in which all the cells are either constituents of the vascular wall or of the adventitia. Considered in this light, it is easy to see how this angioma-like thecal tissue would enter into the growing corpus luteum to form its blood-vessels and its connective tissue without the necessity of transformation or regression of the specialized theca cells into fibroblasts, as described by Sobotta. So far as is known to me, no subsequent investigator has confirmed the view of Stratz, all others being agreed that the theca interna consists of a distinct layer of highly specialized cells derived from the mesenchymatous elements of the ovarian stroma, and containing a network of blood-vessels, supported by cells and fibrils of connective tissue.
A third theory as to the fate of the theca interna is proposed in the important papers of 0. Van der Stricht, of which the first appeared in 1901. The study was carried out upon the ovaries of large numbers of European bats, chiefly Vesperugo noctula. As with the two previously cited investigations, the animals were collected primarily for the study of the ova and the early embryos of the species used, and the series is therefore accurately controlled by the condition of the ova. Van der Stricht demonstrates beyond doubt that in these species the granulosa layer persists in situ after rupture of the follicle, and that its cells enlarge, acquire granules of lipoids staining black with osmium tetroxide, and finally become the typical lutein cells. Contrary to Sobotta, he thinks that mitotic division may occasionally occur in these cells, so that the filling of the follicular cavity is brought about by a slight increase in their number as well as by the vast increase in their individual bulk. The connective tissue of the corpus luteum arises chiefly as Sobotta described it in the mouse. After rupture of the follicle the membrana propria disappears, and fibroblasts invade the metamorphosing granulosa layer. Van der Stricht thinks that these are the spindle-cells of the theca interna or their descendants. Of the distinctive cells of the theca, some seem to disappear, but others remain, chiefly about the periphery of the new corpus luteum, or enter a short distance into the granulosa, and here they remain almost in their original condition. After a few days, however, when the deposition of fatty droplets in the granulosa cells has progressed, the two types of cells so closely resemble each other that Van der Stricht could no longer distinguish them in his Flemming-fixed tissue. He believes, in fact, that they have become identical, and therefore that in the fully formed corpus luteum most of the lutein cells are of granulosa origin; a few of them, however, are from the theca interna.
A nearly identical theory is that proposed by Rabl ('98), who studied human corpora lutea (the youngest estimated at ten days), and found in them about the periphery a layer of cells differing from the rest of the lutein tissue; this, he suggested, might be the theca interna, which he supposed to persist in its original position until its cells were lost to view, some by becoming converted into lutein cells, others by degenerating.
During these five years from 1896 to 1901 there was no lack of publications restating the total loss of the granulosa before rupture, in opposition to the descriptions of Stratz, Sobotta, and Van der Stricht. A few of these investigations were carried out upon the ovaries of swine, and will therefore be discussed more fully later in this paper. As Sobotta pointed out in his resume of 1902, not one writer among those who taught the non-participation of the granulosa in corpus luteum formation had been able to prove that his specimens were normal mature follicles and corpora lutea by presenting the ova which had proceeded therefrom.
From 1901 to 1917 there have been about thirty-five more publications upon the question, of which some twenty-five represent actual original investigations. As the subject has not been brought up to date in any publication in English, it may be as well to take up in some detail the work of the past sixteen years, especially as the old differences of opinion still persist.
Jankowski ('04) reports studies upon a series of ovaries of sows and guinea-pigs, collected without an attempt to learn the reproductive cycle of the animals or to test the normal conditions of the specimens according to the postulates of Sobotta (which he says he had found impracticable to apply and whose value he questions). He believes the granulosa to be intact until the rupture of the follicle and even afterward, but to degenerate before the ingrowth of the theca interna, to which he ascribes the origin of the lutein tissue. The value of his results with the pig will be discussed below; Sobotta has presented directly opposite evidence and a vigorous criticism with regard to his work on the guinea-pig ('07).
The contributions of Pottet ('10) on the human corpus luteum and Delestre ('10) on that of the cow are based on evidence which can hardly be considered conclusive. Delestre had no bovine corpora lutea of pregnancy at an earlier stage than two and a half months. He had twelve corpora lutea from non-pregnant animals, four of which he thought to be in the first stages of formation, but there was no effort, by observing the animals alive or by searching for the ova, to determine that ovulation had actually been recent. Pottet studied twenty-two human corpora lutea of pregnancy, the youngest already six weeks old. Both of these authors speak for the degeneration of the granulosa before rupture.
We cannot judge the work upon the human ovary very critically until the relation of ovulation to menstruation is better known or some other method of estimating the age of young human corpora lutea and of obtaining really young specimens is at hand. Buhler ('00) collected ovaries of rabbits according to Sobotta's methods, but found the distinction between theca and granulosa so difficult that he turned to the human corpus luteum. The only specimen of importance described by him is one from an operative case, without menstrual history or other means of estimating its age, except that it showed a point of rupture in process of healing (see below, p. 179, as to the possibility of error on this point) . In this supposedly early corpus luteum the granulosa is degenerating and a 'typical lutein tissue' appearing in the place of the theca interna. Cristalli ('03), a pupil of Paladino, whose peculiar views will be quoted below, believes also in the total degeneration of the granulosa layer before rupture, but gives no data as to his specimens. Teacher, in discussing the TeacherBryce-Kerr case of early ovarian pregnancy ('08), states that he had been studying corpus luteum formation in the human, and interprets his preparations to indicate quite clearly that "whatever the source of the cells (of the corpus luteum) may be in the lower animals, they do not in man arise from the membrana granulosa," which latter membrane he thinks is probably shed with the ovum. Hegar, in 1910, reported an examination of six human ovaries removed four and two days before the onset of menstruation, all containing corpora lutea which he supposes to be fairly young. While admitting the epithelial origin of the structure in mammals lower than man, he is inclined to view his preparations as indicating a thecal origin of the lutein cells in the human species. J. Whitridge Williams, whose text-book of obstetrics ('03-' 17) is based to so great an extent upon original study that it is regularly quoted in scientific literature as authoritative, retains in his last edition the views of the preceding writers, of whose correctness he feels convinced by the study of several hundred corpora lutea.
This completes the list of recent authors who see in the corpus luteum a structure of connective-tissue origin alone. All other investigators of the past sixteen years uphold in general the epithelial origin of the lutein cells, but among themselves they vary according to their views as to the fate of the theca interna. The rabbit has been studied in 1897 by Sobotta, who found the process exactly as in the mouse, but Honore, three years later, in the same animal, found that not all the theca interna cells are converted into fibroblasts, but that some of them linger about the periphery of even the fully formed corpus luteum. Cohn, in 1903, repeated the work, apparently without study of the ova, but dating his specimens from an observed copulation, (in the rabbit ovulation occurs only after coitus), and confirmed the results of Sobotta. Marshall, in the next year, described the corpus luteum of the sheep, dating his specimens from observed copulation. He did not seek the ova, but as in this species ovulation and coitus can occur at no time except during a short oestral period, so that coitus dates the time of ovulation within a very few hours, the presumption is great that Marshall possessed normal corpora lutea of ages accurately known. He found the granulosa to persist and to be vascularized by sprouts from the theca interna, all the cells of which were finally used up, having been converted into spindle-cells of connective tissue. A part of the connective tissue of the corpus is contributed also by the theca externa, which is drawn inward in places by the folding of the follicular walls. O'Donoghue ('12, '14, '16) has given a confirmation of Sobotta's views for the marsupials (which I believe were first studied by Sandes ('03), whose paper was not accessible to me). Strakosch ('15) is the last to repeat the Sobotta theory in its original purity, basing his statements upon the human ovaries which were used by Robert Schroeder in his study of the time relation between ovulation and menstruation ('14).
Van der Stricht's belief that the theca interna cells are not converted into fibroblasts, but remain in the corpus luteum, no longer distinguishable from other lutein cells, found support in the study of L. Loeb ('06) upon the guinea-pig. The specimens were collected in a series dated from copulation without study of the ova. The theca interna cells, after a few hours, could no longer be distinguished from the granulosa lutein cells. This work is open to the criticism that haematoxylin and eosin (the only staining combination used) do not accentuate differences between cells of the types met with in this problem. In 1908 Van der Stricht himself repeated his ideas as the result of researches upon the ovaries of dogs, carefully checked up by examination of the ova, and in 1912 he repeated his findings in the bat.
A somewhat different view has found exposition in the very careful work of Volker ('05) upon Spermophilus citellus, a European marmot allied to the gophers of the western United States. The ova and embryos were recovered and examined in all cases, and there appears to have been a sufficient number of stages, though the author does not state the number of specimens studied. The theca interna cells were found to persist unchanged between the granulosa and the theca externa, even until the end of pregnancy. The few spindle-cells found in the fully formed corpus luteum are said to proceed from the theca externa. Practically the same view is presented in the thesis of Niskoubina ('09), who worked on the rabbit, but does not give an account of the methods used. Cohn ('09) studied the human ovary, but appears to have seen no really young stages. His descriptions agree with those of Rabl, and he thinks the layer of theca cells is not destined to persist, but is a 'matrix' or source of origin for the newly forming connective tissue of the corpus luteum.
With this group should be placed one of the most ambitious of the recent attempts to work out the origin of the human corpus luteum, that of R. Meyer ('11 a). The paper describes five corpora lutea in process of formation, of which one is claimed by the author to be the youngest ever obtained in the human. The appearance of the structures and the menstrual histories were the only guides to their age. The specimens show first a proliferative stage, during which the granulosa cells swell and acquire granules of a fatty substance, and, second, a stage of 'glandular metamorphosis' through vascularization of the granulosa layer. The first spindle-cells seen in the lutein layer arise from the bloodvessels, which are sprouting inward. The wall is thrown into folds, in which the larger fat-infiltrated theca interna cells are crowded. Here they remain until the pressure of the swelling lutein tissue crushes them out of existence, an event which may be early or late according to the internal conditions of pressure. Groups of them, serving as sources of nutrition for the growing organ, may be seen about the periphery of the corpus luteum and in the folds of its wall, until fairly late in the life of the corpus luteum. The name theca-lutein cells has been given them.
As an example of the difficulty of proving anything about the origin of the corpus luteum by specimens 'whose age can only be guessed, it may be mentioned that the genuineness of Meyer's first and supposedly youngest corpus luteum has been sharply attacked. Ricker and Dahlmann ('12) have hinted that it is not even a naturally ruptured follicle, and J. W. Miller ('11) believed it was an atretic follicle, because Meyer had stated the granulosa cells to contain 'Fett,' while, according to Miller, neutral fat is never found in the normal fresh corpus luteum. It must be admitted that this criticism was rescinded when Meyer ('lib) stated that 'Fett' meant merely lipoids in general, and that Miller is himself no opponent of Meyer's views. But after all we shall never be certain of the early human corpus luteum until skill and good fortune enable someone to obtain the tubal ovum with the ovary.
Four recent writers upon the human ovary have repeated the same views as Meyer with but slight modifications. Elizabeth Wolz ('12), from the study of a few specimens, believes that none of the theca interna cells suffer change into connective tissue. Some degenerate by atrophy, others remain in situ a long time. Timofeiev ('13) l and Wallart ('14) appear to have given as accurate and modern a description as is possible in the face of the particular difficulties of the human material. Careful menstrual histories are given, and both used varied and interesting histological methods. According to both, the theca interna cells remain in groups about the periphery, of the corpus luteum for a long time, as described by Meyer, and they slowly atrophy. None of them are converted into spindle-shaped connective-tissue cells. Timofeiev describes also the deposition of lipoid bodies in the granulosa lutein cells during the first days of the new corpus luteum. Lastly, Novak ('16) reports five early corpora similar to those of Meyer, whose conclusions he follows.
Previous Work on the Corpus Luteum of the Sow
It is said that von Baer's celebrated monograph ('27) announcing the discovery of the mammalian ovum, contains a description of the early corpus luteum of the sow. The first account of the histological development in swine which has come into my hands, however, is that of Zwicky ('44), entitled "De corporum luteorum origine." Zwicky was a medical student who was set to work by the distinguished Henle to study the formation of connective tissue in fibrin clots, Henle being under the mistaken impression that the corpus luteum represented the conversion of the clotted follicular haemorrhage into scar tissue. The student was acute enough to correct his master's error, even though he found haemorrhage into the follicle in two-thirds of the early corpora lutea of swine. As a result of his studies, he announced himself as on the side of von Baer and Bischoff in favor of the granulosa origin of the corpus luteum, but his description shows that he Mas probably including the theca interna as part of the granulosa. The little dissertation must remain as a not uninteresting example of the effect produced upon the work of an active student by the recently announced cellular theories, rather than as an important contribution to its subject.
- 1 This Russian dissertation seems to me the best contribution to the origin of the human corpus luteum yet presented. As it was abstracted for me by a Russian-speaking scientific colleague, not especially acquainted with histological methods, I quote it with some slight hesitation, but I believe our interpretation is correct.
The next to use the sow for study was Paladino (79, '80, '81, '87), who collected about 500 corpora lutea of 100 sows. His extensive papers propose a peculiar theory of his own, namely, that the entire granulosa is lost before rupture of the follicle, and that the theca externa, carrying blood-vessels, proliferates inward, to form the corpus luteum tissue, pushing the theca interna before it to form a central connective-tissue core. In 1900 and 1905 he repeated his views of twenty years before in criticism of Sobotta, who pointed out in return that Paladino 's writings contain no evidence at all, in text or plates, that he had ever seen developing stages of the corpus luteum; a just criticism, as study of the originals has convinced me.
Far different is the work of Benckiser ('84), who has given a very careful description of a small series of stages. He had, without doubt, normal and mature Graafian follicles just prior to rupture. These contained the membrana granulosa intact. In his recently collapsed follicles, containing large clots, the granulosa had been torn off in places; when there was no hemorrhage, this layer remained very largely in situ. His next stage is much further developed, its wall showing only one homogeneous layer, and the gap was bridged by the assumption that the granulosa had degenerated during the interval. The account is clearly written, its author was describing what we can now state to be normal specimens, and it was only lack of sufficient intermediate stages that led him into an error of interpretation.
One of the most frequently quoted works is that of Clark ('98), whose material consists of ovaries collected at random in the slaughterhouse. It is said that among the sows used by the butchers there were many undergoing oestrus, but it is not stated that any of those ovaries used in the research were known to be from the animals in heat. Clark gives a good description of the immature follicle at growing stages. For study of the mature follicle he selected large follicles, without discovering whether or not they contained normal maturing ova. From these large follicles all the granulosa cells had disappeared. The author was willing to consider the possibility that they might be atretic, but inclined to rate them as normal because they seemed logical predecessors of his next stage. Much is made of the fact that the theca interna cells of ripening follicles contain granules of yellowish fat, which are taken to be the 'lutein' already present in the future lutein cells before rupture. This assumption rather overreaches itself, as one glance at fresh corpus luteum tissue of the sow will show that there is no microscopic yellow pigment present in the so-called lutein cells. In the one pair of ovaries next described, some follicles were ruptured, others were not. In the latter, the granulosa was no longer visible, except for a few cells lying in the cavity. The theca interna was thickened and closely resembled lutein tissue. In a later stage there was a central cavity rimmed by connective tissue, supposed to represent the membrana propria pushed before the thickening theca interna. Sobotta ('99 b) has given a vigorous criticism of Clark's specimens, explaining his so-called mature and just-ruptured follicles as cases of atresia. Volker ('05) has also pointed out what he considered errors of interpretation of the specimens.
However, Doering ('99) came to the defense of Clark, also using material collected at random. He states that the wall of a recently ruptured follicle shows no granulosa. His principal evidence, however, is from one corpus luteum of the sow, which shows, near the center of the section he figures, a flattened circle of granulosa cells. This he interprets as the granulosa of the same follicle in which the corpus luteum formed, which had been pushed inward by the proliferating theca interna, and which for some reason had not degenerated. I have seen very much the same appearance when a young growing follicle had crowded itself into the side of an old corpus luteum, so that a tangential section appeared to show a follicle within a corpus luteum.
Jankowski ('04), using the same method of collection, would seem to have had normal mature follicles of swine; at any rate, the granulosa was intact. The ova were not seen. His veryearly corpora lutea, showing stigmata at the point of rupture, also contain the granulosa in situ and completely preserved, except that the cells are swollen, irregular in form, and contain vacuoles. The theca interna cells are large, contain lipoid granules, and resemble lutein cells. No specimens between this and the solid corpus luteum are presented. Upon such evidence he confirms Clark's account.
Kopsch ('01) demonstrated at a meeting of the Anatomische Gesellschaft certain preparations by Menzer of the corpora lutea of swine three, six, and ten days after copulation. This contribution appeared by title only, and our sole information as to its nature is the statement of Sobotta that Menzer's specimens are in general agreement with his own views.
It is fair to say that the theory of the origin of the corpus luteum from the theca alone, though it still holds a place in current literature, has no good evidence in its favor. Every investigator whose methods assure us that accurately dated specimens of a sufficient number of stages were in his hands has declared the persistence of the granulosa cells and their transformation directly, with little or no mitotic division, into the characteristic large 'lutein cells' of the corpus luteum. The problem has shifted during the sixteen years whose progress I have reviewed; the present point of interest is as to the fate of the theca interna. Are its cells all converted into connective tissue; do they persist as a special peripheral layer of the corpus luteum; do they assume an impenetrably close resemblance to the granulosa lutein cells; or can we find some other and clearer explanation of the problem of their disappearance?
The following pages contain the results of an attempt to answer these questions. Choice of the species to be studied was influenced by several reasons. There is a frequently expressed idea that perhaps the larger and smaller animals differ in the formation of their corpora lutea, as they do in many other features of their reproductive cycles; the sow is large and has an ovulation cycle not unlike the human species. Other considerations in elude previous experience of the author with the ovaries of swine; certain presumed histological advantages of the species, to be explained later, and, above all, the fact that the previous work on swine has been much quoted by writers in confirmation of the thecal-origin theory. Here, if anywhere, the application of modern methods of research should settle the old difference once for all.
Material and Methods
As it has been pointed out that the only hope of trustworthy results in this problem depends upon the possession of an unbroken series of normal specimens of known ages, a description of the material in the author's hands and the methods of obtaining it will be given in detail. The first step was a preliminary investigation to obtain exact knowledge of the period in the reproductive cycle at which the ova are shed from the ovary in order that mature follicles and very early corpora lutea might be obtained. The results of this study have already been published (Corner and Amsbaugh, '17) and will not be repeated in detail here. We were able to confirm the current supposition that in swine ovulation is coincident with the oestral period, and by this fact we are at once provided with the means of obtaining the desired stages of corpus luteum formation.
The females of the wild swine of Europe are monoestrous, according to Kaeppeli ('08), having but one period of heat in the year; but under domestication the sow becomes polyoestrous, coming in heat at intervals of two to four weeks, usually about every twenty-one days, as all breeders agree. The period of heat commonly lasts three days and is characterized by sexual excitement and in some individuals by swelling, reddening, and slight eversion of the vulva, or even at times by a serous, mucous, or partially sanguineous discharge from the genital orifice. If a boar be present, the sexual excitement is made apparent by ready acceptance of coitus (commonly on the second or third day of oestrus) ; if none but females are in the pen, the sow in heat will be seen to sniff at the genitals of her neighbors and 'ride' them in imitation of coitus. Frequently the sow is the recipient rather than the donor of these attentions. The period is not terminated by coitus, but continues until the end of three days.
For the purpose of the present investigation, the condition of oestrus was observed while the animals were alive in the yards of the packinghouse. The sows were marked, and on the day of killing they were traced through the processes of the abattoir and the internal genitalia received from the hands of the eviscerator. The Fallopian tubes were then removed by cutting across the upper portion of the uterine horns, were carried to the laboratory in 0.7 per cent saline solution, and there washed out by inflating them with salt solution through a slit in the wall near the fimbriated extremity. After inflation with the fluid, the tubes were gently 'milked' into a Syracuse dish, and the washings examined with the dissecting microscope. This simple and almost infallible method of finding the ova was suggested to us by Professor Evans as an improvement upon Martin Barry's practice of milking the tube without injected fluid ('39). As we have subsequently found, it had been used by Sobotta (in the rabbit) and no doubt by others as well.
We found that ovulation occurs on the first or second day of oestrus, and that the stimulus of copulation is not necessary to cause rupture of the follicles. The ovaries of all sows killed during heat contain mature Graafian follicles ready to rupture or just ruptured, in which latter case the ova are in the tubes and may be recovered therefrom for study by the method described below. Little or nothing has been known of the mature ovum of the sow, and we have found no record of any previous observation of the unsegmented ovum from the tube. We measured fourteen fresh tubal ova from nine sows and found the diameter, including the zona pellucida, to vary from 155^ to 165m, the zone being about 10^ in thickness. The ova are plainly visible to the naked eye if placed against a strong light. We have not noticed a radial striation of the zona pellucida either in fresh or fixed ova. The ovum is filled with yolk granules of varying sizes, usually about 3m to 5m in diameter, which are so numerous and so retractile that they quite conceal the nucleus.
The author has presented ('17 c) a brief study of the maturation of the pig's ovum, based upon some of these specimens, which indicate that the sequence of events is the same as in other mammals. The first polar body and the second polar spindle are formed in the ovary just before rupture. After the entrance of the spermatozoon, which occurs in the tube, the second spindle completes its division, and the presence of two polar bodies is therefore a sign of fertilization. If the ova are not fertilized, they degenerate in the tube with the second spindle undivided. Just how long they survive is not known, but by analogy with the smaller and better-known mammals, we may assume that after three or four days they are no longer capable of segmentation; the degenerating ova may be found in the tubes a few days longer. It is said that pregnancy is more likely to result when the sow is served on the second day of oestrus. The number of ova extruded at one ovulation, and consequently the number of fresh corpora lutea in one animal, may be quite large. One prolific sow is known to have given birth to twenty-three pigs in one litter. However, in the mixed stock, not especially adapted for breeding, which is found in the abattoirs, small litters are the rule. Records of 128 sows raised in Maryland, presented in my paper of 1915, show that the corpora lutea of pregnancy in both ovaries numbered one to sixteen, averaging eight, and that the number of foetuses in the uteri of the same sows varied from one to ten, averaging six. Failure of fertilization, abortion, and resorption of embryos dying in utero account for the fact that not all the eggs of one ovulation proceed to full development.
About 133 embryos of the sow younger than two weeks, taken from twenty-three sows, have been observed and described in the literature. The youngest of all are the three ova found in three different sows by the present writer and Amsbaugh ('17), in which conjugation of the pronuclei had not occurred. It was not possible to know the exact time of insemination in these animals, but in one case it is believed that the animal had not been in heat, and consequently had not copulated, more than forty hours before killing. R. Assheton ('99) studied about 100 specimens during the first ten days, the youngest stage being that of two blastomeres. It would seem that fertilization may occur about the end of the first day or may be postponed until two or three days after copulation — a conclusion which he draws from finding embryos of the same stage in two sows killed on the fourth, fifth, and sixth day post coitum. Likewise, embryos in the same uterus may vary rather markedly as to their state of development, for instance, one uterus contained ova of two segments, of nine segments, and completed morulae. For this reason it is possible to give only an approximate time schedule of early development. The ova pass down the tube rapidly and enter the uterus about the fourth day post coitum. Assheton did not find any stage further advanced than four blastomeres in the Fallopian tubes. (A specimen found by the present writer and Mr. Felix H. Hurni contained ova of two, four, and six blastomeres, all in the tube.) Assheton found that various sows killed on the sixth day presented uterine embyros from the stage of six blastomeres to fairly well-developed blastodermic vesicles. By the seventh day the zona pellucida has usually disappeared and the inner cell mass of the early vesicle has differentiated into two layers, the epiblast and the hypoblast. By the twelfth day the great elongation of the blastodermic vesicle which is so characteristic of the pig, is well under way and the vesicle is already 10 to 12 mm. long. By the fourteenth day each vesicle may measure 20 cm. ; in the embryonic area the primitive streak is well developed and there are from one to three somites. In addition to Assheton's studies, thirty embryos of the ninth, tenth and eleventh days have been described by Weysse ('94), and from the fourteenth day to about the twenty-fifth we have the accurate tables of Keibel ('97). For older (foetal) stages, no good age-length ratios have been determined. The period of gestation is usually 116 to 120 days. It is stated that sows undergo oestrus and may become pregnant again five weeks after littering.
During the progress of this investigation the ovaries and uteri of several thousand sows have been examined macroscopically, and the corpora lutea of about 300 have been studied under the microscope. The permanent preparations upon which the following description is based comprise sections from the Graafian follicles and corpora lutea of 171 sows of which there are records sufficient to determine the stage of the reproductive cycle. In 162 of them the ova, developing embryos, or foetuses were examined and recorded.
Twenty-four were killed during the oestral period or within the first week after the onset of heat. Some of the tubal ova found were unfertilized, others were fertilized and were in stages from the one-celled to the six-celled embryo. Five of the twentyfour sows mentioned were obtained before a method of discovering the ova had been acquired, and the ova were therefore not sought, but as the dates of copulation were noted at the University of California Farm by Professor Thompson, it seems proper to include them, since their corpora lutea agree with the others in structure.
Six sows were taken in the second week after ovulation. As the ova were unfertilized, they had degenerated, and were not found, except shriveled eggs in two of the sows. It chanced that none of those sows which had copulated were killed during this period, and thus the opportunity to obtain embryos of the second week did not fall to my lot.
Fifteen contained embryos of the third week, from five to thirty-nine somites. The ovaries of the eleven youngest of these were given me by Prof. F. R. Sabin; some of the embryos to which they were related are described and pictured in her recent contribution to the early vasculogenesis of the pig (Carnegie Institution of Washington, Contributions to Embryology, No. 18, 1917).
One hundred and twenty-four compose a complete series from animals containing embryos of the fourth week to the end of pregnancy, the embryos or foetuses being measured in each case. Two were obtained from sows which had littered seven and ten days before killing, respectively. Most of the older corpora lutea of pregnancy were prepared in the Anatomical Laboratory of Johns Hopkins University, and formed part of the material for my previous monograph ('15). They have been restudied in the light of the results gained from the specimens of the first fourteen days after ovulation, all of which were obtained in California.
The collection of this material would have been impossible without the special and unusual cooperation which has been extended to this laboratory by the Western Meat Company of San Francisco. I refer to their donation of permanent laboratory quarters in their West Berkeley plant (Oakland Meat and Packing Company) to the Anatomical Laboratory of this institution. I owe especial thanks to Mr. J. 0. Snyder, general superintendent of the Western Meat Company, and to Mr. Ralston B. Brown, superintendent of the Oakland Meat and Packing Company, and to many other members of the staffs and employes of both these establishments; to Dr. H. H. Hicks, U. S. Supervising Inspector, Dr. G. R. Ward, and other members of the U. S. Inspection Service at the South San Francisco plant, and to Dr. Thomas Presst, of the California State Inspection Service. To Mr. R. B. Brown in particular I owe the opportunity of observing living animals and of obtaining their pelvic organs, often at the cost, I fear, of some inconvenience to the routine of his plant. The permanent laboratory space provided by him at the packinghouse has been invaluable during the prosecution of this work. I am further indebted to Professors Evans and Sabin for the contribution of ovaries with the corresponding early embryos; to Prof. J. I. Thompson, of the Department of Agriculture of the University of California, for observing and marking five animals, and to Messrs. A. E. Amsbaugh and Felix H. Hurni for assistance in the collection and preparation.
In general the younger specimens were fixed in Bouin's fluid, the older in 10 per cent formol, these fluids being selected to secure the advantage of fixation in slow aqueous coagulants, as will be explained in the next section; small pieces of many ovaries were placed in osmium tetroxide for study of the lipoids. Blocks were imbedded in paraffin and celloidin. The chief stains used with the specimens herein described comprised haematoxylin and eosin, Heidenhain's iron haematoxylin, Mallory's triple connective-tissue stain, Van Gieson's mixture, and several lipoid-soluble dyes (Nile-blue sulphate, Sudan III, Scharlach R), besides many special procedures applied to fresh and fixed tissues.
Special Cytology of the Lutein Cells of the Sow
Four years ago the writer undertook, at the suggestion of Professor Mall, to study the corpus luteum at different stages of pregnancy, with the aim of learning through the varying appearances to standardize the stages as a means of determining the ages of embryos and foetuses (Corner, '15). It was very good fortune that led to the choice of the pig for the first studies, for a useful peculiarity of cytoplasmic structure was found to occur in this species. If we take a section of the corpus luteum of a pregnant sow whose foetuses are perhaps 100 mm. long, fixed in formol, and stain it with any strong cytoplasmic stain, study of the lutein cells shows that the cytoplasm contains unstained areas which are roughly concentric to the nucleus, and which appear to form canal-like paths in the cell (fig. 1) . In the younger corpora the canals grow more and more complex, assuming the form of wide V-shaped spaces, long clefts, and circles in the cytoplasm, so extensive that the nucleus is surrounded only by a narrow zone of endoplasm. But it is in the corpora lutea of pregnancies under 30 mm. that the highest development of the exoplasmic zone is found. Here the entire outer part of the cell is occupied by a curiously elaborate system of vacuoles, almost every one of them in turn containing a spherule of substance which, although it takes the same stain as the cytoplasm, yet has a more hyaline appearance, and is seen in the section as a bright ring. Within many of the spherules is found another and tiny vacuole (fig. 2, r). Corpora lutea of pregnancies with foetuses more than 140 mm. long contain no trace of this system (figs. 23 and 24), and by careful attention to the degree of its development it is possible, therefore, to estimate the age of the corresponding embryo with some accuracy. Taking other histological features into consideration, I find myself able to detect the stage of pregnancy within close limits by examination of the corpus luteum alone. The same bodies are present in the corpora lutea of dogs, and were seen in the lutein cells of rabbits by Cohn ('03), who undertook certain microchemical studies upon their nature, which I have been able to extend. It was tentatively suggested, in my former paper, that they represent an elaborate modification of the Golgi-Holmgren intracellular apparatus. This view I have had to discard as a result of work which had led to the correct interpretation (Corner, '17 a, b). The spherules are due to the presence of a lipoid, probably of phosphatid nature, which is sufficiently oily to round up in the presence of water. The round droplets thus produced usually surround the preexisting globules of neutral fat present in considerable numbers in the early corpus luteum cells of swine (fig. 3). After immersion in alcohol, xylol, ether, or other lipoid solvents, both the fatty center and the phosphatid substance of the spherical droplet are dissolved out, leaving only a hollow sphere (appearing as a ring in thin sections), probably composed of proteid constituents of the cytoplasm precipitated in the spherules during fixation. The bodies are not seen in fresh tissues nor in material fixed with very rapid coagulants like osmium tetroxide, which precipitate the proteids before the oil droplets round up. The microchemical evidence of these conclusions is given in the articles cited.
Fig. 1 Cells of corpus luteum of pregnant sow (foetuses 100 mm. long), showing spaces in cytoplasm. Mallory's connective-tissue stain. Formol fixation. X 810.
Fig. 2 Cells of corpus luteum of pregnant sow (embryos 20 mm. long). Formol fixation. Mallory's connective-tissue stain. X 810. r, vacuoles in lutein cell; th.l.c.l, theca lutein cell, type 1; th.l.c.2, theca lutein cell, type 2.
Fig. 3 Cells of corpus luteum of pregnant sow (embryos 20 mm. long). Formol fixation followed by osmium tetroxide. X 810.
The appearances in question, therefore, are simply the result of methods of fixation which do not preserve certain obscure lipoids in* their natural diffused state. But the artifact is a useful one. In the first place, it enables us to follow the changes in amount of the phosphatid substance during the advance of pregnancy. It also allows us to estimate the age of a corpus luteum of pregnancy from the histological appearance alone, and it gives us a constant (even though artificial) cytological characteristic of the cell which can be used in determining the early history of the lutein cells. Due no doubt to different physical state of the cell lipoids, the phenomenon does not occur in the human and bovine corpora lutea (a former statement of the author to the contrary).
The Mature Follicle
As the reader has perceived, one of the crucial points in this debate has been as to the condition of the granulosa of the mature follicle. Some investigators think that this layer degenerates before rupture, others that it remains intact. It would seem, offhand, an easy matter to obtain mature follicles and settle the question at once. To be certain that a given follicle is really mature is very difficult, however, and particularly so in some species. Mere size is no criterion, for full-sized follicles are not infrequently in a state of advanced atresia. The presence of maturation processes in the ovum is no more certain a sign, for the formation of the polar bodies, as was pointed out by Flemming ('85), is a frequent occurrence in early atresia. As atresia may set in at any time in the life of a follicle, even up to the last, it is obvious that we can never state with complete assurance whether a given Graafian follicle is doomed to degeneration or is about to rupture and give rise to a corpus luteum.
We shall probably not be in error, however, in assuming that a follicle is normal and mature if it is taken from the animal at a time when ovulation is known to be imminent, and if it contains a normal ovum in which the process of maturation is under way.
To satisfy these requirements is easy when the animal is small enough to be under observation in the laboratory, when an impending ovulation can be predicted (as, for instance, in the rat and mouse, which are now known to ovulate about eighteen hours after littering) and when the small size of the ovaries and tubes readily permits serial sectioning. In animals like the hog, however, it is more difficult to observe these two criteria of the mature follicle, and no previous investigators of this species have watched the animal during life in order to determine the imminence of ovulation, nor have any taken the pains to find and study the ova of the follicles which they described as ripe.
In the author's material, of sixteen animals known to have been in heat when killed, only two were taken early enough in oestrus to contain unruptured follicles. In one, all the follicles were still unruptured. Three of them were successfully sectioned ; two of them contained ova with nuclei presenting 'germinal vesicles,' the third showed the first polar body and the second polar spindle. In the second sow, one of the follicles had ruptured; the tubal ovum could not be found; one of the remaining follicles, upon sectioning, showed its ovum to be in the matured state, with the second polar spindle formed. There can be little doubt, therefore, that the follicles in question were perfectly normal and would have immediately shed their ova and developed into corpora lutea had the sows not been slaughtered.
These follicles possessed clear, translucent, almost spherical walls, protruding a great part of their bulk from the ovary, as is characteristic of the species. They all measured about 7 mm. in diameter, the measurement ranging from 6 to 8 mm. in some which were distorted by crowding. 2 The surface presented no 'stigma' or other sign of impending rupture. On section, they were found to possess the usual three layers, and the membrana granulosa was present and intact, showing no sign of degeneration. The wall of that part of the follicle lying deepest in the ovary presents a slightly wavy contour toward the cavity.
The cells of the granulosa (fig. 4, a) form a layer about six to nine cells deep, or about 0.13 to 0.17 mm. thick. Those cells nearest the membrana propria form an irregular columnar layer, but the upper cells show less semblance of order in their arrangement. The cells are round or polyhedral, from 8 x 8/* to 10 x 16^ in diameter (in celloidin sections), with round nuclei 5n or 6/x in diameter. The cytoplasm appears homogeneous after the usual fixing reagents, except for a few vacuoles due to the presence of lipoid substances, as will be explained later. Often the cells possess short processes which meet those of the neighboring cells so as to make the tissue resemble a syncytium.
The theca interna is about 0.09 to 0.10 mm. thick, or a little more than half as thick as the granulosa. Its most striking characteristic is the presence of three to five layers of large 'epithelioid' cells, usually from 10 x 17/x to 12 x 17m in diameter, but occasionally reaching larger sizes, up to perhaps 16 x 24/x (fig. 4, b). On section, they are oval, spindle-shaped, or almost rectangular, with their long axes in the circumference of the follicle, so that they usually lie at right angles to the columnar layer of the membrana granulosa. Between the larger cells, and especially along the inner border of the layer formed by them, are others of small size (though still somewhat larger than the granulosa cells), which are similar in appearance to the large theca cells. In material fixed in Bouin's fluid the theca cells are notable for the presence in their cytoplasm of a number of vacuoles, giving them a striking honeycombed appearance (fig. 4, b). These vacuoles are due, at least in part, to the presence in the fresh tissue of granules of fat-like substance, packed closely into the theca cells, whose chemical nature has not been determined (fig. 5, 6). In some species they are quite yellow, since they hold in solution some of the lipochromes common in the ovaries of certain animals, but in the pig they are practically colorless. It is of course the appearance of these large fatty cells of the theca which has helped establish the belief that they are the precursors of the ' lutein cells' of the corpus luteum. The granules are soluble in alcohol; in osmium tetroxide they take a color varying from gray to deep black; they take a decided reddish color with Herxheimer's alkaline Scharlach Rot, but appear not to stain at all with Nile-blue sulphate; they are not anisotropic. From these reactions we may assume that the substance is of lipoid nature, but is perhaps not a neutral fat. The granules are variable in diameter, from 0.5/x to 1.5ju, a few even reaching 2.5/*. Many of the theca cells contain, instead of lipoid granules, vacuoles which are not stained even in osmium preparations, and which therefore must contain either a modified form of the lipoid or some other substance which is not rendered insoluble by combining with Os0 4 (fig. 5, b). The smaller cells of the theca interna mentioned above usually have finer granules, but there are all transitions between the large and small types. The cells of the granulosa contain a few very small granules, uniformly black after osmium fixation; these are usually more numerous in the basal layer of the follicular epithelium.
- 2 In a previous publication ('15) I fell more or less into the same error of method which I am now imputing to others, and attempted to determine the size of the 'ripe' follicle without knowing the state of the enclosed ovum. The larger follicles there mentioned, however, agree histologically with the accurately known specimens described in these pages, and it is therefore likely that my previous conclusions were correct, namely, that the normal mature follicle may attain a diameter of 10 mm.
Fig. 4 a, Portion of wall of unruptured Graafian follicle (sow in heat, ova maturing). Iron haematoxylin. X 380. b, A few cells from theca interna of same specimen. X SOU. gran., membrana granulosa; th.int., theca interna; th.ext., theca externa; sp.cz., spindle-cell zone of theca interna; b.v., bloodvessels.
Between the granulosa and the cellular layer of the theca interna just described is a narrow zone (0.03 to 0.04 mm.), which contains chiefly spindle cells without fatty inclusions or vacuoles (figs. 4, a, and 5, a). These cells appear to be of two types: first, the endothelium of the blood-vessels which lie in this zone and, second, the fibroblasts of the perivascular tissue, which are part of a light network of connective-tissue reticulum supporting the theca and forming a base for the granulosa, for I agree with J. G. Clark that the membrana propria is nothing more nor less than a network of these fibrils applied closely to the base of the granulosa layer.
Fig. 5 a, Portion of wall of unruptured Graafian follicle (sow in heat, ova maturing). Osmium tetroxide fixation without further stain, showing distribution of i'ats. X 380. b, A few cells from theca interna of same specimen. X 800. gran., membrana granulosa; th.int., theca interna: th.ext., theca externa; sp.c.s., spindle-cell zone.
The theca externa consists of a layer of long spindle-shaped cells, shading off into the stroma of the ovary (or into the capsular connective tissue, over that part of the follicle which is jutting out from the ovary). It is composed chiefly of collagenous fibrils and their associated fibroblasts, but it is highly interesting to note in connection with the subsequent collapse of the follicle, that there are also a good many smooth muscle fibers, as is readily seen by the use of Van Gieson's stain. There are no elastic fibers, except in the walls of the larger bloodvessels.
Just before rupture there are many mitotic figures in the cells of the theca externa, but only occasional signs of cell division in the theca interna and the granulosa.
Injected specimens show the blood- vascular distribution to be as described by His and J. G. Clark (fig. 6). Large vessels form a network in the theca externa, sending twigs inward to form a generously anastomosing plexus which lies in the abovedescribed spindle-celled zone of the theca interna.
I have found a curious arrangement of the blood-vessels in the ovum-bearing area of the follicle. The discus proligerus is a cone-shaped or rounded projection of the granulosa bearing the ovum near its apex, which until shortly before maturation of the ovum is composed of densely packed granulosa cells. At its base, in this particular species, are found a number of little vascular loops sprouting up from the vessels of the theca interna well into the granulosa of the discus, and pushing before them the cells of the basal columnar layer (fig. 6, loops). The basal cells appear as if radiating from the loops, and like all the cells of the area occupied by the loops are enlarged and have a much less dense cytoplasm than the other granulosa cells. Those loops which are near the center of the discus are longer than those toward the periphery. Such vascular loops penetrating the granulosa have apparently not been mentioned previously. They are not to be found in the rat and mouse, the only other species which I have studied in this regard. It would seem that the large size of the discus proligerus (as large as the entire follicle of the mouse) places the ovum at such a distance from the vascular bed that special vessels are needed for its nutriment.
Fig. 7 Portion of wall of mature Graafian follicle (sow in heat), showing mature ovum in situ, and discus proligerus in process of dissociation. X 50.
Be this as it may, by the time maturation of the ovum is in progress, the character of the discus proligerus has become considerably modified (fig. 7). Its cells are very much swollen by a vacuolization of the cytoplasm, so that they stand farther apart from each other, and in many places seem to be no longer in contact. About the ovum the cells of the corona radiata still hold together, but the rest of the discus has nearly crumbled away, and the slightest disturbance must complete the freeing of the ovum, bound as it now is to the parietal granulosa only by a few strands of cytoplasm. This rearrangement of the discus proligerus was long ago shown by Bischoff ('78) to be a trustworthy sign of impending rupture of the normal follicle. Certain subsequent observers, wondering how the ovum could be freed from its apparently secure moorings, were led to conjecture that there is a total desquamation of the granulosa— an error in which they were confirmed by the fact that for lack of the proper stages they did not see the mechanism for cutting off the ovum described by Bischoff, but did see, on the other hand, the complete dissolution of the granulosa, in follicles which we now know to have been atretic.
The Freshly Ruptured Follicle
Four animals of my series contained follicles which had ruptured very recently. One of these sows had shown the first signs of heat at some time between thirteen and twenty-two hours before killing, another between sixteen and thirty-nine hours before killing, and the other two were in the second or third day of oestrus (probably the second). As we have shown on page p. 132, rupture of the follicle occurs on the first or second day of oestrus. In all four of these cases, unfertilized ova were found in the tubes, there having been no copulation.
Apparently the act of rupture begins by the production of a small slit in the exposed part of the follicle, through which the ovum escapes to enter the tubal fimbria (figs. 8 and 9). A varying amount of the follicular fluid, usually a considerable portion, is extruded with the egg, and the follicle collapses as the volume of its contents suddenly lessens. It seems that an important part in this collapse must be taken by the fibers of involuntary muscle which lie in the theca externa; through their contraction the follicle is greatly diminished in all dimensions. At the point of rupture, the muscle fibers draw the theca externa away from the torn area, and the result is a slight eversion of the wound, through which the theca interna and granulosa protrude, forming a small reddish papule 1 mm. or less in diameter, the so-called stigma (fig. 12). The eversion of the inner layers appears at times to close the orifice immediately, but in other cases the follicular walls do not come together about the opening at once; the tiny slit is first plugged by fibrin, and later closed permanently by proliferation of its edges, much as described by Strakosch ('15) in the human corpus luteum. From the minute blood-vessels whose torn ends lie in the stigma there is often a slight oozing of serum or of blood, so that the surface of the ovaries at this time may be roughened by tags of pale or bloody
Fig. 8 Diagram of ruptured Graafian follicle (sow in heat, ova in tubes), illustrating partial collapse without great infolding of walls. X 14. (Compare with figure 9.)
fibrin, sometimes forming temporary adhesions to the fimbriae of the tubes. The entire ovaries and the tubes are usually much congested during oestrus.
On section, the follicular cavity is collapsed to a mere slit in some cases, in others it is still partially distended, owing to the continued presence of more or less of the follicular fluid. For this reason, the size of the structure varies, but in general it is much smaller than the mature unruptured follicle, its diameters varying from 3.5 to 6.5 mm. when not distended by hemorrhage. Most frequently the ruptured follicles are ovoid in form, about 4x4x5 to 5x5x6 mm. in diameter. Owing to the collapse of the follicle and to the contraction of the theca externa, the inner walls of the cavity are no longer smooth, but are thrown into folds whose complexity varies from that of low ridges in those follicles where much follicular fluid still lingers (fig. 8) to elaborately interwoven folds such as those shown in figure 9.
Fig. 9 Diagram of ruptured Graafian follicle (sow in heat, ova in tubes), illustrating complete collapse with much infolding of walls. X 14. (Compare with figure 8.)
Microscopically, the follicular wall is found to consist of the same layers as before rupture (fig. 10). There is no sign of any degeneration of the granulosa, which is now somewhat thicker, since it lines a smaller cavity than before. The individual cells are about the same size as formerly, but in many places are now elongated into oval or spindle forms by the stresses of the collapse, appearing to have slid upon each other as the granulosa thickened. The theca interna is the layer most affected, in a mechanical way, by the sudden collapse, for in some of the
Fig. 10 Portions of wall of recently ruptured Graafian follicle (sow in first clay of oestrus, ova in tubes). X 330. a, Mallory's connective-tissue stain, Bouin's fixation, b, Osmium tetroxide after formol fixation, showing distribution of fats, gran., membrana granulosa; th.int., theca interna; Ih.ext., theca externa; memb. prop., membrana propria.
folds it is violently torn apart, so that there are many wide spaces either within the theca interna or between the two thecae (fig. 11, tear). These spaces are occupied either by networks of fibrin, which may be altogether devoid of cells, or contain an occasional theca interna cell, or a leucocyte; or the space may not be an actual tear, but merely an oedematous area in which the cells, connective-tissue fibers, and blood-vessels of the thecae interna and externa are held apart by the tissue fluids. However, over many of the folds, and always in the depressions between the folds, the theca interna is neither torn nor oedema tous (fig. 11, dep.).
Fig. 11 Portion of wall of recently ruptured Graafian follicle (sow in heat first day of oestrus, ova in tubes; same animal as in figures 9 and 10), showing torn area in theca interna in a fold of the wail. X 80. gran., membrana granulosa; th.int., theca interna; tear, torn area in theca interna; dep., depression or recess between folds of wall.
The tears do not separate the theca interna from the granulosa; these two layers are everywhere in apposition, and the boundary between them is still marked at most points by the slight wall of condensed connective-tissue fibers, originating in the innermost layer of the theca interna, the so-called membrana propria (fig. 10). The theca externa is of course drawn into the folds of the wall, and where these folds are verydeep, long spindle-cells of the externa may thus penetrate almost to the center of the former follicular cavity — though of course they are walled out by the inner layers (fig. 9). Dividing cells are found not infrequently in the theca externa, but are quite rare in the inner layers. The distribution of lipoid substances, as indicated by the use of osmium tetroxide and Herxheimer's stain, is exactly as in the mature unruptured follicle (fig. 10, b). Leucocytes are found in the walls of all developing corpora lutea. The blood-vessels are exactly as in the unruptured follicle, the picture presented by them being modified only by the elaborate infolding of the walls (fig. 12). At the point of rupture the torn vessels of the thecal plexus present to the outside, and within a few days of rupture have sprouted into a little rosette of capillaries about the stigma, which helps to make this spot conspicuous by its redness. In the production of the curious torn spaces of the theca interna, described above, vessels of the theca interna are not infrequently ruptured, with resultant haemorrhage into the theca. If the loss of blood is very slight, the broken-down blood is taken up by the large cells of the theca interna, in which the phagocyted golden-brown pigment may remain for some days at least (fig. 13) . In one of my cases there was a single local haemorrhage into the theca externa, and the nearest cells of the theca interna were full of blood-pigment granules. However, when the thecal haemorrhages are large, the resultant haematomata may burst through the granulosa into the cavity. I am inclined to think that we have here the source of most of the bleeding into the early corpus luteum cavity. The now almost forgotten doctrine of Henle and Paterson, that the corpus luteum is formed from the blood clot of the newly ruptured follicle, naturally led to investigations into the importance and constancy of the haemorrhage in various species, which have been summed up by Sobotta in his paper of 1896. In the pig, Zwicky ('44) held that bleeding is frequent, Paladino ('80) that it occurs in two-thirds of the cases, Benckiser ('84) that it is inconstant, Spiegelberg ('65) that it is important, and Bonnet ('91) that it is constant and marked in extent. Sobotta ('96), reviewing the evidence, is inclined to the last view. Pfluger ('63), in an experimental investigation, found that in cats and rabbits killed violently there was much more frequent bleeding into young corpora lutea than in animals killed without struggle and very carefully autopsied. In my own specimens, out of sixteen sows whose ovaries contained very early corpora lutea, dressed at a packinghouse using the relatively gentle method of scraping by hand, four showed more or less blood in the corpora lutea and twelve were entirely free of macroscopic haemorrhage. In another establishment, where the carcasses are conveyed 150 feet dangling from a chain and are scraped by engine-driven revolving vanes (so that in the bodies of pregnant sows, young foetuses frequently suffer an effusion of blood into the amniotic sac), there chanced to be a somewhat higher proportion of haemorrhagic follicles; but even there, in spite of such excessive violence, it is common enough to see delicate corpora lutea one or two days old come through with no blood at all in their cavities. Again, trauma at the time of killing does not explain awayall the haemorrhages; for instance, in those cases in which among a number of solid, bloodless corpora lutea several days old, one or two others are found distended with dark clotted blood to a size exceeding the normal corpora. I feel that the present evidence indicates that haemorrhage into the corpus luteum of the sow, while not uncommon, is the exception rather than the rule, and is of no anatomical or physiological importance. Indeed, the arrangement of the follicle seems well adapted to prevent any considerable loss of blood into the cavity, for the tiny vessels at the place of rupture are promptly directed outward toward the peritoneal cavity, while the follicle is provided with smooth muscle, which keeps the walls tensely contracted, even after rupture. When small haemorrhages occur, undoubtedly they are readily resorbed, and the corpus luteum then goes on to develop normally. When great enough to distend the follicle and compress the growing wall, inhibition of corpus luteum formation presumably occurs, and we have here one of the causes of corpus luteum cysts, which are very common in swine.
Fig. 12 Recently ruptured Graafian follicle (ova found in tubes), bloodvessels injected with India ink. X 15. gran., membrana granulosa; th.int., theca interna; th.ext., theca externa; stig., stigma (blood-vessels at point of rupture).
Fig. 13 Cells from theca interna of recently ruptured follicle (sow in heat, ova in tubes). Iron haematoxylin stain, showing pigment and broken-down erythrocytes in theca cells. X 1000.
Invasion of the Granulosa
The next stage is represented by seven animals in my collection, all of which were killed during oestrus, as normal ova were found in the tubes. Moreover, four of them were observed during life, and were actually seen to be in the second or third day of oestrus. In three, copulation had not occurred; in three others, fertilization had taken place, the ova showing the pronuclei approaching conjugation; and in the seventh, the ova were segmented into two, four and six blastomeres.
The first sign of an advance upon the previous stage consists of the breaking-down of the membrana propria, at first at the apices of some of the folds, later over the entire follicle, so that the former sharp line of division between granulosa and theca interna is no longer present (fig. 14). Wherever the membrana propria is disappearing, slender spindle-cells are seen to be insinuating themselves between the still closely packed granulosa cells (fig. 14, sp.c). The nature of the inwandering cells is difficult to decide. In places there can be no doubt that they are endothelial in nature and represent the first sprouts from the walls of the thecal capillaries, growing inward to the granulosa. In the endothelial cells mitoses are not uncommon at this time. It seems quite likely that all the early invading cells are of endothelial nature, but at some few points, however, it is impossible to convince oneself that the spindle-cells have any connection with the vessels, for they are not always arranged in tubular form and are sometimes well disseminated throughout the granulosa in advance of any circulation of blood. It cannot be denied absolutely, therefore, that some of them may be inwandering cells of the perivascular spindle-cell zone of the theca interna. During these early changes the large cells of the theca interna remain in their place, and I have never seen convincing evidence of their conversion into spindle cells.
Fig. 14 Portion of wall of young corpus luteum (ova found in tubes), showing swollen cells of granulosa with inwandering spindle-shaped cells. Mallory's connective-tissue stain, formol fixation. X 810. gr.l.c, granulosa lutein cells; th.int., theca interna; th.ext., theca externa; sp.c, spindle-shaped cells.
Practically all of those observers who have been convinced of the persistence of the granulosa have described such an early invasion of the innermost layer by spindle-cells, a stage which was called by Robert Meyer the stage of proliferation, but as in the pig there is doubt as to the interpretation of the observed facts. Sobotta ('96) holds that all the cells of the theca interna are converted into spindle-cells (fibroblasts), and wander into the granulosa, dividing frequently, to form the connective-tissue framework of the corpus luteum. In this view he is supported by Marshall ('04) in his work on the sheep, and by O'Donoghue ('16), who studied the marsupial ovary; but several authors, including Volker ('05), Loeb ('06), and R. Meyer ('11), working, respectively, with the corpora lutea of the marmot, the guineapig, and man, are inclined to consider the first inwandering cells as endothelial, and deny the conversion of the theca interna cells into fibroblasts.
It has been mentioned that the breaking-down of the raembrana propria and the invasion of the granulosa by spindle-cells does not take place at once over the entire inner surface of the collapsed follicle, but begins first at the apices of the folds, where the structure has presumably been subjected to the greatest mechanical strain. Because of this very important fact, we are able to observe a definite stage at which, while in places there is an actual intermingling of the two layers going on in part of the structure (fig. 15 a, X), in other parts the two inner layers of the wall maintain their original relations. During the same period there is a marked and rather sudden change in the granulosa cells (fig. 15 b). Their cytoplasm increases in volume so that the cells are now much larger in size, varying from 9.5 x 11/x to 14 x 21 \i in diameter in sections, or half again as large in diameter as before rupture. The nuclei become rather more vesicular. Most important is the fact that many of the larger
Fig. 15 a, Part of wall of developing corpus luteum in stage of spindle-cell invasion (ova in tubes). Formol fixation, Mallory's connective-tissue stain. X 110. X, area of active invasion by spindle-cells (at apex of fold in wall).
cells now have in their cytoplasm large spaces containing rounded bodies resembling rings in section, which we have already seen (pp. 137, 139) to be characteristic of the so-called 'lutein cells' of the young corpus luteum in swine and to be due to the presence in the cytoplasm of an oily lipoid substance.
Furthermore, these changes in the granulosa cells are found to occur in all parts of the wall, as well in those areas as yet uninvaded as in those where granulosa and spindle-cells are already intermingled. To sum up the evidence, there is a time in the development of the corpus luteum, about three days after the rupture of the follicle, when changes of structure within the organ are already under way, and when many of the cells have begun to acquire adult characteristics, but in some parts of the organ the original relations of granulosa and theca interna are still intact; in these areas we find that it is the granulosa cells alone which have assumed the appearance of the large cells of the corpus luteum, commonly called lutein cells.
Fig. 15 a, Enlarged view of portion of same as shown by rectangle. X 1000. gr.l.c, granulosa lutein cells; th.c, theca cells; b.v., blood-vessel.
The breaking-down of the membrana is followed by a rapid sprouting and branching of the blood-capillaries throughout the entire granulosa (figs. 16 and 17). This stage is represented, in my material, by seven sows, of which one contained fertilized ova (some unsegmented and some with two blastomeres) ; one was killed about five days after the onset of heat, no ova being found, probably having degenerated; one was killed about six days after the onset of heat, one degenerate ovum being found; the other four were among those received from the University Farm School, in which the ova were not sought, but in which the dates of copulation were accurately known, in two on the third day and in two on the fifth day before killing.
Coincidently with the spread of the blood-vessels in a network throughout the granulosa, there continues a marked swelling of the cells of this layer, which double or more than double in diameter, thus making an eightfold increase in volume; some of them reach dimensions of 30V to 35^- The nuclei are larger and more vesicular. I have never seen a mitotic figure in a cell of the granulosa at this or later stages, and feel sure that the generalization of Sobotta on this point is correct. In the formol- or Bouin-fixed specimens, the periphery of the cells is studded with the striking ringlike phosphatid artifacts of fixation (fig. 18, gr.l.c). The rest of the cytoplasm is thin and contains irregular vacuolar spaces, due partly perhaps to the shrinking away, during fixation, of the cell-substances which form the ring bodies, and partly to the solution in the alcohols, xylol, or ether, of other lipoid substances, which osmic preparations show as small black globules in the center of each ring and also scattered about the nucleus or throughout the cytoplasm. I have not been able to apply other microchemical tests to the tissues at this stage, but the globules are morphologically like the neutral fat of later stages (fig. 3), and I assume that they are indeed the neutral fat or its forerunner.
Fig. 16 Young corpus luteum (segmenting ova with one and two blastomeres found in tubes); blood-vessels injected with India ink. Compare with figure 12. X 15.
Meanwhile, changes have also been taking place in the large cells of the theca interna. Mitoses are more common, and the former definite internal limit of this layer has been blurred by the breaking-down of the membrana propria and the ingrowth of the capillaries at all points of the follicular wall. Within the cells there are changes in the lipoid inclusions which are their chief distinguishing characteristic. In some cells of osmic preparations the granules are larger, in others smaller than before; in some they do not form an insoluble black compound with osmium tetroxide, but leave vacuoles of varying sizes, between which a few stained granules may remain giving a characteristic foamy appearance to the cytoplasm (fig. 19) ; and in others practically all the fatty bodies and vacuoles have disappeared, leaving a smooth homogeneous cytoplasm. In ordinary stained sections, then, the theca interna cells are of about the same size as before rupture, their nuclei are perhaps slightly more vesicular, and the cytoplasm is either homogeneous or contains many densely packed vacuoles, usually uniform in size within any one cell, resulting in the foamy appearance.
Fig. 17 Enlarged view of small part of figure 16 as indicated by rectangle, showing blood-vessels of theca interna branching throughout granulosa. X 4").
Fig. 19 Theca interna cells of corpus luteum in stage of invasion, osmium tetroxide fixation without further staining, showing varying degrees of fatty inclusion. X 1000.
Fig. 18 a, Part of wall of developing corpus luteum in stage of invasion (ova in tubes). Bouin fixation. Mallory's connective-tissue stain. X 110. b, Enlarged view of portion of same as indicated by rectangle. Mallory's connectivetissue stain. X 1000. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells; th.int., portion of original theca interna, near a fold in wall of corpus luteum; b.v., blood-vessel.
It will be obvious that the previous clean-cut distinction between the two layers is now lost. Heretofore we have been able to distinguish them by position, size, and content; there has been a wall of connective-tissue fibrils between the layers; the cells of the granulosa have been smaller than those of the theca interna ; and the former have contained but small numbers of lipoid granules, the latter considerable amounts. But now the membrana propria is done away with, the granulosa cells have increased in size and are becoming rich in lipoids, while the theca interna cells are losing their lipoids. It is not strange that investigators have become involved in uncertainty regarding the further fate of the theca cells. I have seen the abrupt ending, at stages similar to those now being described, of two careful attempts, by students in our histological courses, to follow the theca cells of the mouse and rat by means of their osmiumstaining inclusions, owing to failure to observe further distinctions between the two cell types. In the pig, however, we possess a peculiar advantage in the tendency of the phosphatid material to form the previously mentioned cytoplasmic rings when fixed with slow aqueous fixatives, giving the granulosa cells a distinctive appearance. There are other less regularly present criteria, which when added together afford the practiced observer means of partially distinguishing the two cell types; these are a tendency of the cytoplasm of the theca cells to take acid stains somewhat more deeply than the granulosa cells (perhaps this indicates merely a denser cytoplasm) and also the regularity in size and closely packed disposition of the lipoid granules or the vacuoles left when they disappear or are dissolved. Following these clues, we find that many of the theca interna cells remain in their original location about the periphery of the follicle, running into the interior of the folds produced by the collapse; but also that many of them, as the blood-vessels grow inward, are carried or wander with the vessels, and become disseminated among the cells of the membrana granulosa, where they are finally lodged, either singly or in small groups, frequently along the capillaries (fig. 18). It must be remembered that a general scattering of the theca cells among the granulosa in this way will not require a longer journey for any single cell than the thickness of the inner layer, which is not more than 0.2 mm. The cells thus immigrating resemble in every way their mates left behind at the periphery and in the folds, some of them containing large granules, staining black with osmium tetroxide, others showing almost no fatty inclusions (fig. 19).
They are often bioadly spindle-shaped or irregular in form, sometimes compressed between the granulosa cells or applied demilune-fashion to one of them.
Further changes are brorght about by the great swelling of the granulosa cells, which proceeds so far that the contents of the follicle begin to equal and finally to exceed the capacity of the contracted theca externa. The first effect of the internal pressure is to fill whatever remains of the original follicular cavity solidly with new tissue, then to compress the thecal cores of the folds of the walls so that all fibrin-containing cavities and
Fig. 20 Diagram showing outline of section of a corpus luteum about four days after ovulation. X 5. pr., 'Pfropf or hernia-like bulging of contents through point of rupture.
oedematous spaces are obliterated, and the folds become merely connective-tissue septa containing the remains of the theca interna in the shape of a diminished number of theca cells enmeshed by reticular fibrils; in the bases of the folds the bloodvessels of the young corpus luteum enter, usually accompanied by fibroblasts and fibrils proceeding from the theca externa. In many young corpora lutea the swelling of the granulosa cells finally causes a bulging of the contents through the outer pole of the wall, at the point previously weakened by the rupture; which in the prolific ovaries of the sow may be exaggerated by the pressure of the many neighboring corpora lutea of the same crop, until there is produced a knoblike hernia of corpus luteum tissue sometimes containing a tenth or more of the whole corpus luteum (fig. 20). This appearance is sometimes called by the handy German name 'Pfropf.' In some species, as, for instance the cow, it seems to occur invariably and to persist throughout pregnancy, but in swine the hernia is not always produced, the whole wall of the corpus distending evenly instead; and later it seems to subside, as in most corpora lutea in more advanced pregnancy there are no 'Pfropf en.'
The Fully Formed Corpus Luteum and its Morphological Changes until the Termination of Pregnancy
Invasion of the granulosa by the thecal vessels and cells begins about the third day after the onset of oestrus (or about the second or third day after rupture of the follicle) and is completed about the sixth or seventh day. My series contains five sows killed during the second week after ovulation and a large number from all stages of pregnancy from fifteen days after ovulation on to full term and into the period of lactation, so that altogether there is an unbroken series representing almost every day of the entire reproductive cycle.
By the seventh day the corpus luteum may be considered to have completed the first stage of its metamorphosis. It is solid (unless there has been a decided haemorrhage into the cavity), and it is already larger than the follicle in which it arose, reaching diameters of 8 and 9 mm., although a slow increase in size is yet to go on until the second or third week, by which time the full diameter, 10 to 11 mm., is reached. The blood-vessels have grown into a very narrow-meshed plexus, reaching every cell. The remains of the former great folds of the walls are seen as thin septa of connective-tissue fibrils running radially into the corpus luteum, carrying the larger blood-vessels of the organ. In some specimens, just inside the theca externa capsule and along the septa is a layer of theca interna cells or sometimes a few scattered clumps of them which have not chanced to invade the granulosa. These clumps may be found as late as the second month of pregnancy or may disappear long before (fig. 21). In appearance the cells of these clumps resemble the theca cells of the stage of invasion. In osmium-tetroxide preparations, some retain many lipoid globules, others have lost all of their fatty inclusions; but many present the previously noted foamy appearance of the cytoplasm, due to the presence of many small evenly packed vacuoles with a few tiny black granules interspersed.
Fig. 21 Portion of corpus luteum from second month of pregnancy (foetuses 35 mm. long), showing a clump of theca interna cells still in their original position. Formol fixation. Mallory's connective-tissue stain. X 450.
In the substance of the corpus luteum are now found two types of cells whose differing characteristics become well marked after the beginning of the third week. One type is that whose identity with the granulosa is fully demonstrated by the presence of the peculiar lipoid spherules about the periphery of the cytoplasm (figs. 2 and 21). From now on these cells grow slowly in size, until just before delivery some of them have attained the immense size of 30 x 45^. During the first few weeks the neutral fat increases until it greatly exceeds the small amount found in the cells of the granulosa before rupture, and then grows progressively less, finally almost disappearing by the 110th day of pregnancy. The phosphatid substance which produces the artifacts of fixation, so frequently referred to, disappears also, and therefore during the last third of pregnancy the cells, in ordinary formol preparations, possess a homogeneous cytoplasm, strikingly different from the greatly vacuolated cell substance of the earlier stages (fig. 23). Just before delivery, however, great globules of an osmium-staining material, presumably a fat, appear about the periphery of some of the cells.
Fig. 22 Cells of corpus luteum of pregnancy eighteen to twenty days old (embryos of twenty-seven somites). Osmium tetroxide after formol fixation, a, Diagram of a small portion of the periphery, showing a clump of theca cells still present at the periphery at the site of a fold in the follicular wall, and indicating by small rectangles the location of the cell groups in b and c. b, Cells from persisting theca interna. X 1000. c, Cells from interior of corpus luteum, showing two granulosa lutein cells and a third cell exactly resembling those of the theca. X 1000.
The cells of the other type are those described in my contribution of 1915 as "additional cells of the corpus luteum, type 1." They are found throughout the corpus scattered between the larger cells or in small clumps along blood-vessels and connective-tissue septa. They are smaller than the granulosa lutein cells, having diameters of 15^ to 20/*. In form they are adapted to their interstitial position, being rounded, almost rectangular, or at times compressed into polyangular shape (figs. 23 and 24). Their cytoplasm is either finely granular or contains regular vacuoles so closely packed as to give a foamy appearance. Indeed, in form, size, and in intracellular characteristics they present a most striking resemblance to those cells of the theca interna which in the same preparations are still belatedly situated at the periphery of the corpus luteum (fig. 22). Especially in osmium preparations is the similarity so great that one is forced to the hypothesis that we have scattered throughout the organ, among the granulosa lutein cells, the multiplied and immigrated cells of the theca interna. In the light of the apparent origin of these cells, it would seem well to give them the name, already established in the literature, 'theca lutein cells,' for though there are certain just reasons for criticism of this term and the name 'granulosa lutein cells' as applied to the other great class of corpus luteum elements, there would seem to be no better names at hand.
While the cells derived from the granulosa lose their lipoids after the first few weeks, the smaller cells just described again gradually increase their content of osmium-staining lipoids during the span of gestation, and some of them come at last to be laden with these bodies, which, how r ever, do not altogether resemble the lipoid granules of their earlier days (fig. 24). At the end of pregnancy the cells of this type are still present among those derived from the granulosa, apparently having maintained separate identity during the entire term of gestation. Even those which remain for a while in clumps or a definite layer about the periphery are not found to degenerate, but seem to be drawn in among the neighboring granulosa cells as the corpus grows older.
Fig. 23 Cells of corpus luteum of advanced pregnancy (foetuses 230 mm. long). Formol fixation. Mallory's connective-tissue stain. X 810. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells.
Fig. 24 Same stage as in figure 22, osmium tetroxide fixation without further staining, showing distribution of fatty inclusions in cells. X 810. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells.
However, when an attempt is made to classify all the elements of the fully formed corpus luteum, the picture is complicated by the fact that numerous cells are found which are intermediate in size between the two classes described above, and whose cytoplasmic vacuoles and fatty inclusions are of nature too indifferent to place them definitely with either granulosa or theca derivatives. The evidence, therefore, is not yet conclusive as to the exact fate of all the theca lutein cells. Either the intermediate forms represent genuine transitional stages in the formation of 'lutein cells' from theca interna cells or else they are merely cells, actually of one line or the other, in which the all too slight distinguishing features of the type (size, form, lipoid inclusions and vacuoles) have not been obvious. Toward the latter view 7 — the intermingling of the two cell lines without actual conversion of one into the other — the author is inclined to lean, without more positive evidence than has already been given.
As a digression, it may be mentioned that some few of the theca lutein cells retain the primitive characteristics of the theca interna, even exaggerating them at times; they are variable in shape and size, they have a cytoplasm which stains deeply with acid stains, becoming dark blue, brown, or even orange with Mallory's triple stain and very dark with iron haematoxylin; the cytoplasm is usually somewhat shrunken, and contains clear vacuoles about 1^ to 2/x in diameter, which are quite uniform in size, are closely packed, and which either stain intensely black with osmium tetroxide or remain as vacuoles. The nuclei are often very dense, even pyknotic, and sometimes stain bright orange with Mallory's stain. The cells are often spindle-shaped, branched, or compressed in such a way that they give the appearance of amoeboid motion, as if they were active wanderingcells. These are the cells described in my paper of 1915 as "additional cells of the corpus luteum, type 2" (fig. 2). Although their origin is now explained, I have no more light upon their nature or possible function than before. They appear to be more common in the earlier half of pregnancy, but their number varies greatly from animal to animal.
The cells of the young fully formed corpus luteum are supported by a reticulum of delicate connective-tissue fibrils with denser strands along the septa. In sections stained by Mallory's anilin-blue mixture and by the Bielschowsky technique as modified by Ferguson ('11), it is clear that neither the granulosa nor the theca lutein cells are intimately related to the fibrils, which form dense baskets about them, but are not found within the cytoplasm of the 'lutein cells' of either type (fig. 25). In other words, the activities of the theca interna cells have become so far modified in the process of their differentiation from the primitive 4 mesoblast of the ovarian stroma that they no longer exercise the function of fibril formation. What new activities they may have assumed, whether they may not now share in producing the hypothetical internal secretions of the corpus luteum, are questions for speculation and further research.
Fig. 25 Corpus luteum of pregnancy (embryos 20 mm. long). Formol fixation. Bielschowsky's silver-impregnation method, showing reticular fibrils. X 1000. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells; cap., capillary blood-vessel containing an erythrocyte.
Fig. 2G Diagrams of small portions of walls of corpora lutea at different ages, showing increasing density of connective tissue. Dianol red X after Bouin's fluid. X SO. 1, young corpus luteum (ova in tubes); 2, foetuses 115 mm. long; 3, seven days after birth of young.
Having found that the theca lutein cells are not the fibrilproducing elements, one is further surprised to find that the corpus luteum of the sow contains very few fibroblasts of the conventional type. It is impossible to convince oneself that there are in the organ any cells other than the two types of lutein cells beside the endothelial cells of the capillary wall, except here and there along the greater vessels which run in from the periphery. Since there are multitudinous fibrils, and no 'fibroblasts' to produce them, one is forced to suspect that, as in the liver, the endothelial cells themselves lay down the reticular fibrils. This evidence by elimination would appear to be supported by observation of the actual fact, but the question needs fuller investigation/ 1
Whatever be their source, the amount and density of the reticular fibrils of the corpus luteum increases steadily from the nativity of the organ until retrogression, when the organ is entirely replaced by scar tissue (fig. 26). It is along the large septa that the fibers first become dense, perhaps because a few connective-tissue cells of the theca externa are often drawn into the folds produced by the collapse following follicular rupture.
Retrogression of the Corpus Luteum
Owing to conditions of the meat-packing trade, it is difficult to obtain ovaries of sows within a few days after parturition. Study of the two specimens from the seventh and tenth days after littering shows that regression of the corpus luteum is as rapid as its appearance. By the seventh day the structure, which was a flesh-colored body 10 or 11 mm. in diameter before parturition, is only half this size; it has already shrunken until it is almost buried in the ovarian stroma, and it has acquired a yellow-brown color. By the time another ovulation occurs, the former corpora lutea are dense scar-like nodules of connective tissue rendered a pale yellowish brown by the presence of pigment in the shrunken cells caught in the meshes of the scar. The microscopic changes during this process are obscure to me for lack of material, and therefore until a series including every day of the first week after parturition can be obtained, the ultimate fate of the theca interna cells of the sow must remain unknown.
3 The resuRs of a study of the question made since the completion of this paper have proved that our hypothesis was correct, and that in the corpus luteum and a number of other organs part or all of the reticular framework is laid down by the cells of the capillary endothelium. (See a contribution by the author to the forthcoming volume in memory of Dr. F. P. Mall in the Publications of the Carnegie Institute of Washington, no. 272.)
It is hardly necessary to point out that the evidence just given completely negates the results of previous investigators in this species, and that it therefore removes one of the main supports of the theca-origin theory. But since this view has been held by so many anatomists, the reader will ask more than mere denial; stating their errors, we must also explain them. The reply, already pointed out by Sobotta, has become emphasized to the present writer during the course of his own studies. It would be a simple matter to select from this material a number of specimens which would duplicate the figures of Clark, Jankowski, or Doering, but placed in proper position in the series, the same specimens lead to far different interpretations. The small, pale, and inconspicuous nature of the corpora lutea at their earliest stages has allowed them to escape the notice of investigators seeking, under preconceived notions, for brightly haemorrhagic structures in the ovaries, and thus the all-important stages of the first four or five days have not been at hand to explain the more difficult later conditions of development. Insufficient data, leading to confusion with the process of atresia and to failure to obtain the earlier stages, and insufficient numbers of specimens, leaving important gaps to be filled by the imagination, are the two great sources of error in the study of this difficult problem.
In the investigation of the human ovary the obstacles to the acquisition of attested material are still greater than in the mammals subject to experimental methods. With our obscure knowledge of the reproductive cycle, the only guide to the age of a corpus luteum is its appearance, and we have seen what a useless aid this can be. Even the presence of a healing point of rupture, which is so characteristic of early corpora lutea in other animals, is not entirely trustworthy in human ovaries.
The specimens have nearly all been obtained at operation upon gynecological patients, who before operation are usually subjected to palpatory examinations, often none too gentle. Any gynecologist will know that the rupture of a 'small ovarian cyst' by the examiner's hands is a not infrequent occurrence; such cysts, were they actually large follicles, immature or in early atresia, if removed a day or two after the artificial rupture, might present the anatomist with all too convincing specimens of 'early corpora lutea.' Other possibilities of error might be suggested, none of which can be ruled out until the ova are studied with the specimen. While awaiting that almost impossible outcome, we shall be wise to follow those workers with the human corpus luteum whose findings are most nearly confirmed by the evidence of comparative anatomy.
The interpretation of the origin and morphology of the corpus luteum given in these pages does not represent a wide divergence from previous views. Nearly all observers now agree in describing the persistence of the membrana granulosa and its invasion by elements arising in the theca interna. The present work, so far as it traces the fate of the theca cells, is in accord with the best of recent investigations, and the author's hypothesis of the persistence of all the theca cells as distinct elements would, if finally proved, explain the remaining difficulties.
It is not so easy to align the findings in the sow's corpus luteum with the conceptions of Sobotta and his followers, who believe that the theca interna cells revert to mesoblastic type and by division give rise to a strain of fibroblasts which lay down the connective-tissue frame of the corpus luteum. This view implies a notion of the structure of the adult corpus which differs from that found in swine, and will necessitate a new study of the cell types and the relation of the reticular fibrils to fixed cells in corpora lutea of animals such as the mouse and sheep before the discrepancy can be understood.
In conclusion, it is a pleasure to express my thanks to Professor Evans for his encouragement and generous provision of aid and materials for this work.
- In swine the membrana granulosa is retained intact after the rupture of the Graafian follicle. Its cells increase in size without division, their cytoplasm becomes laden with lipoid substances, and they become the larger elements commonly called 'lutein cells' in the fully formed corpus luteum.
- The membrana granulosa is invaded by blood-capillaries from the theca interna, which ramify to form an extensive vascular plexus throughout the new structure.
- The large lipoid-laden cells of the theca interna are increased in number by mitotic division, lose many or all of their fatty inclusions, and pass into the corpus luteum to become lodged between the granulosa cells throughout the whole structure.
- There is no evidence that cells of the theca interna are ever converted into fibroblasts of the usual spindle-cell type or that they lay down the fibrils of the close-meshed reticulum which is present in the corpus luteum.
- There appears to be good evidence that some of the theca interna cells persist throughout pregnancy as distinct elements of the corpus luteum, but the exact fate of all of them cannot be learned by present methods because of a confusing resemblance between some of the theca and some of the granulosa derivatives.
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