Studies in Spermatogenesis Volume Ii Part 1

Studies in Spermatogenesis.

by Nettie Maria Stevens.

INTRODUCTION.

In Part I of this series of papers, the spermatogenesis of five species belonging to four different orders of insects was considered. In two species of Orthoptera an "accessory chromosome" was found; in _Tenebrio molitor_, one of the Coleoptera, an unequal pair of chromosomes was described; in the other species no heterochromosomes were discovered.

The apparent bearing of the chromosome conditions in _Tenebrio molitor_ on the problem of s.e.x determination has led to a further investigation of the germ cells of the Coleoptera. One of the Hemiptera h.o.m.optera and two of the Lepidoptera have also been examined for comparison with the Coleoptera and the Hemiptera heteroptera.

METHODS.

As a result of previous experience with similar material, only two general methods of fixing and staining have been employed: (1) Fixation in Flemming's strong solution or Hermann's platino-aceto-osmic, followed by either Heidenhain's iron-haematoxylin or Hermann's safranin-gentian staining method (Arch. f. mikr. Anat. 1889). (2) Fixation after Gilson's mercuro-nitric formula, followed by iron-haematoxylin, Delafield's haematoxylin and orange G, Auerbach's combination of methyl green and acid fuchsin, or thionin.

The iron-haematoxylin with either mode of fixation gives by far the most satisfactory preparations for general study. The other stains were used mainly for the purpose of distinguishing between heterochromosomes and plasmosomes in resting stages of the nucleus.

COLEOPTERA.

Trirhabda virgata (Family Chrysomelidae).

Two species of _Trirhabda_ were found in larval, pupal, and adult stage on _Solidago sempervirens_, one at Harpswell, Maine, the other at Woods Hole, Ma.s.sachusetts. The adult insects of the two species differ slightly in size and color, the germ cells mainly in the number of chromosomes, _Trirhabda virgata_ having 28 and _Trirhabda canadense_ 30 in spermatogonia and somatic cells.

In _Trirhabda virgata_, the metaphase of a spermatogonial mitosis (plate VIII, fig. 3) contains 28 chromosomes, one of which, as in _Tenebrio molitor_ is very much smaller than any of the others. The maternal h.o.m.ologue of the small chromosome is, as later stages show, one of the largest chromosomes. In _Tenebrio_ the unequal pair could not be distinguished in the growth stages of the spermatocytes. In _Trirhabda_ it has not been detected in the synizesis stage (fig. 4), but in the later growth stages (figs. 5-7) this pair is conspicuous in preparations stained by the various methods cited above, while the spireme is pale and inconspicuous. The size of the heterochromosome pair varies considerably at different times in the growth period, and in some nuclei (fig. 7) both chromosomes appear to be attached to a plasmosome. The ordinary chromosomes a.s.sume the form of rings and crosses in the prophase of the first maturation mitosis (fig. 8), but usually appear in the spindle as dumb-bells or occasionally as tetrads (fig. 10), or crosses (fig. 11). The unsymmetrical pair is plainly seen in figures 9 and 11, but is not distinguishable in a polar view of the metaphase (fig. 13). In the anaphase (figs. 14-16) the larger and the smaller components of the pair separate as in _Tenebrio_. This is, therefore, clearly a reducing division as far as this pair is concerned, and probably for all of the other pairs, though neither the synapsis stage nor the prophase forms are so clear on this point as in some of the other species studied. Figures 17 and 18 show metaphases of the two cla.s.ses of second spermatocytes, the chromosomes varying somewhat in form in different preparations and even in different cysts of the same preparation. An early anaphase of this mitosis is shown in figure 19; here the small chromosome is already divided. It was impossible to find good polar views of the daughter plates in the two cla.s.ses of second spermatocytes, but it is evident from figure 19 and other similar views of the second spermatocyte spindle that, as in _Tenebrio_, one-half of the spermatids will contain one of the derivatives of the small chromosome, the other half one of the products of its larger h.o.m.ologue.

Sections of male pupae were examined for equatorial plates of somatic mitoses. Figure 1 is a specimen of such plates. As might be expected, this figure resembles quite closely the spermatogonial equatorial plate (fig. 3) in number, form, and size of chromosomes, the small one being present in both. Figure 2 is from the follicle of a young egg; here we find 28 chromosomes, but no small one. The chromosome corresponding to the larger member of the unequal pair in the male evidently has a h.o.m.ologue of equal size in the female. The chromosome relations in the male and female somatic cells are therefore the same as in _Tenebrio molitor_, and must have been brought about by the development of a male from an egg fertilized by a spermatozoon containing the small chromosome, and a female from an egg fertilized by a spermatozoon containing the larger heterochromosome.

Trirhabda canadense.

In _Trirhabda canadense_ the spermatogonial chromosomes are invariably smaller than in _T. virgata_, but similar size relations prevail. The spermatogonial plate (fig. 21) contains 30 chromosomes, 29 large and 1 extremely small. In the growth stages the a.s.sociation of the two unequally paired chromosomes with a rather large plasmosome is more evident than in _T. virgata_ (figs. 22-23). In this species the unequal pair is more often found at a different level from the other chromosomes in the early metaphase of the first maturation mitosis (fig. 24), but it later comes into the plate with the other chromosomes (figs. 25-27), and divides earlier than most of the other bivalents (fig. 27). In a polar view of this metaphase the largest chromosome often appears double (fig.

28); in a front view it is a tetrad as in _T. virgata_, figure 10.

Figure 29 is the equatorial plate of a metaphase in which the larger component of the unequal pair has been removed in sectioning. The daughter plates of a first spermatocyte in anaphase (fig. 30) show the separation of the components of the heterochromosome pair; and equatorial plates of the resulting two cla.s.ses of second spermatocytes (fig. 31) show the same conditions. Figures 32 and 33 are prophases of the second division, figure 33 showing the small chromosome ready for metakinesis. It was impossible here also to get good drawings of daughter plates of the second spermatocytes to show the content of the two cla.s.ses of spermatozoa, but there is no doubt that all of the chromosomes divide in the second mitosis, giving one cla.s.s of spermatids containing the small chromosome, the other cla.s.s its larger h.o.m.ologue.

No male somatic cells were found in mitosis, but they would, if found, show the same conditions as in the spermatogonia. One of many good equatorial plates from egg follicles (fig. 20) shows 30 large chromosomes, indicating an equal pair in place of the unequal pair of the male.

Chelymorpha argus (Family Chrysomelidae).

This species was found in larval and adult stages on _Convolvulus arvensis_ at Harpswell, Maine, in July and August. It shows the same conditions as _Trirhabda_ and _Tenebrio_, so far as the unequal pair of chromosomes is concerned, and is especially favorable for study of synapsis stages. The number of chromosomes in the spermatogonia (plate IX, fig. 36) is 22. Here the components of the unequal pair are the small spherical chromosome and one of the several chromosomes third in size, forming a comparatively small unsymmetrical bivalent (figs.

47-49). The spermatogonia occupy the outer end of each follicle, and next to them comes a layer of cysts in which the chromosomes from the last spermatogonial division are closely ma.s.sed in the form of short deeply staining loops at one side of the nuclear s.p.a.ce (fig. 37).

Following this synizesis stage comes one in which some of the short loops have straightened, their free ends extending out into the nuclear s.p.a.ce (figs. 38 and 39). Figure 40 shows the nucleus of a slightly later stage in which the free ends of two straightened chromosomes are on the point of uniting. In figures 41 and 42 the point of union of h.o.m.ologous chromosomes is indicated in some cases by a k.n.o.b, in others by a sharply acute angle. In a slightly later stage (fig. 43), when all of the short loops have straightened and united in pairs, the point of union is no longer visible, all of the loops being rounded at the bend and of equal thickness throughout. My attention was first called to this method of synapsis by the conspicuous difference in number and length of loops in the synizesis stage compared with the later bouquet stage just before the spireme is formed. Following the synapsis stage shown in figure 43 comes one in which the loops lose their polarized arrangement and unite to form a continuous spireme (figs. 44 and 45). In this form, the heterochromosome pair could not be distinguished until the spireme stage, and it is, therefore, uncertain whether these chromosomes remain condensed after the last spermatogonial divisions and are hidden among the ma.s.sed and deeply staining loops of the synizesis and synapsis stages, or whether they pa.s.s through the same synaptic phases as the other chromosomes, condensing and remaining isolated at the beginning of the spireme stage. An early prophase of the first maturation mitosis (fig. 46) shows segments of the spireme longitudinally split, and in some cases transformed into crosses which show a transverse division also. Most of the equal bivalents have the dumb-bell form in the spindle (figs. 47-49). One is ring-shaped, the ring being formed by union of the free ends of the segment so that the spindle fibers are attached to the middle of each univalent chromosome (fig. 49). This method of ring formation, like that described by Montgomery ('03) for the Amphibia, is of very frequent occurrence in the spermatocytes of the Coleoptera. The dumb-bells are so bent at the ends (fig. 52) that the spindle fibers, here also, are attached at or near the center of each univalent component of a bivalent chromosome, and the separated, univalent chromosomes go to the poles of the spindle in the form of Vs.

As in _Tenebrio_ the heterochromosome pair is late about coming into the equatorial plate (figs. 47-48), but it does finally take its position with the others (fig. 49) and separates into its component parts somewhat earlier than the other bivalents (figs. 52, 53). Figures 50 and 51 show polar views of the metaphase, the smaller element (_x_) being the unequal pair. The chromosomes in late anaphase are too much crowded to give clear drawings. As in all the beetles so far studied there is no rest stage between the two maturation divisions, but the late anaphase of the first mitosis pa.s.ses over quickly into the second spindle.

Figures 54 and 55 are typical equatorial plates of the second division, one showing the small chromosome (_s_), the other its mate more nearly spherical than the others (_l_). An anaphase including the small chromosome is shown in figure 56. As in the species previously described the spermatozoa are evidently dimorphic.

Female somatic equatorial plates from egg follicles are shown in figures 34 and 35; 22 chromosomes are present and no one is without an equal mate.

Odontota dorsalis (Family Chrysomelidae).

_Odontota dorsalis_ is a small leaf-beetle found on _Robinia pseudacacia_. The chromosomes are comparatively few in number, 16 in the spermatogonia (figs. 58 and 59), and of immense size when one considers the smallness of the beetle. In some of the spermatogonial cysts many of the chromosomes are V-shaped as in figure 58, while in others all, with the exception of the small one, are rod-shaped as in figure 59, which looks like a hemipteran equatorial plate. The spermatogonial resting nucleus (fig. 60) contains a large plasmosome (_p_), but no condensed chromatin. The synizesis and synapsis stages are similar to those in Chelymorpha (figs. 61 and 62). The spireme stage (figs. 63, 64) contains, in addition to the pale spireme, a very conspicuous group consisting of a large plasmosome with a large and a small chromosome attached to it. In the prophase, before the nuclear membrane has disappeared, this group is easily distinguished from the other dumb-bell and ring-shaped bivalents (figs. 65-67). In preparations much destained (fig. 67) the small chromosome component of the group retains the stain longer than the larger one. The spindle in prophase (fig. 68) is much elongated and the 8 chromosomes are often spread out upon it so as to be easily counted. In the early metaphase the parachute-like heterochromosome group is always nearer one pole of the spindle (plate X, figs. 69 and 70). The equatorial plate often shows both the larger component of the pair and the plasmosome (fig. 71). Figures 72-74 show the metakinesis of the heterochromosome bivalent. In figure 74 the two unequal elements are completely separated and the plasmosome has disappeared. The equatorial plates of the two resulting kinds of second spermatocytes appear in figures 75 and 76. In the anaphase of the second division all of the chromosomes are divided quant.i.tatively as may be seen in figures 77 and 78. A few dividing male somatic cells were found in the walls of the testis. Figure 57 (plate IX) is an equatorial plate from one of these. The chromosomes are like those of the spermatogonia (figs. 58 and 59), 15 large and 1 small. No dividing female somatic cells were found.

A few drawings of developing spermatids are given to show the transformations of a peculiar body which seems to be characteristic of insect spermatids. Figure 79 is a very young spermatid showing only diffuse chromatin in the nucleus. The nucleus soon enlarges (fig. 80) and a large dense body (_n_) appears which stains like chromatin with various staining media. A little later (fig. 81) the chromatin forms a h.o.m.ogeneous, more or less hemispherical or sometimes crescent-shaped ma.s.s which stains an even gray in iron-haematoxylin. In addition the nucleus contains a body (_n_) smaller than in the preceding stage, but staining the same. As the nucleus condenses and elongates to form the sperm head, a light region containing this deeply staining body is seen on one side (figs. 82, 83). A little later the body is divided into two, which appear sometimes spherical (fig. 84), sometimes elongated (fig.

85). As the sperm head elongates still more, approaching maturity, these bodies diminish in size (figs. 86, 87) and ultimately disappear. A cross section of the sperm head at such a stage as figure 87 shows the chromatin in crescent shape with material which stains very little within (fig. 88). The chromatin-like body described above was observed in _Tenebrio_ in a stage corresponding to figure 81, and it was thought that the larger body seen in some cases and the smaller one in others might be the larger and smaller heterochromosomes, but a study of this element in more favorable material disproves that supposition by showing that the different sizes are merely different phases in the evolution of the body. Throughout its history it stains like dense chromatin, and my only suggestion as to its origin is that it seems, from a study of this and other species of beetles, to be a derivative of the chromatin of the spermatid, increasing in size for a time, then decreasing, and finally breaking up into granules and dissolving in the karyolymph. Whether it has any function connected with the development of the spermatozoon, or whether it is merely material rejected from the chromosomes, as in many cases in oogenesis, one can only surmise.

In one testis a peculiar abnormality was found. In all of the perfect spermatogonial plates two small chromosomes were present (figs. 89, 90).

Nineteen such plates were counted in five different cysts. All of the equatorial plates of the first spermatocytes showed 8 chromosomes, as usual. In a few favorable growth stages (fig. 91) the two small chromosomes were seen to be combined with the larger heterochromosome and a plasmosome, and one first spermatocyte spindle was found in which the same combination could be clearly seen (fig. 92). All of the second spermatocyte metaphases in which a small chromosome occurred, contained two small ones, making 9 in all (fig. 93). The others contained 8 large chromosomes, as usual. The only explanation suggested by the conditions is that somewhere in its history, the small chromosome had undergone an extra division, and that ever afterward the two products behaved like the one small heterochromosome of a normal individual. The chief interest in this abnormality centers in the fact that the two small chromosomes of this specimen behave exactly like the usual single one, emphasizing the individuality of this particular heterochromosome. Both evidently have the same individual characteristics and affinities as the one in other cases.

Epilachna borealis (Family Coccinellidae).

_Epilachna borealis_ was found in abundance on squash vines at Woods Hole, Ma.s.sachusetts, in September. The testes, unlike those of most of the Coleoptera, consist of many free follicles similar to those of the Orthoptera. The germ glands were rather far advanced, but some good spermatogonial and spermatocyte cysts were found. In figure 94, a spermatogonial metaphase, the small chromosome is shown with 17 larger ones. The heterochromosome pair appears in condensed form in the spireme stage (fig. 95), and again in the first maturation spindle (figs. 96, 97). The varying forms of the ordinary chromosomes are shown in figure 98. Figures 99 and 100 are equatorial plates of the first mitosis. The unequal pair is shown by itself in figure 101, and the separation of the heterochromosomes is seen in figure 102. Equatorial plates of the second division, one containing the small chromosome (_b_), are shown in figure 103. A prophase of the same division (fig. 104) proves that the small chromosome divides quant.i.tatively like the others. It was interesting to find here and there in this material whole cysts in which the nuclei were like those described by Paulmier ('99) for _Anasa tristis_ (plate XIII, fig. 14) as cells which were being transformed to serve as food for the glowing spermatids (figs. 105, 106). The only occasional appearance of these cysts seems to me to preclude their being a special dispensation to furnish the spermatids with nutrition during their transformation. Their appearance and size make me suspect that they are giant spermatids due to the failure of one of the spermatogonial or spermatocyte mitoses. The smaller chromatin body seems to correspond to that described for the spermatids of _Odontota dorsalis_.

Euphoria inda (Family Scarabaeidae).

Of _Euphoria inda_ only one male was captured, but the numerous testes furnished abundant material in desirable stages. The spermatogonial equatorial plate (fig. 107) contains 20 chromosomes of which the two smallest (_l_ and _s_) form the unequal pair. The resting spermatogonium contains a two-lobed plasmosome (fig. 108). The growth stages are similar to those in _Tenebrio_ in showing no distinct bouquet stage, but there is a spireme stage in which the heterochromosome pair is clearly seen (fig. 109). Figure 110 (plate XI) is an early prophase, and figure 111 one in which the unequal pair appears with a tetrad and several dumb-bell forms. The prophase of the spindle, as in _Odontota_, is much elongated (fig. 112). In figures 113-116 the small heterochromosome pair is shown in various positions with reference to the other chromosomes of the metaphase of the first spermatocyte. Figure 117 shows it more deeply stained than the others in the equatorial plate. This pair divides in advance of the others, and the larger and smaller elements are plainly seen nearer the poles in anaphase than the other univalent chromosomes (figs. 118-120). Daughter plates of the first spermatocyte are shown in figure 121, and equatorial plates of the second spermatocyte in figure 123. Figure 122 shows the telophase of the first division with the spindle for the second division forming. In figures 124 and 125 we have daughter plates of the two cla.s.ses of second spermatocytes, showing the content of the two equal cla.s.ses of dimorphic spermatozoa, as this was shown in _Tenebrio_. Figures 126 and 127 are anaphases showing the division of the heterochromosomes (_l_ and _s_). Figures 128-130 are early stages in the development of the spermatid showing the chromatin nucleolus (_n_) in various phases.

Blepharida rhois (Family Chrysomelidae).

The testes were rather too far advanced when this material was collected and no dividing spermatogonia were present. The growth stages (figs.

131, 132) show a faintly staining spireme and a heterochromosome group similar to that of _Odontota_, a large and a small chromosome attached to a large plasmosome. The spireme appears to go directly over by condensation and segmentation into the dumb-bell-shaped figures seen in the first maturation spindle (figs. 133, 134), though cross-shaped bivalents occasionally occur (fig. 135). The heterochromosome pair, slightly separated by plasmosome material, is usually found at the periphery of the plate (figs. 133-136). Figure 137 is an exceptional anaphase in which the heterochromosome elements are not mingled with the polar ma.s.ses of chromatin. Figures 138 _a_ and _b_ are equatorial plates of the second mitosis, and figures 139 and 140 are pairs of daughter plates from second spermatocytes showing again the dimorphism of the spermatozoa as to their chromatin content. As in several of the forms studied, material was collected for examination of the somatic cells, but no favorable cases of mitosis were to be found.

Silpha americana (Family Silphidae).

Only one male of this species was secured, but the large testes gave all stages in abundance. The chromosomes, however, were very small and too numerous, 40 in the spermatogonia (fig. 141). The small chromosome is, nevertheless clearly distinguished in many of these plates (_s_). The resting spermatogonium contains one very large plasmosome and often one or two smaller ones (fig. 142, _p_). The unequal pair is seen in the growth stages (figs. 143, 144), and may frequently be seen outside of the equatorial plate of the first spermatocyte spindle (fig. 146). In favorable sections it may also be found in the plate among the other bivalents (fig. 147). Figure 145 is a prophase showing the bivalent chromosomes still connected by linin fibers. An equatorial plate of the first division is shown in figure 148, and a pair of corresponding plates of the second spermatocyte in figure 149. The small heterochromosome divides in the second spindle in advance of the others as seen in figure 150. Therefore, although this form is not especially favorable for detailed study on account of the large number of small chromosomes, the conditions are evidently the same as in the other species described--an unsymmetrical heterochromosome bivalent in the first spermatocyte, giving rise by the second maturation division to equal numbers of dimorphic spermatozoa, one cla.s.s receiving the large heterochromosome, the other cla.s.s the small one.

Doryphora decemlineata (Family Chrysomelidae).

_Doryphora decemlineata_ has been the most difficult one of the collection to work out satisfactorily. The chromosomes in the spermatogonial plates were in most cases much tangled, and the behavior of the heterochromosome pair was such as to suggest an "accessory chromosome" rather than an unequal pair. Abundant material for the study of somatic cells was at hand, but nothing favorable could be found in the sections.

Two spermatogonial plates, containing 36 chromosomes, are shown in figures 151 and 152 (plate XII). The small heterochromosome (_s_) is slightly elongated. The synizesis and synapsis stages are especially clear. The chromosomes, after the last spermatogonial mitosis go over immediately into a synizesis stage consisting of a polarized group of short loops, which later straighten and unite in pairs (figs. 153 and 154). From these loops are formed the spireme (figs. 155-158), which splits and segments, producing various cross, dumb-bell, and ring forms (figs. 159-163). As in most of the other species of Coleoptera, the unequal pair is not distinguishable until the spireme stage. Figure 162 is an unusual prophase in which all of the equal pairs show a longitudinal split as well as a transverse constriction, and the larger heterochromosome (_l_) is also split. Figure 163 shows a somewhat later and more common prophase in which the unequal pair, one ring, crosses, and dumb-bells may be seen. This figure, as well as figures 164-168, show the unequal pair in various relations to the other chromosomes.

This pair in _Doryphora_ consists of a large V-shaped chromosome with a small spherical one attached to it in different positions. When the small one is behind the V, the group has the appearance of an orthopteran "accessory."

Figures 169-171 show the separation of the two elements outside of the equatorial plate, while in figure 168 the unequal pair is in line with the other chromosomes. In figure 172, an anaphase, the unequal elements are barely separated, while the metakinesis of the other pairs is much further advanced.

Figures 173 and 174 are equatorial plates of the first division, one showing only the larger element of the heterochromosome pair (fig. 174, _x_), the other both elements (fig. 173, _l_ and _s_). In the late anaphase (fig. 175) the larger heterochromosome is often seen outside of the polar ma.s.s, reminding one again of the "accessory" in the Orthoptera. Occasionally it is found in some other isolated position (fig. 176). Equatorial plates of the second division show the same conditions as in the other species; some contain the larger heterochromosome, others the smaller one (fig. 177, _a_ and _b_). It was impossible to draw anaphases of the second division from a polar view and the lateral view showed nothing unusual, merely the longitudinal division of all of the chromosomes.

The spermatids show some interesting variations from the other species which have been examined. In figures 178 and 179 we have telophases of the second spermatocyte, showing centrosome and archoplasm (fig. 178) and certain ma.s.ses of deeply staining material in the cytoplasm (fig.

179, _a_{1}_). Figures 180 and 181 are young spermatids showing the archoplasm from the second spindle (_a_{2}_) and a smaller, more deeply staining ma.s.s (_a_{1}_), derived from the irregular ma.s.ses of the earlier stage (fig. 179, _a_{1}_). In figures 182 and 183, the axial fiber has appeared and the larger ma.s.s of archoplasm (_a_{2}_) is being transformed into a sheath. The other body remains unchanged. During the following stages this smaller archoplasmic body (_a_{1}_) lies in close contact with the axial fiber and sheath (_a_{2}_), and gradually decreases in size (figs. 184-186) until it disappears in a slightly later stage. The acrosome seems to develop directly out of the cytoplasm. The enigmatical body (_a_{1}_), which is probably archoplasm from the first maturation spindle, as it is not found in the cytoplasm of the first spermatocyte, may serve as nutriment for the developing axial fiber. The sperm head has a peculiar triangular form, staining more deeply on two sides.