Resumen por el autor, Jean Firket Escuela M6dica Johns Hopkins Sobre el origen de las c6lulas germinales de 10s vertebrados superiores En el testfculo y ovario del pollo existen dos generaciones de c6lulas germinales: las c6lulas germinales primarias, que aparecen en estados muy tempranos del desarrollo, antes de formarse la cresta genital, y las cklulas germinales secundarias, que se derivan del llamado “ epitelio germinal.” Las primeras c6lulas pueden transformarse en espermatocitos y ovocitos y aunque degeneran en su mayor parte no es posible determinar si algunas de ellas producen c6lulas germinales definitivas, porque en ciertos estados es imposible distinguirlas de esta tiltima clase de c6lulas. En el macho de la rata blanca aparecen las mismas dos generaciones, per0 las c6lulas germinales primarias degeneran antes de alcanzar el period0 de crecimiento y solamente las c6lulas germinales secundarias se transforman en las definitivas. La desaparicih de las c6lulas germinales primarias de 10s mamiferos durante la ontogen6sis en estados mas tempranos del desarrollo que en las sves parece indicar que se las debe considerar como cklulas en “ regresi6n filogen6tica.” Translation by Jose F. Nonidez Csrnegie Institution of Washington A U T E O R J hB-TH4CT O F T H I S P 4 P E R IS S U ED B Y T H E BIBLIOGRAPHIC SERVICE, MARCH 20 ON THE ORIGIN OF GERM CELLS I N HIGHER VERTEBRATES JEAN FIRKET Anatomical Laboratory, Johns Hopkins Medical School The opinion that a continual germinal path exists in all animals and especially in the higher vertebrates is gradually replacing the more conservative idea that germ cells are derived only from Waldeyer’s germinal epithelium. Many investigators now hold that, in the early stages of the ontogenesis of any species, cells occur which are absolutely distinct from the soma cells and which are the only cells capable of giving rise to definitive germ cells during the later development. These cells are called primordial germ cells. Such a continual germinal path, the so-called ‘Keimbahn’ of German authors, has been described in Ascaris, by Boveri; as early as the fifth segmentation division, one is able to recognize one of the blastomeres as being the only progenitor of all future germ cells. It would be certainly valuable, if all animals could be shown to have this development in common. Many embryologists have accepted this theory for higher vertebrates, but only a few have attempted to prove it; the principal among these are Rubaschkin and Swift. Everyone knows that, in the amniotes, primordial germ cells have been shown to occur in very early stages of ontogenesis before the differentiation of soma cells, and therefore well before the appearance of the genital ridge. The early history of these elements has been carefully studied during the past ten years, but the later history of their evolution, when they are embedded in the genital gland, is not so clearly settled. Three opinions regarding the later development of these germ cells in higher vertebrates are now defended. 309 310 JEAN FIRKET 1. Some investigators claim that the primordial germ cells in the genital glands are the only possible germ cells, and that in accordance with this fact the existence of a ‘continual germinal path’ is a reality (Rubaschkin and Swift). 2. Others believe that the so-called ‘primordial g e m cells’ are really germ cells, but that most of them degenerate and that a second generation of germ cells is derived from the so-called .‘germinal epithelium’ (Waldeyer) or the sex cords which it produces (Felix, Dustin, Allen, and Firket). These authors call the germ cells belonging to the first generation ‘primary germ cells’ V.S. to the germ cells derived from the germinal epithelium which are called ‘secondary germ cells.’ 3. Still others assert that the ‘primordial germ cells’ are not true germ cells, but are temporary hypertrophied cells which disappear later and that all definitive germ cells are derived directly from the germinal epithelium (de Winiwarter et Sainmont, von Berenberg-Gossler). .During the five years preceding the war, I studied, in the Department of Anatomy of the University of Liege, the changes in primary germ cells during the development of the genital glands in birds. Only one part of this work has been published.1 The second part dealt chiefly with the development of the chicken’s testis. It was entirely written when the war broke out,’but because I was on duty in the Belgian army, these last observations could not be published. The Archives de Biologie, printed in Brussels and suspended during the war, will publish my work in 1920. As regards primary germ cells in the chick, my opinion is as fo1lows:z They are free cells embedded in the mesenchyme tissue of the splanchnopleure and of the radix of the mesentery before the genital ridge appears. In these early stages and in the first days of the developing genital glands, they are migrating cells, and their migration is ensured by their own movements Recherches sur l’organogenese des glandes sexuelles chez les oiseaux. Ch. I B VI. Arch. de Biol., T. 59, 1914. * These principal results were published in a preliminary note. Anatomischer Anzeiger, 1914, Bd. 46, S. 422. GERM CELLS IN HIGHER VERTEBRATES 311 until they are embedded and fixed in the sex cords of the genital glands either in the medullary and cortical cords of the ovary or in the sex cords of the testis. Some of them do not reach the sex gland, but they continue their evolution outside of it, becoming oocytes or spermatocytes while still embedded in the mesentery or rete. It seemed to me most important, in order t o prove their sexual nature, to observe, outside the genital gland, the transformation of these cells into well-characterized oocytes, for the oocytes observed inside trhe genital organs can always be said to be elements derived, in situ, from the germinal epithelium or the epithelial derivatives. Most of the primary germ cells in the testis as well as in the ovary subsequently degenerate, and the great majority of the definitive ova or spermatogonia are derived from the epithelial organs of the sex glands and must consequently be called secondary germ cells; nevertheless, there is no reason why certain of the primary germ cells may not produce some of the definitive ova or spermat,ogonia, as it is impossible, at one stage of the ontogenesis, t o distinguish between primary and secondary germ cells. In addition t o the above interpretation of these observations in the chick, the following hypothesis was derived: primary germ cells must be considered to be “un rappel phylogenique des gonocytes ddfinitifs des classes infdrieures notament des cyclostomes et des Acraniens.” Since this work was concluded, other papers on this subject have appeared. Another account dealing with the ovary and the testis of the chick has been written by Swift. Though Swift confirms most of my observations on the organogenesis of the indifferent gonad and ovary, he does not agree with me in regard to the existence of secondary gonocytes. His articles and others, which have been publi’shed during the war, will be discussed later in a more complete paper with a fuller review of the literature, where the details of my study on the testis of the albino rat will be given. Notwithstanding the last articles which conclude that only primordial germ cells form definitive sex cells, the evidence does not seem to me sufficient to establish this opinion. M y own 312 JEAN FIRKET research, started in 1914 on the testis of albino rats, has reinforced my fornier opinion and brought out a new argument in favor of the hypothesis previously stated regarding the morphological value of the primary germ cells. Before relating briefly my observations on albino rats, it is a pleasure to acknowledge the kindness of the Department of Anatomy of the Johns Hopkins Medical School and especially that of Doctor Sabin and Doctor Weed for their good welcome and the great opportunity for work which Ifound in their laborat ories. The material studied consisted of the testis of young albino Tats of all stages from birth until seven weeks and also of a few embryonic stages. The organs were fixed in different fixing fluids, particularly those of Flemming, Meves, Regaud, and mixture). The three Bensley (bichromate-osmic-acetic-acid latter were employed for showing the mitochondria (chondriosomes), because Rubaschkin stated that these methods were elective for studying the primary germ cells in other species.3 The structure of the foetal sex cord of the testis has been known for a long time; two kinds of cells have been described-large and small-their nuclei lying regularly in a single row in the peripheral portion of the cord. The larger cells are characterized by their size, their well-outlined cytoplasmic body, and their clear and round nuclei, while the nuclei of the small cells are smaller, more deeply stained, and are oval in shape with their long axes at right angles to the basal membrane. The occurrence of these two kinds of cells brings up the problem as to whether the larger or the smaller gives rise t o the future spermatogonia. This question has become still more interesting since it is evident that the fo&er are not derived from the latter, as is stated in many text-books and even by recent investigators (Hoven), but that the largest cells of the sex cord 8 I have used for this work material belonging to the embryological collections of the Anatomical Department of Liege. First collected by Doctor Duesberg, afterward by Doctor Hoven, and then by myself; I am still enlarging it, due to the kindness of Dr. Milton J. Greenman, of The Wistar Institute in Philadelphia. GERM CELLS I N HIGHER VERTEBRATES 313 of the foetal testis are exactly the same as the so-called ‘ primordial or primary germ cells’ which appear in the early stages of ontogenesis. This has been definitely established by the work of Rubaschkin on mammals and that of Firket and Swift on the chick. The structure of the sex cord of the albino rat, the first day after birth, is just the same as the typical one described above. The large cells, which we may now call ‘primary’ germ cells, are easily observed in sections fixed or stained with various histological methods. In these cells a very striking cytological feature may be observed; the mitochondria are grouped in a complete ring surrounding the nuclei, all of them are thick, darkly stained, and very sharply outlined granules; this aspect of the mitochondrial substance differentiates them sharply from the smaller cells (ordinarily called indifferent germinative cells), in which the mitochondria are short undulated filaments. This cytological aspect of mitochondria has been already described in the primordial germ cells of the guinea-pig by Rubaschkin; he attached great importance to this latter feature, because he thought that a granular shape of mitochondria was evidence of the undifferentiated nature of the cells containing them, in contrast with the soma cells in which their filament-shaped mitochondria show their differentiated nature. I have already discussed in a previous paper that interpretation, and have said that I could not accept it; however, it is a valuable cytological method for following these cells in their development. What is the evolution of these cells in the developing testis of albino rats? During the days following birth, the gonad increases in size, because of the active multiplication of the small epithelial germinative cells of the sex cord. Among them foci of mitosis take place in which the long axis is most often in a line parallel with the long axis of the sex cord itself. The primary germ cells in this stage do not, or at least very infrequently, divide. This difference in the distribution of the foci of cell division explains the fact that in earlier stages an average of three or four small epithelial cells were found to separate the two nearest primary 3 14 JEAN FIRKET germ cells, but that in the five-day-old rats this number has increased to nine or ten. In the following stages, from the fifth to the tenth day, this number increases still more rapidly, according to a new phenomenon of the evolution of the primary germ cells : during the first five days their relative number seemed to decrease because they were not dividing while the sex cords were increasing; after the fifth day their number diminishes absolutely, but not relatively, because they degenerate. A special type of their degeneration was striking and frequent, characterized by a great hypertrophy of the cells, in which cytoplasm and nucleus seem to be filled with water; the chromatin is shriveled into four or five irregular blocks staining weakly and lying next to the nuclear membrane; the mitochondria lose their sharp outlines and seem to be dissolved in the general cytoplasm. This type of degeneration seems to be a typical one in the evolution of primordial germ cells, as has been shown in the ovary and testis of the chick;4 this may be compared to the type of degeneration described by Champy as ‘ degenerence ovif orme.’ I n the eighth-day stage, the ratio of the cells in a sex cord is one primary germ cell to eighteen small epithelial cells, while in a rat of ten or eleven days old the primary germ cells become very rare. In this stage there are only a very few cells which may represent the remains of the primary germ cells, they are interspersed in the peripheral portion of the sex cord among the small epithelial cells, from which they are not easily distinguished, although their protoplasm is more deeply stained, their nuclei have a more round shape and seem to contain less chromatin. The number of these cells is so small, two or three in the whole transverse section of the testis, that we may surely conclude that, as regards their relation to future spermatogonia, the, primary germ cells have disappeared entirely in the albino rat’s testis from the tenth t o the fifteenth day after birth. A t that time the first spermatogonia appear; they are easily recognizable by the texture of their nuclei and are very numerous. 4 J. Firket, Recherches sur l’organogenese des glandes sexuelles des oiseaux. Anat. Anzeiger, Bd. 46, S. 422. GERM CELLS IN HIGHER VERTEBRATES 315 This has been shown very distinctly in the same species by Hoven. Let us insist that these spermatogonia can only be derived from the small epithelial cells, as they are at this stage the only type of cells present in sufficient number in the sex cord. The spermatogonia must then be called ‘secondary germ cells.’ It is important t o insist that these results have been obtained by studying the sections where the choddriosomes were stained as well as those prepared for the study of nuclear texture. That mitochondria have a very definite granular shape in the firstday stage has been previously noticed; in the later stages, from the tenth t o the fifteenth day, no cells containing such mitochondria can be found. All the cells composing the sex cofds contain mitochondria which are not deeply stained and which have the shape of short filaments. The principal conclusion derived in this study of the testes of young rats is as follows: two generations of germ cells exist which can very easily be distinguished from each other because the cells of the second generation (the so-called ‘secondary germ cells’) only arise when those of the first generation (the so-called ‘primary germ cells’) have disappeared. This is in interesting contrast with what I have described in the chick, where it is impossible t o separate the first from the second generation of germ cells when they are present together. In general, the primary germ cells in the chick are able t o develop at least until the period of growth; some of these having become oocytes and spermatocytes reach the well-characterized stages of preparation for maturation divisions. This is not true in the albino rat; no primary germ cells ever reach the period of growth, all of them degenerating at an earlier stage. If we take the chick as the avian type and the rat as the mammalian type, the following conclusion may be drawn: the primary germ cells disappear in the ontogenesis of mammals earlier than they do in the ontogenesis of birds, and can therefore be considered as being cells in a ‘phylogenetic regression. 316 JEAN FIRKET Let us insist, however, that the lack of function or, at the most, the very low importance of function which the primary germ cells seem to have in amniotes does not diminish the importance of their morphological nature which is asserted by their ancestral character. BIBLIOGRAPHY ALLEN, B. M. 1904 The embryonic development of the ovary and testis of the mammalia. Am. Jour. Anat., vol. 3, p. 86. 1906 The origin of the sex cells of chrysemys. Anat. Anzeiger, Bd. 29. VON BERENBERQ-GOSSLER 1914 Ueber Herkunft und Wesen der sogenannten primiiren Urgeschlechtszellen der Amnioten. Anat. Anzeiger, Bd. 47, S. 241. 1912 Die Urgeschlechtszellen des Huhnerembryos am 3. und 4. Bebrutungstage. Arch. f. mikr. Anat., Bd. 81. S. 24, Abt. 11. DUSTIN,A. P. 1910 L’Origine e t l’evolution des gonocytes chez les reptiles. Arch. de Biol., T. 25, p. 411. FELIX1906 Die Entwickelung der Keimdrusen. Hertwig’s Handbuch. FIRKET,JEAN1914 Recherche5 sur l’organogenese des glandes sexuelles des oiseaux. Arch. de Biol., T. 29, e t Anat. Anzeiger, Bd. 46, S. 413. JORDAN, H. E. 1917 Embryonic history of the germ cells of the loggerhead turtle (Caretta caretta). CarnegiePublication 251, vol. 11, pp. 313-344. REAGAN, F. P. 1916 .Some results and possibilities of embryonic castration. Anat. Rec., vol. 11, p. 251. RUBASCHKIN, W. 1910 Chondriosomen und Differenzierungsprozesse bei Saugetier embryonen. Anat. Hefte, Bd. 41, 8. 399. 1912 Zur Lehre von der Keimbahn bei Saugetieren. Anat. Hefte, Bd. 46. SWIFT, C. H. 1914 Origin and early history of the primordial germ cells in the chick. Am. Jour. Anat., vol. 15, p. 483. 1915 Origin of the definitive sex cells in the female chick and their relation t o the primordial germ cells. Am. Jour. Anat., vol. 18, p. 441. 1916 Origin of the sex cords and definitive spermatogonia in the male chick. Am. Jour. Anat., vol. 20, p. 375. VON WINIWARTER,H., AND G. SAINMONT1909 Nouvelles recherches 8ur l’ovogenese e t l’organogenese de I’ovaire des Mammiferes. Arch. de Biologie, T. 24, p. 1.