Resumen por el autor, Ludwig A. Emge Notas sobre el estudio de las mitocondrias en el amnios humano. Los grhulos mitocondriales aparecen en las c6lulas del amnios humano en todos 10s estados de la gestaci6n. Bajo el punto de vista morfol6gico son semejantes a las mitocondrias que se encuentran en otros 6rganos en todos sus rasgos, con excepci6n de su afinidad por las materias tint6reas, que es menor que la de las mitocondrias en otros tejidos. Su disposici6n estA influida por la forma de la chlula, pero es constante para grupos definidos de ciertos tipos de c6lulas. El period0 de’ gestaci6n no influye sobre el tipo, forma, tamafio, disposicih o nGmero de las mitocondrias, con la excepci6n de 10s amnios muy j6venes que el autor ha estudiado. Translation by J o d F. Nonidez Cornell Medical College, New York AWIEOR’S ABSTRACT OF TEIS PAPER IMUED B Y THE BIBLIOGRAPHIC SERVICE, DECEMBER 12 NOTES ON THE STUDY OF MITOCHONDRIA I N THE HUMAN AMNION LUDWIG A. EMGE Division of Obstetrics and Gynecology, Stanford University School of Medicine, San Francisco, Colijornia TWO FIQURES The search for mitochondria in the human amnion was undertaken for purely anatomical reasons, in order to prove or disprove their presence and to establish such morphologica1 variations which may facilitate future investigations. It is well known that certain types of these granules exist in other units of the gestation product. Van Cauwenberghe found mitochondria in the cells of the chorion and the placental villi during the first half of gestation. Later, Kervily demonstrated them also in the same structures during the second half of pregnancy, laying Van Cauwenberghe’s failure to find them during the second half to insuficient chroniization of the tissues studied. Kervily concluded that morphologically these granular structures were alike at the various periods of gestation. While studying the amnion I have had occasion to verify Kervily’s findings, and can confirm his contention that mitochondria of quite similar appearance are present in the cells of the chorion and the placental villi at all stages of gestation. On the other hand, our own technical observations do not confirm Kervily’s assumption that Van Cauwenberghe’s failure was due to imperfect chromization, since we saw these granular structures equally often if chromization was materially shortened. TXCHNIQUE The study of the material includes twenty human amnions fully mature and five younger amnions of 13, 4, 5, 7 , and 8 months of age, respectively. Of the first group five were obtained 343 344 LUDWIG A. E M G E at caesarean sections before the membranes had ruptured. The location of the tissue removed from all membranes was carefully noted, and during the early part of the study various locations were studied in each amniotic sac. This latter procedure has proved t o be of greatest importance, since there is a definite difference in the appearance of groups of cells within one amniotic sac. The greatest uniformity is present in the area covering approximately the inner two thirds of the placenta. For a comparative study of human amnions this is the only area which will give sufficiently uniform results to permit conclusions, and this rule for selecting tissues should be observed for all amnions more than five months old. Since Kervily emphasized prolonged chromization of the tissues in order to arrive at the best results, small pieces of amnion obtained directly at birth were fixed in Regaud's neutral formaldehyde-bichromate mixture, which was changed daily for six days. The tissues were then chromicized in 3 per cent potassium bichromate for one week. (Osmic acid could not be obtained at the time of this study.) All tissues were collected in body-warm fluids and the containers kept in an ice-box for twenty-four hours. They were then transferred to a dark locker and kept at room temperature for the remainder of the period. The exclusion of light, and especially sunlight, is of importance in obtaining even penetrations with fixing agents containing potassium bichromate. I arrived at this conclusion by purely physicochemical reasoning. As a rule, such fixing fluids remain clear in the ice-box, and to some degree when light is excluded at room temperature, while, when they are exposed to bright light or sunlight or when they are kept in the incubator at 37"C., the fluids show a heavy precipitate. This means that certain rays and certain temperatures rapidly reduce the potassium bichromate. When this occurs, one finds a heavy water-insoluble layer around the tissue which prevents or materially retards the further process of fixing and mordanting. Since I have observed this occurrence, I have strictly adhered t,o fixing under the exclusion of direct light, and I am convinced that I have obtained tissues more uniformly penetrated by these fixing agents. MITOCHONDRIA I N THE HUMAN AMNION 345 In the course of this study the period of fixing and chromicizing was gradually reduced to twenty-four and forty-eight hours, respectively, and it was found that, this time is entirely sufficient to obtain satisfactory results. I, therefore, cannot join Kervily in his contention that Van Cauwenberghe’s failure was due to an insufficient period of chromization. In this laboratory we practice secondary chromization as advocated by Bensley if we feel that poor staining results are due to imperfect mordanting. This method of chromicizing the mounted section for a few minutes before staining has proved very valuable in our hands. Nevertheless, staining difficulties will be encountered continually when it comes to staining the amniotic cell. This holds true for both vital and other stains. The only explanation that can be offered is that the plasma of this cell has a low chemical or physical affinity for the conventional fixing agents as well as for the mitochondria1 stains. The prohibitively high price of other metals used in fixing fine cell structures has kept me from looking further into this question. The staining of the amniotic cell for mitochondria, either in the vital or fixed state, is a difficult problem indeed. I have not been able to discover the reason, but it remains a fact that this cell as well as all of its finer details have a poor affinity for dyes. After trying out various methods, it was found that modified acid fuchsin mixtures, such as described by Altmann and by Bensley, would give the best results to demonstrate mitochondria, although even these ‘best results’ were not very satisfactory. It is of no use to describe the modifications, as they vary with each individual piece of tissue and depend upon the ingenuity of the morphologist in knowing how to adapt staining methods. Vital dyeing was found t o be unusually difficult. The vital dyes diffuse the cell plasma so quickly that it is often impossible to make out individual structures. To obtain a clear cell picture is a matter of luck in spite of the greatest care in not allowing the tissues to dry and in attempting to keep them at an even temperature. The saline solutions which were used as solvents for the dyes were varied in their composition, but this did not materially affect the end results. There was no better success when amni- 346 LUDWIG A. EMGE otic fluid was used as a solvent. After finding thatalldyesrelated to neutral red and methylene blue were useless on account of their tendency to stain granules of various nature, Janus green B in 1:4000 Ringer’s solution was used as a criterion. I n tissues exposed to this dye, basal striations, apparently made up of mitochondria, could be seen occasionally in certain types of cells; in other types, masses of what seemed to be mitochondrial substance were observed. But the really finer types of granules could not be made out on account of the difficulty stated above. The only definite statement that one can make in regard to vitally stained amniotic tissue is that mitochondria1 granules are present at all stages of gestation described here. A comparative study between fresh and fixed tissue beyond the question of existence of these granules in the amnion proved to be fruitless. A STUDY O F THE FIXED MATERIAL Among twenty-five amnions studied there were twenty mature membranes, five obtained at caesarean sections, before rupture, and one each from a pregnancy terminated therapeutically at I+, 4, 5, 7, and 8 months. The outstanding factor is that there are several types of mitochondria in the cells of all these membranes, but that there is no essential differencein the morphological appearance of the individual types of granules in corresponding types of cells at the various periods quoted. A slight variation is found only on comparing the granules of the earliest to the fully mature membranes. Wide the relative number of mitochondria in the given cells of these membranes is apparently proportionally the same, one does notice a greater uniformity in the size and the shape of these granules as a whole in the earliest three amnions described here. The explanation for this may be the greater uniformity of amniotic cells as such at these periods. In the second half of gestation groups of cells within one amnion vary a good deal in appearance, which finding is most marked ‘at term.’ In the mature membrane there are found, side by side, groups of high and low columnar and cuboidal cells, each group varying somewhat in the detail architecture of its individual cells. MITOCHONDRIA I N T H E HUMAN AMNION 347 Nevertheless, after allowing for these slight variations, one can establish a mitochondrial criterion which is applicable to practically all groups of cell types in the individual membranes. I n the first half of pregnancy the globular and cocci-shaped mitochondrial granules are the most common forms present. In the second half rod-like and filamentous shapes become more conspicuous. and in the fully mature membrane masses suggesting mitochondrial substance are common. While the types of niitochondria are fairly uniform under similar conditions, the distribution of these granules varies within individual cells of the same type. Only in the earliest amnion studied a uniform distribution of the mitochondrial granules was found throughout the individual cell. I n the cells of all other amnions the distribution depends definitely upon the shape of the cell and its location in regard to certain points of pressure. I call attention to the fact that certain locations in the amnion do not represent the true general type of the amniotic cells and its internal architecture. For instance, each membrane has certain areas in which cell life is either interrupted or actually at its end, if one may judge from morphological appearances. The extreme of this trophic disturbance is best seen in an area varying from 8 to 10 or more cm. in diameter, which apparently represents the point of greatest amniotic pressure. Here the cell structures have lost most of their staining affinity. If one may use the expression, it is only the shell of the cell that accepts some dye. TJsually the cells of this area are flattened out. Their nuclei are indistinct or necrotic. Mitochondria1 structures are commonly absent or exceedingly scarce, and in the latter case will not hold the dye. M y first impression was that this area was always located over the internal 0s of the cervix uteri, since it was found here in all of the unruptured membranes obtained a t caesarean sections. That this assumption is not correct was demonstrated later by a comparative study, when it was found that this area may be situated at higher levels and that it corresponds to the site of the spontaneous rupture of the membrane. Usually there are several of these areas demonstrable which vary in degree of trophic changes. It is true that in multiparous THE I N I T O M I C A L RECORD, VOL. 22, NO. 5 348 LUDWIG A. EMGE women such an area is commonly present over the internal 0s of the cervix, although this may not be the future point of rupture, and therefore at least two atrophic areas may be encountered in one membrane. I n primiparous women the location of these areas may be found anywhere. The presence of these areas is constant in all membranes and suggests that the spontaneous rutpure occurs at the point of greatest disintegration of cells. Since the outline of this area or areas is irregular, it was found advisable t o secure tissues a good distance away from the point of rupture. The best and uniform tissues were obtained from that part of the amnion which covers the inner half of the placenta. I have dwelt so lengthily on this factor because if this rule is not observed future studies rriust end in conflicting observations. The distribution of mitochondrial structures in the membranes of the second half of gestation is relatively uniform in corresponding types of cells, as pointed out above. Here one often notices a distinct bipolar distribution of mitochondria with a rather marked difference in the appearance of the granules at each pole. The largest amount of mitochondria is seen in the basal third of the cell regardless of what shape the cell may be. The closer the granular structures approach the actual base of the cell, the oftener they are seen to assume a formation of basal striation. This is most pronounced in the high columnar and piriform cells. This striation is made up of short parallel thin rods of equal length. The individual rods which bulge slightly at their midpoints rarely traverse more than one-sixth of the entire length of the cell and most commonly occupy about onetenth of the long diameter of the cell. When the cell approaches the lower types of columnar shape, the striation begins to move away from the base. At the same time the staining intensity decreases and some of its definition is lost until, when the cell becomes very low, it is lost entirely. Mitochondria1 structures are then replaced by a conglomerate mass that has all the staining characteristics, vital and fixed, of mitochondrial substance. Janus green B is taken up more readily by this substance than it is by defined mitochondrial structures. When exposed to any MITOCHONDRIA I N T H E H U M A N AMNION 349 of the mit,ochondrial solvents t8hemass disappears, which leads me to believe that it is either a massing of mitochondria prior to dissolution or that it is a direct and very early mitochondrial derivative. This mass also has a marked affinity for fat dyes Fig. 1 Photomicrograph with oil-immersion lens. Represents the rod-shaped mitochondria of the columnar cell types. Note the regularity with which they are arranged in basal striation. The cupolas of the cells are conncrted by fine plasma bands. Few spherical mitochondria can be seen in several cell cupolas. Fig. 2 Photomicrograph with oil-immersion lens. Represents the low columnar and cuboidal cell type in which the mitochondrial substance has lost its definition and forms a conglomerate mass away from thc base of the cell. about in the same proportion as mitochondria have when they undergo dissolution. Such masses are rarely seen in the high types of cells and are also uncommon in the very lowest types. Most commonly they are seen in the cuboidal and low columnar types. 350 LUDWIG A. EMGE Towards the mid level of the cell mitochondrial structures become very scarce. The only exception to this is an occasional very slender cell standing distinctly alone and supported only by very delicate plasma strands bridging to adjacent cells. In this type of cell long, slender filamentous mitochondria traverse the entire length of the cell. Individual filaments gracefully embrance the nucleus, leaving a very narrow clear zone in it,s direct vicinity, and enter the cell cupola to form a densely interlaced labyrinth. In all other cells there is, as a rule, a fairly wide nuclear zone which is free from mitochondria. This zone varies in size and position with that of the nucleus. It is widest if the long axis of the nucleus is parallel to that of the cell and narrowest if the two axes intersect at right angles. A fair appreciation of this can be gained only if the cell is seen in its true profile. Most often this zone occupies the upper end of the middle third of the cell, but it may actually move into the upper third and even enter the cell cupola. While in the basal third of the cell rod-shape types of mitochondria are the outstanding structures with the globular and filamentous forms in the minority, the reverse is true of the upper third of the cell. In fact, here rods are so rare that they are negligible and filamentous forms occur onIy in the certain slender cells described. Most commonly, one finds the spherical, small, cocci-like types, but in contradistinction to mitochondria in the basal third these globular and cocci-like structures are ill-defined. They are few in number, vary considerably in size, usually are located in the joints of the cellular network, which renders them still more indistinct, and tends to form a very thin conglomerate layer directly under the membrane of the cupola. Occasionally, one sees strands of these conglomerates enter the intercellular bridges. Aberrant forms of mitochondrial structures may be found in any type of cell, but their number appeared to be so small that it seemed to be insignificant to describe them. No attempt has been made at this time to make a very close comparison of the earlier amnions in regard to mitochondria, because the material is too scanty to obtain definite comparative pictures. Thc MITOCHONDRIA I N THE HUMAN AMNION 35 1 description of these, therefore, has been confined to the most general statements made in the first part of the report. CONCLUSIONS In conclusion iGis stated: That mitochondria1 granules occur in the cells of the human amnion at all stages of gestation; That, with the exception of their staining affinity, which is less than in other tissues, they morphologically resemble in every respect mitochondria found elsewhere; That their arrangement is influenced by the shape of the cell, but that this arrangement is quite constant for definite groups of certain types of cells; That, with the exception of the very earliest amnion studied, the period of gestation has no influence on the type, shape, size, arrangement, or number of mitochondria seen. I wish to express my appreciation to Miss Ella Wing, who looked after the cutting and mounting of the material studied. Stanford University Hospital, &n Francisco. California. BIBLIOGRAPHY KERVILY, MICHELDE 1916 Les mitochondries du syncytium dee villositka placentaires chez la femme. Cpt. Rd. de la SOC. de Biol., T. 79, p. 226. VAN CAUWBNBERGHE, A. 1907 Recherches s u r l e r61e du ayncytium dans la nutrition embryonnaire dans la femme. Arch. d e Biol., T. 23, p. 13.