Paper - The interstitial cells of the mammalian ovary (1914)
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The Interstitial Cells Of The Mammalian Ovary: Felis Domestica
B. F. Kingsbury
DeparimenL of Histology and Embryology, Cornell University
It is a rather striking fact that while the occurrence of relatively large epitheloid cells, with clear vesicular nucleus, which are generally termed 'interstitial cells' has been known for a number of years to exist in the ovaries of the common mammals including man, they generally receive but scant mention in text-books of anatomy and histology. Herein accounts of the structure of the ovary usually contrast with those of the male reproductive organ, the testis, the interstitial cells of which are practically always fully described. On the other hand, contrasting with this conservative attitude we have the tendency on the part of many recent writers to see in these cells formers of an ovarian 'internal secretion,' even designating them as the 'internal gland' of the ovary. Yet, on the physiological as on the morphological side there is evident the same contrast between the definiteness of statement as to the interstitial cells of the testis and the vague and uncertain attitude as to the basis of the 'internal secretion' of the ovary. ^
^ In recent works summing up results upon the "Organs of internal secretion," this contrast is revealed. Biedl ('10) concludes that the evidence strongl}' supports the secretory character of the interstitial cells of the testis, while stating that it is as yet inconclusive for the interstitial cells of the ovary. Swale Vincent ('12) quotes conclusions su])porting the importance of the interstitial cells of the testis; he but incidentally mentions the interstitial cells of the ovary. Gushing ('12) says (p. 276): "In the testis there are two factors to be considered, the interstitial cells and the cells of Sertoli or spei'matogenous epithelium. The former are undoubtedly related to tlie acquirement of secondary masculine characters of sex." Of the ovary he says (p. 279): " . . . . the interstitial cells of the gland which, in all probability, play a role similar to that of the cells of Lej'dig in the male. The existence of such cells in the ovary has been fully described by Limon."
A priori the recognition in the testis of specific cells as constituting morphologically a gland of internal secretion conveys the assumption of a morphologically comparable gland in the ovary. Indeed, the application to them of the term 'interstitial cells' carries with it the acceptance of their homodynamy with the interstitial cells of Leydig in the testis, which is provisionally adhered to in the present article.
The literature dealing with these cells of the mammalian ovary, specifically or incidentally, is extensive and is found in numerous papers on the structure and development of the ovary. From their mode of origin (discussed subsequently) more specific descriptions occur in connection with the theca foUiculi, atresia folliculi, origin and nature of the corpus luteum, etc. Historical summaries have been well presented by a number of writers (Schulin '81; Harz '83; Limin '02; Schaeffer '11). A concise statement of the growth of knowledge of these cells will nevertheless be helpful at this point.
By Pfliiger ('63) and by Schron ('63) it would appear that these cells in the ovary were first described and figured, but His in 1865 was the first to descril^e them in detail (in the ovary of the cat) and discuss their significance. Their occurrence in the midst of the spindle-shaped stroma cells and an apparent close association with the blood-capillary network led him to regard them as developed out of the stroma cells (spindle cells) while at the same time he suggested a genetic relationship to the blood capillaries. Between these two mutually irreconcilable modes of origin of these cells he made no definite choice. He found them best developed in the 'membrana folliculi interna' (theca interna) which one is led to infer he believed developed out of them. He designated them as granule cells (Kornzellen) and recognized their lipoid content, as did Pfliiger. Waldeyer, in his classical paper on the ovary ('70) briefly mentions the 'Kornzellen' of His which he interprets as 'Wanderzellen.' Subsequently ('74) therefore in the paper in which he establishes his group of 'plasma cells,' although the interstitial cells of the testis were included in that group no mention is made of these ovarian cells. The next important paper dealing with these cells is that of Tourneux ('79), earlier workers (Born '74; Slavjansky '79) seeing the cells but not identifying or discussing them. Tourneux compared them directly with the interstitial cells of the testis, applying to them — apparently for the first time — the same name. He compared them also with the cells of the suprarenal gland, coccygeal gland, carotid gland and the decidual cells of the uterus, adding them to the list of cells already grouped together by Mihalkovics (73) and Waldeyer ('74). The term 'interstitial cells' (of the ovary) was immediately applied to them by MacLeod ('80) and has persisted up to the present. Some seven w^orkers dealt with the ovarian interstitial cells between 1880 and 1898 when the papers of Kolliker, Clark and Rabl appeared and since then the interstitial cells of the ovary have received an increasing amount of attention. Four aspects of the historical growth of knowledge of these cells seem to the writer worthy of emphasis : (a) their relation to the tunica thecae interna and atresia follicuh; (b) their comparison with the cells of the corpus luteum (lutein cells) ; (c) then- granular and lipoid content; (d) their significance and their interpretation as constituting an 'interstitial gland.'
An association of the interstitial cells with the tunica interna of the follicular theca was recognized almost from the time of their first observation although the relation given was the reverse of that which subsequent observers determined to be the correct one. His ('65) made the tentative suggestion that his 'granule cells' give rise to this layer of the follicular wall. Harz ('83) inclined to the same view. Less emi)hatically or clearl}^ stated, in the contributions of workers before 1898 (i.e., Paladino '88, Janosik '88, Schottlander '91, '93, Storkel, and others) the derivation of these cells from the internal theca of immature follicles undergoing regression, has been shown beyond all question for a large number of mammals of several orders — including man — through the investigations of KolHker ('98), Rabl ('98), Clark ('98), Van der Stricht ('01, '12), Limon ('02), Allen ('04), Seitz ('06), Wallart ('07), Fellner ('09), Regaud and Dubreuil ('07) and others, some of these writers laying their emphasis on the atresia folliculi, others on the interstitial cells. It was to be expected that in an organ as complicated as the ovary other modes of origin had been advanced. Hence we find the strands of interstitial cells interpreted as degenerating egg-tubes of Pfitiger (Schulin '81} or derived from the Wolffian body (Balfour 78, Harz '84). From the medullary cords of the ovary in the fetal, new-born or young animal the strands of interstitial cells were not readily distinguished and the two structures were confused, notably by Balfour. Genetic relationship to the blood vessels was advanced by His and Mihalkovics. Tourneux and Janosik regarded them as connective tissue cells. Miss Lane-Claypon ('05) has more recently derived them from the germinal epithelium as cell cords with the potentiality of primordial ova. The appearance of the interstitial cells and their epitheloid arrangement led a number to assume an epithelial origin for them. Finally, their resemblance to the cells of the suprarenal organ (cortex) had been commented on as suggestive by Mihalkovics, Janosik, Limon, MacLeod, Van Beneden and Tourneux. It should be noted that while it is quite clear from the literature alone that the interstitial cells of the mature or maturing ovary come from the internal layer of the theca of degenerating follicles, those in the fetal or postpartum ovary can have no such origin. This will be discussed subsequently.
The relation of the problem of the interstitial cells to that of the corpus luteum is to be found in the view that has l^een more or less definitely expressed by many workers — that no fundamental difference exists between the regressive changes that occur in the ruptured and in the unruptured follicle when they undergo degeneration. First definitely expressed, as far as the writer knows by Schulin ('81) who believed he could find intermediate forms between the typical atresia and corpus luteum formation, the fundamental similarity of the two processes has been insisted on by Paladino ('88), Schottliinder ('91), Hoelzl ('93), Kolliker ('98), Clark ('98), Bouin ('02), Allen ('04), Van der Stricht ('02), Limon ('02), Fellner ('09) and others. The atresia foUiculi has been designated as a corpus luteum atreticum (Kolliker, Van der Stricht), false corpus luteum (Schottlander and others), while by Fellner the interstitial cells are called 'theca lutein cells' — a name carrying with it the interpretation of similarity but not genetic identity. Entirely comparable is Pinto's Taralutein cells.' On the other hand, the homodynamy or comparability of structures arising out of the ruptured and unruptured follicles is emphatically denied by many ^notably of course, Sabotta — and the problem on this side merges with that of the origin and significance of the corpus luteum. The comparison made by Bouin and Ancel is considered subsequently.
One of the most striking characteristics of these cells is their granular content of a lipoid nature. Recognized by Pfliiger and His, this has been commented on by most later writers. Degeneration products derived from the follicular epithelium or from the theca; unutilized nutritive material; nutritive material for the developing ova; secretions, etc.; these are among the interpretations made of their presence, but no decisive evidence has been brought forward in favor of any one of these views. A yellow or orange pigment (a lipochrome) may or may not be present, with or without the lipoid granules, seemingly somewhat characteristic of some forms and of certain periods of life in other forms. A rather close association of pigment and lipoid appears to exist, the condition in the interstitial cells clearly paralleling closely that in the corpus luteum.
A number of recent writers have united the interstitial cells of the ovary under the collective name of 'interstitial gland' (Bouin '02, Limon '02, Regaud and Dubreuil '07, Aime '07, Wallart, '07, Bianchi '07, Mulon '10, Van der Stricht '12). The term has been objected to by some (e.g., Friinkel '05, Biihler '06) as has the assumption of the secretory character of these cells. Fellner accepts the secretory interpretation without making use of the term 'interstitial gland.' It will be necessary to discuss this interpretation subsequently.
The different mammals in which ovarian interstitial cells have been described and their origin and significance considered comprise a relatively large number of species, almost exclusively from the orders of Chiroptera, Insectivora, Rodentia, Carnivora, Ungulata and primates — particularly man. To the ovary of the rabbit, guinea-pig, bat, cat and man most attention has been devoted. The conclusions in favor of the 'interstitial gland' interpretation have perhaps been largely based on the conditions in the rabbit and bat. Only a few observations have, therefore, been made as far as known to me on the lower orders of mammals (marsupials and edentates) by Benthin ('11) and Schaeffer ('11). Within the range of animal forms examined, interstitial cells are present in very varying numbers. Thickly massed in the rabbit, they are present in large amount in the cat's ovary, scantily developed in the human ovary, they appear to be practically absent in certain other forms, such as the pig, sheep, cow. In general, it would seem that they are abundantly present in the small mammals examined and few or absent in the larger mammals in the adult.
The great variability in their development certainly calls for careful consideration in drawing any general conclusions as to their significance, as has been insisted upon by Friinkel ('05). Furthermore, it would appear that the same variability is found in the period of life at which the interstitial cells are present. Upon this basis Aime divided the mammals into four groups: (a) those in which the 'interstitial gland' occurs in fetal life only (e.g., the horse); (b) those in which the 'interstitial gland' is present only in postpartum (essentially, adult) life (Chiroptera, Insectivora, rodents); (c) forms possessing no 'interstitial gland,' wherein he included man, pig, sheep, goat, boar, dog; (d) mammals in which an 'interstitial gland' occurs both antepartum and postpartum, as an example of which group he gives the cat. It might be added therefore that Aime accordingly distinguished two groups of interstitial cells or two forms of 'gland:' (a) the fetal, derived from the 'connective tissue;' (b) the adult, derived from the theca interna. This grouping has been essentially followed recently by Popoff ('11). Any discussion of the strict applicability or value of these distinctions will not be entered into now. It may be pointed out however that a full study of the developmental history of the ovary and of the interstitial cells in particular, has been made in no form save the cat, by Saimont ('05) and v. Winiwarter and Saimont ('08).
The ovary of the cat is well suited for a study of the genesis and significance of these cells by reason of their abundant presence and discriminate arrangement, as well as because of the availability and convenient size of the material. It is therefore rather interesting that it was apparently in this form that the interstitial cells were first seen and described by Pfliiger in 1863, figured by Schron in the same year, and discussed by His in 1865 (his Kornzellen). The interstitial cells of the cat's ovary have since received attention from Creighton ('78), Harz ('83), Janosik ('85), Plato ('97), H. Rabl ('98), Coert ('90), Ganfini ('07), and Saimont and v. Winiwarter and Saimont as above mentioned.
My interest in these cells dated from 1908 when I began an investigation to satisfy my curiosity as to their structural characteristics and mode of origin in the cat's ovary. The material collected and made use of in this study comprises 63 complete or partial sets of serial sections through the ovary, from the fetus (9 series), after birth (25), before sexual maturity (15), before, during and after pregnancy (i.e., in lactation) (14). Most of these series illustrating well the general morphogenesis of the cat's ovary a special study of this aspect of the development of the cat's ovary was made and has been separately published. ^ Indeed, it soon became apparent that any explanation of the interstitial cells was intimately linked with that of the developmental processes taking place in the ovary as a whole, and that they could not be separately interpreted.
^ B. F. Kingsbury: The morphogenesis of the mammalian ovary (Felis domestica). Amer. Jour. Anat., vol. 15, no. 3, pp. 345-379. November, 1913.
^ This is stated with the reservation that the ultimate source of the germ cells may be from the entoderm, in accordance with the evidence therefor adduced by several recent writers. The stromal cells appear to be derived from the mesothelial covering (cf. Allen '04; Whitehead '04; Rubaschkin '12). This origin, I believe, would not affect their essential connective tissue nature.
A brief statement of the general features of the morphogenesis will facilitate the discussion of the interstitial cells subsequently. The ovary in the cat increases mainly by peripheral growth, the ultimate source of the germ cells and indifferent or follicle cells being the mesothelial covering. These and the stroma ovarii constitute the characteristic tissues of the ovary. The earUest parenchymal growth^as usually termed, the medullary cords, constituting the first proliferation — occupy therefore the center and mediastinal portion of the ovary. The medullary cords at first are free from distinguishable germ cells, although later definite ova seem to appear in their midst. Later proliferations from the surface epithelium produce cell cords in which the reverse of the conditions characteristic of the medullary cords holds — the germ cells become the dominant cells and the indifferent cells are distinguishable with difficulty. The differential growth of the ovary is centrifugal, and hence older stages of the oogenesis are more centrally located. The zone of oogonial multiplication is peripheral, the proliferation of new oocytes ceasing soon after birth. Connection of the egg cords with the surface epithelium becomes lost two to three weeks after birth, and by separation of the oocytes composing the outer portions of the egg cords, resting or primary follicles are formed, marking out a definite cortex. While the peripheral growth changes are leading to the establishment of the definite cortex, within the medulla the differentiation takes the form of follicle-formation. The first follicles are apparently formed within the medullary cords themselves, later ones include ova derived from the inner portions of the egg cords. The early or medullary follicles are very irregular and largely pluri-ovular. The follicle formation follows the general plan of centrifugal differential growth, so that, in general, less advanced follicles are more peripherally located. Many of the large, irregular pluri-ovular follicles attskin a marked development so that in old kittens with the greatest development of these follicles the ovary reaches a size greater than at a subsequent period, since these, as well as all the early formed medullary follicles degenerate. Such degeneration is particularly profound as sexual maturity is approached, although degenerations are constantly present in the ante- and postpartum periods, in less mature ovaries, and subsequently throughout the period of sexual maturity. The degenerations of the presexual period are of a somewhat different type. The early medullary follicles degenerate; later ones that attain a large size as Graafian follicles are exceedingly irregular, atypical, and ultimately undergo atresia. The follicular growth processes and conditions are different in the immature ovary, itself undergoing rapid growth. In the growth of the ovary the stroma obviously plays an important part, and in nowise is to be thought of as playing a passive, purely supportive role.
In the above exceedingly brief outline of the morphogenesis of the ovary I have presented the conclusions reached in a study of the later development of the ovary. In general there has been full confirmation of the monographic investigation by v. Winiwarter and Saimont. In certain respects, however, the emphasis, point of view, or interpretation has been different. These riiatters of divergent interpretation have been discussed in the previously published article on the morphogenesis.
The growth of the ovary is a continuous process, and the recognition of periods is rather artificial; however, it is convenient to distinguish (a) period of early (1st) proliferation; (b) the period of later (2d) proliferation; (c) postpartum period — up to the establishment of definite cortex of resting follicles; characterized by development of medullary follicles; (d) the presexual period; growth of the pluri-ovular large medullary follicles; (e) the period of profound degeneration; (f) sexual maturity; (g) senescence.
The interstitial cells first make their appearance during the second period, being largely associated with the medullary cords, and hence central. Small groups of them, however, occur constantly in the stromal septa between the egg cords in the primitive cortex (figs. 1 and 2). From this period on, the interstitial cells are present in the ovary of the cat into extreme old age. How early they are present in the fetal ovary I cannot say. The youngest ovary in which they were identified was from a 95-mm. fetus. Inasmuch as the series of younger ovaries were not prepared by a method^ that rendered their identification easy (Flemming's fluid fixation, the sections bleached and stained with iron hematoxyhn) some few interstitial cells may have been present and not detected. This is easily possible from the mode of their formation. Saimont ('05) has described and figured them as occurring in the basal connective tissue nucleus and in the stromal lamellae between the medullary cords in fetuses of 29 to 52 days age (ca.-25 to 91 mm. length). He has recognized in the cat's ovary three periods of development of interstitial cells, (a) the period just referred to; (b) a second period, from about 58 days postcoitum (ca. 120 mm. length) to sixty or sixtyseven days postpartum, in which they occur in the neighborhood of the rete and in the zone of the medullary cords; and (c) a third period, extending from about 50 days postpartum into adult life in which they occur as the large thecal cells in follicles in process of development (en voie de developpement) . The interstitial cells of the first period seem to completely disappear before the second period sets in. These cells I have not studied, and hence cannot discuss their mode of origin. The distinction of the second and third periods is one of convenience only. As will appear subsequently my conclusions as to the origin of cells of the 'third period' and the interpretation for all periods are quite the reverse of Saimont's.
Two methods were used with advantage: (a) Flemming's fluid (Hermann's or Benda's) fixation, the chloroform paraffin imbedding method, the sections being mounted unstained and without coverglass under a thin fihu of chloroform balsam. In toto staining with carmine might, I believe, have been advantageously employed in this method. The lipoid granules are completely preserved by this technique; (b) fixation in modified Zenker's fluid (1 per cent of acetic, or less), followed by a dichromate mordantage, paraffin imbedding, and a Weigert hematoxylin stain, as described in the Anatomical Record, vol. 5", p. 313.
As to the origin of the interstitial cells, the statement of my conclusions is brief. They arise as a modification of stroma cells, and the stroma is therefore the parent tissue for this type of cell. This conclusion is but a confirmation of the results of nearly all later workers upon the subject (Tourneux, Allen, Janosik, and others).
By increase in the amount of cytoplasm and the appearance of free lipoid therein, the typically spindle shaped stromal cell increases largely in size, and is altered in shape, becoming thus recognizable as an interstitial cell. The correspondingly elongated and densely staining nucleus enlarges to a spherical form,
clearer, with a well defined 'nucleolus.' No special cytological study of nucleus or cytoplasm was made, hence the nature of this nucleolus was not ascertained, whether true or false.
The problem of the interstitial cell, on this side, therefore, resolves itself into an examination of the conditions under which this transformation occurs, and the part that the stroma plays in the ever-continuous processes of growth within the ovary.
That the stroma plays an important part in the development of the ovary as a whole, in the transformations that take place in the medulla of the developing ovary, and in the phenomena of follicle development in the adult ovary, becomes quite evident from the examination of such a series of ovaries as has been at my disposal. The histological growth changes are so complex however that their analysis and broad comprehension are exceedingly difficult. Stromal strands and lamellae penetrate the primitive cortex, but any particular correlation with the egg cords is not apparent. Oocytes that have either earlier or later become separated by the breaking up of the egg cords, are surrounded by a follicular epithelium, and there is usually recognizable a concentric stromal investment. In the primary follicles of the cortex in the adolescent and adult periods, the follicular epithelial cells are so thin that careful examination is necessary for their recognition. Such follicles possess no evident theca. As soon, however, as the growth of the follicle begins, and the follicular epithelium becomes cuboidal, columnar, and then stratified in the well known method of growth of a Graafian follicle, a concentric arrangement of the stroma is evident, becoming as growth proceeds, the theca of the Graafian follicle. The so-called medullary cord of the fetal and post partum ovary possesses a stromal sheath as a form of theca (figs. 11 and 12), which becomes very evident in the larger masses. The concentric arrangement of the stroma is found whether an ovum is enclosed or not. Naked ova, lying free in the stroma are not found save occasionally. Such ova are apparently always degenerating or degenerated. The growth correlation which leads to the formation of a definitive theca in the development of a Graafian follicle, appears to be more directly between stroma and the indifferent or follicle cells rather than directly between stroma and developing ovum.
During the first three months after birth great irregularity characterizes the growth changes of the parenchyma within the interior of the cat's ovary, causing morphological relations of considerable complexity and irregularity, which are differently interpreted by myself (in the previous paper) and by v. Winiwarter and Saimont ('08). The series during this period indicate that it is one of marked growth of the stroma as well as of the parenchyma so that it seemed to me the irregular and atypical structures expressed, morphologically an attempt at follicle formation during rapid growth of the ovary as a whole. While the stroma contributes to the general complexity, a relation to the masses of indifferent cells and irregular follicles, is nevertheless evident, as appears in some of the photographs illustrating the previous paper.
Many of these follicles develop into the large, irregular pluriovular follicles previously mentioned. These possess very definite thick stromal thecas, including, in fact, most of the stroma in this zone of the ovary.
The period of developing medullary follicles is likewise the most complex in the distribution of the interstitial cells, which occur in smaller or larger masses in the midst of the stroma strands, which show in general the same relation to the follicular masses as does the stroma out of which they are developed.
The zones occupied by the bulk of the interstitial cell groups in four typical stages — 6 days, ca. 5 weeks, ca. 3 months, and in the adult ovary — may be illustrated by figures 1 to 4, in which the dense black of the osmic acid stained lipoid locates the interstitial cells in which it is contained.
It may be noted that the interstitial cells occur in the zone where growth (of the indifferent cells) is apparently most active — about the medullary cords, before and after birth; in the zone of the medullary follicles, during their stages of growth, etc.
Hence it might appear that they had to do particularly with those growth changes and possessed a trophic 'function.' This was the interpretation of Saimont ('05) and of v. Winiwarter and Saimont ('08) ; but I believe when closely examined, the evidence for this interpretation is not only inconclusive, but on the contrary points in quite the reverse direction. This will be discussed presently.
Before taking up the question of the interpretation of these cells, I wish to confirm the conclusion of Saimont and others that these cells are transcient structures in the ovary; that they, as interstitial cells, possess no permanency; that they come and go. This conclusion would be a priori self evident from their mode of origin in the adult ovary. I further agree with Saimont that their fate is not one of degeneration, but that they revert to the cell type out of which they were developed, that is, the stroma cells. Saimont accordingly recognizes (a) young interstitial cells, (b) transitional forms, (c) adult interstitial cells, (d) degenerated interstitial cells; i.e., those reassuming their character as stroma cells, and (e) hypertophied cells. These stages, he finds to be somewhat characteristic of the three periods that he recognizes. Thus the stage (a) he thought limited to the first period; the stage (b) occurs only after birth, etc. It might be pointed out, however, that, obviously, from their very evident continous formation, when once fully established, essentially all stages must exist in the ovary at the same time — as in the adult. The distinction of stages is but a matter of convenience in description, and the terms are likewise of but metaphoric value.
The interstitial cells thus possess no morphological individuality and hence do not, strictly speaking, deserve recognition as a distinct kind of cell. They are stroma cells, which under certain conditions, undergo changes, the most striking and characteristic histological alteration being the accumulation of lipoid granules within their cytoplasm. Apparently with the passing of these conditions thej' again become stroma cells.
In considering the question of the interpretation of these cells, therefore, the above definition must be kept in mind, since what may be true of a stroma cell need not necessarily be also true of an 'interstitial cell.' This is particularly true of the 'trophic function' interpretation as advanced by Plato and accepted by Saimont ('05). As has been indicated the conditions within the ovary during growth might well be considered to support this interpretation. The morphological relations during growth are of considerable complexity whereas during the adolescent and adult period (Saimont's 3rd period), the genetic relations of these cells are of the clearest, and show conclusively that these cells arise from the theca interna during the degeneration of Graafian follicles as atresia folliculi. During the growth of a follicle from the primary resting follicle to the appearance of the antrum, the cells of the theca, which becomes progressively more distinct, retain their spindle shape and the general histological appearance of stroma cells. They are almost or quite free from demonstrable lipoid. Next the follicular epithelium the cells are more densely arranged and larger (theca interna). In Graafian follicles of different sizes and degrees of development, the theca interna cells are largely free from demonstrable lipoid. Frequently, lipoid granules occur and in some cases, particularly in the larger follicles that are evidently nearing maturity, the amount of lipoid granules within the cells of the internal theca is quite marked. The cells, while somewhat larger, still retain their spindle shape which is doubtless largely an expression of the mechanical conditions under which they exist. Such follicles showing lipoid content in the theca cells are apparently normal. It is this, I believe, that led Saimont to ascribe to the interstitial cells a 'trophic function.' The amount of free lipoid in such cells is small and they do not correspond to his figures of -'adult' cells.
With the onset of atresia, a succession of changes occurs that markedly increases the fat content in the theca cells and determines their transformation to the so-called interstitial cells. From a series of nine photographs showing successive steps I select four stages to illustrate atresia folliculi (figs. 5-8). So well have the salient histological features of atresia folliculi been described, for the cat particularly by H. Rabl, that any extensive description is unnecessary. One of the earliest indications of the onset of degeneration is an alteration in the liquor folliculi. Instead of coagulating on fixation as a loose flocculant mass, it is more granular, seemingly dgiser and becoming more dense and homogeneous as the atresia progresses. The ovum early shows an alteration, loses its spherical shape, becoming ellipsoidal, as though affected by an increased intra-follicular pressure. The follicular epithelum and theca, in early atresia show in the cat no marked alteration. In later stages the follicular cells become markedly spindle-shaped or stellate. Lipoid granules which (in the cat) are only scatteringly demonstrable in the follicular epithelium become more numerous, typical karyolysis sets in, of the type early described by Flemming in this kind of cell as chromatolysis, and the cells subsequently completely disappear. There is an undoubted 'invasion' of stromal connective tissue and possibly of leucocytes as well. The liquor becomes completely absorbed, and the antrum thus obliterated. The egg cell disappears, the egg membrane (zona pellucida) persisting for a long time (fig. 8). The last recognizable trace of the follicle is the irregular ring of interstitial cells derived from the theca interna. Ultimately this arrangement is broken up under the stress of the growth changes within the ovary, and there are but isolated groups of interstitial cells dispersed apparently at random in the stroma. These finally, according to the observations of Saimont and myself, revert to their original stromal cell form.
The relation of the interstitial cells to the atretic follicles is strikingly shown in preparations in which osmic acid fixers, such as Flemming's fluid, are used. The reproduction of typical sections is given in figures 4, 9 and 10, from which, -as in figures 5 to 8, of which they give the complementary 'lipoid picture,' the relation of the interstitial cells to atresia is illustrated. Several degrees of atresia are there shown.
So consistently has this mode of thecal origin of the interstitial cells of the adult ovary been supported by the observations of nearly all who have devoted careful study to this phase of the subject, that it may be accepted as clearly proven.
I find therefore no support for the interpretation of Saimont that (in his 3rd period) the interstitial cells are found as large thecal cells in follicles that are undergoing development," but that they are the thecal stroma cells of Graafian follicles that have undergone or are undergoing degeneration. Saimont seems to have based his conclusions upon the conditions in the theca of the larger Graafian follicles. In atresia there are formed as interpreted by him, hypertorphied interstitial cells. Reproduction of such cells shown in figures 13 and 14, however agree rather with his figures of 'adult' cells. He published no figures illustrating specifically this period, nor have v. Winiwarter and Saimont as yet published descriptions or figures of the follicle formation in the adult cat's ovary. If it were stated that the thecal cells were intimately associated with the growth changes of the maturing follicle and it were considered advisable to so express this by ascribing to them a nutritive or trophic function" there would, it seems to me, be little basis for a disagreement, since there is much that indicates the importance of the stroma in the growth processes within the ovary. The interstitial cells might thus prove to be an expression of the arrest or abeyance of a trophic function.
Whatever may perhaps be ultimately shown to be the exact nature of the correlation, the phenomena of atresia show that the interstitial cells stand as an expression of an altered metabolism associated with the (degenerative) processes that occur within the atretic follicle, although the occurrence of free lipoid in thecal cells of apparently normal follicles indicates both the delicacy of the metabolic balance between theca and follicular epithelium and that the appearance of free lipoid in the thecal cells is not necessarily an indication of irreversible degeneration.
The ovarian parenchyma (egg cells and indifferent or follicle cells) is characterized by the high evident lipoid content of the cells. Oocytes of the primary resting follicles show little or no free lipoid with osmic acid as an indicator. The application of a mitochondrial technique, however, reveals the numerous mitochondrial granules which undoubtedly represent lipoid in masked or combined form. With the growth of the ovum numerous globules of free lipoid make their appearance in the cytoplasm. The follicle cells of normal follicles show little or no free lipoid. Mitochondria are however present in abundance, particularly in the cytoplasm toward the ovum in the cumulus, or toward the antrum in other portions. With the degeneration of the ovum in atresia, numerous lipoid granules appear in the follicle cells.
In the fetal and postpartum ovary lipoid granules are abundantly present in the medullary cords. They were recognized first in my series in the 95-mm. fetus and from that time on appear to be constantly present in the indifferent cell masses which do not immediately surround an ovum until such disappear from the ovary before sexual maturity. In the period of development of the peculiar medullary follicles (ca. 5 weeks) , it is interesting to observe that in the irregular follicle, such as shown in figures 18, 24 and 25 in my previous paper, the epithelium immediately surrounding the ovum is particularly clear from such free lipoid, whereas the follicular epithelium of the remainder contains it in abundance. This is easily demonstrable by means of osmic acid fluids, and in preparations fixed and stained by the Weigert hematoxylin method, the portion containing the greater amount of lipoid stains much more intensely than the sheathing follicular cells. In the 3 months ovary (fig. 3), containing the large irregular pluriovular follicles, the follicular epithelium bordering the antrum and ova mainly lacks the free lipoid content. Many of these Graafian follicles however possess irregular tubular extensions of the follicular epithelium, and the follicular cells composing these contain free lipoid droplets.
It is in the medulla in particular association with the free lipoid containing medullary cords and irregularly developing medullary follicles, that the groups of interstitial cells are found. Figures 11 and 12 show medullary cords of a 3 to 4 day kitten, illustrating the stromal investment, the 'loose' character of the cells and the free lipoid content, some of which had doubtless been dissolved out before it was drawn, since it was from a stained and covered series (No. 11). They likewise show the modified, free hpoid-containing stroma cells, or in other words, interstitial cells, and their close relation to the cells of the medullary cord.
During the period of growth of the medullary follicles, to the interpretation of which considerable discussion was devoted in my previous paper, the relation of the interstitial cells to the follicular masses is likewise evident, although much more intricate and less exact, due in part, I believe, to the shiftings during a period of marked growth of the ovary as a whole. In illustration of the relations at this period two line drawings (figs. 15 and 16) are introduced -from which the location of interstitial ceU groups may be seen.
Finally, from figure 3, the location of the interstitial cells in the period of large irregular pluriovnlar follicles may be seen. It will be noted that the theca of these follicles contains abundant lipoid containing cells. These follicles are quite atypical, aside from their irregularity and pluriovular character, in the thinness of their follicular epithelium, and as v. Winiwarter and Saimont pointed out, in the 'abnormal' appearance of contained ova, one element of which, the scanty amount of lipoid contained, interests us here.
From the above it may be seen that the follicular growth processes within the medulla of the immature ovary depart widely from those of the adult period, and it is undoubtedly in correlation with the irregular and essentially atypical ('abnormal') character of the growth processes rather than with growth itself that the interstitial cells make their appearance. In my previous paper, I have interpreted the growth relations encountered as essentially successive 'attempts' at normal follicle formation, becoming more typical as the presexual development of the ovary advances. All these early formed follicles, as v. Winiwarter and Saimont first recognized, are doomed to degenerate. There succeeds the period of large irregular pluriovular Graafian follicles a period of profound degeneration, and it is at this presexual period that the cat's ovary contains the greatest relative number of interstitial cells. From an ovary of this period figure 9 is taken, and for it might have been substituted reproductions of sections from several ovaries in which the mass of interstitial cells was much greater.
No fundamental difference is thus felt to exist between the conditions of interstitial cell formation in the adult and in the growing ovary.
In the fetal (112 mm.) and newborn kitten small groups of interstitial cells were mentioned earlier in this paper as occurring in the stromal lamellae between the egg cords of the primitive cortex. Similar small groups occur in the primitive cortex of older kittens. In the adult in the definitive cortex (zone of resting primary follicles) I did not find undoubted instances of their presence. I have been unable to find any clear correlation of these with growth or degenerative processes. Correlations doubtless exist which continued study of the changes in the primitive cortex may reveal.
]\I3' observations do not support the conclusions of Aime and of Popoff, that there are two morphologically distinct 'interstitial glands/ the one fetal and medullary in position, the second adult and 'cortical,' that is, formed in the atresia folliculi; the first possibly, as Popoff believes it to be, homodynamous with the testicular interstitial apparatus, corresponding to a medullary cord — seminal tubule homodynamy. I cannot ascribe to the interstitial cell groups any morphological value, as is evident from the consideration of them given above.
I desire next to consider briefly the appropriateness of the term so generally used by a number of recent writers — that of 'interstitial gland.' The term 'gland' is avowedly employed somewhat indiscriminately and subject to abuse, particularly as applied in the group of 'internal secretory glands,' or 'endocrine glands.' The term may be used in a morphological or in a purely physiological sense. In the morphological sense, namely, as a differentiation of definite cells in development for the elaboration of specific chemical substances, and possessing accordingly corresponding characteristic structure, morphological arrangement and relations — the interstitial cells of the ovary certainly do not deserve the designation of gland. They are clearly more or less transient transformations of stroma cells or theca stroma cells under altered conditions of metabolic correlation.
There are involved here somewhat the same considerations that led Kohn ('00) to reject the name as applied to the various groupings of phaeochrome cells.
There is no constant or characteristic arrangement of the interstitial cells, nor does there appear to be any marked or peculiar relation to the blood or lymph vascular system. This is however a distinctly disputed point. Since the time of the paper of His, a peculiar relation of the interstitial cells to the blood vessels has been described by Limon, Aime, Athias and Saimont. This, on the other hand, is denied by Cohn.
Perhaps the most suggestive evidence of a glandular arrangement of the interstitial cells and a definite relation to the vascular system is that presented recently by 0. Van der Stricht ('12) in his studies of the ovary of the bat, Vespertilio. He adduces arguments and evidence to show that the 'secretion' finds its way into the lymphatic channels and therein is transported from the ovary. I fail to find in the cat any unusual or peculiar relation of the interstitial cells to either the blood vessels or lymph vessels.
In fact, the tunica thecae interna seems to become markedly less vascular in atresia folliculi. Two conditions should I think be borne in mind in considering this phase of the subject: first, that the theca interna of the maturing Graafian follicle is markedly vascular — in very obvious correlation with the growth of the follicle, so that the thecal cells naturally possess a quite vascular environment; second, that in atresia folliculi a relatively large amount of fluid and products of histolysis must leave the ovary by some channel, and in some forms and at growth periods where the degeneration was extensive, a great secretory activity on the part of the interstitial cells might be simulated. In the reversion of the interstitial cells to the stromal cell type also a relatively large amount of substance must pass from them without necessarily possessing any ulterior significance as a secretory phenomenon.
The great variability in the presence and development of the interstitial cells in different forms is a third count against their possessing any morphological value as a gland. This evidence appears clearly from the studies of Aime, FrJinkel, Schaeffer and others. I cannot see any escape from the force of Frankel's argument. A gland existing for the specific 'purpose' of forming substances of very distinct value to the organism as a whole, would be as constant in its presence and development.
Most biological problems, particularly those attempting to explain the genetic significance of a structure or organ, possess two quite distinct aspect ; (a) the determination of the processes that underlie the structural appearance, their correlations and explanation in terms of cause and effect, (b) the part they play in the bodily economy — for the individual or the race; as com-" monly expressed, the 'purpose' for which they exist, their 'function' in the organism; as the writer prefers to put it, their contribution to the complex pattern of the bodily activities and whose dominant component is adaptation. The existence of the double character of developmental problems and the significance of this, is not, I believe, sufficiently appreciated at the present day. If it is possible to ascribe a 'function' to an organ, the end and aim of its investigation is frequently believed to be attained, without full comprehension of what has been gained in the determination of 'function.' It is largely due to the dominance of the conception of the animal organism as a colony of organs each with its specific function or functions, that the interstitial cells are seized upon as an internal secretory gland, because there are no other cells in the ovary which meet the requirement of 'gland cells.' It is I beheve the analogy of a superficial resemblance that here, as in other instances, has led to a false morphological grouping.
Apart from the question of the recognition of the interstitial cells as a gland in the morphological sense, is the quite distinct question as to whether they constitute a gland in the physiological sense; that is, whether from them, as interstitial cells, come substances formed as a result of their interstitial cell metabolism, which reaching the circulation, produce characteristic effects in other parts of the body. Any adequate consideration of this question lies beyond the scope of this paper and outside any morphogenetic study of the ovary. I desire therefore simply to offer a few comments upon certain aspects only. The striking characteristic of the interstitial cell is the large lipoid content. This apparently consists of phosphatids, fats and cholesterol, together with a hpochrome (lutein).^ The suggestion is close therefore that the lipoids' are either closel,y concerned with the ovarian effect upon other parts of the body, or otherwise important. We have thus the interpretation of Regaud and Dubreuil that the secretion is hpoid in nature; and that of Loisel that the hpoid is associated with an activity in the neutrahzation .of poisons formed in the bodily metabolism. He there*fore looks with favor on the comparison of the interstitial cells and the cells of the suprarenal cortex made by Mihalkovics and others. As a source of 'internal secretion' he considers the degeneration of egg and follicle cells as well as the interstitial cells, and if I interpret him rightly, the undegenerating follicle and egg cells as well. Mulon ('10) holds similar views of the antitoxic action and of the comparison with the suprarenal cortex.
However attractive the view that the interstitial cells are the formers of specific substances that are correlated with the appearance of the secondary sexual characters, it should be borne in mind that there is no direct evidence in favor of the view, nor does it seem possible from the character of their morphological position and relations that such evidence could be obtained. The circumstantial or indirect evidence seems to the writer, far from conclusive on this point. The following comments are offered from a purely morphological point of view. First, the same argument holds against the acceptance of these cells as constituting an internal secretory gland in the physiological sense as against their recognition as a morphological gland, namely, the variability in the amount and in the time of appearance, mentioned and briefly discussed above and in the introductory paragraphs of the paper. In many forms, after birth at least, the interstitial cells appear to be lacking; in others lacking before a certain age (e.g., rabbit). It should of course be borne in mind that a full history of the development of the ovary in most of these forms has not been worked out. In the cat, from my series, interstitial cells appear to be continuously present in the ovary from a 95-mm. fetus to extreme old age, and Saimont has described them in younger fetuses, stages that I lacked. They are markedly abundant before the time of sexual maturity, and are likewise abundantly present during adult life. The number of series is too limited to determine whether they are more abundant during pregnancy, but have given no indication that such is the case. The large numbers of interstitial cells in the ovary of the maturing kitten might be suggestive, were it not for their presence in both ovaries of a 17-year-old cat. One ovary was atrophic, oocytes had completely disappeared, but interstitial cells were relatively abundant as were cords and groups of the indifferent or follicle cells. The other ovary was hypertophied, due to the large increase in the indifferent cells giving a superficial resemblance to a cryptorchid testis. Interstitial cells were present in relative abundance. Hence the distribution throughout the different periods of the life cycle gives no support to their secretory nature.
5 Cf. Regaud et Dubreuil; Pargon, Dumitresca and Xissipesco, Wallart; Holin and Staedeler; Mulon.
The stromal cell might with more likelihood be seized upon as the former of ovarian 'hormones' than the interstitial cell which is but a transitory development out of it of variable occurrence. Still more likely, from the writer's point of view, if he may be pardoned for intruding it, is the suggestion that end-products of the metabolism of the parenchyma itself may be a source of 'hormones' upon which depends the effect of the ovary upon the bodily metabolism. These possibly are set free in the degeneration of ovum and follicle.*' Certain it is that one of the most striking features of ovarian morphogenesis is the constant degeneration of egg cells and follicle cells throughout fetal and postnatal development until the onset of sexual senility. Before the onset of sexual maturity the degeneration is most pronounced (cat, man). The assumption that the end-products of metabolism may stimulate growth, is not I believe opposed to the facts of general physiology but rather the reverse. Whatever may be the outcome of future investigations as to the source and nature of the substances through which the ovary affects the organism, the study of the origin of the interstitial cells indicates clearly that considerable hesitancy should be observed in declaring them specific glandular elements.
•^ Coini)arc Loiscl, 1905, p. 89.
The corpus luteum and the corpus atreticum have been repeatedly compared, and despite marked differences there appears to be much fundamental resemblance. Both express essentially reactions of degeneration. In both relatively large amounts of lipoid appear in their cells attended by the presence of lipochrome. In the one it is the follicle cells that apparently undergo hypertrophy — although this is still a disputed point, and it is easily conceivable that the process may differ in different forms. In the other the cells of the theca alone hypertrophy and become charged with lipoid. The older describers of the corpus atreticum compared it with the corpus luteum — sometimes perhaps confused the two — or described intermediate forms (Schulin '81). Bouin and Ancel have more recently revived the comparison, placing it on the modern 'functional' basis of the internal secretions. The mammals are divided by them into two groups: those in which ovulation is spontaneous, at definite periods, and those in which it occurs at coitus. Those of the first group possess, accordingly, two varieties of corpus luteum, a corpus luteum periodicum (menstruationis), and a corpus luteum gestativum (gra\dditatis), w^hile the forms of the second division have but the last kind of corpus luteum. These forms possess, however, an 'interstitial gland' which takes the place of the corpus luteum periodicum and is not present in those mammals which possess the two varieties of corpus luteum, among which Bouin and Ancel include man, dog, cow, mare, sow. Both classes have thus two kinds of internal secretory gland; the first associated with the uterine changes at pregnancy, the second subserves other forms of ovarian influence.
While there may be an ultimate significance in the comparison of corpus luteum and corpus atreticum, on the morphological side this particular comparison fails, if for no other reason, because in one member of the forms of the first group (man), interstitial cells are present, and in the other forms it is quite clear that corpora atretica occur and differ in no fundamental characteristics in their formation Benthin ('H); Schaeffer ('11). There ha\'e l^een a number of investigators whose work has shown clearly that in man atresia folliculi, and interstitial cell formation follows the same plan as in the cat, for example. Grohe ('63) showed that before puberty Graafian follicles were continuously formed. Slavjansky ('74) described atresia in ovaries from childhood, the sexually mature and during pregnancy. Schottlander ('91, '93), and Hoelzl ('93)— the latter in a series of 60 cases from 1 year to 71 years of age — added several histological details, calling attention to the prominence the large cells of the theca (i.e., interstitial cells) may attain. H. Rabl ('98) made a careful study of atresia (25 cases) and supplemented it by comparative observations. He recognized the interstitial cells as "nothing more than hypertrophied stroma cells." Clarke, Boshagen, Pinto, Friinkel, Seitz, Fellner, Wallart and Schaeffer have given consistent descriptions of atresia foUiculi and the interstitial cells of the human ovary. Wallart from an examination of 67 ovaries from fetal life (8 months) up to ninety-one years, concluded that the interstitial cells (termed by him the interstitial gland) are best developed and most closely massed during early years (up to puberty). During sexual maturity they are present but not so abundant, save during pregnancy. He recognized their origin from the theca interna of atretic follicles. A greater amount of atresia during the period of pregnancy and in the puerperium as indicated by the observations of Sinety, Wallart, Seitz, Pinto and Fellner, would be expected a priori. Whether or not the interstitial cells so formed should be regarded as an internal secretory gland having peculiar relations to pregnancy is of course another question. The more follicles reach maturity and rupture (forming corpora lutea) the less the degeneration, hence the appearance of balance and vicarious development that appealed to Aime, and Ancel and Bouin. From my personal observation I may state the atretic follicles and their lipoid-containing theca cells (interstitial cells) are easily demonstrated in the human ovary by appropriate methods. The latter are more insignificant than in the cat, and doubtless relatively less persistent (as such) in the larger ovary. The time the cells remain in the interstitial cell state, the amount and rapidity of the follicular degeneration and the size of the ovary, and possibly the type of 'metabolism' are evidently determining factors. At one extreme are such ovaries as the rabbit's, at the other those of mare or cow.
A brief discussion of the interstitial cells of the testis may be permissible even though no personal work has been done by me on their structure or origin. They appear to be quite abundantly present in the fetal testis (numerous papers), less developed during childhood, becoming numerous again at puberty and demonstrable in the active testis, thence forward during life. Their connective tissue origin appears to have been quite clearly shown. Their most striking histological feature is their lipoid and granular content. In histogenetic origin and structure they resemble closely the interstitial cells of the ovary, and like these are connective tissue (stromal) elements which for some causes have undergone the typical hypertrophy and lipoid change. In the testis, however, the analysis of the conditions and relations that underlie the appearance of these cells is far more difficult than in the ovary. In the latter organ the oogenetic growth processes are more distributed in time and place; in the testis, in close proximity are spermatocytes, spermatogonia, spermatids, maturing spermatozoa; and the indifferent or Sertoli cells — comparable to the ovarian follicle cells — are undergoing their progressive and regressive changes in association with the maturation of the male reproductive elements. In the anura, reptiles, birds and mammals this is the case. It is necessary therefore to turn for definite morphological clues to the lower forms, where the processes, progressive and regressive, occur in distinct portions of the organ. Despite the excellent studies of Allen, Whitehead and others I believe that there has not been made a full study of the development of the testis with a view to determining the growth correlations that lead to the appearance of the interstitial cells, and such an investigation might add much, even in the complex mammalian testis. That there is such a correlation between the tubule and interstitial cells would appear from the fact that the interstitial cells are not recorded as existing by themselves— in the absence of seminal tubules, although tubule and interstitial cells seem to be often in reciprocal relation — atrophic changes in the tubule being accompanied by increase in the number of interstitial cells (Ancel and Bouin, Voinov; Biedl, p. 3G5). There is no evidence, therefore, that the factors that determine the transformation are intrinsic, that is, in the cells themselves. From the condition in the fetal testis, cryptorchid testis, the experimental results of vaso-ligation, and action of Roentgen rays (Biedl), it is clearly evident their presence is not directly correlated with the spermatogenetic process as such, l)ut with the indifferent cells alone.
In fact, the absence of the reproductive cells — ^or the alteration in the processes in the indifferent cells correlated therewith, possibly of a distinctly 'degenerative' character — ^seems to particularly determine the appearance of these cells (fetal testis, cryptorchid testis, diseased condition).
Plato, in an excellent paper, attempted to correlate the presence of the interstitial cells in the reproductively active testis with the Sertoli cell and concluded that they fulfilled an essentially 'trophic function.' It may be mentioned, however, that during spermatogenesis the indifferent cells are continuously and alternately undergoing regressive as well as progressive changes, and that the largest part of the cytoplasm of the spermatids undergoes degeneration. Hence, it is rather difficult to determine with which phase of the cyclic activity of the Sertoli cells the correlation might be — the entire evidence seems to me to point to the regressive rather than the progressive. In the testis as in the ovary, therefore, it seems to me that the evidence of others indicates that the interstitial cell formation is related to processes that take place within the indifferent cells of the tubule, not directly correlated with spermatogenetic cells and in their absence, and of a fundamentally regressive character. With full cessation of activity within the tubule the interstitial cells would accordingly more or less completely disappear; as, it may be suggested, in extreme atrophy (Cushing's case xxxii, p. 277), and in hibernation (Tandler).
As to the quite distinct question — whether the interstitial cells of the testis, as such, form specific chemical substances which determine the development of the secondary sexual characters, or the appearance of sexual instincts — I only venture to suggest that since there is no direct evidence of their being such specific producers of 'hormones' of internal secretion, and since they never occur separately as far as known to me from the indifferent cells of the seminal tubules, considerable reluctance should be felt in deciding that they alone are the 'gland' that produces the substances with which the influence of the testis in the organism is correlated.
To Prof. S. H. Gage, the writer is indebted for numerous suggestions which are here gratefully acknowledged.
1. The interstitial cells are modified stroma cells, and hence of connective tissue origin.
2. Their origin in the adult as an hypertrophy of theca cells during atresia folliculi is fully confirmed.
3. In the fetus, new born and immature kitten, they appear associated with the irregular so-called medullary cords and follicle formations of these periods.
4. Free lipoid granules appear in the indifferent cells of the atretic follicles, medullary cords and irregular medullary follicles in parts not associated with the ova.
5. The development of interstitial cells appears to be correlated with the activity of the indifferent or follicle cells in the absence of germ cells. The suggestion is strong that an element of degeneration is involved.
6. The zone in which they occur conforms to the centrifugal march of differential growth in the ovary.
7. No morphological value is believed to attach to the distinction of a fetal (or presexual) from an adult grouping of the cells.
8. No evidence is found for regarding the interstitial cells as constituting morphologically an intra-ovarian gland.
9. The recognition of them as constituting a physiological gland of ovarian secretion is regarded as without sufficient evidence.
10. A conii)arison with the interstitial cells of the testis is briefly presented. It is suggested that the same conditions determine their appearance in the testis as in the ovary.
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EXPLANATION OF FIGURES
1 Photograph of section of ovary, kitten 6 days P. P. Flemming's fluid fixation; no stain; no coverglass. X 30. The groups of interstitial cells, appearing black from the contained fat, occupy a central position in the ovary about or close to the medullary cords. Two groups of few cells each occur in the stromal septa of the primitive cortex.
2 Photograph of ovary, kitten, about 5 weeks P. P. Flemming's fluid fixation; no stain; no coverglass. X 20. Interstitial cell-groups appearing black. One small group in the primitive cortex.
3 Photograph of ovary, kitten, about 3 months P. P. Flemming's fluid fixation; no stain; no coverglass. Interstitial cell-groups black. It shows the relation of the interstitial cells to the irregular follicles of this period. Small groups of interstitial cells occur in the cortex. X 15.
4 Photograph of ovary, cat, adult; Flemming's fluid; saffranin stain; interstitial cell-groups black.
EXPLANATION OF FIGURES
5, 6, 7, 8 Four stages of atresia folUculi, to show the origin of the interstitial cells from the theca interna. In the first figure the cells of the internal theca are hardly discernible. The shape and position of the ovum indicates beginning atresia. In the last figure the ovum has disappeared save the zona pellucida, and the antrum is nearly obliterated. Intermediate stages appear in figures 8 and 9. Photographs.
9 Photograph of section of ovary, young virgin adult; Hermann's fluid; no stain; interstitial cell-groups black. To show the typical picture of atresia, and its prevalence at this period. All the follicles save one are in some stage of atresia.
EXPLANATION OF FIGURES
10 Photograph from a section of ovary of an adult cat; Flemming's fluid; saffranin. Four atretic follicles are shown.
11 Camera lucida drawing of a medullary cord; ovary of kitten 3 to 4 days P. P. Fat granules in the cells of the medullary cord are shown, as also the intimate relation of the interestitial cells to the parenchyma.
12 Camera lucida drawing of medullary cords; ovary of kitten, 3 to 4 days P. P.; two cords shown in transection.
13 Photograph of an interstitial cell-group from an adult ovary; cat; Flemming's fluid; saffranin; to illustrate the lipoid content.
14 Photograph of an interstitial cell-group. Zenker's fluid; iron hematoxylin. The lipoid content is therefore dissolved out; some of the nuclei are apparently pyknotic.
15 Drawing, showing the location of interstitial cell-groups in relation to the irregular medullary follicles. Kitten, about 5 to 6 weeks old. X 100. The interstitial cell-groups are shown in black.
16 Drawing, as in figure 15.