Development of the Sex Glands of Calotes. I. Cytology and Growth of the Gonads prior to Hatching. By Cleveland Sylvester Simkins, Division of Anatomy, University of Tennessee, School of Medicine, Memphis and J. J. Asana, Department of Biology, Gujarat College, Ahmedabad, India.

With Plates 4, 5, 6.

THERE are two dissimilar and opposing views regarding the origin of the sexual elements in the vertebrates in general, and these dissimilar views are held by workers upon the origin of the reproductive elements in the Reptilia as well. The pro- visional conjectures are these : first, the forerunners of the propagative elements, the so-called primordial germ-cells, originate outside the fundament of the gonad and migrate to that incipient structure throughout the course of early onto- genesis ; second, the definitive germ-cells arise in the gonad, concomitant with its formation from units directly derived from the germinal epithelium. The first view was clearly set forth in the pioneer work of Nussbaum (1880), and the second was probably first enunciated by Waldeyer (1870). Both men worked on animals other than the reptiles. It was not until Allen (1906) traced the migration of the germ-cells in Chrysemys that work was specifically done upon the Eeptilia. Allen maintained that the primordial germ-cells of the turtle arose in the hypoblast and migrated through the entoderm of the gut, and then through the meso- derm of the dorsal mesentery to the incipient gonad. He considered these primordial germ-cells to be extra-regional fore- runners of the definitive sexual elements and were essentially 134 C. S. SIMKINS AND J. J. ASANA alike during the indifferent stage of embryonic development, but they suffered different fates in the course of sexual differen- tiation. In the male the migratory cell became incorporated into the sex-cords and developed into sperms, while in the female the early sex-cords degenerated along with the primordial ele- ments that were incorporated into them; those primordial germ-cells, however, that did not become involved in the de- generating sex-cords developed into ova. According to Allen the primordial germ-cells give rise to both somatic and germinal stuff. Dustin (1910) called the extra-regional forerunners of the definitive germ-cells gonocytes, and explained that they degenerated after migrating to the gonad. Other cells, he believed, arose from the germinal epithelium which developed into the procreative elements. He divided the generations of germ-cells into two, primary and secondary. The primary generation of germ-cells, which are probably the same things as the primordial germ-cells, arose before the anlage of the gonad was even formed, in a region that was not clearly defined. These primary cells remained in the medial edges of the lateral meso- dermal plates until the anlage of the gonads was formed. Immediately the primary germ-cells began to migrate into the fundament and degenerate. The secondary generation of germ- cells arose from the germinal epithelium, bearing no relation whatever to the first, and differentiated into the definitive ova and sperms. Von Berenberg-Gossler (1914) found, in the lizard (La cert a agilis), only the secondary generation of gonocytes, those that arose from the germinal epithelium. He ventured the assertion that the Urgeschlechtzellen, the so-called primordial germ-cells, or the cells of Dustin's primary generation, were not germ-cells at all, but were wandering entoderm cells moving out of the entoderm to swell the ranks of the mesenchyme cells. Jordan (1917), from the loggerhead turtle, adduced evidence that supported a part of Allen's original contention, especially that part dealing with the indifferent stage. Jordan believed that the primordial germ-cells, extra-regional forerunners of the propagative cells, originated early in some unknown region of SEX GLANDS OF CALOTES 135 the embryo and migrated over a route similar to that of C h r y - semys to the gonads, where they directly transformed into either ova or sperms. This is the most extreme contention that has been reported, for there was recognized neither a primary nor secondary generation of gonocytes, and no difference was recognized in the fate of the primordial cells in the two sexes. Simkins (1925), in Trionyx, was unable to find evidence supporting the assumption that the cells of the germ-line have an extra-regional origin. It is quite doubtful whether there is such a cell as the primordial germ-cell at all; in the sense that such cells are the forerunners of the definitive sexual ele- ments that arise in regions remote from the gonad, and migrate thereto over a longer or shorter period of time. It was found in Trionyx that the definitive sexual cells arose from the germinal epithelium. There is no general agreement, among advocates of the view that the primordial germ-cells are segregated early, either upon the time of their origin or as to just where the cells arise. There is likewise no agreement concerning their fate. The preponderr ance of opinion now supports the contention of Waldeyer that the reproductive elements arise from the germinal epithelium, by a process of differentiation of somatic cells into germ-cells. The opinion is growing that both the large and small units in the early gonad share in the process of differentiation. In the female sex the large cells often go into the formation of ova while the small cells in the male transform into spermatogonia. The origin of the definitive procreative elements involves the cytological differentiation of the gonad and does not necessarily involve the primordial germ-cell, since such a cell, if present at all, degenerates in most instances and hence cannot be taken as a forerunner of the sexual elements.^ The postulation of such a cell, or collection of cells, becomes as unnecessary as the degeneration, since the actual units used in building the repro- ductive gland are derived in the last analysis from that portion of the peritoneal epithelium immediately covering the genital thickening. Granting that such is the case, the problem of the embryonic differentiation of the gonads is in many cases un- solved. The present paper deals with the origin of the gonad 136 C. S. SIMKINS AND J. J. ASANA and its cytological differentiation from the first day of laying to hatching. The material was collected and preserved by the junior author.

MATERIALS AND METHODS. The materials used in the present study consist of a collection of embryos of the Agamid lizard, C a 1 o t e s, collected by the junior author in the vicinity of Ahmedabad. The eggs were incubated in the laboratory, and at the desired time the em- bryos were removed and fixed in a variety of fixatives, of which the modified Bouin gave the best results. The incubation period was found to be slightly in excess of six weeks, so for convenience of treatment the collection was arranged into six groups, representing intervals of one week. In the early stages the interval was found to be too large, so the early embryos are regarded as being so many days old, i. e. following the time of laying. Such accurate timing was rendered possible by frequently surprising the female on the nest and recovering the eggs at once. At other times gravid females were captured in the field and the eggs removed by slitting the oviduct.

OBSERVATIONS. The eggs of Calotes undergo considerable development before they are laid. Embryos obtained from eggs immediately after laying, and from eggs removed from the oviduct as well, vary from 2 to 2-5 mm. in crown rump length. In these early embryos the fundaments of most of the organs are well advanced. The neural tube is closed except the anterior and posterior neuropore, the optic vesicle is formed, and the fore- and hind- gut folded off. The mesonephric body extends along the dorsal roof of the body-cavity from the posterior region of the lung- buds to the caudal limit of the coelom. Along the ventromedian border of this mesonephric body there is a thickened layer of cells which extends from the cephalic to the caudal end. The anterior portion of this thickening may be considered the anlage of the cortex of the suprarenal gland and the posterior, SEX GLANDS OF CALOTES 137 the anlage of the sexual gland. It is impossible, at this early age, to tell where one leaves off and the other begins. Transvers- ally this thickening extends from the root of the mesentery to the crest of the mesonephric swelling. The epithelium of the mesenophros, other than over the thickened area, is composed of a single layer of cells, which merges laterally into the epithe- lium of the body-wall and medially into the radix mesentericus. Everywhere the nuclei of the cells composing the epithelium and the thickened layer beneath are ovoid or spherical, of small size, deeply staining, and crowded closely together. The cells of the thickened area are of the same general types, but indicate a more active mitosis by the increased frequency of division figures. This increase in the rate of proliferation is responsible for the thickening, which in some places bulges medially far enough to impinge against the dorsal mesentery. Associated with the small cells of the dorsal body-wall are to be found large masses of yolk incorporated into more or less definite limits. In such units an irregular structure resembling a nucleus can be discerned, whose size, however, is no greater than that of surrounding nuclei. These large masses (fig. 11, PI. 5) are to be found anywhere from the cephalic end of the thickening to the caudal longitudinally, and from the mesentery to lateral body-wall transversally. They are as numerous in the cortical portion of the swelling as they are in the genital, and so cannot be considered special types or even forerunners of the sexual elements. Compared in size to the smaller units they are enormous, and contain more or less closely aggregated granules of material that stains intensely red in eosin and intensely black in osmic acid (fig. 16, PL 6, and fig. 1, PI. 4). In this manner they resemble cells of the wall of the yolk sac. Their peripheral limits are not always distinctly demarcated and rarely regular, although the contour is usually rounded, even in cases where the form is plainly pyramidal. The large granules, which are probably modified yolk spherules, occur always in that part of the cell occupied by the cytoplasm whose ground-work stains lavender with haematoxylin, as does the structure identified as the nucleus. Of particular interest are these tenta- tive nuclei; they are rarely regular in outline, always pale and 138 C. S. SIMKINS AND J. J. ASANA dimly seen, and in many places do not seem to have a nuclear membrane at all. The red (or black, depending on whether eosin or osmic is used) spherules have been no doubt derived from the yolk and represent modified vitelline materials which have been incor- porated into them by direct contact with the yolk-mass. Be- cause these spherules have been conceived as yolk and are included in elements resembling cells, they have been called nurse-cells, with the reservation, however, that they may not conform to a rigid definition of a cell. The nurse-cells differ so markedly from the others among which they lie that they can be identified, quite easily, even under low-power magnification. These nurse-cells have been found in embryos of the first day in the nephrocoel, in the lumen of the fore- and hind-gut (never in the epithelial walls), in the epithelium of the mesonephros, and in the swelling that may either give rise to the cortex of the suprarenal or the genital anlage. There may be an important significance in the fact that the nurse-cells found in the lumen of the gut are much smaller than those found elsewhere, and they stain a more brilliant red in eosin. It is singular, too, that the nurse-cells occur more numerously in those regions of the embryo which are in open communication with the mass of yolk in the yolk-sac. The entire thickening on the ventromedian border of the mesonephros is shown in fig. 1, PI. 4. The smaller and larger units entering into its structure are shown, and is typical of any section cut through the mesonephros from, the caudal to the cephalic end. One is unable to differentiate the suprarenal from the genital anlage. On the second day of incubation the ventromedian swelling is somewhat thicker and the nurse-cells occur in bunches, and are rarely met with outside the thickened area. The smaller units are more numerous and the mitotic figures occur more often in the periphery of the thickening. On the third day the nurse-cells have all disappeared from regions outside the ventromedian thickening, and many found within the thickening show unmistakable signs of retrogression. They are smaller, the granules are not so large, stain feebly and SEX GLANDS OF CALOTES 139 diffusely. The smaller cells of the thickening have multiplied until the layer is now several cells in thickness, probably showing a slightly thicker posterior than anterior end. In embryos of the fourth day the nurse-cells are confined to the thickening, and for the next three days very little change takes place in the shape of cytological constituents of the cortical and genital anlagen. The nurse-cells continue to dissipate until their size is very little greater than the somatic elements derived from the epithelium. As the nurse-cells become progressively smaller and smaller and poorer and poorer in yolk materials, they take more and more of the haematoxylin and less of the eosin. During the first week of incubation the epithelial thickening consists of a single part having more than one layer of cells. The nurse-cells are most frequently found in the peripheral part of this layer, which during all this time undergo no divisions but progressively dissipate. The number of smaller cells is augmented by the division of cells mainly derived from the germinal epithelium, and this division progresses outward, centrifugally, not inward or centripetally. Up to this time, too, the longitudinal mass presents no differentiation into a strict cortical and genital area, but this condition is soon changed. The anterior part of the ventromedian thickening does not keep pace with the posterior part and, also, the cephalic portion increases so as to involve more and more of the root of the mesentery and does not extend so far laterally on the meso- nephric body. This cephalic part has been taken to be the cortex of the suprarenal gland, while the thicker more caudal part must be the anlage of the gonad. One part merges imper- ceptibly into the other, but they begin to acquire characteristic differences which will be brought out later on. The sexual part of the thickening, the gonogenic mass, now tends to become differentiated into medullary and cortical layers (fig. 11, PI. 5). At the close of the first week there is little difference between the cortex and the medulla of the gonad: the chief distinction is that the medulla is more compact and appears of slightly greater density than the cortex. The 140 C. S. SIMKINS AND J. J. ASANA cells are of equal size, though one finds the nurse-cells only in the cortical layer (fig. 11, PI. 4). During the second week of incubation the nurse-cells diminish in size and number and are confined strictly to the cortex of the gonad and of the suprarenal. The yolk-spherules, which were at first large globular masses, fragment, and in embryos of eleven days appear as fine granules surrounding an inde- finitely delimited nucleus (fig. 12, PL 6). Later, as the yolk- granules become more finely divided, the mass takes on a cloud- like effect and is diffused into the cytoplasm. By the end of the second week the nurse-cells have become so reduced that one meets them only occasionally. One still finds them in the lumen of the gut, in the walls of the yolk-sac, and in the pri- mordium of the liver. The close of the second week witnesses another important development. A new type of cell begins to make its appearance and stands out so distinctly that some space will be devoted to its description. This new cell arises in the cortex and is clearly demarcated from the surrounding cells. Its cytoplasmic mem- brane is absent, but the nucleus is much larger than other cells and the cytoplasm immediately around it is pale and lightly staining (figs. 5, 7, 15, Pis. 4 and 6). This cell is undoubtedly a primitive germ-cell and first appears during the second week of development. Such cells have been found in the cortex of em- bryos of the eleventh day (fig. 15, PL 6), and from that time on to hatching their role in the formation of the gonad can be traced successfully. These primary germ-cells are not to be confused with the so-called primordial germ-cell. They have been designated here as primary in the sense that they may not transform directly into the definitive eggs and sperms, and also their behaviour in the two sexes may not be the same. They arise from enlarged cortical cells and are confined, for the most part, to the posterior part of the cortical thickening, although one cannot tell for sure whether any may be in that part of the thickening which forms the cortex of the suprarenal gland. By the end of the second and well into the first part of the third week the cortex of the gonad can be definitely told from the cortex of the SEX GLANDS OF CALOTES " 141 suprarenal by the presence of these germ-cells; for they are then strictly confined to the cortex or germinal epithelium (fig. 7, PI. 4). It is impossible to recognize, in embryos earlier than the eleventh day, a pregonocytic progenitor of these primary germ-cells. No cells that compare with them in general or particular appearance exist anywhere in the embryo prior to their origin on the eleventh day. Some of them may form earlier than we have discovered, and in a few instances we have noticed indications of them as early as the ninth day, but always in the cortex and never in regions remote from the fundament of the sex gland and suprarenal cortex. The only conspicuous elements along the dorsal body-wall before the primary germ-cells arise are the nurse-cells, and we shall show later on that these units bear only an indirect relation to the germ-cells. The origin of the primary germ-cells is con- ceived to be a gradual one ; they do not spring suddenly into being but enlarge slowly until they assume the proportions sufficient to render them conspicuous. Somatic cells derived from the germinal epithelium proliferate to form the cortex, and these cortical cells enlarge throughout the entire period of incubation to form the primary germ-cells. At the end of the third week the nurse-cells have all dis- appeared from the genital swelling, and the fundament enlarges by multiplication of cells in the medulla and cortex. The medulla is the thicker (fig. 7, PI. 4). The rate of growth shifts during the third week and the greater activity takes place in the cortex towards its close: the shift in growth soon results in a fundament whose medullary and cortical parts are of equal thickness. There are no nurse-cells to be found in the fundament and the number of gonocytes has greatly increased, which causes the anlage to bulge into the body-cavity. The disappearance of the nurse-cells can be followed with precision; as a general rule the units decrease in size. The granules of yolk that were at first quite large begin to fragment and apparently to dissolve until they appear as diffuse clouds about a more or less indistinct nucleus. Finally, the clouds are no longer detectable and the nurse-cell ceases to be identified in any part of the embryonic body, except perhaps in the 142 ' C. S. SIMKINS AND J. J. ASANA fundament of the liver. The stages in the dissolution of the nurse- cells can be better understood and graphically visualized if the figs. 16, 20, 17,12, and 13, PI. 6, are compared in the order given. During the fourth week the rate of growth is greater in the cortex than in the medulla, and it takes on cytological characters that differentiate it completely from the core of the fundament. The cells of the cortex are large (fig. 8, PI. 4), dispersed, and surrounded by very clear areas and cytoplasmic mantles that are denser away from rather than proximal to the nucleus. The medulla, on the other hand, is composed of much smaller cells, closely packed, darkly staining, and containing only an occasional large cell. Prom the periphery of the cortex certain cells are seen to lie detached and directed towards the body-cavity as if they had been pushed out by the enlarging fundament. This condition is constantly met with in the development of the gonad of C a 1 o t e s, and it is so striking that it suggests the possible direction of growth of the entire fundament. In a former paper (Simkins, 1925), the senior author mentioned certain down-growths of cells from the ger- minal epithelium of Trionyx in a centripetal manner that gave rise to the primary germ-cells. If there is a movement of cells in the opposite direction, then the fundament increases by a centrifugal and not a centripetal growth, and the correct expres- sion would be an outgrowth rather than an ingrowth. The identity of the germinal epithelium, that is, its identity as a distinct and additional layer of the cortex, is lost. Indeed, the entire cortex seems to be a thickened germinal epithelium which reposes as a sort of cap over the medulla. The mesenchyme cells lying between the germinal epithelium and the tubules and glomeruli of the mesonephros proliferate to form the core of the distending cortex. Certain cells from the inner surface of the cortex become involved in the medulla and help increase its bulk. From the fourth week onwards the distinction between cortex and medulla is so well marked, and the preponderating types of cells are so markedly different, that a very extensive interchange of cells between the two portions of the sexual primordium seems quite unlikely. Both portions pursue independent rates of growth by multiplication of their cells. SEX GLANDS OF CALOTES 143 In embryos of the fifth week the genital anlage increases in size, the lateral and medial folds become more marked, and the attach- ment to the mesonephros is reduced to a relatively narrow nieso- varium (rnesorchium). The cortex does not change greatly, it is somewhat thicker in some cases and in others does not seem to have increased much. The medulla exhibits a tendency to diverge along two lines of differentiation. In one type the cells are arranged in irregular rows from the hilus outward (fig. 10, PI. 5). These rows end in masses of cells against the space that separates the medulla from the cortex. In the second type (fig. 2, PL 4) certain cells of the medulla arrange themselves into incipient rods, or solid cords, not at all unlike the beginning stages in the forma- tion of the seminiferous tubules as observed in other forms (Simians, 1923, 1928). In the earlier stages (fig. 8, PL 4) this cord-like arrangement was not discernible, hence the solid massed arrangement of the medullary cells is taken as the more un- differentiated condition and the cord-like arrangement as the tendency towards higher differentiation. But there are embryos, also of the fifth week, in which no cord-like arrangement of the medullary cells can be seen. The explanation of this seeming anachronism is possibly due to a delay in differentiation, presaging the formation of the female sex. In general, the cortex shows very little change in structure; in those cases, however, where the medulla tends to form the incipient tubules it is not so thick as in cases where no such tubules or cords are present. Its cells rapidly divide as shown by the frequency with which mitotic figures are met, and in those cases where no tubules are found in the medulla there is formed a layer of very small cells around the periphery of the cortex (fig. 10, PL 5), which are in striking contrast to the germ-cells. This is taken to be an epithelial layer and may be germinal, although the activity seems to be in the cells below it. During the sixth week the embryos hatch, and at that time the gonads are quite large and hang from the dorsal body-wall by the mesentery, but the gonads at hatching are not all of the same size and all do not present the same cytological picture in cross- section. In one type the gonad is smaller, the medulla relatively thick with many incipient tubular of cord-like structures. The 144 C. S. SIMKINS AND J. J. ASANA other type has fewer cords and the cortex is very thick and the gland as a whole is larger (fig. 9, PI. 5). One is struck by the absence of a well-defined germinal epithelium around the cortex. Indeed, the whole cortex (fig. 9, PL 5) appears to be a greatly hypertrophied germinal epithelium. It is quite sharply marked off from the medulla and is composed, for the most part, of large cells actively engaged in dividing. The smaller cells are incon- spicuous in both number and size. Sexual differentiation is not completed at hatching, but there is a definite tendency for the gonads to lean decidedly towards the male or female sex. This tendency is revealed in the two types of gonads that can be recognized at hatching, one type with a relatively thin cortex and a thick medulla whose small cells surround certain larger cells in the form of incipient cords, and a second type with thick cortex and relatively thin medulla (fig. 9, PI. 5) in which the sex cords are reduced to a minimum or are not present at all. The former type probably results in the formation of a male and the second a female. In a bird's-eye view of the development of the gonads of C a 1 o t e s one notices that the anlage of the cortex of the supra- renal gland and of the genital thickening are intimately related, so intimately in fact that several days of incubation must pass before one can be sure which is which, and in which there are large conspicuous nurse-cells that dissipate during the third week and seem to take no part in the formation of the cortex of either the suprarenal or the gonads. Along with the disappearance of the nurse-cells the primary germ-cells arise from the somatic cells which were products of the germinal epithelium. As the gonads grow in size, which seems to be accomplished by cells pushing out centrifugally, they differen- tiate into two portions, a medullary and cortical, both of which may play an important role in the process of sexual differentiation later. It is suggested that the further development of the cortex at the expense of or with the diminution of the medulla forms a female, while the augmented medulla at the expense of the cortex results in a male. SEX GLANDS OF CALOTES 145

DISCUSSION. The presence of yolk in the epithelium of the body-cavity during the very early stages of development may have some significance other than indicating that the tissues including such yolk once enjoyed a close contact with the yolk-sac and yolk-mass. If it is permissible to look upon the genesis of the gonad as an expression of the rate of growth, the nurse-cells might conceivably be looked upon as serving to supply the necessary fuel for such increased metabolism. The fact that the nurse-cells were found to remain longer in the genital thickening might favour such a view, and yet the disappearance of the nurse-cells from the cortex of the suprarenal precedes that of the gonads so shortly that increased metabolism must be extended so as to include the formation of both cortices. There might have been an accidental incorporation of the nurse-cells into the epithelium of the body- cavity that has no particular significance at all. It is quite definitely shown that the nurse-cells have no relation whatever with the primordial germ-cell. There is no evidence that the nurse-cells ever move about. They are impassively caught and remain in that place where they are entrapped until their substance dissolves and the nucleus fragments. In a former study the senior writer (Simkins, 1925) pointed out that the reten- tion of yolk by cells for a longer or shorter time was not a reliable characteristic of germ-cells. And, also, the presence of large nurse- cells so widely scattered, in such remote parts of the embryo, leads one to believe that they were passively incorporated there and destined to become resorbed in situ without ever moving anywhere else. There is no evidence whatever that the nurse- cells are migratory. The germ-cells that arise in the cortex come from no more remote regions than the germinal epithelium. If the figs. 15, 19, 14, and 18, PI. 6, are studied in the order mentioned the origin and growth of the germ-cell can be appreciated at once, and if such upward growth is compared with figs. 16, 20, 17, 12, and 13, PI. 6, which show the dissolution of the nurse-cell, the differences in the two processes and their independency can be correctly seen. Whether or not the large cells of the early gonad, designated NO. 293 L 146 C. S. SIMKINS AND J. J. ASANA the primary germ-cell, ever metamorphoses into definite ova and sperm has not yet been demonstrated. It is possible that they do not, because in many forms the smaller units of the gonads (Hargitt, 1925, 1926) have been shown to be the direct forebears of the definitive reproductive cells. The significance of the relation of the medulla to the cortex may be of consequence, in that it would serve as a basis for the explanation of sex intergrades embryologically, in which case a sex intergrade could be looked upon as consisting of an equal proportion of cortical and medullary portions more or less in- timately intermingled. The fate of the two portions, cortex and medulla, will be dealt with in detail in the forthcoming study on the development of the sex-gland from hatching to maturity. The general hypothesis that definitive ova and sperms have extra-regional forerunners in far-off places of the embryo is not supported by the experimental evidence now accumulating. Janda, 1929, has reported unmistakable evidence that in the segmented worm, Criodrilus, both male and female germ- cells regenerate from somatic cells. Stohler, 1928, finds that there is seasonal regeneration of the germ-cells from the peritoneal epithelium in the European toad, and the junior author has determined a like seasonal regeneration of the germ-cells in C a 1 o t e s. The evidence from castrations is conflicting. Hanson and Heys, 1929, report some cases of regeneration of the ovaries in the rat. The conflict seems to be one of definition of the word and concept of regeneration. If regeneration is denned as the restoration of the original form after mutilation, then there can be no doubt that in the mammals there is a high percentage of regeneration, but if regeneration of ovaries is defined as the formation or transformation of differentiated peritoneal epithe- lium into an ovary, then no such thing occurs. The excellent work of Humphrey, 1928, by no means clinches the solution of the problem in favour of the primordial germ-cell. In his series of experiments on the embryos of Amblystoma, the mesoderm containing the early gonads was removed along with the whole lateral window in the abdominal wall. After the lapse of a certain length of time he could find no evidence that germ- glands reformed on the operated side. If the embryos had been SEX GLANDS OF CALOTES 147 reared to sexual maturity and found sterile or lacking one gonad on the operated side, greater weight could be attached to the conclusions. On the other hand, there is no assurance that the germinal epithelium, which is the sole source of the germ-cells, was left intact. Of course, if the germinal epithelium is removed, the somatic source of the germ-cells is gone, and there could be no sex-cells or sex-gland formed. The evidence now building up concerning the phenomena of sex reversal does not support the belief that sex is irrevocably fixed, or that the germ-cells are set apart early in ontogeny. Such a view denies the influence of the hormones and the rate of meta- bolism on the determination of sex and the formation of the sexual elements. The forerunners of the definitive sexual cells arise from the germinal epithelium after the anlage of the gonad is formed, and during the course of development some become modified into eggs, some degenerate, and in some cases the smaller cells of the reproductive gland directly change over into the procreative elements. SUMMARY. 1. The ventromedian thickening on the mesonephric body gives rise to the cortex of the genital gland and the suprarenal. 2. Nurse-cells are to be found in this thickening, which disappear during the third week of incubation, without influence on the germ-cells. 3. The primary germ-cells arise in the anlage of the gonad while the nurse-cells are degenerating, from elements derived from the germinal epithelium. 4. The gonad at the fourth week is composed of a medullary and cortical portion, the former composed of small cells, some of which begin to arrange themselves into incipient cords before hatching; the latter is composed of large -cells and probably represents the thickened germinative epithelium. 5. There is a tendency towards sexual differentiation at hatching, but sex is not yet definitely established.

NO. 293 L 2 148 C. S. SIMKINS AND J. J. ASANA

BIBLIOGRAPHY. Allen, B. M. (1906).—" Origin of the sex-cells of Chrysemys ", ' Anat. Anz.', Bd. 29, S. 218-36. Asana, J. J. (1928).—' Observation on the incubation of the eggs and the development of the embryo of Calotes versicolor Boulenger ', Indian Science Congress, Calcutta, 1928. von Berenberg-Gossler, H. (1914).—" tJber Herkunft und Wesen der sogenannten primaren Urgeschlechtszellen der Amnioten", ' Anat. Anz.', Bd. 47, S. 241. Dustin, A. P. (1910).—'L'origine et revolution desgonocytes chez reptiles", 'Arch, de Biol.', torn. 25, pp. 495-534. Hanson, F. B., and Florence Heys (1929).—" On ovarian regeneration in the albino rat", ' Proc. Soc. Exp. Biol. and Med.', vol. 25, pp. 183-4. Hargitt, G. T. (1925).—" The formation of the sex-glands and germ-cells of mammals. I. The origin of the germ-cells in the albino rat ", ' Journ. Morph. and Physiol.', vol. 40, pp. 517-57. (1926).—" The formation of the sex-glands and germ-cells of mam- mals. II. The history of the male germ-cells in the albino rat ", ibid., vol. 42, pp. 253-305. Humphrey, R. R. (1928).—" The developmental potencies of the inter- mediate mesoderm of Amblystoma when transplanted into ventro- lateral sites in other embryos, the primordial germ-cells of such grafts and their role in the development of a gonad ", ' Anat. Rec.', vol. 40, pp. 67-101. Janda, V. (1929).—" tJber einen besonderen Fall von Heteromorphose und andere kiinstlicherzeugte Abnormitaten bei Criodrilus lacuum Hofi ", ' Arch. f. Entwick. Mechanik. der Organ ', Bd. 114, S. 587-92. Jordan, H. E. (1917).—' Embryonic history of the germ-cells of the logger- head turtle, Caratta caratta.' Pub. no. 251, Dept. Marine Biol., Carnegie Inst., Washington, vol. 11, pp. 315-44. Lloyd, J. H. (1929).—" Hermaphroditism in the common frog ", ' Amer. Nat.', vol. 63, pp. 130-8. Simkins, C. S. (1923).—" On the origin and migration of the so-called primordial germ-cells of the mouse and rat ", ' Acta Zool.', Arg. 4, Heft. 2-3, S. 241-84. (1925).—"Origin of the germ-cells in Trionyx ", 'Amer. Journ. Anat.', vol. 36, pp. 185-213. (1928).—" Origin of the sex-cells in man ", ibid., vol. 41, pp. 249-93. Stonier, R. (1928).—" Cytologische Untersuchungen an der Keimdriisen mitteleuropaischer Kroten (Bufo viridis Laur., B. calamita, B. vulgaris)", ' Zeitschr. f. Zellforsch. mikr. Anat.', Bd. 7, S. Waldeyer, W. (1870).—' Eierstock und Ei.' Leipzig, W. Englemann, 174 S., 6 Taf. SEX GLANDS OF CALOTES 149

EXPLANATION OF PLATES 4-6.

ABBREVIATIONS. art.ren., renal artery; cl.g., germ-cell; cl.mt., mitotic cell; cl.so., somatic cell; cl.vt., nurse-cell; cr.t cortex of the gonad ; e'th.g., germinal epithelium ; e'th.pi'th., peritoneal epithelium ; m., medulla of the gonad ; sph.vt., yolk spherule ; ibl., incipient tubule.

PLATE 4. Fig. 1.—Cross-section of the genital ridge, first day after laying, x 1000. Fig. 2.—The gonad, 40 days of incubation. X 750. Fig. 3.—Cortex of the gonad during the fourth week of incubation. X 750. Fig. 4.—Germ-cells in the cortex at hatching, x 1500. Fig. 5.—Germ-cells from the cortex, 16 days' incubation. X 1500. Fig. 6.—Enlarged cell from the medulla, 16 days' incubation, x 1500. Fig. 7.—Cortex of the gonad at 16 days' incubation. The medulla is not shown but would occupy the space in which fig. 5 is placed. X 750. Fig. 8.—A portion of the cortex and medulla of the gonad at 30 days' incubation. Free cells moving centrifugally. x 750.

PLATE 5. Fig. 9.—Cross-section of the gonad at hatching, x 750. The enlarged germ-cell at end of leader cl.g. X 1500. Fig. 10.—Cortex and medulla of a gonad at 30 days' incubation. X 750. Fig. 11.—Cortex of the gonad at 7 days' incubation. The stroma and that part destined to form the medulla, not shown, x 1000.

PLATE 6. All figures drawn to the scale of 1500 diameters magnification. Fig. 12.—Nurse-cell at 12 days' incubation. Fig. 13.—Nurse-cell at 20 days' incubation. Fig. 14.—Germ-cell at 28 days. Fig. 15.—Germ-cell at 11 days. Fig. 16.—Nurse-cell on the first day after laying. Fig. 17.—Nurse-cell on the eleventh day. Fig. 18.—Germ-cell at hatching. Fig. 19.—Germ-cell at the sixteenth day. Fig. 20.—Nurse-cell on the seventh day. Quart. Journ. Micr. Sci. Vol. 74, N. S., PL 4

C. S. Simkins, del. Quart. Journ. Micr. Sci. Vol. 74, N.S., PI. 5

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C. S. Simians, del. Quart. Journ. Micr. Sci. Vol. 74, N.S., PI. 6

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C. S. Simkins, del.