Paper - The corpus luteum of pregnancy, as it is in swine (1915)
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The Corpus Luteum of Pregnancy, as it is in Swine
Contributions to Embryology, No. 5.
By George W. Corner With three plates.
At the instance of Professor Mall, I have undertaken the studies to he presented in this paper, in the hope of gaining such knowledge as would enable us to draw up a standard description of the corpus luteum throughout its history. If we knew the exact appearance of the human corpus luteum at all its stages, especially during pregnancy, we should have an instrument of the greatest value in the solution of many complicated problems of embryology and physiology, and even of clinical gynecology and organotherapy. Since the path of such a research as I have here begun is as yet unblazed, and because studies of the human tissues are so hampered by pathological changes, faulty histories, and difficulties of collection and preservation, for the beginning we have limited ourselves to the ovary of the domestic sow, and have started out with the simple hope of obtaining an answer to one question: What is the appearance of the corpus luteum at each and every stage of pregnancy? To this query as simple an answer has been found, but the work has led in such unexpected directions and into such interesting by-ways that the reader will no doubt feel as much disappointment in reading as the author has in writing a paper which answers one question, only to raise a score as yet unsolved.
During the months of October 1913 to May 1914 I have collected from the hundreds of sows' uteri and adnexa obtained at the slaughter-house adjacent to this laboratory, 128 pairs of ovaries from pregnant animals, the embryos being in each case examined and measured. The youngest embryo studied had 5 somites, and its age was estimated at about 15 days. The specimen is preserved as pig embryo No. 10 in the embryological collection in this laboratory. The oldest fetus in my series was 290 mm. long, or just about at term. In this paper, where lengths are given, they represent careful crown-rump measurements. Since the age-length ratio of embryos of the pig has never been fully worked out, the ages given are only approximate, and were obtained, for the younger stages, by comparison with Kernel's lists and figures (1897), and for the older stages from a table found in Strangeways' "Veterinary Anatomy," and said to have been compiled from the works of Gurlt, Leyhs, Franck, and others. The entire uteri and adnexa were received at the laboratory within an hour after killing, generally within a few minutes, and while yet warm. The fixing agent used was 10 per cent aqueous solution of formalin (40 per cent formaldehyde). The preparations were made by cannulating the ovarian artery, or the uterine, in which latter case it was advisable to tie off a few branches. The blood-vessels of the ovary were then injected with the formalin solution heated to 40° to 50° centigrade, and were placed in the same fluid. In a few cases where injection was not possible or not successful, the corpora were sliced with a razor-blade and the ovaries immersed in the warm formalin. For the study of the fat-content of the tissue, slices were cut from certain of the fresh corpora lutea and fixed in 2 per cent osmic acid for 24 hours. In all cases the ovaries were numbered consecutively, the right and left ovaries being indicated, the embryo pigs were measured, and the number of them in each horn of the uterus recorded.
The specimens were left in the formalin 24 to 48 hours, the solution having been allowed to cool an hour or two after immersing the specimens. After fixing they were washed in running water 24 hours, then dehydrated by passing through graded alcohols for 24 hours each (30, 40, 50, 60, 65, 70, 75, 80 per cent). The whole ovaries were then placed in 80 per cent alcohol for preservation, and blocks cut from them for sectioning were put for 24 hours in each of the following fluids: 85, 90, 95, 98 per cent alcohol, equal parts 98 per cent alcohol and ether, celloidin solutions 2, 4, 6, 8, 10, 12, 14 per cent. Finally the celloidin blocks were hardened with chloroform. As a routine, from one corpus luteum of each ovary or pair of ovaries two sections were made, 10 or 15 microns in thickness (the sections often passed through two adjacent corpora lutea), and in addition sections were made of any unusual or puzzling structure. Of these two routine sections one was stained with eosin and Ehrlich's hematoxylin, the other with Mallory's triple connective-tissue stain (acid fuchsin, phosphomolybdic acid, aniline blue, and orange G) . The latter staining method is particularly valuable, since it not only gives beautiful pictures of the connective tissue, but also of those very structures of the lutein cells which are most important for the present study.
Breeding Habits of the Sow
The females of the wild swine are monestrous and give birth to one litter a year, according to Kaeppeli (1908). But in domestication the sow undergoes an estrous cycle lasting 2 to 4 weeks, generally 3 weeks (Kaeppeli, Fleming: Veterinary Obstetrics). If the animal becomes pregnant, she may conceive again 5 weeks after delivery. The period of gestation is 16 to 17 weeks, usually between 116 and 120 days. Farmers commonly arrange to have litters produced in the spring and autumn, but in the material received at this laboratory, from stock raised for slaughter, fetuses are found in all stages of development, without great regard to the time of year.
The domestic sow is a very prolific animal, sometimes giving birth to as many as 19 pigs in one litter. Wentworth (1914) mentions an extreme case in which a sow gave birth to 23 pigs in one litter. John Hunter kept a record of the total progeny of one sow, in order to have a control for another, one of whose ovaries he had removed. During her life the normal animal gave birth to 13 litters, numbering in all 162 pigs, making an average of 12.5 pigs for each birth. In animals sent to the city for slaughter such high figures do not occur, for several reasons. The animals are sold young, often while bearing their first litters, which are commonly small in number; or if they are multipara, they are sold for the very reason that they are not profilic breeders. Moreover, the stock sent to the slaughter-house is the general product of the country-side, not always from selected droves. My records show that 6 is the commonest number of pigs in one litter, the extremes being 1 and 10.
General Features of the Corpus Luteum
On examining the ovary of the pregnant sow, the first objects to strike the eye are the extraordinarily prominent corpora lutea, which project nearly all their volume from the surface of the* ovary. In each ovary of the domestic sow I have found from 1, or none, to 10 recent corpora lutea, and in both ovaries from 1 to 16, most commonly 8. For the reasons which I have given, these figures are lower than would be found in well-bred, selected stock. It will be noticed also that the pigs are commonly fewer than the corpora lutea; in other words, that frequently not all the ova expelled at one time develop into pigs. The corpora lutea are generally ovoid or spherical in form, with outside diameters of 8 to 10 mm., most often about 10x10x10 mm. The full size is attained when the fetuses are about 10 mm. long, and does not change until retrogression of the corpus luteum is well advanced after delivery ; in other animals the corpus luteum is said to increase slowly in size throughout pregnancy. (For instance, the bat, as studied by Van der Stricht, 1912.) The customary name, corpus luteum, does not apply to swine, for the color of the cut surface of the fresh organ is a light pinkish gray, which only changes to yellow in the old corpora.
Besides the 1 to 10 corpora lutea in a given ovary, which experience shows mark the site of the ovulation which led to the pregnancy, there are often a number of smaller corpora lutea, which are bright yellow in color, and on section prove to consist mainly of dense connective tissue. In some ovaries there is still another older generation, dating from the second previous ovulation, which are seen as small white bodies nearly buried in the ovary. The remains of corpora lutea older than the second previous generation are not usually visible on gross examination, except as minute opaque bodies tucked in between the follicles and appearing in sections as the familiar hyaline areas. Since the corpus luteum remains as a distinct structure through the two succeeding ovulations, it follows that during the sexually active period of life the ovary should never be free from corpora lutea. This was true of the specimens examined by Kaeppeli (1908), but in our material there are many which contain nothing but follicles and what are apparently corpora lutea several periods old. Either these are atretic follicles, resembling corpora lutea, the sexually active period not having begun, or else it is not uncommon for the sow to pass an cestrual period without ovulation.
If, then, the corpus luteum is still evident after two successive ovulations following its formation, it becomes important to the progress of our study to know whether there is any likelihood of confusing the corpora lutea oi two different generations. When two or more generations are present, by microscopical study we always find such marked histological differences that there is no possibility of confusion, but on naked-eye examination it is not uncommon to find corpora lutea apparently intermediate, in size and color, between the two obviously different groups present. I have submitted all such cases, 8 in number, to microscopical study, which showed that in 3 cases corpora lutea markedly smaller than their mates presented the same histological appearances. A typical instance is given by a specimen in which there were two corpora lutea of pregnancy 8 to 9 mm. in diameter, and another of the same histological appearance only 4 mm. in diameter. In another case it was necessary to section every corpus luteum of both ovaries to find how many of them were recent. The moral is that critical points regarding the age of corpora lutea can not be decided without microscopical evidence.
Important also are the questions as to whether the corpora lutea of both ovaries, dating from the same ovulation, present the same histological appearance, and whether all the corpora of one ovary, dating from the same ovulation, are alike. To answer these questions, I have examined sections taken from corpora lutea of both ovaries of 19 sows. and a larger number taken from two or more corpora lutea of single ovaries, with the simple result that all the corpora lutea of the same pregnancy, in both ovaries are in cytological structure absolutely identical. Sometimes the central cavity of the follicle lingers longer than in its mates, or there is a minor difference in size or other gross characteristic, but in microscopic structure never.
HISTOLOGY OF THE CORPUS LUTEUM OF THE SOW. It is necessary, for the sake of clearness, to discuss briefly the perennial question of the ovary — that of the origin of the lutein cells— which above almost all other anatomical questions is notable for radical differences of opinion among the many capable investigators who have attacked it. The reader will recall that there are three views as to the source of the lutein cells of the mammalian corpus luteum. The first suggestion was that of von
Baer (1827), who believed that the lutein cells are derived from the connective tissue about the follicle, the theca interna; and the second is that of Bischoff (1842), who concluded that the cells come from the epithelial layer of the follicle, the membrana granulosa. The chief arguments in favor of the first view are that, firstly, as the Graafian follicle ripens, the theca cells show marked changes; they swell in volume, become rounded, and in short come to resemble the lutein cell very closely (see below, p. 86, and figures 16 and 18) ; secondly, the membrana granulosa of large follicles is often degenerated, and is believed to be cast off at the time of rupture; thirdly, such follicles as do not rupture are filled up by proliferation of the theca interna, and in this process of atresia become very much like corpora lutea. It is interesting to note that the latter view happens to have for prominent defenders those who have worked most carefully upon the sow's ovary, Benckiser (1884), Jankowski (1904), and Clark (1898). Great doubt has been cast upon it, however, by the careful and patient work of Sobotta (1896 ff.) and his followers, who maintain the origin of the lutein cells from the granulosa alone, and do not admit the participation of theca cells except to form the connective tissue reticulum.
A third theory, due I believe to Schron (1863), but upheld recently by Rabl (1898), Seitz (1896), Leo Loeb (1906), R. Meyer (1911), Van der Stricht (1912), and others, is that both layers of the follicle form lutein cells. The upholders of this view do not deny one of the arguments of their opponents, that regarding the changes of the theca interna during the ripening process, but they think it is not permissible to draw conclusions from the resemblances between follicular atresia and corpus luteum formation. Moreover, it is not true that the granulosa is cast off from normal follicles before or at the time of rupture, as is well shown in figure 1. Many of those who believe that the theca cells take part in lutein-cell formation describe some of the "theca lutein cells," as they call them, remaining for a time in little groups about the periphery of the corpus luteum and along the septa of connective tissue which penetrate it.
I think it may be said with fairness that most of those who have really devoted themselves to accurate studies of the corpus luteum with modern methods maintain the origin of the lutein cells either from the granulosa or from both layers of the follicle. With only a few specimens of very early corpora lutea at hand, I can not presume to enter this controversy, except to mention that in the sow the granulosa undoubtedly persists after rupture of the follicle, and becomes vascularized by vessels from the theca interna. After this there is a gap in my series until the corpus luteum becomes solid. However, from some new points in the histology of the corpus luteum, which I am about to mention, it seems ! that in the sow, at least, there is a fourth possibility to be reckoned with by the investigator who is to clear up this baffling question — namely, that the granulosa and perhaps part of the theca enter into the formation of true lutein cells, while some of the cells of the theca interna remain as distinct cells of special nature in the fully formed corpora lutea.
It is apparent at first glance at a well-prepared section of the corpus luteum of the sow that it is not the simple structure composed of a parenchyma of lutein cells and a framework of connective tissue which is figured in the manuals of histology. The lutein cells are indeed the chief element, but lying between them are other cells of at least two sorts, which can be found at all stages of pregnancy. For want of better terms I shall denote them as additional cells of the corpus luteum, types 1 and 2.
Type 1 (fig. 2, b). — About the periphery of the corpus luteum, and along the septa of connective tissue which penetrate it, in many sections one sees small groups of cells whicli have the following characteristics: round or oval nuclei containing a moderate amount of chromatin (more than the true lutein cells), and smoothly staining, very finely granular cytoplasm. The cells are round, or slightly elongated in one direction, and are smaller than the lutein cells, having a diameter of 15 to 20 microns. Now, if we examine the figures of some upholders of the compromise or joint-origin theory of the corpus luteum, for instance, those of R. Meyer (1911), we find that they show cells of this same appearance in this same situation, which are described in the text as cells of the theca interna, not yet changed into lutein cells or connective-tissue cells. Are these cells which I have described in the pig identical with the so-called theca lutein cells? The evidence for this view lies in the facts that they are present in the early stages of pregnancy, are then very distinct from the ordinary lutein cells, and lie mostly in the position in which the theca lutein cells are described. Against it is the fact that when the corpus luteum undergoes certain changes to be described later as distinctive of the latter part of pregnancy, these cells seem to increase in number, they follow the growing connective-tissue reticulum into all parts of the corpus luteum, and they come to resemble the true lutein cells very closefy; indeed, at times it is almost impossible to distinguish them; whence it might be inferred that the two sorts of cells are different only in state of activity. The origin of these cells must be left without further definite statement, although we shall come back to them later, as they form part of our criteria for determining the age of the corpus luteum by microscopical examination. Type 2 (fig. 3, b). — These are cells of varying form and size, generally rather smaller than the lutein cells, ranging in shape from spindles to branching and spherical forms, having a cytoplasm which stains deeply with eosin, and takes a dark brown or purple color with Mallory's stain, but sometimes takes instead the aniline blue ingredient of the mixture. Very often the CNdoplasm is filled with minute vacuoles which appear as bright, highly refractile granules about 1 to 2 microns in diameter. No matter what fixing reagent and stain is used, they remain vacuoles, and may even be found in osmic-acid material. The same vacuoles may occasionally be found in the lutein cells. I have not seen these cells in fresh teased preparations. They are not numerous; in one section from a very few to a hundred may be seen scattered among the hundreds of lutein cells, but they are not found in all ovaries, although they are easily seen, because of their dark stain and the fact that their cytoplasm is usually slightly shrunken, and hence they stand out distinctly. They seem to have been observed previously only by Delestre, who saw them, if I interpret his description aright, in the corpora lutea of cows (1910). As to the nature of these cells, it is difficult to say whether they represent a modification of the lutein cells or of the connective tissue. There seem to be transitions in both directions. But there is perhaps a clue in the specimen illustrated in figure 1, of a very recently ruptured follicle, in which the layer of theca interna cells nearest the granulosa is partly composed of cells similar in size, shape, appearance, and staining reactions to the cells in question. The significance of the cells is totally obscure to me.
Cytology of the Lutein Cell
The lutein cell possesses the elementary structures found in all animal cells. The nucleus, which in the active stages of the corpus luteum is round, vesicular, and poor in chromatin, has one or more large nucleoli. The cytoplasm, at certain stages to be specified below, is differentiated into two portions: an inner dense zone, the endoplasm, which in fixed specimens appears to be very finely granular, and an outer zone, the exoplasm. This latter zone, whether studied in fresh preparations or in carefully fixed sections, is of the most astonishing complexity. It is so full of granules and globules of diverse substances that the nucleus of the fresh cell can sometimes hardly be seen. Some of these granules are mitochondria; this may very easily be confirmed by making a preparation of fresh teased cells with Janus green; this dye gives a vital stain of the mitochondria, as shown by Bensley (1911). Other globules are fatty and are selectively stained, for the purpose of analyzing the cell elements, by any of the usual methods for neutral fats, such as Sudan III and osmic acid. The nature of the fatty substance of the lutein cell, as found in the horse, cow, and sow, has been studied microchemically by Cesa-Bianchi (1908), who thinks it is lecithin; the existence of this substance in the corpora lutea had previously been declared by Loisel (1904) on the basis of test-tube analyses. A striking contrast to the statements just made is offered by the report of J. W. Miller (1910) upon his studies of the human ovary, in which he states emphatically that there is no substance in the fresh corpus luteum of pregnancy which gives the reactions of the neutral fats, and that such reactions are obtained only during the puerperium; that is to say, in retrogressive corpora lutea. On the other hand, the lutein cell of the cat is often so honeycombed with fat that the vacuoles resulting from the action of alcohol and ether obscure the finer structure of the cell, so that I found it impossible to use some cats' ovaries which were at hand, in studies to corroborate the results of this research.
The most comprehensive description of the fatty inclusions in the lutein cells is that of Van der Stricht (1912), who worked with the ovaries of several species of bats. He finds the cells of young corpora lutea loaded with osmic-blackening material, but that there is a gradual decrease in amount of this substance until the latter part of pregnancy, when globules again begin to be deposited. It is his opinion that the early deposit of fat is epithelial in nature, representing a secretion of the corpus luteum, but that the later reappearance of presumably fatty material is a sign of senescence of the tissue. I shall detail my findings in the sows' lutein cells later; it suffices to say here that my preparations agree fully with those of Van der Stricht, in spite of the great difference in zoological position of the animals used.
In celloidin sections of corpora lutea from animals containing fetuses less than 40 mm. long, fixed with such common solutions as formalin, absolute alcohol, and bichloride of mercury, in which neither the fat nor mitochondria are preserved; and in osmic acid preparations, in which the fat is distinguished by its intensely black color, we find the peripheral part of every cell, the exoplasm, occupied by a most curious maze of clear spaces, of protean form, alternating with rings and rod-shaped masses of cytoplasmic material, which give the cell an almost indescribable but striking appearance (fig. 4).
Since these peculiar structures are found to be important guides in following the history of the lutein cell, it will be advisable to review briefly the conclusions of other investigators regarding similar appearances in other cells. In 1898 Golgi reported that in preparations of nerve cells from the central nervous system, made by a method of chrome-silver impregnation, he found the metallic substance deposited in the form of a network which lay in the cytoplasmic portion of the cell. This structure he called the "Apparato reticulare interne" About the same time Nelis and Holmgren both described net-like or strand-like appearances in the cytoplasm of nerve cells, which were seen as unstained areas in sections colored by dyes. Nelis called these structures "etat spiremateux du protoplasm" and Holmgren applied the term "Saftkanalchen" and later "trophospongium" to the essentially similar structures which he had found.
As these canals began to be discovered in nerve cells of all kinds, and then in cells of many other organs, their importance was recognized, and the many questions connected with them were vigorously discussed. Golgi denied the identity of his reticular apparatus with the trophospongium, but Holmgren affirmed it, at least in part. The latter view has taken the ascendant. Ram6n y Cajal believes them to be identical (1908). Cowdry (1912) , from the study of specimens prepared by the methods advocated by both discoverers, points out the great similarity of the two phenomena.
The functional importance assigned to the reticular apparatus by different authors is varied. It seems well established that the appearances are not artifacts, but are present during life. Some have thought them excretory channels, others circulatory paths for cell juices. Holmgren thinks they are often entered by processes of the connective-tissue cells and that they are the morphological expression of vital processes such as the formation of secretion droplets and the like. According to this view the system of canaliculi is an unstable, constantly changing structure, like any other physiologically active organ, a view which explains the variability of the histological appearance. Most investigators have conceived the reticular apparatus to be a constant component of the cells in which it is found, but von Bergen (1904), who studied the canaliculi in a great variety of cells from many organs, maintains the transitory nature of the structure. This latter opinion is certainly borne out by the study of the lutein cells, as will be shown later.
Cohn (1903), in a careful paper on the histology of the corpus luteUm, states that he was unable to see the trophospongium in the lutein cell, although he had followed the methods of Holmgren; but Vastarini-Cresi (1904), in a short note, mentions, without describing, certain preparations which he had demonstrated at Naples in 1904. Cesa-Bianchi (1908) describes and figures lutein cells of swine which show appearances resembling the trophospongium as observed in other cells. By the Golgi method Riquier (1910) demonstrated an intracellular reticular apparatus in the lutein cells of the cow and, what is very important in connection with the findings to be described in this paper, he showed that the structures undergo marked changes with the advancing age of the cell.
Now, if we take a well-prepared section of the corpus luteum of a pregnant sow, whose fetuses are perhaps 100 mm. long, and stain it with any strong cytoplasmic stain (Mallory's does excellently), careful 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. 8). If we take younger corpora lutea, we find the canalicular apparatus growing more and more complex. It assumes the form of wide V-shaped spaces, long clefts, and circles in the cytoplasm, so extensive that the nucleus is surrounded by only a narrow zone of endoplasm (fig. 7). But it is in the corpora lutea of pregnancies under 30 mm. that the highest development of the exoplasm is found (figs. 4 and 5). Here the exoplasm is occupied by a most curious and elaborate system of vacuoles, almost every one of which in turn contains a spherule of substance which, although it takes the same stain as the cytoplasm, yet has a more hyaline appearance, and is seen on section as a bright ring. Within many of the spherules is found another and tiny vacuole.
Curiously enough, the only investigator who has previously described this remarkable state of the lutein cells is F. Cohn (1903), who is mentioned above as denying the existence of the trophospongium in lutein cells. He observed all the appearances which have just been mentioned, in the corpus luteum of the rabbit at the height of its development, and undertook certain microchemical studies on their nature. He found that the innermost tiny vacuole contains a substance which blackens with osmic acid, presumably fat. In the sow's corpus luteum (fig. 6, a) I have been able to confirm this observation of Cohn, but not his other observation, namely, that the outermost vacuole, or clear space about the hyaline ring, is stainable with one of the methods for demonstrating myelin, Plessen and Rabinowitz's modification of Weigert's hematoxylin. I have seen the ring-like structures in tissue fixed with formalin, osmic acid, saturated mercuric chloride, Zenker's fluid, and absolute alcohol. Although I have not made an extensive search for these formations in various species, I have looked through a number of specimens prepared for other purposes which chanced to be at hand in the laboratory, and found the ring-like bodies in corpora lutea of the human species, the dog, and the cat. Add to these Cohn's original description in the rabbit, and we have seen the peculiar exoplasmic rings in the corpora lutea of ungu- lates, carnivora, rodents, and primates — surely a general distribution among the higher mammalia. Van der Stricht, in his long paper on secretory appearances in the corpora lutea of bats (1912), does not mention them, but the lutein cells of the bat (as are often those of the cat) are heavily loaded with fatty matter which would obscure the other details of the cytoplasm.
To avoid making an unproved assumption, in this paper I shall refer to these formations merely as exoplasmic formations, but I think there can be very little doubt that they are really one stage of the canalicular apparatus of Golgi-Holmgren, although granules within the canaliculi have never been described in other cells. In the first place, they can be traced back directly through all transitions from cells containing the classical form of trophospongium (figs. 4 to 10). In the second place, ring-like canaliculi are not new. Cattaneo (1914), in a paper on the canalicular apparatus of ovarian ova, figures appearances not unlike those under discussion, except that he shows no bodies within the canaliculi; and Bensley (1910) has shown very similar spaces in the root-tip cells of the onion. The ring-like structures are so beautiful and so striking that one wonders why they have not been emphasized long ago by some of those who have studied the ovary. Still, they require the best of fixation, and in the usual eosin stain are not as visible, as with Mallory's connective tissue stain and others which give a strong coloring of the cytoplasm. Yet I think they have been seen before and misunderstood. For instance, J. G. Clark (1898) states that the lutein cells of swine become full of vacuoles and their cytoplasm becomes shrunken, but he was probably actually describing the exoplasmic vacuoles, which we shall see to be evidences of cellular activity, not of senescence.
There is still another important histological element of the lutein cell to be considered, namely, certain granules of the endoplasmic zone which were described by Cesa-Bianchi in 1908. He found, in the perinuclear portions of the cell, a large number of densely packed granules about 1 micron in diameter. To demonstrate them, since the multitude of cytoplasmic structures makes them difficult to see in the living cell, he fixes the tissue in Zenker's fluid or saturated mercuric chloride, sections it in paraffin, and stains with any one of a number of staining combinations, especially hemalum-safranin light green, iron hematoxylin, and Mann's complicated methyl-blue-eosin stain. I have repeated the Zenker safranin light green method, but I find that the granules show up fairly well in formalin tissue stained with hematoxylin and eosin, and very well with Mallory's triple connective-tissue stain; indeed, I had observed them in my preparations before I knew of Cesa-Bianchi's work. In tissue fixed with osmic acid they appear as dark-gray granules against the light-gray endoplasm (fig. 9, c). With Mallory's stain they generally take the orange constituent of the mixture and appear as bright round bodies against a blue-gray background (fig. 7, c) ; but when the sections take the aniline blue ingredient of the stain more strongly— as they sometimes do, with the result that the nucleus becomes blue— it is interesting to note that the endoplasmic granules also stain blue. Cesa-Bianchi's figures, which are rather diagrammatic, show the granules clustered closely about the nucleus, bul I generally find them more or less diffusely scattered through the endoplasm. When the endoplasm reaches to the periphery, as it does in the later stages, then consequently the granules are seen throughout the cell. As to their nature and function we can only guess. As they are not dissolved from fixed tissues by ether, alcohol, or water, and because they are preserved by mercury salts, Cesa-Bianchi thinks they are of albuminous nature. I see no reason to think otherwise, although one hesitates to speculate on the chemical nature during life of minute objects which one does not see until they have been through •">(• to 70 changes of reagents. It has impressed me strongly that the staining reactions of the endoplasmic granules are the same as those of the peculiar rings found in the exoplasm, Now, in the life-history of the lutein cell, as we shall see later, the exoplasm recedes as tin' endoplasm becomes wider, and the granules seem to increase in proportion to the amount of the endoplasm. It occurs to me that possibly the two structures are but stages in the same process. Cesa-Bianchi presumes the granules to be connected with the supposed phenomena of internal secretion of the corpus luteum, and that his findings support the theory of an internal secretion; a view based of course on analogy, but one which must be kept in mind.
In the corpus luteum of the sow I do not observe the yellow granules of pigment which are said to be plainly visible in the corpus of the cow, and concerning which the remarkable discovery has recently been made by Escher (1913) that in chemical composition the substance is identical in all reactions with the lipochrome body carotin, found in carrots and green leaves.
Life History of the Lutein Cell of Pregnancy
In the earliest corpus luteum of known age of pregnancy in my series of sections, the lutein cells are fully formed and contain the peripheral ring-apparatus in a high state of development. In discussing them at an earlier stage we tread upon debatable ground. In a specimen to be described at length (p. 88, figs. 21, 22, 23), as an illustration of the interesting anomaly known as partial accessory lutein-cell formation, the cells of the granulosa of the follicle in question, at the point where the supposed lutein-cell formation is taking place, are swollen, possess vesicular nuclei, and contain in their cytoplasm very definite canals having within them a few minute hyaline rings. The resemblance to the fully formed lutein cell is striking.
In corpora lutea of pregnancy of 20 to 25 mm., all the lutein cells are of about the same size, are rounded in outline, and possess round vesicular nuclei relatively rich in chromatin. The endoplasmic zone is almost nil, being crowded out by the great development of the exoplasmic formations. The latter are composed of the ring-like structures previously described, in a high stage of perfection. At this time the cells contain considerable quantities of osmic-blackening substance, which almost fills some of the cells with good-sized globules of varying magnitude, besides occurring, as shown by Cohn, in tiny droplets at the centers of the spherical structures of the peripheral vacuolar apparatus (fig. 6, a).
As the cells grow older the following changes take place, as shown in figures 4 to 15:
(1) The nuclei become paler by reason of the lessening of the chromatin.
(2) The ring-like formations give place to less elaborate forms; that is, to simple clefts and V-shaped spaces in the cytoplasm, arranged concentrically to the nucleus. In corpora lutea of old pregnancies (150 to 290 mm.) no sign of the once elaborate structure is to be found in most cells.
(3) The fat content changes, at first decreasing. In the cells of the type most abundant at the middle of pregnancy, in which the endoplasmic zone occupies half or more of the cytoplasmic area, the globules of osmic-blackening material are found in amount much less than at previous stages, and clustered at the margin between exoplasm and endoplasm. Toward the end of pregnancy, when the fetuses are 200 mm. or more in length, the lipoid material has practically disappeared from the cell, being found, if present, as a few globules at the periphery. But still later, in the corpora lutea of pregnancies of 270 mm. or longer, many cells contain one or two globules twice as large as those previously seen, and which are often observed in ordinary sections as large vacuoles, sometimes as large as the nucleus, in the cytoplasm. This later deposition of fat I take to be a sign of senility of the tissue, whereas the large amount of lipoid in the early lutein cell would seem to be correlated with physiological activity. I have already mentioned the similar conclusion of Van der Stricht regarding the corpora lutea of the bat. It is very interesting to note that the occurrence of fatty substances in the connective-tissue cells and in those cells which I have called type 1 is in inverse ratio to that of the lutein cells. At 20 mm. only relatively few and very small osmic-blackening granules are found in the spindle cells, but as pregnancy advances fat is deposited in larger and larger masses until at 250 mm. the nuclei of nearly all the interstitial connective- tissue cells are hidden by the large, closely packed fat globules.
(4) Another change with advancing age of the fetuses is in the chromatic granules of the endoplasm, which appear to grow larger and more apparent, reaching sometimes a diameter of 2 microns.
Throughout all this change the size of the lutein cell remains practically the same.
Previous Observers on the Corpus Luteum at Various Stages of Pregnancy
While it has been recognized in a vague way by some authors that the corpus luteum is not the same at all stages of pregnancy, others hold that after the organ is formed it maintains its structure in the same state until delivery or at least until retrogression sets in, as, for instance, Ravano (1907) and R. Meyer (1911). At any rate, the attempt has never been made to trace the changes carefully and to relate the corpus to the stage of the fetus at all times of pregnancy. In general, investigators have recognized merely three stages of the corpus luteum, namely formation, maturity, and retrogression. Robert Meyer would divide the first stage into two, proliferation and vascularization. Cesa-Bianchi, on the basis of his work on the evidences of internal secretion in the corpus luteum of swine, mares, and cows, mentioned above (1908), finds three stages of the lutein cells. In the first or preparatory period, the lutein cells are of relatively small dimensions (15 to 20 microns), with clear and regular contour, round or oval form, nucleus almost central, with rich chromatic net and marked nucleolus, protoplasm homogeneous or uniformly granular, not presenting granules, vacuoles, or inclusions. This description does not fit the stage of formation as I find it in the sow, but it is not easy to say at what time of pregnancy Cesa-Bianchi places his first stage, as he does not give full measurements. His second period is characterized by the presence of abundant granulations in the cytoplasm. The lutein cells are 30 to 40 microns in diameter; in form they are irregularly polyhedral, with excentric nuclei which are vesicular, poor in chromatin, and possess numerous nucleoli. The cytoplasm presents two zones: the endoplasm, directly encircling the nucleus and filled with the chromatic granules described by him; and the exoplasm, presenting vacuoles of varying size. The third period is characterized by the presence in the cytoplasm of numerous drops of fat.
In 1910 Delestre published, from the Clinique Baudeloquo, the results of his studies in this direction, using the cow as the subject of his work, on account of the fact that the term of pregnancy is very nearly that of the human, being 284 to 285 days. Unfortunately his youngest corpus luteum of pregnancy dated from about 2\ months, the snout-rump length of the calf being 12 cm., so that he missed the very stage at which the exoplasmic apparatus is at its height, if the sow's corpora lutea are like the cow's. At this stage he describes four cellular elements of the corpus luteum: (1) The lutein cells, presenting no particular feature. (2) Cells somewhat larger than the lutein cells, having lightly staining protoplasm. I do not know what these may be, as I have not seen them in the sow. (3) Plasmodial masses with 5 or 6 nuclei, destined to form young lutein cells. I have not seen these in the sow. (4) Cells which, if I interpret the description rightly, are like those mentioned above as additional cells of the corpus luteum, type 2. Delestre states that at 4 months the plasmodial masses have disappeared, giving rise to young lutein cells, which are smaller than adult lutein cells, and are grouped, several together, in one mesh of the connective tissue. There are now two sorts of lutein cells, one containing fat, the other free of fat. At 4^ months the number of cells containing fat is augmented and the young cells are rare. At 5 months all the young cells have disappeared and the adult features predominate, most of the cells containing fat. At this time signs of degeneration appear, in the form of vagueness of outline of some of the cells, poor staining reaction, and dried-up skeleton-like nucleus.
Delestre gives a comparative table of the characteristics of the corpus luteum at the beginning and end of pregnancy, which in most particulars applies equally well to the sow, although I can not confirm his account of the formation of young lutein cells from plasmodial masses, nor the description of degenerating cells at such an early time. With these simple outlines of the life-history of the corpus luteum as described by previous investigators, and with the extensive cytological data now at hand, we have the tools for the main part of our task, which is to relate the changing anatomy of the corpus luteum to the stages of advancing pregnancy.
Characteristics of the Corpus Luteum at the Various Stages of Pregnancy
I had hoped that the gross characteristics of the corpus luteum would be of help in the problem at hand, but I have been forced to the conclusion that the differentiation of corpora lutea of varying ages is only to be made with the oil-immersion lens. The corpus luteum of the youngest pregnancy in my series is already solid, so we gain nothing from the size of the central cavity, as we might in other species, such as the human, in which the cavity lingers longer. The blood-vascular system changes little during pregnancy, since there is the same amount of tissue to be supplied. The color is variable and gives no distinction of age. Sometimes in early corpora lutea the rapid increase in volume of lutein tissue causes a bulging of the inner substance of the corpus luteum through the point of rupture, the so-called "Propf" of the German writers. If we find an ovary in which the corpora lutea possess "Propfen," or several of the corpora contain slight remains of the former central cavity in the form of small spaces containing old blood, then probably the ovulation was fairly recent; but, on the other hand, in pregnancies of the first month the corpora lutea are often solid and homogeneous.
We must resort, then, to the immersion lens and a study of finer histological details. Because it will make the results more convincing, I shall mention briefly the method by which the material was studied. As stated in the introduction, all the specimens had been prepared in the same way. All the specimens from pregnancies of 20 to 25 mm. were taken together and carefully studied, then all those from pregnancies of 50 to 55 mm., and so forth; and in this way was obtained an idea of the nature of the corpus luteum at a series of stages from an early period to term. Next the specimens of intervening length were examined, and finally from these observations it was found possible to divide the life of the corpus luteum into a number of definite periods according to the length of the fetus. When I was sure of being on firm ground, I began to go through box after box of sections, registering a diagnosis of the age of each corpus luteum, and then looking it up in the records. With increasing experience it became possible to place the specimen in its proper period practically every time, never varying from the correct stage by more than one of the seven periods of pregnancy. Of course these periods into which I have found it possible to rank my specimens are purely empirical, and represent lines which I have drawn at the limits of my ability to distinguish the stages of the cytological changes. I had at first several more periods interpolated between those given, but found that I could not regularly distinguish them. In short, it can generally be told, by examination of a section of a corpus luteum, on the basis of distinguishing characteristics to be given below, in which one of the following periods of pregnancy the specimen falls:
Exoplasmic development, first part. . Exoplasmic development, seeond part..
Endoplasmic development, first part Endoplasmic development, second part Beginning retrogression
Less than 20 20 to 30 30 to 55 55 to 140 140 to 170 170 to 220 220 to 290
Needless to say, the corpus luteum, being an organic body, presents considerable variety of structure, so that these periods overlap, and pass gradually into each other, in such a way that, besides the specimens which fall near a border-line and hence are naturally difficult to place, a small number give an appearance which experience shows is normal to corpora lutea of older or younger age, just as in a series of embryos a few will exceed or fall behind the others in development.
Criteria of Age of the Corpus Luteum
First period - preparation
Pigs less than 20 mm. long, duration of pregnancy less than 25 days (fig. 4). The corpus luteum of pregnancy does not attain full size until the twentieth day or thereabouts, but variation in size is so marked that no dependence is to be placed on dimension as a sign of early corpora lutea. On section the following features may be noted : The tissue is very densely packed, so that the nuclei of the slender connective- tissue cells seem relatively numerous. The septa of connective-tissue fibers are rather marked, but are narrow and compressed, and still preserve the radial arrangement in which they invaded the space. Between them are packed the lutein cells, which are polymorphic, but usually elongated in the direction of the radius of the corpus luteum; they are much more varied in size than in the succeeding stages. The nuclei are more chromatic than they will be found later. The ring-forms occur in every lutein cell, and are so large and numerous that there is practically no endoplasm. Fat is present in greal quantity in the lutein cells, as in the next stage, under which it will be described. In the interstitial connective-tissue cells of the corpus luteum there is a moderate quantity of fat in fine granules.
Second period - height of exoplasmic development
Pigs 20 to 30 mm. long, approximate duration of pregnancy 25 to 30 days (fig. 5). The striking feature of this stage is that all the lutein cells are in the same state. All tend to be rounded in outline, all are of the same size, and show a uniform development of the exoplasmic apparatus, with ring-forms in every cell. The endoplasm is still very limited in amount. The nucleus is not so chromatic as in the previous period. Osmic-blackening substance is present in considerable amount in the lutein cells, in some cases almost filling the cells with globules of diameters varying from 2 to 5 microns. In the most typical cases the globules are thickly clumped at one end of the cell, or at both, but there is often a cluster of fat globules about the nucleus; indeed, at this stage the distribution of fat is almost general, at least in the lutein cells. However, the connective-tissue cells show little fat; in osmic preparations they show at most a few small black granules (fig. 6).
Third period - height of exoplasmic development, second part
Pigs 30 to 55 mm. long, approximate duration of pregnancy 30 to 40 days (fig. 7). The previous stage passes insensibly into this, in which in many cells the exoplasmic structure is more varied in form; some cells contain the ring-forms (fig. 7, a), others present irregular channels and clefts (fig. 7, b). The endoplasmic zone about the nucleus can now be seen, and in a very few cells reaches to the periphery, in which case there are no peripheral spaces in the cytoplasm. In the endoplasm are seen the chromatic granules described by Cesa-Bianchi (fig. 7, c).
Fourth period - transition
Pigs 55 to 140 mm. long, approximate duration of preg- nancy 40 to 75 days (fig. 8). The ring-structures are disappearing, and become rare in the latter half of this stage. I have seen them but once or twice in ovaries from pregnancies above 140 mm., and therefore set that as the upper limit of the period. It is noted that the progressive changes of the cells are a little earlier at the periphery of the corpus luteum than at the center, and hence estimates of the age of the corpus luteum must be based upon a study of the whole area, giving more weight to the state of the peripheral tissue. In the earlier half of this period one sees an occasional cell without any exoplasmic clear areas or channels; in other words the entire cell, from nucleus to border, is occupied by the homogeneous endoplasm. Such cells become fairly common toward the latter half of the period, and in some fields of the lens may form half of the lutein cells. Fat has decreased in quantity in the lutein cells, where it is found chiefly at the border between endoplasm and exoplasm. In the lutein cells it occurs in globules of varying size (fig. 9, a), but in the connective-tissue cells in smaller granules only (fig. 9, b).
Fifth period - endoplasmic development
Pigs 140 to 170 mm. long, approximate duration of pregnancy 75 to 105 days (fig. 10). A marked change has taken place in the corpus luteum, although it is not strikingly shown in the figure — a change which has for its most obvious feature a great increase in the diversity of the cells. Although the ring-forms have disappeared from all but a rare cell or two, yet many cells show considerable peripheral canalization. Next to such a cell may be a lutein cell which shows only endoplasm (fig. 10, a). Moreover, there has been an increase in the amount of connective tissue, so that in many sections the lutein cells are somewhat spread apart. Between them, adding to the diversity of appearance, lie the several kinds of cells which we have mentioned as members of this complicated structure, namely, the branching or spindle cells of the connective tissue, with their product, the reticular fibrils; the darkly staining cells which I have called type 2; and, most notable of all, many cells of that type which I have called additional cells of the corpus luteum, type 1, which, with their nuclei, more chromatic than that of the lutein cell, and their homogeneous cytoplasm, resemble the "theca lutein cells" of other writers (fig. 10, b). With the increase of the connective tissue, penetrating between and spacing apart the true lutein cells, these smaller, homogeneous cells are found much more generally scattered through the corpus luteum than before. In osmic preparations they are often loaded with large densely-packed black granules (fig. 11, a). Others of the interstitial cells of the corpus luteum, of all types, contain only fine, diffusely scattered black granules (fig. 11, b). In the lutein cells themselves the amount of osmic-staining matter is progressively lessening; the globules are found in a narrow zone at or near the periphery of the cell. I think that most of the vacuoles seen at the border of the cell in Mallory's material at this stage are due to the fatty substance, and that the lesser number only are due to remains of the once extensive peripheral canalicular system.
Sixth period - endoplasmic development, second part
Pigs 170 to 220 mm. long, approximate duration of pregnancy 105 to 110 days (fig. 12). The diversity of staining reaction is less marked than in the preceding stage, and the diversity of size of the cells is less noticeable, because in the first place the lutein cells have lost the peripheral vacuolization, while, secondly, the smaller interstitial cells of the corpus luteum have grown in size, and therefore the two types of cells so resemble each other that it is difficult to distinguish them. Fat is very slight in amount and limited to the periphery of the lutein cells, where a few granules are found. The interstitial cells of the corpus luteum are often loaded with large fat globules (fig. 13).
Seventh period - beginning retrogression
Pigs 220 to 290 mm. long, approximate duration of pregnancy 110 days to term (fig. 14). The only difference between this stage and the preceding is that here the lutein cells frequently contain one or more fat globules larger than any previously seen, which are found near the periphery of the cell, and at first in cells near the periphery of the corpus luteum (figs. 14, 15). The oldest corpus luteum of my series (290 mm.) contains many such vacuoles.
Theoretical Considerations concerning the Life-History of the Corpus Luteum
I have purposely omitted theoretical discussion from the foregoing description, because I do not wish false analogy or faulty deduction to belittle the value of these observations as criteria for determining the age of lutein cells of pregnancy. I may be entirely wrong in ascribing to the cells which I have called type 1 an independent existence and a classification different from the true lutein cells, and in thinking that they resemble the theca lutein cells of the descriptions of Rabl, Cohn, and R. Meyer. Those who believe that the origin of the lutein cells from the theca interna alone is incontrovertibly established will simply say that, as all the cells of the corpus luteum arise from one source, what I have described are merely stages or variants of one type of cell. On the other hand, those who adhere to the now widely accepted view that both layers of the follicle enter into the formation of the lutein cells should see no difficulty in my tentative proposition that, in the sow at least, the theca cells perhaps persist partly as distinct cells throughout pregnancy. For, as I have mentioned, Rabl, Cohn, and R. Meyer have thought them to persist well into pregnancy, and Van der Stricht indeed thinks that they persist throughout pregnancy as cells of internal secretion, perhaps less active than the true lutein cells.
As to the lutein cell itself, the present findings, that the cells are apparently in a high state of organization from early pregnancy to term, would seem to give some strength to the contention that the corpus luteum has a function of importance in pregnancy. Empirically it has been shown clearly that extracts and powders of corpus luteum have a beneficial effect in certain disturbances of the reproductive system in women (see Burnam (1912) for full account and literature to date) ; and it has been found that extracts of corpus luteum when injected into dogs promptly cause a lowering of the blood-pressure. None of those who have worked in this field, so far as I know, have tried the effects of corpora lutea of definite age. There is, however, certain evidence that the corpus luteum during the first half of pregnancy has a different potency than in the second half, namely, the fact discovered by Fraenkel (1903) and confirmed by Marshall and Jolly (1905), Niskoubina (1909), Dick and Curtis (1912), and others, that extirpation of the corpus luteum during the first half of pregnancy is almost invariably followed by abortion. This fact, which seems so true for guinea-pigs and rabbits, has been flatly denied to hold good in animals by Daels (1908), and for human patients by Essen-Moller (1904), Graefe (1905), Flatau (1907), and Sokoloff (1913), while Puech and Vanverts (1913) propose the compromise statement that the corpus luteum is useful in an accessory way, but not absolutely necessary to the embedding and early development of the human embryo. Unfortunately for our present discussion, the experiment has not been tried on sows, which are not convenient laboratory animals. I agree with Cohn and Van der Stricht that the microscopic appearance of the lutein cell seems to favor the theory of an internal secretion of the corpus luteum, and I would add that the corpus luteum, while apparently in a high state of activity from the beginning to the end of pregnancy, shows greatly different forms of cell-structure in the earlier and later months. We may hope that this hint will be put to further test by experiment and clinical observation.
Distinction Between the Corpus Luteum of Pregnancy and of Ovulation
The history of the corpus luteum for 350 years has been a curious mingling of truth and error. Discovered by Volcherus Coiter in 1573, the corpus luteum was thought by Regner de Graaf to be an evidence of pregnancy or of previous child-bearing. Abernethy and Sir Astley Cooper swore away the innocence of a dead woman in a court of law because one of her ovaries was found at post-mortem to contain a corpus luteum. About the earlier decades of the nineteenth century it became known that every ovulation is followed by the formation of a corpus, and then arose the still unsettled discussion as to whether the corpus luteum of ovulation can be distinguished from the corpus of pregnancy. Interesting accounts of the earlier phases of this question are found in Dalton's Prize Essay of the Philadelphia Academy of Sciences for 1851, and in Taylor's "Medical Jurisprudence." Next the microscope was called to aid, with little effect. De Sin6ty (1877) believed the two sorts of corpora to be exactly alike. The same statement is made by Ravano (1907). Marshall (1910) sums up his opinion in the statement that the two kinds of corpora are in the earlier stages identical, and otherwise essentially similar. Niskoubina (1909) thinks that the corpora lutea of pregnancy of rabbits contain more fat than the corpora of ovulation, but Fenger, working on the ovaries of cows, analyzed hundreds of corpora lutea, and found the fat content equal (1914). Miller (1914), in the latest publication upon this subject, holds that the corpora lutea of pregnancy (human) may be distinguished from those of ovulation by the absence of fat-reaction, of colloid degeneration, and of deposition of calcium. It is plain that the divergent opinions upon the question are due to incomplete knowledge of the corpus luteum at all its stages. Meanwhile the clinicians are beginning to put the matter up to the anatomists anew, for instance, with such statements as that of Dannreuther (1914), that the dried corpus luteum of pregnancy is much more efficient therapeutically than the corpus luteum of ovulation.
I have sections of ovaries from about 25 uteri which did not contain embryos, many of them having been carefully examined by another investigator who was searching for early embryos. No doubt a few of them are from pregnant animals after delivery, so that the corpora lutea may not all be those of ovulation. At any rate, it can be said with certainty that the presence of embryos in the uterus is to be diagnosed by microscopic examination of the ovary. The difference between the corpus luteum of pregnancy and that of ovulation is like the difference between an army and a mob. In the corpus luteum of pregnancy there is a regularity of structure which is lacking in thai of ovulation. For instance, in the latter we find cells having highly developed exoplasm side by side with others having a smooth undifferentiated cytoplasm and large fat-vacuoles in them (fig. 17). The fat-vacuoles of the connective-tissue cells are usually much larger and more numerous than in the corpus luteum of pregnancy, so that the sections are riddled with holes. It appears likely, though it can not be proven, since we have no way of determining the age of a non-pregnant corpus luteum from the slaughter-house, that retrogression sets in before the corpus is fully formed, and affects the cells in an irregular way, so that they do not all progress through the stages of their life history at one time. Whether or not there are differences of note in the first 15 days I can not say, as I have no data for corpora lutea of pregnancy in the first two weeks. After this period the two sorts of corpora lutea can be distinguished from each other.
Influence of Pregnancy on the Structure and Function of the Graafian Follicles
It has been known for a long time that pregnancy interrupts the function of the corpus luteum; that in the presence of an embryo in the uterus there is normally no ovulation and therefore no formation of new corpora lutea. In animals which undergo rut or menstruation these phenomena are also interrupted by pregnancy.
To understand the causes underlying the non-occurrence of ovulation during pregnancy, we must know the life-history of the normal follicle. Fortunately this is a subject upon which there is substantial agreement between all observers. As is well known, the fully formed resting follicle consists of three layers: the membrana granulosa, of presumably epithelial origin; the theca interna, probably of mesenchymal origin; and the theca externa, of mesenchymal origin. The exact histological characteristics of the three layers vary slightly in different animals, but in the pig the granulosa is composed of 3 to 10 layers of rounded cells (the lowest layer, next the theca, is columnar) with round, darkly staining nuclei and little cytoplasm (fig. 16, a). The theca interna is composed of 5 to 10 layers of short, spindle-shaped cells, with moderate amount of cytoplasm and round, or oval, nuclei, not so chromatic as those of the granulosa (fig. 16, b). The theca externa is made up of definitely spindle-shaped cells of connective-tissue derived from the ovarian stroma (fig. 16, e).
Now, one of two fates awaits the unripe follicle in the course of time. It may never attain ripeness, but instead may undergo degenerative changes leading to obliteration.
The process of destruction, which is called atresia, consists of the degeneration of the granulosa cells, shrinkage and disappearance of the ovum, and filling of the cavity by enlarged cells of the theca interna, so that the atretic follicle resembles a corpus luteum, and like the latter is finally replaced by a hyaline scar. On the other hand, the follicle may proceed to ripeness, in which case the granulosa tends to range its cells in rather marked layers, and the cells of the theca interna increase in number, show mitoses, become polygonal, swell in size, and have deposited in them numerous fat granules, so that altogether they show a notable resemblance to lutein cells (fig. IS, b). Next, through causes not yet understood, the follicle bursts, the ovum is extruded, and a corpus luteum is formed. Should rupture not occur, atresia may take place in the ripe as well as the unripe follicle. As one of the causes of the non-ovulation during pregnancy, it has been pointed out, especially by De Sinety (1877), Stratz (1898), and Schulin (1881), that atresia of the follicles tends to be very marked during pregnancy. Sandes has confirmed this in the marsupial Dasyurus (1903). In the pig it is certainly true. I have not a sufficiently large number of non-pregnant ovaries to make a numerical comparison, but it can be stated that a great part of the larger follicles in pregnant animals are degenerating. Yet in very many ovaries of pregnancy fairly large intact follicles are found. Very interesting in this connection is the observation of Pearl and Surface (1914) that ovulation in the domestic fowl can be inhibited by the administration of corpus-luteum extracts.
It has been suggested by some that the follicles do not grow to full size during pregnancy, and that hence the crop of follicles which gave rise to the embryos in utero is not followed by another crop until pregnancy is terminated. This view has been studied by Leo Loeb (1906). He reports that in many of his animals the follicles went on to full size during pregnancy, but that they did not rupture, as they would in the absence of pregnancy. It is unfortunate that microscopic studies were not made, for, as pointed out by Robert Meyer, in the human ovary mere size of the Graafian follicles is a faulty criterion of their state; those which appear large and ripe may in fact be in a stage of cystic atresia.
The diameter of the ripe follicle of the sow is said by Kaeppeli to be 5 to 8 mm., and in this statement he is followed by Schmaltz (1911), but I believe that normal ripe follicles may grow much larger than 8 mm. Eenckiser (1884) included in his studies on the origin of the corpus luteum a perfectly normal ripe follicle 11 mm. in diameter. I collected 24 pairs of ovaries at random, without selection except to exclude specimens containing corpora lutea and cystic formations. The largest follicles in each ovary varied in diameter from 1 to 10 mm. ,On microscopical examination even the largest follicle of these ovaries proved to be normal and ripe. The lower limit of ripeness is difficult to set. I have seen 3 mm. follicles which were apparently ripe; and a recent corpus luteum of ovulation may be as small as 5 mm., showing that the follicle from which it proceeded must have been no larger. In the hundreds of ovaries I have examined I have seen many such large follicles, and I have no doubt that had they been examined microscopically they would have proved normal.
As a contrast to this, in my entire series of ovaries from pregnant sows no follicles were found having an outside diameter of more than 6 mm. with two exceptions, and I did not find ripe follicles in any ovary of the series. The conclusion is simple: in pregnancy the Graafian follicles may attain or maintain a size at which, in the absence of pregnancy, ripening and rupture might occur. But they do not usually attain the larger diameters reached by follicles in the non-pregnant ovary; and when they do happen to reach large size, they are found to be either unripe or atretic.
Fellner (1909) believes that in the human ovary a few follicles may remain ripe or become ripe during pregnancy. Ravano (1907) has taken the still more advanced standpoint, based on the study of ovaries from 60 pregnant women who came to the operating table or to post-mortem during pregnancy. He found many follicles "approaching ripeness," and also thinks that he had three cases in which ovulation had occurred during pregnancy. These were merely cases in which two corpora lutea were found without twin pregnancy ; and the microscopical details given are so slight that I do not think these cases are sufficient evidence on which to base such a statement as Ravano's that ovulation and corpus luteum formation subsequent to that which gave rise to the fetus occur in 5 per cent of all human pregnancies. In spite of these cases I retain the opinion that the rupture of a Graafian follicle in an ovary containing a corpus luteum of pregnancy during term is an extremely rare occurrence. Keller (1913) did not find a case in 24 human cases, Fellner ( 1909) in 13 cases, nor Seitz (1905) in 36. Ruge (1913) studied 106 human ovaries without finding a freshly ruptured follicle, and he thinks the presence of a recent corpus luteum excludes the possibility of the rupture of a follicle. The case which I have to report seems to be one of those classic exceptions which prove a rule.
The uterus of specimen No. 70 contained three fetuses whose crown-rump length measured 70 to 75 mm. The left ovary contained no corpora lutea; the right ovary contained 8 prominent corpora lutea about 11 mm. in diameter and a number of follicles measuring 5 mm. in diameter. Between one of the corpora lutea and one of the follicles was found a body of ovoid shape about 2 by 2 by 3 mm. in size, which presented on its surface a crater-like scar covered by a few tags of fibrin, resembling exactly the healing point of rupture of an ordinary corpus luteum, but only about 0.5 mm. in diameter (fig. 19, a). A section of this structure showed that it was indeed a recently ruptured follicle (fig. 20). The granulosa was several layers thick, of normal appearance, and intact except at its point of rupture, where it and also the theca were replaced by a recent scar. The granulosa was rather wavy in contour (fig. 20, a), and beneath it lay the theca interna, thicker than usual (fig. 20, b). If I interpret the appearance aright, neither layer showed signs of conversion into lutein cells. I believe that this unusual specimen is due to an accidental rupture of a Graafian follicle, probably by pressure between its neighboring growing follicle and corpus luteum.
Abnormalities of the Lutein Tissue
An interesting anomaly of the ovary is partial accessory lutein-cell formation, to which attention was called by R. Meyer (1913) . This consists of the formation, during pregnancy, of lutein cells in atretic follicles or even in ripe follicles, which in some human ovaries is said to go so far that a full corpus luteum may be formed which is finally indistinguishable from the true corpus luteum proper to the pregnancy, which is found in the same ovary or its mate. This phenomenon, at least in its advanced state, must be very uncommon. In the sow I have seen a case of what is apparently the same partial lutein formation in a normal ripe follicle, not during pregnancy, but in the presence of a corpus luteum of ovulation.
The right ovary of specimen No. 136 contains 4 corpora lutea 5 mm. in diameter, which appear on section to be young corpora lutea of ovulation. In addition there is a follicle 10 mm. in diameter, to be described. The left ovary contains 3 corpora similar in gross appearance to those of the right ovary, except that one of them is slightly cystic. On section of the large follicle in the right ovary, it is found that over almost the entire circumference the wall presents, as would be expected, the structure of a normal unripe follicle. At one point, however (fig. 21, a), for a space of 3 mm. the whole theca interna is doubled or tripled in thickness, its cells show the appearance of ripeness, and have begun to invade the granulosa (fig. 22, a). The granulosa cells, in turn, are 20 to 30 microns in diameter and many of them have vesicular nuclei with prominent nucleoli; they are filled with fat- vacuoles, and some of them have in their cytoplasm the ring-formations of young lutein cells. In short, the granulosa cells are turning into lutein cells (fig. 23).
Seitz (1905) has emphasized the transformation of theca-interna cells of atretic follicles into lutein-like cells as a physiological phenomenon in pregnancy. While I do not consider this process as uniformly present in the sow, as Seitz maintains it is in the human, still we do occasionally find follicles lacking the granulosa, but with the theca interna cells full of fat-vacuoles and enlarged just as in ripe follicles. It is hard to see how it is to be decided whether these were normal ripe follicles at the beginning of pregnancy, which are merely becoming atretic, or whether they were formerly atretic follicles in which the formation of theca-lutein cells is taking place, as Seitz would have it.
Here we come to the border-line between normal and pathological anatomy of the ovary. While my specimens of ovarian cysts were not collected with a view to formal study, still I shall risk presenting a few incidental observations in the hope of calling attention to the unusually promising opportunity offered by the sow's ovary for the study of the pathology of the Graafian follicle and the corpus luteum. Cysts are extremely common, are often multiple, and are frequently found in ovaries containing normal corpora lutea of ovulation, so that comparisons can be made. It needs only time and patience to collect a great number, and the result would undoubtedly give us explanations of much that is obscure concerning these tumors.
In glancing over the 13 specimens of ovarian cysts lined by lutein cells which I have collected, it at once appears that they fall into two classes. First, there are cases in which one of a crop of corpora has merely retained its original cavity beyond the normal time, the cavity has become fixed, and may have become much enlarged. Second, there are cystic cavities in the ovary which are lined by lutein cells, but which we can not as surely say came from the corpora lutea, as they may merely represent atretic follicles in which lutein-cell formation has taken place. One's view of the origin of these cells will depend entirely upon the view of the origin of the lutein cells of normal corpora. As will have been gleaned from this paper, the writer has tentatively come to the belief that the true lutein cells are derived from the granulosa cells, but that the theca interna gives rise to cells resembling lutein cells, which seem in part to remain as special cells in the fully formed corpus luteum, but most of which are lost, either by reverting to connective-tissue cells of the ordinary kind or by becoming themselves genuine lutein cells. On this theory, we should have the following kinds of lutein-cell cysts:
I. Arising in corpora lutea which have remained cystic or have undergone cystic degeneration. II. Arising in follicles:
a. Accessory lutein-cell formation from both layers of normal follicles, as described by Meyer.
b. Accessory lutein-cell formation from the theca alone, as in atretic follicles.
It is of clinical importance to know something about the life-history of lutein-cell cysts— for instance, whether such a cyst may persist and by its activity inhibit ovulation, as in certain women, in whom persistent corpora lutea seem to cause sterility. Now, the lutein cells in cystic tumors are not often as well preserved as in normal tissue, but I believe that in all the specimens in hand the lutein cells are of the same age as those of the normal corpora in the same ovary. I have not seen lutein-cell cysts containing active-looking cells of different age from the corpora lutea present. Presumably, then, the lutein cells of cysts are as transient as normal lutein cells, and in cases where the cystic formation persists, the lutein cells lining it probably revert or disappear, so that finally the cysts can not be distinguished from an old Graafian follicle cyst.
In the 128 specimens from pregnant sows of which I have record, there was only one case of lutein-cell cyst. In the same number of non-pregnant ovaries, containing corpora lutea of ovulation, we should find 5 or 10 lutein-cell cysts. For comparison, I have looked up the cases indexed as corpus-luteum cysts in the records of the gynecological service of the Johns Hopkins Hospital. There were 19 cases available, all discovered at operation for other conditions. One patient had passed the menopause (61 years) ; the others were from 16 to 42 years.
8 had never been pregnant.
4 had their last child or miscarriage 6 or more years before.
5 had their last child or miscarriage 22 months to 4 years before. 1 had miscarried 4 months before, at the second month.
1 had miscarried 2 weeks before, at the sixth week.
Apparently, then, the corpus luteum of pregnancy is a comparatively less frequent seat of cysts than that of non-pregnant animals, a fact in accord with the general rule that abnormal alterations are less frequent in organs exercising a needed function.
Migration of the Ovum in the Sow
.Since the uterus of the sow is bifid nearly its whole length, it is very easy to count the number of fetuses on each side. When this is done, it is often observed that there are more pigs on one side of the uterus than there are corpora lutea in the corresponding ovary. In a series of 117 uteri, I found 28 in which there was 1 more pig than corpora lutea on a given side, 13 cases with 2 more pigs than corpora lutea on one side, and 2 in which the pigs num- bered 3 more than the corpora lutea of the corresponding ovary.
This phenomenon may be explained in three ways: (1) corpora lutea may not have formed at the site of the follicle which gave rise to the supernumerary pigs; (2) there may have been two or more ova in one follicle, which therefore produced two or more embryos, but only one corpus luteum; (3) one or more ova may have crossed the abdominal cavity and entered the opposite tube (external migration), or passed down to the junction of the uterine horns on its own side and into the opposite tube from below (internal migration).
(1) The failure of a corpus luteum to form at the site of a ruptured follicle is not unthinkable, but must be very rare. In hundreds of sows' uteri I have never seen a case of pregnancy without corpora lutea; nor do I know of any really unexceptionable cases reported from the human or any other of the mammalia which give birth to a single infant, in which such a phenomenon would be more easily detected. Fellner (1909) has mentioned one case in a woman who died on the day of delivery, Ravano (1907) cites four, also from women prac- tically at term, and Miller (1914) one from a case of eclampsia. Although these cases are of interest otherwise, the absence of a corpus luteum at term can not be taken as evidence that one had not been present earlier in pregnancy, especially since all these cases were complicated by disease.
(2) In the sow, however, I have found a uterus in which there were T> fetuses, hut only 4 corpora lutea in both ovaries. Such a case, if our first hypothesis he excluded, can be explained only by the second, namely, that two of the pigs originated from a polyovular Graafian follicle, and this latter explanation is based on anatomical facts. Follicles con- taining more than one ovum have been known for many years, and have been recorded from man, the cat, dog, sow, certain bats, and other animals. Ancel (1903) thinks they are especially common in the dog, in which he has found follicles with 2, 3, 4, 5 ova. Arnold (1912), in an interesting report with review of the literature, describes a human case in which a large proportion of follicles were polyovular, some of them having as many as 18 ova. The anatomical origin of the condition is still obscure. The question arises whether the phenomenon is sufficiently common to account for the fact that in swine 37 per cent of uteri have more pigs in one horn than there are corpora lutea in the corresponding ovary. Schmaltz (1911) states that polyovular follicles are seen in almost every preparation of the sow's ovary, sometimes having as many as 6 ova, 4 to a follicle being frequent, but he thinks that such follicles are usually doomed to become atretic and rarely attain maturity. In sections I personally have found only 2 polyovular follicles, one of them with 2, and the other with 3 ova, and these follicles were both small and undeveloped. The members of a class in histology were requested to watch for the condition when studying the ovary of the sow, and one of the students found and showed me 2 ova which he had discovered in a large ripe follicle. The conclusion is that some of the 43 cases under discussion may have been due to the fertilization of ova from polyovular follicles, but that the latter certainly do not occur frequently enough to explain all the cases, since for this it would be necessary for at least one follicle to be polyovular in one-third of all the groups of follicles maturing at once.
(3) On the other hand, migration of the ovum is a well-attested fact, in the human subject, and has been produced experimentally in animals by Leopold (1880), who excised one ovary and the opposite tube, and found that the animals could still become pregnant. Howard A. Kelly performed the same operation in a human patient, for the cure of disease, and the woman later became pregnant. Other interesting examples have been mentioned by J. Whitridge Williams, who found the corpus luteum in one ovary and the embryo in the opposite tube, in 5 out of 30 carefully recorded cases of extra-uterine pregnancy. These are all cases of external migration. The occurrence of internal migration has never been conclusively proven. The frequency of external migration of the ovum is difficult to esti- mate in the human subject, since it is demonstrable only in the presence of some abnormal condition, such as the lack of one tube and the opposite ovary, a bicornate uterus, an extra- uterine pregnancy, or the like. Mayrhofer (1876) estimates tentatively that migration must occur at least once in every ten ovulations in the human female.
That it can take place in the sow I think is proven by four uteri of my series, in each of which one ovary contained no corpora lutea, and yet in two of the cases the side of the uterus corresponding to the ovary without corpora lutea contained 1 fetus, in the other two cases 3 fetuses. In all four cases the total number of corpora lutea in both ovaries accounted for the total number of pigs in both sides of the uterus. In all likelihood we can explain most of our cases by external migration. The point which I wish to bring out is not that migra- tion is a possibility — that has been shown many times — but I want to emphasize its great frequency. For every time it can be demonstrated in the sow, there must be many more times when it can not be detected; for instance, when ova migrate from both ovaries, and hence the condition is not apparent. It is a very conservative estimate if we suppose that one or more ova migrate in 50 per cent of all ovulations in the sow. In a sense, the term migration is a misnomer, for the ovum does not make any great journey to reach the oppo- site tube. I have not had the opportunity to study the relations of the pelvic organs in an adult sow, but in large fetuses the ovaries may be quite close to each other; I have even found them touching. It is likely that the fimbria? of the Fallopian tubes, which in the sow are widely spreading and patulous, frequently reach both ovaries, and hence make it a matter of chance which tube receives a given ovum.
The question of the migration of the ovum has two aspects of immediate practical interest. To the gynecological surgeon it is important to know that the extirpation of one tube and the opposite ovary will not necessarily cause sterility. For the biologist the subject brings up certain questions concerning the determination of sex, which will be mentioned briefly. It is a very ancient theory that the production of two sexes of offspring and their practically equal distribution in number are due to the formation of males by one ovary, of females by the other, the left ovary usually being honored by the ascription of female-producing potencies. This simple hypothesis is capable of proof or disproof by numerous methods, which have not been wanting a trial, the most recent of such experi- ments being those of G. H. Parker (1914), who adopted a very neat and ingenious plan of experimentation. The method was to take an animal possessing a notably bifid uterus, namely, the sow, and count the number of unborn pigs on each side with reference to sex. Parker recognized the possible disturbing effect of migration of the ovum, though I think to a far less degree than the facts warrant; and therefore he took large litters, and omitted from his counts the pigs in the middle of the uterine horns, which may be thought most likely to be the mixed product of the uterine horns, and counted only the two fetuses next to each ovary and the two next the junction of the horns. The result, from the tabulation of 1 ,300 pairs of fetuses, gave an almost absolutely equal distribution of the sexes on the two sides. Now, at first sight it might seem that the great frequency of migration of the ovum, which I have shown to exist, renders Parker's conclusions false, since they are based on the general assumption that the embryos on one side of the uterus come from the corresponding ovary. But, on the other hand, if it is true, as I believe, that the embryos in either horn of the uterus are an inextricable mixture of the products of the two ovaries, then Parker's figures can only be explained by the far-fetched assumption that exactly 50 per cent of the ova migrate, or else that both sexes are equally represented in each ovary; the latter being of course the conclusions already reached by Parker.
In the foregoing paper the writer has given an account of the histology of the corpus luteum of the domestic sow, remarking the presence of cells differing from the typical lutein cells.
A description is given of the canalicular apparatus and granules found in the lutein cells, and it is shown that these structures undergo progressive changes during the course of pregnancy.
The microscopic appearance of the corpus luteum is described as it varies with the advance of pregnancy.
The corpus luteum of pregnancy is distinguished from that of ovulation by the more regular and uniform morphology of the former, and the greater infiltration of fat in the latter.
During pregnancy the Graafian follicles do not undergo the process of ripening, or change of the theca interna which is preparatory to rupture.
External migration of the ovum is a normal and very frequent occurrence in the sow.
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1. The wall of a recently ruptured Graafian follicle, showing the granulosa intact. Hematoxylin and eosin. X "• '00.
(6) Granulosa, (c) Theca interna.
2. Additional cells of corpus luteum, type I. Mallory*s stain. X 400.
(a) Lutein cells. (A) Additional cells, type I.
3. Additional cells of corpus luteum. type 2. Mallory's stain. X 650.
(a) Lutein cell. (A) Additional cells, type 2.
4. Corpus luteum, first period. Mallory's stain. X 650.
5. Corpus luteum. second period, Mallory's stain. X 650.
6. Corpus luteum, second period, osmic acid stain. X 650.
(a) Droplet of fat inside one of the peripheral vacuoles.
7. Corpus luteum. third period. Mallory's stain. X 650.
(a) Lutein cell containing ring-form of canaliculi. (A) Lutein cell containing cleft-like canalicular apparatus, (c) Chromatic granules.
8. Corpus luteum. fourth period. Mallory's stain. X 650.
9. Corpus luteum, fourth period, osmic acid stain. X 650.
(a) Fat-globules in a lutein cell. (A) Fat-globules in a connective tissue cell, (c) Chromatic granules.
10. Corpus luteum, fifth period. Mallory's stain. X 650.
(a) Lutein cell showing no peripheral vacuoles. (A) Additional cell, type I . I I. Corpus luteum, fifth period, osmic acid stain. X 650.
(a) "Additional" cell containing large fat-globules.
(A) Connective tissue cell containing minute fat-granules.
12. Corpus luteum, sixth period, Mallory's stain. X 650.
13. Corpus luteum, sixth period, osmic acid stain. X 650.
14. Corpus luteum, seventh period, Mallory's stain. X 650.
15. Corpus luteum, seventh period, osmic acid stain. X 650.
16. Wall of unripe Graafian follicle, hematoxylin and eosin. X 400.
(a) Granulosa. (A) Theca interna, (c) Theca externa.
17. Corpus luteum, of ovulation (non-pregnant sow), hematoxylin and eosin. X 650.
18. Wall of ripe Graafian follicle, hematoxylin and eosin. X 400. (A) Theca interna.
19. Specimen No. 70, showing a Graafian follicle ruptured during pregnancy. Hematoxylin and eosin. X 3.
(a) The ruptured follicle. (A) An unruptured follicle, (c) A corpus luteum of pregnancy.
20. Wall of follicle shown in Figure 19. Hematoxylin and eosin. X- ca. 100.
(a) Granulosa. (A) Theca interna.
21. Specimen No. 136, a Graafian follicle showing partial accessory lutein-cell formation. Hematoxylin and eosin. X 3.
(a) Point at which the accessory lutein formation occurs.
22. Showing the area marked a. Fig. 21, enlarged 70 diameters. Hematoxylin and eosin. X 70.
23. Four cells of the granulosa of the same specimen, showing ring-like vacuoles in the cytoplasm like those seen in lutein cells. Mallory's stain. X600.
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