III. Observations.
(a) the nucleus.
The chromatin in Gryllus behaves very much as Henking (’91) has described for Pyrrhocoris, and Paulmier (’99) for Anasa. At the end of the anaphase the chromosomes are crowded together at the poles of the spindle. ([Fig. 1.]) A clear space begins to be formed around them, especially on the equatorial side. ([Fig. 2.]) They then separate and are scattered somewhat evenly over the nuclear membrane when it is formed, as seen in [figs. 4, 5, and 6]. Soon protuberances appear on the surface of the individual chromosomes, making their outlines irregular. At about this time they begin to show a granular structure. The chromosomes break up more and more, and soon the chromatin appears in irregularly arranged patches or blotches of granules of various sizes. ([Fig. 8.])
Meanwhile the nucleus has slowly increased in size. It reaches its maximum, which is probably about treble its original diameter, before the cell begins to elongate. The granules have been breaking up into finer and finer pieces, until, a little after the cell has reached its maximum, the chromatin has largely disappeared, or, to be more exact, has lost its affinity for stains. ([Fig. 16.]) As the cell begins to elongate, the nucleus goes to one end, namely, that toward the periphery of the cyst. ([Figs. 14 and 15.]) The chromatin again frequently appears in patches as the cell decreases in size. ([Figs. 33 and 36.]) In many cells the patches never disappear. When it has diminished to less than half its maximum diameter, the nucleus begins to elongate, becoming elliptical, and finally forms the long, tube-like head of the spermatozoon. ([Figs. 43 and 45.])
I found here that the nucleus was hollow, or, better, that it showed a clear space within. For a long time it contains the many chromatin granules, as seen in [figs. 39, 40, and 41]; but at last the walls seem to attract all these granular masses, and the center is entirely clear, as in [fig. 45]. In [fig. 44] are shown cross-sections of spermatozoa heads, of the stage of [fig. 43]. In [fig. 45] we have a mature spermatozoon head.
The accessory chromosome in Gryllus lags behind during the spermatogonial divisions, just as in Xiphidium (McClung, ’99), and retains its identity all through the growth period, in many cases becoming quite large. At first it cannot be distinguished in the spermatid, but soon its stronger stain and exemption from disintegration make it apparent. ([Fig. 4.]) It is flattened against the nuclear wall and, unlike Paulmier’s “small chromosome,” it does not break up but remains intact, as Henking and McClung have described. ([Figs. 14, 18], [32, 37], and others.) It gets larger and then smaller, and is finally lost in the rest of the chromatin, as the latter condenses to form the spermatozoon head. I was not able to see that it occupied one side of the elongating head, as McClung describes it in Xiphidium. The darker stain and regular contour, and sometimes its greater size, made it in most stages quite evident.
(b) cytoplasmic structures.
It is in the cytoplasmic structures that Gryllus shows some things that have not been described, so far as I know; yet I almost hesitate to enter the discussion concerning the nebenkern, the mitosome, the idiozome, the archoplasm, the attraction sphere, the acrosome, the “mitochondrion körper” and other bodies which have been described in the extra-nuclear parts of the germ-cell. But because I have been unable to find in any of the papers a description of a structure like the one in Gryllus, I shall proceed to describe the nebenkern as I find it. I shall interpret the appearances as far as possible, leaving to others, or to later efforts, the complete harmonizing of this element with the structures of other species.
Before I proceed, I would add my protest against the name “nebenkern,” as voiced by Calkins (’95), Erlanger (’96), Meves (’00), and others. It is inapt and not at all descriptive or distinctive. But since we have the word, and investigators will use it to mean something, I think it would be best to restrict the word, as Calkins and Erlanger suggest, to the remaining spindle fibers and connective fibers which go to each spermatid and which have an important part in forming the tail membrane. I shall retain the name for a further reason, which St. George (’97) gives for first using it, “weil sie die Frage nach der Herkunft dieses Gebildes offen lässt.” A comparison of the literature will convince any one that the origin of this element is still a much-disputed question.
1.—Nebenkern.
There are, in Gryllus spermatids, two quite permanent and prominent extra-nuclear bodies. The larger of these I shall call nebenkern, because I believe it originates from the spindle remains, and goes to form the tail covering. Its behavior is as follows: At the beginning of the telophase the fibers which had connected the centrosomes with the chromosomes bulge out like barrel staves, as Henking (’91) has described. These break loose from the chromosomes and centrosomes as the clear space begins to form around the chromatin mass ([fig. 2]); but they are not separated into two rings, as in Anasa and Pyrrhocoris. As the dividing cell membrane is formed, the middle portions of the fibers are drawn together and the so-called “mid-body” is formed. Some of the fibers unite, producing thicker ones, as seen in [figs. 1, 2, and 3]. In the last-named figure some of the central fibers seem to be still united with those of the companion spermatids.
As the daughter-cell shortens and the nucleus takes a more central position these fibers unite still more, shorten, bend together ([figs. 4, 5, and 6]), and finally assume the shape shown in [fig. 7]. I shall call it for the present the “striated condition,” or simply “striated nebenkern.” The shape resembles that of an egg with rather sharply drawn-out ends. The surface is occupied at intervals with deeply staining lines which look much like hoops. In most cases the long axis of the nebenkern is at right angles to the axis of the spindle from which it was formed, while the striæ, or dark lines, are in planes parallel to it, as is seen in [figs. 7 and 8a]. In a few cases the lines are parallel to the long axis of the nebenkern, as shown in [fig. 8b]. [Fig. 9] exhibits a partial end view of the nebenkern, while [fig. 26] represents a polar view in all planes.
The fibers in [figs. 4 and 5] are not all in the same plane, giving frequently, with changing focus, the impression that the lines are the edges of plates. Even in [figs. 7 and 8], the appearances shown made me hesitate for some time before I dared say that the lines represent striæ on the surface and not plates extending through the body. But the end views ([figs. 9 and 26]) and the two drawings in [fig. 19] show clearly that we are dealing with encircling hoops and not dividing plates. Just how or why the fibers bend and assume the shape of [figs. 7 and 8] I am not able to say, but I am sure it is not an artefact, as might be supposed, because I have found them occupying whole cysts of several testes, and nearly all of them were as regular and definite as [fig. 7].[A]
I have found the nebenkern in its perfect condition ([fig. 7]) only in material fixed in Flemming; although Zenker’s fixation showed some of the breaking-up stages. This may be due partly to poorer fixation in other mixtures and partly to chance, as not so many testes fixed in other agents were examined, and the appearance evidently represents a very brief stage. These fibers now begin to break up, and we get figures that remind one of Meves’s “mitochondrion körper”; that is, a darker staining center and radiating lines to the outer ring. ([Fig.11.]) Sometimes stages a little later recall Paulmier’s “blackberry stage.” ([Figs. 12 and 25.]) [Fig. 19a] is a surface view and [fig. 19b] an optical section of a nebenkern that is in the process of breaking up. [Figs. 20 and 21] have a part of one end cut off, while [fig. 23] is a polar view. [Fig. 22] is probably a forming blackberry stage.
All the above are brief stages, and we soon get the appearance of [figs. 13-16]. Here we have the darkly staining central ball surrounded by a clear space, enclosed by a ring, as Meves (’00) found it in Pygæra. The central ball sometimes shows vacuoles ([fig. 16]), but most frequently stains as one mass. In some cysts with cells resembling [fig. 16], it is difficult to distinguish between nucleus and nebenkern, yet the latter’s stain is never as intense as that of chromatin. The stages shown in [figs. 13 and 14] persist for a long time, and it is probable that this is the end of the telophase and the beginning of the spermatid transformations. I have treated the above more in detail and shown more drawings than its importance may deserve, but it is in these stages that Gryllus seems to differ from other species, and I have tried to describe and illustrate the appearances and changes fully.
As the cell elongates, the axial filament grows out, the nebenkern approaches it, and a junction is effected in such a way that the axial filament runs over the surface of the nebenkern. ([Figs. 18], [27, 28, and 30].) At first the nebenkern is still almost round, but it begins to elongate and the dark inner ball sends out a protrusion to the nucleus, and sometimes back to the point of separation between the external envelope and the axial filament. The outside ring disappears and the dark mass moves down the axial filament. ([Fig. 31.]) In many cases it breaks up into several small drops, which appear at intervals on the axial filaments. ([Figs. 34 and 36.]) In fig. 38 is seen such a hanging drop. In this way the nebenkern material is distributed over the axial filament and forms a sheath around it.
[A] Professor McClung, Mr. Sutton, and Mr. Blackman, of Kansas University, and Doctor Child and Mr. Harper, of Chicago University, have seen my preparations, and all confirm my statement that the appearance is not an artefact.
2.—The Acrosome.
The second extra-nuclear body I shall call the acrosome, as it forms the point of the spermatozoon head. I could not trace its origin, because the fixation was not definite in some of the early stages. [Fig. 4] shows a small, darkly staining body, which may be a centrosome, but more likely is the beginning of the acrosome, of which [figs. 7, 9 and 10] show developing stages, and [figs. 11, 13 and 15] more advanced ones. Sometimes the central portion stains darkest ([figs. 11], [32, and 36]), but more frequently there is a small clearer space in the center surrounded by a ring or band of darker material. ([Figs. 10, 13], [27, and 30].) This ring usually stains darker on one side, very often on the side toward the nucleus.
The position, which is, with few exceptions, in the angle between the nucleus and the nebenkern, induces me to consider the differently staining bodies as the same organ of the cell. For some time after the nebenkern has disappeared the acrosome keeps its position; then it approaches the nuclear wall, flattens against it, and later wanders to the apical end, where it forms the tip of the spermatozoon. ([See figs. 32-36 and 39-42].) [Fig. 16] shows the acrosome back of the nebenkern. It is not an isolated example, still, I think, it is an abnormality. [Fig. 37] shows an apparent division of the acrosome. I did not see enough instances to consider it a regular occurrence.
3.—The Axial Filament.
As the cell is elongating, the axial filament is seen apparently growing out of the nucleus. I do not mean to say that it grows out of the nuclear substance, but in almost every cell observed, where the axial filament was incomplete, there was rather a large mass of chromatin gathered at the place where the axial filament was attached to the nucleus. It may be that the smaller one of the extra-nuclear bodies in [figs. 4, 7 and 9] is a centrosome which is passing to the equatorial region of the nucleus, where it later develops the axial filament. As already noted, the axial filament does not pass through the nebenkern, but only over its surface. [Fig. 29, a, b, and c], shows different cross-sections through the elongating cell. In b the axial filament was cut at an angle.
4.—The Centrosome.
I have not followed the centrosome through its migrations. In [fig. 2] one of the two bodies is the centrosome, but I did not trace its changes farther.
5.—Cell Body.
The cells, up to the stage of [figs. 13 or 14], have been scattered promiscuously through the cyst; but as the cell begins to elongate, the one end becomes the anterior-nuclear end, and it shifts to the periphery of the cyst. The central part of the cyst now shows the so-called central lumen. As the lengthening goes on the heads of the forming spermatozoa are all turned toward the distal end of the cyst. The cyst becomes very narrow and long, apparently preparing to contain the long, slender spermatozoa. Near the rachis the spermatozoa seem much twisted, and I surmise that they turn so as to have the head foremost when set free into the vas deferens. [Fig. 42] may indicate some such behavior.
(c) the spermatozoon.
The mature spermatozoon consists: (1) of a sharp, spear-like point; (2) a tube-like head about .02 mm. long, composed of a densely staining outer wall and a clearer central cavity; and (3) a filamentous tail about .5 mm. long. The point comes from the acrosome, the head from the nucleus, and the tail from the axial filament and nebenkern. While I do not deny the presence of a middle piece, my observations do not warrant me in describing one, although [fig. 40] would suggest it.