Chapter 2

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.[A]

[Footnote A: Prof. E. B. Wilson has recently found a similar dimorphism in the spermatozoa of _Lygæus_ and other of the _Hemiptera heteroptera_.]

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.

In the prophase of the first maturation division this element divides (figs. 233-234), and in metakinesis the two elements are found in various positions with regard to the spindle (figs. 235-237), often as conspicuous as in these figures, but sometimes concealed among the chromosomes. Before the spindle for the second division forms, this element divides again and one of the products goes into each spermatid (figs. 238-241).

As _Sagitta_ is hermaphrodite, there would appear to be no question of sex determination by any special chromatic element. The size of the element _x_, its evident chromatic nature, its division before each mitosis, and its presence in mitosis and in the spermatids, with the same staining qualities as in the previous rest stages, certainly indicate some important function, either in the whole process of spermatogenesis or in the formation of the sperm-head, of which it finally becomes a part. In _Sagitta_ this element certainly can not be regarded as a specialized spermatogonial chromosome, or as chromatin rejected from the spireme. No such element is present in the ovogenesis of _Sagitta_ (Stevens, '03), nor has any been detected in connection with fertilization. It is certain that none is present in the first segmentation spindle of the egg.

GENERAL DISCUSSION.

THE "ACCESSORY CHROMOSOME."

The literature bearing on the "accessory chromosome" of McClung, the "small chromosomes" of Paulmier, and the "chromatin nucleoli" of Montgomery has been fully discussed by McClung in the paper entitled, "The accessory chromosome--sex determinant?" ('02), and will therefore be considered here only in its relation to the several forms studied. The present status of the question has been well summarized more recently by Montgomery under the heading "Heterochromosomes" in the paper, "Some observations and considerations upon the maturation phenomena of the germ cells."

Three theories as to the function of the "heterochromosomes" have been advanced: (1) That of McClung that they are sex-determinants, since in the forms which he has examined these chromatin bodies occur in only one-half of the spermatozoa, and the sex-character is the only character which divides the individuals of a species into two approximately equal groups. (2) That of Paulmier and Montgomery that they are degenerating chromatin. Montgomery regards them as "chromosomes that are in the process of disappearance in the evolution of a higher to a lower chromosome number." (3) That of Miss Wallace, who suggests that in the spider only the one out of each four spermatids which contains the accessory chromosome is capable of developing into a functional spermatozoön, while the other three degenerate, as do the polar bodies given off by the egg. McClung is inclined to believe that the accessory chromosome is an element common to all of the male reproductive cells of Arthropods, and probably to vertebrate spermatocytes as well ('02).

Of the insects considered in this paper _Aphis_ and _Termopsis_ have no "accessory chromosome" or "heterochromosome" of any kind. The fact that no males develop from the fertilized eggs of _Aphis_ might be offered as a reason for its absence, but such an argument would not apply to _Termopsis_. The sex-character may indeed be represented in the chromatin of some one of the pairs of paternal and maternal chromosomes of the spermatocytes, but there is no evident peculiarity by which one-half of the spermatozoa can be said to be different from the other half. As to McClung's statement ('02) "that if there is a cross-division of the chromosomes in the maturation mitosis, there must be two kinds of spermatozoa, regardless of the presence of the accessory chromosome," it appears to me that in a case like the aphid, where the paired elements of the five bivalent chromosomes are separated in the first maturation mitosis, there may be as many as seventeen kinds of spermatozoa instead of two. If, however, we suppose that the sex characters are segregated in the first maturation mitosis, there would, of course, be two equal classes of spermatozoa with reference to that character.

In _Stenopelmatus_ the element _x_ in certain stages closely resembles the "accessory chromosome" of McClung, and especially that described by Baumgartner for _Gryllus domesticus_, but its origin and fate are different. It first appears attached to an end of the spireme in the growth stage of the young spermatocytes, where it is much smaller than in later growth stages. It gradually increases in size, is a conspicuous element in the first maturation spindle, goes into one of each pair of spermatocytes of the second order, and there degenerates during the rest stage between the two maturation mitoses. The whole history of this element suggests that it may be rejected chromatin analogous to that observed in the ovogenesis of many forms. In _Sagitta_, for example, a considerable quantity of chromatin granules is given off by the chromosomes and cast out into the cytoplasm near the close of ovogenesis (Stevens, '03). Rückert ('92) has described a similar casting out of chromatin material by the chromosomes of the oöcytes of _Pristiurus_.

The spermatogenesis of _Stenopelmatus_, therefore, differs from that of the other Orthoptera which have been described in having (1) a larger number of chromosomes (46), (2) an even number in the spermatogonia, (3) an accessory chromatin structure in the spermatocytes of the first order, which disappears before the second maturation division.

In _Blattella_ we have a typical "accessory chromosome," according to McClung. It appears (1) in all resting spermatogonia closely associated with a nucleolus, (2) in the spermatogonial mitoses as an odd chromatin element, making 23 in all, (3) in the growth stage of the spermatocytes connected with an end of the spireme and also with the nucleolus. It becomes separated from the other chromatin in the tetrad-stage, remains nucleolus-like in form, and later appears in the first maturation division either among the chromosomes or in a more or less aberrant position. It passes into one of each pair of spermatocytes of the second order, persists during the rest stage, appears in the second mitosis as a dyad and then divides, going into one-half of the spermatids. The spermatids, however, as in _Stenopelmatus_, all have the same appearance: each has in the center--not against the nuclear membrane--a small element that stains like chromatin. Occasionally a mass of chromatin is found outside the nucleus, but this is not constant enough to support the contention of Moore and Robinson ('05) that the "nucleolus" of the related form, _Periplaneta americana_, is fragmented and cast out into the cytoplasm. The spermatids all appear to develop equally well for some time, but as they approach maturity a varying proportion of them become degenerate. This can not, however, be due to absence of the accessory chromosome, as Miss Wallace supposes, in the spider; for in some follicles no degenerate spermatozoa are found, and in others more than half may be degenerate. All attempts to study fertilization stages of the egg have so far failed, and the chromosomes in the female somatic cells have not proved favorable for counting. Twenty-three have been counted in several cases, but there was always some chance of error. If 23 is the somatic number in both sexes, it must be maintained by union of sex-cells containing 11 and 12 chromosomes, respectively, the same unequal number occurring in the maturated eggs as in the sperm. Under such conditions it is difficult to see how the odd chromatin element of the spermatozoa can determine sex.

The brief description of the chromatin element _x_ in _Sagitta_, introduced here because it behaves like the accessory chromosome in many particulars, serves as an example of the occurrence of such an element in the spermatogenesis of a hermaphrodite form, where it can not possibly be conceived of as a sex determinant. In _Sagitta_ it is known to be confined to the male germ-cells. No such element occurs in the ovogenesis, in the sperm nucleus in the egg, or in the first segmentation spindle. Its function must, therefore, be confined to the process of spermatogenesis.

From the standpoint of sex determination, we have in _Tenebrio molitor_ the most interesting of the forms considered in this paper. In both somatic and germ cells of the two sexes there is a difference not in the number of chromatin elements, but in the size of one, which is very small in the male and of the same size as the other 19 in the female. The egg nuclei of the female must be alike so far as number and size of chromosomes are concerned, while it is absolutely certain that the spermatids are of two equal classes as to chromatin content of the nucleus--one-half of them have 9 large chromosomes and 1 small one, while the other half have 10 large ones. Since the male somatic cells have 19 large and 1 small chromosome, while the female somatic cells have 20 large ones, it seems certain that an egg fertilized by a spermatozoön which contains the small chromosome must produce a male, while one fertilized by a spermatozoön containing 10 chromosomes of equal size must produce a female. The small chromosome itself may not be a sex determinant, but the conditions in _Tenebrio_ indicate that sex may in some cases be determined by a difference in the amount or quality of the chromatin in different spermatozoa. This is much the most suggestive part of the work, and it will be followed up by the study of related forms.

There appears to be so little uniformity as to the presence of the heterochromosomes, even in insects, and in their behavior when present, that further discussion of their probable function must be deferred until the spermatogenesis of many more forms has been carefully worked out.

BRYN MAWR COLLEGE, _May 15, 1905_.

BIBLIOGRAPHY.

BAUMGARTNER, W. J.

'04. Some new evidences for the individuality of the chromosomes. Biol. Bull., vol. 8, no. 1.

MCCLUNG, C. E.

'99. A peculiar nuclear element in the male reproductive cells of insects. Zool. Bull., vol. 2.

'00. The spermatocyte divisions of the Acrididæ. Kans. Univ. Quart., vol. 9, no. 1.

'01. Notes on the accessory chromosomes. Anat. Anz., bd. 20, nos. 8 and 9.

'02. The accessory chromosome--Sex determinant? Biol. Bull., vol. 3, nos. 1 and 2.

'02_a_. The spermatocyte divisions of the Locustidæ. Kans. Univ. Quart., vol. 1, no. 8.

MONTGOMERY, THOS. H., JR.

'01. A study of the chromosomes of the germ-cells of Metazoa. Trans. Amer. Phil. Soc., vol. 20.

'01_a_. Further studies on the chromosomes of the _Hemiptera heteroptera_. Proc. Acad. Nat. Sci. Phila. 1901.

'04. Some observations and considerations upon the maturation phenomena of the germ-cells. Biol. Bull., vol. 6, no. 3.

MOORE, J. E. S., and ROBINSON, L. E.

'05. On the behavior of the nucleolus in the spermatogenesis of _Periplaneta americana_. Quart. Jour. of Mikr. Sci., n. s., no. 192 (vol. 48, part 4).

PAULMIER, F. C.

'93. Chromatin reduction in the Hemiptera. Anat. Anz., vol. 14.

'99. The spermatogenesis of _Anasa tristis_. Journ. of Morph., vol. 15.

RÜCKERT, J.

'92. Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern. Anat. Anz., vol. 7, no. 4 and 5.

DE SINÉTY, R.

'01. Recherches sur la biologie et l'anatomie des phasms. La Cellule, vol. 19.

STEVENS, N. M.

'03. On the ovogenesis and spermatogenesis of _Sagitta bipunctata_. Zool. Jahrb., vol. 18.

SUTTON, W. S.

'02. On the morphology of the chromosome group in _Brachystola magna_. Biol. Bull., vol. 4, no. 1.

'03. The Chromosomes in heredity. Biol. Bull., vol. 4, no. 5.

WALLACE, L. B.

'00. The accessory chromosome in the spider. Anat. Anz., vol. 18.

'05. The spermatogenesis of the spider. Biol. Bull., vol. 8, no. 3.

WILCOX, E. V.

'95. Spermatogenesis of _Caloptenus femur-rubrum_ and _Cicada tibicen_. Bull. Mus. Comp. Zool. Harvard Univ., vol. 27.

'96. Further studies on the spermatogenesis of _Caloptenus femur-rubrum_. Bull. Mus. Comp. Zool. Harvard Univ., vol. 29.

'97. Chromatic tetrads. Anat. Anz., vol. 14.

'01. Longitudinal and transverse division of chromosomes. Anat. Anz., vol. 19, no. 13.

DESCRIPTION OF PLATES.

[The figures of plates I-VI were all drawn with Zeiss oil-immersion 2 mm., oc. 12, and have been reduced one-third; those of plate VII with oc. 8, not reduced.]

PLATE I.

_Termopsis angusticollis._

FIGS. 1-3. Resting nuclei of spermatogonia, showing division of nucleolus.

4. Equatorial plate of spermatogonial mitosis, 52 chromosomes.

5-6. Young spermatocytes, showing division of nucleolus.

7. First maturation spindle, and two nuclei (6 and 8) in same cyst.

8-10. Skein-stage--so-called synapsis-stage.

11-14. Bouquet-stage, showing two nucleoli, centrosome (_c_) in fig. 11, and loops made up of fine, then coarser granules.

15-17. Stage following preceding; loops straightened out and extending in various directions through nucleus.

18. _a_, Chromosomes much shortened and longitudinally split; _b_, chromosomes contracted to form diamond-shaped figures.

19. Stage between 18_a_ and 18_b_.

20. Stage between 19 and 18_b_.

21. Stage similar to 18_a_, one chromosome in double diamond form.

22. First maturation spindle in metaphase, chromosomes in single and double diamond shapes.

23. Chromosome in single diamond or tetrad form, as they usually come into the spindle.

24. Double diamond-form assumed before metakinesis.

25. The 26 chromosomes of an early metaphase.

26. First maturation spindle in metakinesis.

27. Equatorial plate of first maturation spindle in metakinesis.

28. Another spindle, showing three granules which are probably remains of nucleoli.

29. Anaphase of first maturation mitosis, one centrosome divided.

30. Late anaphase.

31-32. Telophase, exceptional cases of division of the cell.

33-36. Partial rest stage between first and second maturation divisions, two nucleoli present. Chromosomes in fig. 36 in form of double diamonds ready for metakinesis.

37-38. Second maturation spindle in metaphase.

39. Equatorial plate of second maturation spindle, 26 chromosomes.