Part 2

Blattella germanica.

Unlike the spermatogonia of Stenopelmatus, those of Blattella have both a faintly-staining nucleolus and a deeply-staining chromatin element (x), and moreover the two are always closely associated (figs. 95, 96). The number of chromatin elements in the equatorial plate of late spermatogonial mitoses is 23 (fig. 97). Later events indicate that one of the 23 is the element x, but it is impossible to distinguish it here. Figure 98 is a very early stage of the spermatocyte of the first order, showing the element x as a U-shaped body. The centrosome was also conspicuous in all of the cells of this group. The spireme here, as also in figure 99, is fine and closely interwound. In figure 99 and again in figure 100 the element x is joined to the spireme as it is throughout the spireme stage. In the "bouquet" or "polarized" stage the combined nucleolus and element x are always at one side of the group of loops and down very close to the base of the figure (figs. 101, 103). In figure 102 most of the loops are cut across. The stage shown in figures 104 and 105 is a later one than that just described. Here we have again a continuous spireme connected with the element x, making it seem improbable that the bivalent chromosomes are really separated in the bouquet stage. Figure 106 gives some of the variations in form of the combined nucleolus and element x. The last of the five figures was taken from a giant cell containing at least twice the usual amount of chromatin. In one giant cell four unusually large combinations of this kind were found, and a corresponding amount of chromatin in the spireme. In figure 107 one sees the spireme divided into segments still joined by linin bridges. In figure 108 similar segments may be seen, one of them showing a longitudinal split. The element x is present, but the nucleolus has disappeared. In many cases the split, if it appears at all, closes quickly and the chromosome bends in U-shape, as in figure 109, plate IV. This figure also shows two centrosomes (c). In other cases the split persists as in figure 110 and leads to the formation of crosses of a tetrad character (figs. 111, 112, 113), as in Stenopelmatus and many other insects. Figures 114 to 117 show later stages of the U-shaped chromosomes. Perfect rings are rare. All sorts of variations are seen, broad and narrow U-shapes, rings split at one point or the opposite points, a U split at the bottom (fig. 114), pairs of parallel rods (fig. 115), and occasionally rods constricted in the middle and showing a longitudinal split in each half, as in figure 116. Figure 117 shows different views of the split rings. Apparently all of these forms straighten out so that the two components of the bivalent chromosome stand end to end as dumbbells or compressed crosses in the metaphase of the first maturation spindle (figs. 123-125). The element x remains concentrated and more or less spherical in form. Figures 118-122 are equatorial plates, with x absent in figure 120, in the same plane as the 11 other chromosomes in figure 119, far to one side in figure 118, and near one pole of the forming spindle in figure 122. It is also shown in various positions with regard to the spindle in figures 123 to 126 and 128 to 132. In figure 125 it is apparently double, and again in figure 129. In figure 130 one lagging chromosome shows the dyad nature of the products of the division of the tetrad. In this form there can be no doubt that reduction occurs in the first spermatocyte division. The element x is very often concealed by the polar aggregation of chromatin, but it is sometimes as conspicuous as in figures 131 and 132. The spermatocytes of the second order go into a complete resting stage before they are completely separated, and one of a pair shows the element x, while it is lacking in the other (fig. 133). At the close of the resting stage the chromosomes appear as 11 pairs of rods of considerable length, which gradually shorten and thicken and usually bend at the center, forming U's or V's (figs. 134-138). In one stage these double U's look much like tetrads (fig. 138). The rods straighten again as they shorten still more (fig. 139), become more closely approximated, and finally form dumbbells, as in figure 141.

The element x is, of course, present in only one-half of these nuclei. In the equatorial plate, figure 142, it is absent; in figure 143 it is present, but can not be distinguished from the other chromosomes, while in figure 144 it is rendered conspicuous by its spherical form and isolated position. In only a few cases has it been possible to distinguish x in the spindle. Figures 146 and 147 show two of these cases where this element is clearly double and of different form from the other chromosomes. It is probable that it divides and so goes into one-half of all of the spermatids, as in McClung's typical cases of the accessory chromosome. Figure 145 shows the usual appearance of the other chromosomes in metaphase. The two spermatids of a pair are always alike so far as any evidence of the presence of the element x is concerned (fig. 148). Figure 149 is an exceptional case, where one chromatin element (possibly x) has evidently divided late and been left out in the cytoplasm; a smaller chromatin granule is also present in the cytoplasm of each spermatid. All of the spermatids, as in Stenopelmatus, develop a deeply-staining body, which, however, in this case is usually centrally located and often appears double (figs. 150-152).

The spindle-remains (Spindelreste) forms a very conspicuous body at one side of the nucleus in the spermatids, and occasionally a mass of chromatin, probably due to imperfect mitosis, is found near the spindle-substance (fig. 150). The mass of spindle-substance at first appears structureless, but soon assumes the condition shown in figures 150 to 152. In one individual many of the spermatids had two balls of spindle-material (fig. 152), and the resulting later stages were double-tailed (fig. 153). Figure 156 shows how the spindle-substance goes into the tail and gradually disappears as the tail lengthens.

The centrosome is evidently applied to the nuclear membrane, as in Stenopelmatus, and the middle-piece is developed in connection with it, as in figures 156-157, 154-155, 158-160. The element x of the spermatids gradually disappears (figs. 150, 159). An acrosome develops at the anterior end, the head condenses and lengthens, and we have the ripe spermatozoön (fig. 161). The tail is very long and is shown only in part.

Of the forms studied, Blattella alone has many degenerate spermatozoa. Some follicles have none, others a number varying perhaps from one-fourth to three-fourths of the whole number. No evidence of degeneracy was detected among the young spermatids up to the stage shown in figures 154-155, where a few like figure 162 were found. Most of the degenerate forms occur among the nearly ripe spermatozoa or in the sperm-ducts. Such are shown in figures 163 to 168. The chromatin is strangely broken up into irregular clumps, and probably no two of these degenerate sperm-heads can be found which are alike. The tails are always imperfect. The distribution and varying numbers of these degenerate spermatozoa make it impossible to interpret their condition as due to the absence of the accessory chromosome, as Miss Wallace does in the spider. The only probable explanation, it seems to me, is imperfect mitosis. Cases where more or less chromatin is left behind in the cytoplasm, especially in the first spermatocyte mitosis, are very common, and such cases as those shown in figures 149 and 150 are not rare. The giant cells, so far as I have been able to trace them, do not develop into spermatozoa.

The most important points are:

(1) The presence of the element x in the spermatogonia, closely associated with the nucleolus.

(2) The uneven number of chromatin elements in the metaphase of spermatogonial divisions.

(3) The connection of the element x with the spireme up to the stage where the spireme segments to form the bivalent chromosomes.

(4) The varied character of the tetrads, showing the first spermatocyte division to be a reducing division in the sense that it separates whole chromosomes.

(5) The fact that the element x fails to divide in the first maturation division, does divide in the second, but can not be traced beyond the equatorial plate of the latter mitosis.

(6) The similarity of all the normal spermatids, though one-half of them must contain the element x, the other half not.

(7) The varying and often large number of degenerate spermatozoa.

An attempt was made to determine the somatic number of chromosomes. The dividing cells of the follicles of young eggs seemed to afford the most favorable material, but even here there was so much overlapping of the ends of the chromosomes that it was impossible to be absolutely certain of the number. In the two most favorable cases 23 were counted (fig. 94). This differs from McClung's count for similar cases among the Orthoptera, and Sutton's for Brachystola magna. The eggs have so far resisted all efforts to learn what part the odd chromosome may play in fertilization.

Tenebrio molitor.

In the metaphase of all spermatogonial mitoses where it was possible to count accurately, 20 chromosomes were found, 19 large ones of approximately equal size, and 1 small spherical one (figs. 169, 170). There is nothing in the resting nucleus of the spermatogonia which would suggest either a nucleolus or an accessory chromosome. The chromatin stains well during the whole growth period of the spermatocytes, but it is impossible to separate the period into so definite stages as in most other forms.

In the youngest spermatocytes one finds occasionally a cyst containing cells with nuclei like those of figures 171 and 172, indicating that a brief "synapsis" or condensation stage occurs at the close of the last spermatogonial mitosis. During the greater part of the period the chromatin forms a heavy, irregular, and often segmented spireme (figs. 173, 174). Shortly before the first maturation division, such split segments as appear in figure 175 are sometimes found; some of these simulate tetrads with slender connecting bands between the paired elements. Again, one finds a few cases like figure 176, where the spireme is segmented into bivalent chromosomes, each component showing a longitudinal split. This figure also shows the small chromosome. Usually, however, the irregular and much tangled spireme (figs. 173, 174) condenses into a heavy segmented band variously disposed in the nucleus (fig. 177). This band soon separates into the bivalent chromosomes shown in figures 178 and 179, giving 9 symmetrical pairs and 1 unsymmetrical one (fig. 179 s) composed of the small chromosome and a much larger mate. In the prophase of the spindle, in rare cases, some of the chromosomes are longitudinally split and transversely constricted, forming tetrads (fig. 180), but more often they appear as in figure 181. The unequal pair appears in each figure at s. In the metaphase (fig. 182) it is the last to come into the equatorial plate, possibly because of its lack of symmetry. The smaller component of this pair is always directed toward the equator of the spindle. Figure 183 shows a small tangential section of a spindle in metaphase, containing the unequal pair and one equal pair. In figure 184 a polar view of a metaphase is shown, the unequal pair, which was somewhat below the others, being indicated by stippling. Figures 184 a and 185 show that the unequal components of the unsymmetrical pair, as well as the equal components of the symmetrical pairs, are separated in metakinesis, making this clearly a reduction division. Two polar plates are shown in figures 186 and 187, one containing 10 equal elements, the other 9 equal ones and 1 small one. The telophase is shown in figure 188. There is no resting stage, but the new spindle is formed from the remains of the old one, and the spindle-shaped mass of chromatin seen in figure 188 either passes into the center of the new spindle or becomes enveloped by it. The double chromosomes separate as in figures 189 and 190. Figure 190 shows the small dyad, and figure 189 an aberrant one which may be its mate. The spindle in both divisions is peculiar in having outside of the spindle proper a dense mass of fibers which, in osmic material, stain deeply with iron hæmatoxylin. These fibers are shown in all the figures from 174 to 196. Figures 191 and 192 are equatorial plates of the two kinds of spermatocytes of the second order, figure 191 showing the small chromosome. An early anaphase appears in figures 193 and 194, which show both the small and larger chromosomes in metakinesis. Figure 195 is a later anaphase containing the divided small chromosome. In figure 196 are shown the two polar plates of a spindle corresponding to that of figure 195, and in figure 197 the polar plates of a spindle in which 10 equal chromosomes have been divided. In Tenebrio molitor the spermatids are therefore certainly of two distinct kinds, so far as the chromatin content is concerned.

In most of the young spermatids, after the nuclear membrane has formed, there appears an isolated chromatin element, which corresponds fairly well to the large or to the small component of the unsymmetrical pair, separated in the first mitosis and divided in the second. The clear portion of the nucleus containing this isolated element is at first turned toward the spindle-remains (fig. 198), but before the tail appears either the whole nucleus or its contents have rotated 180° (fig. 199). Various stages in the development of the spermatid are seen in figures 200 to 203. The clear region and the isolated element finally disappear (fig. 202 b), and the chromatin breaks up into coarser and then into finer granules within the sperm-head. In the later stages the centrosome is clearly seen at the base of the head (fig. 203).

In order to determine, if possible, the value of the unsymmetrical pair of chromatin elements, very young ovaries and ovaries with egg-tubes were sectioned and the chromosomes counted in the dividing cells of the egg-follicle (Female somatic cells), and in dividing oögonia. In both cases 20 large chromosomes were found. Figure 207 is the equatorial plate from a female somatic cell of a young egg-follicle. Figure 208 a and b shows two sections of an oögonium in the prophase of mitosis. In order to determine the number and character of the chromosomes in the male somatic cells, several male pupæ were sectioned. As in the spermatogonia, 19 large chromosomes and 1 small one were found. Figure 204 shows the equatorial plate of a dividing male somatic cell, and figures 205 to 206 are daughter plates from a similar cell. (Three large chromosomes of the plate shown in figure 206 are in another section.)

From these facts it appears that the egg-pronucleus must in all cases contain 10 large chromosomes, while the spermatozoön in fertilization brings into the egg either 10 large ones or 9 large ones and 1 small one. Since the somatic cells of the female contain 20 large chromosomes, while those of the male contain 19 large ones and 1 small one, this seems to be a clear case of sex-determination, not by an accessory chromosome, but by a definite difference in the character of the elements of one pair of chromosomes of the spermatocytes of the first order, the spermatozoa which contain the small chromosome determining the male sex, while those that contain 10 chromosomes of equal size determine the female sex. This result suggests that there may be in many cases some intrinsic difference affecting sex, in the character of the chromatin of one-half of the spermatozoa, though it may not usually be indicated by such an external difference in form or size of the chromosomes as in Tenebrio. It is important that related forms should be studied in order to ascertain whether the same chromatic conditions prevail in other species of this genus or possibly in the Coleoptera in general.

Aphis oenotherae.

The spermatogenesis of Aphis has been fully described in another paper and will merely be briefly summarized here for the purpose of comparison with other forms.

The spermatogonia contain a large nucleolus, which gradually disappears in the prophases of mitosis (plate VII, figs. 209-211). The youngest spermatocytes closely resemble the spermatogonia (fig. 212). There is no bouquet stage and no such marked spireme stage as in many other insects. The true synapsis occurs, as shown in figure 213, by pairing of like chromosomes side by side. This conjugation of like chromosomes is followed by a stage in which they are massed together at one side of the nucleus (fig. 214). In these latter stages the nucleolus has entirely faded out and nothing suggesting an accessory chromosome is present. Figures 215 and 216 are equatorial plates of the first spermatocyte mitosis. There are 5 chromosomes of different sizes and shapes, and figure 216 shows each one double. The first division of the chromosomes, though apparently longitudinal, is evidently a separation of the elements paired in a preceding stage, and is therefore a reducing division.

The anaphase of the same mitosis is shown in figures 217 and 218; it is peculiar in that one chromosome always divides more slowly than the others, the two elements hanging together at one end. In figure 219 are sister spermatocytes of the second order, the "lagging" chromosomes still connected. The second maturation division is seen in metaphase in figure 220 and in anaphase in figure 221. Figure 222 shows a young spermatid, the five chromosomes still preserving their characteristic form. Figure 223 is the equatorial plate of the first maturation division of the winter egg, showing the same form and size relations of the chromosomes as in the spermatocyte divisions. Figures 224 and 225 are equatorial plates of a polar spindle (fig. 224) and of a segmentation spindle (fig. 225) of the parthenogenetic egg, where 10 chromosomes are present, 2 of each of the sizes found in the sexual germ cells.

So far as an accessory chromosome or any other visible evidence of a sex determinant are concerned, the results are entirely negative. The conditions shown do, however, support Mendel's conception of the "purity of the germ-cells," and also afford evidence in favor of Boveri's theory of the individuality of the chromosomes.

Sagitta bipunctata.

In connection with these insect forms it is of interest to find in the spermatogenesis of Sagitta a body which stains like chromatin and behaves somewhat like the accessory chromosome. It is found in all resting stages of the spermatogonia, closely applied to the nuclear membrane (fig. 226). It divides before each spermatogonial mitosis (fig. 227) and, though not often discernible in the spindle, appears in the next generation. Figure 228 is the last spermatogonial mitosis, and figure 229 shows the element x, and the chromosomes paired at one pole of the spindle. During the various phases of the growth stage (figs. 230-232) the element x is again applied to the nuclear membrane.