Studies in Spermatogenesis.

Part 1.

by Nettie Maria Stevens.

In connection with the problem of s.e.x determination it has seemed necessary to investigate further the so-called "accessory chromosome,"

which, according to McClung ('02), may be a s.e.x 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 oenotherae_.

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 s.e.x 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-haematoxylin, and also with various anilin stains.

Heidenhain's iron-haematoxylin, 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 haematoxylin.

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-grun 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-haematoxylin 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 s.p.a.ce (figs. 15, 16, 17). In fig. 18_a_ a longitudinal split is seen in several chromosomes. Figures 18_b_, 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-haematoxylin 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 a.s.sumed 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.

Figures 69 and 70 show the 23 bivalent chromosomes in metaphase; in figure 69 the element _x_ is shown partly behind the large chromosome and at a different level. In figures 66 and 67 the one exceptionally large chromosome doubtless represents the two larger ones of the spermatogonia. In the anaphase the element _x_ is sometimes as conspicuous as in figure 71; in other cases it is concealed either behind or within the polar ma.s.s of chromatin. In this form there is a distinct resting stage between the two maturation mitoses (figs. 72-75).

The element _x_ is conspicuous in one-half of the cells (figs. 72, 73); it may be included in the nucleus as in figure 72, or it may be partly or wholly outside, as in figures 74, 75, and 76. In the latter case, but not in the former, it is surrounded by its own membrane. As the chromatin begins to condense for the second mitosis, disintegration of the element _x_ becomes apparent. This is most easily made out in cases where the element is isolated, as in figures 75 and 76; but there seems to be little doubt that it disappears before the metaphase of the second maturation mitosis. It is not possible to count the chromosomes in this stage, they are so crowded together, but it is not probable that such a conspicuous chromatin element as that seen in the first division could escape detection, even if it were in the equatorial plate among the chromosomes. No aberrant element is ever seen in these spindles; and, moreover, all of the spindles and all of the spermatids appear to be exactly alike at the same stage. The chromosomes are double in the prophase (fig. 77) and always appear double in the equatorial plate (fig. 78), the paired elements corresponding to those of figure 65.

In figure 80, plate III, a pair of spermatids is shown with nuclear membrane formed and the spindle fibers twisted in a characteristic manner. Figure 81 is a slightly later stage with the spindle-remains ma.s.sed against the nuclear membrane. Curiously enough there appears in the nucleus of every spermatid a body similar to the element _x_ of the spermatocytes of the first order (figs. 82-86). This body is often applied to the nuclear membrane and connected with the spireme (figs.

84-86). It decreases in size and finally disappears (figs. 88-91). The spindle-remains divides (fig. 83), and a small part of it (_a_) goes to form the acrosome at the apex of the head (figs. 85-92). The larger part is probably utilized in the formation of the tail, for it gradually disappears as the tail develops.

The centrosome which, although small, is conspicuous in each mitosis, is seen in figure 83 between the two parts of the spindle-remains, applied to the outside of the nuclear membrane. In figures 85, 86, and 87 the relation of the tail (or its axial fiber) to the centrosome is shown. In figures 87 and 88, instead of the small spherical centrosome of figures 83 to 86, we have a much elongated body, at first (fig. 87) applied for its whole length to the nuclear membrane, but later lying along one side of a middle piece (_m_), as shown in figure 89, and in a later stage in figures 90 to 92. The mature spermatozoon with its forked anterior end appears in figure 93.

The points of especial interest in the spermatogenesis of _Stenopelmatus_ are the development of the aberrant chromatin element _x_ during the growth stage of the spermatocyte of the first order, its distribution to one-half of the spermatocytes of the first order, its disappearance during the rest stage between the two maturation divisions, and the development of a similar, though smaller, element in all of the spermatocytes.

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 a.s.sociated (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 ma.s.s of chromatin, probably due to imperfect mitosis, is found near the spindle-substance (fig. 150). The ma.s.s of spindle-substance at first appears structureless, but soon a.s.sumes the condition shown in figures 150 to 152. In one individual many of the spermatids had two b.a.l.l.s 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 spermatozoon (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 a.s.sociated 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.