Studies in Spermatogenesis (part 1 of 2)

Part 1

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STUDIES IN SPERMATOGENESIS

WITH ESPECIAL REFERENCE TO THE

"ACCESSORY CHROMOSOME"

BY N. M. STEVENS.

WASHINGTON, D. C.: Published by the Carnegie Institution of Washington September, 1905.

CARNEGIE INSTITUTION OF WASHINGTON

PUBLICATION No. 36

FROM THE PRESS OF THE HENRY E. WILKENS PRINTING CO. WASHINGTON, D. C.

STUDIES IN SPERMATOGENESIS WITH ESPECIAL REFERENCE TO THE "ACCESSORY CHROMOSOME."

By N. M. STEVENS.

In connection with the problem of sex determination it has seemed necessary to investigate further the so-called "accessory chromosome," which, according to McClung ('02), may be a sex determinant. This view has been supported by Sutton ('02) in his work on Brachystola magna, but rejected by Miss Wallace ('05) for the spider.

The forms selected for study have been taken from several groups of insects, and are all species whose spermatogenesis has not been previously worked out. They are (1) a California termite, Termopsis angusticollis; (2) a California sand-cricket, Stenopelmatus; (3) the croton-bug, Blattella germanica; (4) the common meal-worm, Tenebrio molitor; and (5) one of the aphids, Aphis oenotheræ.

A brief account of a chromatin element resembling the accessory chromosome in Sagitta has been added for comparison. The spermatogenesis of each form will be described in detail, and a general discussion of the results and their relation to the accessory chromosome and sex determination will follow. The spermatogenesis of the aphid has been included in another paper, but a summary of results and a few figures will be given here for reference in the general discussion.

METHODS.

The testes were fixed in various fluids--Flemming's strong solution, Hermann's platino-aceto-osmic, Gilson's mercuro-nitric, Lenhossek's alcoholic sublimate acetic, and corrosive acetic. Flemming's and Hermann's fluids followed by safranin gave good results in most cases. The mercuro-nitric solution and Lenhossek's fluid gave excellent fixation and were preferable to the osmic mixtures when it was desirable to stain the same material with iron-hæmatoxylin, and also with various anilin stains.

Heidenhain's iron-hæmatoxylin, either alone or with orange G or erythrosin, was used more than any other one stain. With osmic fixation safranin gave better results in some cases, because of the abundance of spindle fibers and sphere substance which were stained by hæmatoxylin. The safranin-gentian combination used by Miss Wallace and others in the study of the accessory chromosome did not prove to be especially helpful with these forms. Thionin was found to be a very useful stain for distinguishing between the accessory chromosome and an ordinary nucleolus. Licht-grün was often used in combination with safranin.

RESULTS OF INVESTIGATIONS.

Termopsis angusticollis.

In the termite it was not found to be practicable to dissect out the testes. The tip of the abdomen was therefore fixed and sectioned, young males whose wings were just apparent being used. The cells are all small, and could not be studied to advantage with less than 1500 magnification (Zeiss oil immersion 2 mm., oc. 12).

In the spermatogonium there is a very large nucleolus (plate I, fig. 1), which in the iron-hæmatoxylin preparations is very conspicuous, but does not stain like chromatin with thionin or other anilin stains, nor does it behave like an accessory chromosome during the maturation mitoses. Before each spermatogonial division it divides as in figures 2 and 3, and the same is true for each maturation mitosis. Figure 4 shows the 52 chromosomes of a spermatogonial division in metaphase. Figures 5 and 6 are young spermatocytes, showing the division of the nucleolus. Figures 8, 9, and 10 show a stage immediately following that shown in figure 6 and evidently persisting for some time. The spireme thread is very fine, stains deeply, and is wound into a dense ball, often concealing one (fig. 10) or both nucleoli (fig. 8). Figure 11 shows the next stage; the bivalent chromosomes are so disposed as to give the familiar "bouquet stage," with the loops directed away from the centrosome and sphere (c). Figures 12, 13, and 14 show the later development of the same stage, the chromatin loops becoming thicker by the concentration of the smaller granules to form the larger ones seen in figure 14. The loops now straighten out and extend in various directions across the nuclear space (figs. 15, 16, 17). In fig. 18a a longitudinal split is seen in several chromosomes. Figures 18b, 19, 20, and 21 show various stages in the contraction of these split bivalent chromosomes to form diamond-shaped tetrads, each side of which is a univalent daughter chromosome. The tetrads come into the spindle in this form (figs. 22, 23), and change to the form shown in figure 24 during the metaphase (figs. 22, 26, 28). Figures 25 and 27 show the 26 bivalent chromosomes, or tetrads, in early and late metaphase, respectively, and figures 29, 30, and 31 in anaphase. This is certainly a reduction division, for the tetrads are always somewhat elongated and come into the spindle with their longer axes parallel with the axis of the spindle. The aberrant bodies in these figures are probably remains of the nucleoli; they are found only in iron-hæmatoxylin preparations. Figures 31 and 32 show exceptional cases where the cell has divided. Usually the two daughter nuclei are formed in an undivided cell. The resting-stage between the two divisions is only partial. The nucleolus appears and divides into two (figs. 33-36), and the chromosomes change into the dyad form (fig. 36), in which they come into the second maturation spindle (figs. 37, 38). The equatorial plate again shows 26 chromosomes (fig. 39). The formation of the spermatozoa is peculiar in that the original spermatocyte cell-body, as a rule, does not divide; but the four nuclei resulting from the two maturation divisions develop into sperm-heads in one cell. All have a nucleolus (fig. 41), and in a slightly later stage (fig. 42) the elongated nuclei have a distinct centrosome and sphere at the posterior end. Later stages are shown in figures 43, 44, and 45.

The points of greatest interest in the spermatogenesis of Termopsis angusticollis are, (1) the fact that no accessory chromosome is present; (2) that the method of tetrad formation and reduction are clear, despite the fact that the cells and the chromatin elements are quite small; and (3) the failure of the cell-bodies to divide and the consequent development of four spermatozoa in one cell.

Stenopelmatus.

The spermatogonium of Stenopelmatus contains from one to three large nucleoli, which stain much less with thionin than does the spireme (plate II, figs. 46, 47, 48). As the distinct chromosomes come into view in the prophase of mitosis, two are seen to be nearly twice as long as the others, but of equal length (figs. 48, 49, 50.) There are 46 chromosomes in the equatorial plate of a spermatogonial spindle (fig. 50). Besides the nucleolus (n), there appears in the young spermatocyte a conspicuous element (x) which stains deeply with all chromatin stains (fig. 51). It is closely applied to the nuclear membrane and is connected with an end of the spireme (figs. 51-54). At first it is quite small, and it gradually increases in size during the spireme stage. There is no "bouquet stage" in this form. Figure 55 shows the spireme segmented and split longitudinally. The segments have begun to open out at the center to give the cross which is the typical tetrad form in Stenopelmatus. Figures 56, 58, 59, and 60 show various stages in the contraction of the split segments to form crosses and diamond-shaped rings. The tetrads usually remain connected by delicate linin threads, as shown in figures 57 and 60, also in figures 62 and 63, the latter taken from the metaphase of the first maturation spindle. If these linin connections persist, as they appear to do, from the segmentation of the spireme to metakinesis, the first division of the contracted tetrads must be longitudinal, corresponding to the split in the segments of figures 55, 57, 58, etc. The chromosomes in the metaphase usually appear as dumbbells (fig. 66) or elongated crosses (fig. 67), but occasionally one can be found which still shows its tetrad nature (fig. 64), so clearly indicated in the quadrivalent crosses of figure 59. In the anaphase the chromosomes are often split as in figure 68, and occasionally the two components can be seen as plainly as in figure 65. Figure 61 shows the various shapes assumed by the element x during the tetrad-stage of the chromosomes. This element x almost invariably appears in a vesicle near one pole of the spindle (figs. 67, 68); in exceptional cases it is found nearer the equatorial plate, as in figure 66, or even in the same plane with the ordinary chromosomes, but always somewhat isolated from them. In position and form this element resembles the accessory chromosomes described by Baumgartner ('04) for Gryllus domesticus; in its mode of origin it seems to differ from the other accessory chromosomes yet described.

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