The Kansas University science bulletin, Vol. I, No. 8, September 1902

by Unknown

IV. OBSERVATIONS.

(a) General Form and Structure of the Testes.

The testes of the Locustidæ are paired structures lying in the anterior dorsal portion of the abdomen. Each organ is made up of numerous short follicles, which are bound together by a connective tissue investment. In adult animals the testes are a bright yellow color, while in nymphs the color varies from white in the youngest to yellow in the oldest. The pigment is lodged in the connective tissue sheath about the testis, and is seen in sections as irregularly rounded masses in the cytoplasm.

(b) The Spermatogonia.

No further discussion of the spermatogonia will be given here than is necessary for an understanding of the derivation of the first spermatocytes. As appears to be universally the case, the second spermatogonia, in their last generation at least, are much reduced in size as compared with the primary spermatogonia that preceded them and with the first spermatocytes that arise from them. The entire cell stains dark with almost all stains and, as the nucleus occupies nearly the whole cell body, the chromatin appears relatively large in amount. A cyst of spermatogonia, therefore, looks as if composed almost entirely of chromatin aggregated into rounded masses—the nuclei.

The chromosomes are of the rod type, and divide longitudinally in each mitosis. The number of chromosomes is large and could not be determined with absolute certainty, but a number of careful enumerations makes it evident that there are most probably thirty-three. In most species of Locustids, one chromosome is easily distinguished from the others by its larger size and tardy division in the act of metakinesis. This is the element as described for Xiphidium, which passes into the first spermatocyte as a formed chromosome, while its fellows break up into the spireme.

In the anaphase the chromosomes are drawn away from the equator, and extend lengthwise of the spindle as long rods. During the telophase the disintegration of the chromosomes takes place rapidly, and, for a time, the individual chromosomes may be distinguished in the loose masses of chromomeres. This distinction, however, is soon lost, and the nuclear vesicle becomes covered with fine and apparently unrelated chromomeres. It is at this point that the transformation of the cells from second spermatogonia to first spermatocytes takes place. So long as the chromosomes are present in the somatic number, we have to deal with spermatogonia, but when the disintegrating process comes upon them and they are lost to view as distinct entities, then is reached the end of destructive spermatogonial changes, and upon their reconstruction they are chromosomes of the spermatocytes.

(c) The First Spermatocytes.

The main features characterizing the next steps in the process are the rapid increase in size of the cell and nucleus, and the arrangement of the chromomeres into a fine thread or threads (figs. 2–4). This is well called the growth stage, for all parts of the cell engage in the work of regaining the ground lost during the period of multiplication in the secondary spermatogonia. As a result of this metabolic activity, the first spermatocytes at the end of the prophase have reached a volume often as much as ten times that possessed by the last generation of the secondary spermatogonia from which they were derived. Nucleus and cytoplasm, in about an equal degree, participate in this enlargement, and, at the end of the period, present an appearance much different from that of the spermatogonia. This consists most strikingly in the greater clearness of all the parts, due to the increased amount of hyaloplasm which separates by greater distances the more solid structures of the cell.

In the nucleus, for instance, the chromatin aggregates are now definitely apparent, and each stands free and clear except for connecting threads of linin. The cytoplasm, likewise, instead of showing a coarsely granular aspect, exhibits a clearly reticular structure, with such large intervening hyaloplasmic areas as to suggest an almost alveolar structure, especially in the later stages (figs. 3–9). This increased amount of fluid becomes evident by an examination of sections under even a low power of the microscope, principally by the lessened density of the general stain in the cell.

A peculiarity of the archoplasm in these early prophases is the persistence manifested by the spindle fibers of the previous generations. Often connecting fibers may be seen, joining cell to cell, as has been described by many writers, but, in addition to this, the spindle remains of more remote ancestral mitoses show themselves. In figure 3 is represented a cross-section through three persisting spindles of as many generations. Their age is suggested by size and intensity of stain, both factors being least marked in the oldest structure.

Centrosomes and astral radiation do not present themselves with the prominence and frequency of such structures in corresponding cells in Hippiscus.

The main interest of these studies, however, attaches to the movements of the chromatin granules. As was suggested in an earlier paper (17), it is only by an understanding of the constructive processes in the prophase that we can appreciate the structure and changes of the chromosomes in the metaphase. It is to this period in the history of the chromosomes that I have given the most attention and to which I will devote the most space in the record of observations.

Apparently the chromomeres resulting from the disintegration of the spermatogonial chromosomes are loosely scattered through the nucleus, so that no formed structure is to be seen. With the increase in size of the cell, however, a linear arrangement of the elements becomes apparent, so that it seems as if a thread is formed. Whether this is continuous or segmented it is not possible to determine. The large amount of chromatin and the tortuous course of the filaments put a solution of the problem beyond the range of assured observation. It is with much regret that this fact is recognized, for one of the most important questions connected with the maturation mitoses hinges upon the method by which the chromosomes, as such, are derived from those of the spermatogonia. Upon this point the evidence of the ordinary chromosomes of these cells would, if anything, tend to confirm the view that there is a possibility of complete rearrangement of the chromomeres in the different chromosomes. Concerning this, however, the accessory chromosome is much more conclusive and convincing, as will be shown later.

Disregarding the relations of the chromosomes of the two generations, it is evident that from the material of the spermatogonial elements there is formed the thread of the spermatocyte prophase. As indicated in figures 3 and 4, this is at first composed of a single series of chromomeres. But in a slightly later stage, represented by figure 5, it becomes plain that the thread is wider and at the same time double. A careful investigation will show that the halves of the thread are exact duplicates of each other, each granule of the one having its mate in the other. There is but one conclusion to be derived from the appearances just described, which is that the double thread is formed by a longitudinal division, granule by granule, of the original filament. The evidence afforded, not only by the Locustids, but by all the Orthoptera, is unequivocal on this point. The cleavage of the thread is not exaggerated in the accompanying figures, and is distinctly in evidence even under ordinary conditions of illumination and magnification.

Much controversy has recently arisen among both botanists and zoologists concerning an appearance of the chromatin in the prophase, which has received the common designation “synapsis,” by which is meant, usually, a one-sided contraction of the chromatin in the nuclear vesicle. No such stage in the nucleus could be found in Hippiscus, and it is likewise absent in the Locustid cells. I therefore repeat the assertion made in the previous paper (17), that in properly fixed material derived from Orthopteran sources the first spermatocyte prophase shows no unilateral massing of the chromatin.

Shortly after the formation of the double spireme, it is to be seen that the thread is no longer—even if it was previously—continuous, but is composed of segments (figs. 5–10). So early as this it is possible to observe that the segments are of very unequal lengths. The extent of this inequality may be gathered by consulting figures 6 and 7. Even in this early stage the real structure of the segments may be determined, and in those favorably situated the quadripartite nature of the future chromosomes manifests itself very distinctly.

This important stage in the history of the first spermatocyte chromosomes first received attention at the hands of Paulmier in his studies upon Anasa. Almost at the same time I found structures in the Orthopteran spermatocytes so nearly identical that it would be impossible to distinguish any marked difference between them. The Locustid material, equally with the Acridian, permits an exact determination of the chromosome structures, which later become so masked as to be indeterminate.

The interest attaching to the construction of the spermatocyte chromosomes is so great as to warrant an account of the process, although, in general, it is largely a repetition of what has been given for Anasa and Hippiscus. As early as the stage represented in figure 6, it becomes noticeable that the chromatids near the middle of the thread tend to diverge from each other, leaving a diamond-shaped space. This becomes more pronounced, and it is soon seen that each half of the thread is broken across at the same level, resulting in the production of a chromosome of four parts. Still retaining their general shape, these segments shorten and broaden until they are almost the size of the metaphase chromosome.

All variations conceivable upon the wider separation of the halves along the longitudinal split, the movement of the parts upon the line of separation at right angles to the original cleft, or of approximation and rotation of the free segmented ends are found. Thus do we get the cross-shaped, the double-V, the figure-of-S, the Y-shaped and ring figures, in figure 11. Many of the rings give the impression, upon superficial examination, of loops with their free ends crossed. A careful examination will always reveal the fact, however, that what appears to be the crossed ends is really the middle portion of the segment, with the chromatids drawn out along the plane of the cross-division. In segments that are favorably placed, there is never any difficulty in correlating the structures with the typical one of a cross-split lengthwise of each arm.

The quadripartite nature of the chromatin segments may be determined, as already indicated, almost as soon as the longitudinal split occurs. From this time on until the chromosomes are divided in the metaphase, it is possible to trace the formation of the tetrad chromosomes and to be sure of the relation existing between the longitudinal and cross planes of separation. As evidence of the existence of a longitudinal division of the chromatin thread and of the sequence of the two divisions, I do not see how more could be asked of any material. In the early prophase the greatly elongated and granular thread becomes twice split, once along its length and once across it. As the cell ages, a continuously closer approximation of the chromomeres occurs, without obliterating the lines of separation between the four parts of the segment; accompanying this, the segment becomes shorter and thicker, and the previously existing linear arrangement of the chromomeres is superseded. When the segments have reached approximately the size of the definitive chromosomes of the metaphase, the nuclear membrane disappears and distinction between cytosome and nucleus is lost. As a coincident step, the formerly granular segments become homogeneous in structure by the disappearance of the chromomeres as individual structures; all lines of separation between parts are lost to view, so that an examination of the formed element would betray no indication of composite structure. But, having traced the formation of the chromosomes in this way, one is at no loss to identify each part of the preexisting quadripartite chromatin segment. This is possible because, while all trace of internal structure is gone, the general outline is retained and the crosses and rings of the early stages are still, even up to the metaphase, crosses and rings.

Having traced the formation of the ordinary chromosomes through the various stages of the prophase, I should like to return to the beginning again and bring up to a like degree of development the aberrant element which I have called the accessory chromosome. This has already been given in general outline in my first paper upon Xiphidium (16), but a number of important observations since made render a general discussion desirable.

I have not yet found it possible to make a detailed study of the spermatogonia of the Locustids, as was done for the Acrididæ by Sutton in this laboratory, but sufficient observations have been made to be assured that the accessory chromosome participates normally in the mitoses of the secondary spermatogonia. It is here distinctly visible because of its large size, which causes it to extend down to the equatorial plate, while the other chromosomes are in a late anaphase.

At the close of the spermatogonial divisions, when the disruptive processes reduce the other chromosomes to masses of chromomeres in which chromosome identities are not apparent, the accessory chromosome, with apparently more cohesive vigor than the others, retains its general form and is at all times distinguishable. It is marked off from the others, not only by persistence of form, but also by the difference in staining reaction, this being such as is usually exhibited by chromatin when concentrated into homogeneous masses. While studying the cells of Xiphidium, I noticed that, at one stage, this color reaction changed somewhat and more nearly approached that of the diffused chromatin. At this time the accessory chromosome had the form of a flattened, apparently fenestrated, plate. I have been fortunate enough, in preparations of Orchesticus, to discover that the accessory is really at this time in the form of a long, coiled thread (fig. 5). It is thus seen that, even in respect to the spireme stage, the accessory chromosome is comparable to the others, the only difference being that the diffusion of the chromomeres is less, and the independence of the element greater, than is the case with the other chromosomes.

As the chromatin segments shorten and thicken, the thread of the accessory likewise increases in diameter at the expense of its length, and is finally observable in various degrees of contortion, as shown in figure 12. By the time the chromosomes are ready for division, the accessory has assumed a form very similar to that it shows in the spermatogonia. With the establishment of the equatorial plate, the accessory moves to one pole of the spindle and there remains undivided during the first spermatocyte mitosis. It is accordingly a member of only one second spermatocyte resulting from the division of each first spermatocyte.

Returning to the group of chromosomes preparing for metakinesis, we find that in their earlier stages they lie so that their longer diameter is in the equatorial plate, while attached to the enlargement in the center of each, representing the point of separation laid out for the second spermatocyte division, are the mantle fibers running to the centrosomes. The changes now ensuing are easily decipherable, because the chromosomes do not all undergo division at the same time. Since the main differences at present existing between insect spermatologists relate to the sequence of the divisions in the spermatocyte mitoses, I shall again describe the process, although it is identical with that already given for Hippiscus.

The necessity for a thorough understanding of the chromosome construction here becomes evident. Knowing how the chromatids were associated in the chromosomes, one can follow understandingly their movements during metakinesis.

It is first to be noted that the chromosomes lie with their longer axis in the equatorial plate. This, as we have seen, is the plane along which the longitudinal cleft occurred, so that a separation in this way means the longitudinal division of the chromosomes in the first spermatocyte. This is, in reality, what occurs. The contracting mantle fibers attached to the middle of the segments drag the adhering chromatids apart without at any time exposing a separating space. It is in this way that in the beginning the longer axes are at right angles to the spindle axis and at the end parallel with it, while during intermediate periods crosses with arms of varying length exist (figs. 13, 14).

The previously disguised lines of separation become at once visible in the daughter chromosomes, for, instead of remaining closely apposed, as formerly, the chromatids spring apart at the free ends and the chromosomes pass through the anaphase as V-shaped bodies instead of as simple rods. The space thus disclosed represents that which separates what would be the ancestral spermatogonial chromosomes, assuming that the reduced number occurs by the end-to-end union of chromosomes of the secondary spermatogonia. As already stated, the accessory chromosome does not divide at this time.

At the end of the anaphase we find the ordinary chromosomes massed at the poles of the cell, and, in addition, at one the undivided accessory chromosome. The second spermatocytes are therefore of two kinds, one possessing the accessory chromosome and the other not. One additional feature of interest that becomes apparent during the migration of the daughter chromosomes to the poles is the retarded division of one of the elements (figs. 22–24). Some cysts contain cells that almost invariably exhibit this peculiarity. The lagging chromosome is always one of the small ones, but whether the same in each case could not be determined.

In the telophase, the main interest is centered in the question as to whether there is a loss of identity of the chromosomes or not. The evidence afforded by the Locustid cells is strongly in favor of the conception of persisting elements. As is usually the case, I believe, the chromosomes, when not under the active influence of the archoplasm, loosen up, and their homogeneous structure gives way to the granular appearance noticeable in the prophase. Although the chromosomes become closely massed and granular, their outlines can usually be distinguished (figs. 23–27). The accessory chromosome does not change its form and structure at this time (figs. 25, 27). The telophase ends with the ingrowth of the dividing cell-wall, and the second spermatocyte mitotic figure is established without any real prophase. Between the two generations it is evident that there exists no such thing as a “rest stage.”

(d) The Second Spermatocytes.

In the metaphase of the second spermatocyte are formed exact duplicates of the chromosomes seen in the anaphase of the first spermatocyte. These arrange themselves radially in the equatorial plate, one chromatid immediately above the other, so that the plane separating the halves is at right angles to the spindle axis. Mantle fibers attach to the inner ends of the chromatids at the point at which, in all probability, the fibers of the first spermatocyte were connected. I am inclined to regard this as true because the opposite ends, during the anaphase, seemed to be mutually repulsive.

The spindle itself is small and weak as compared with that of the first spermatocyte, and does not long survive the anaphase condition. The material composing it, however, persists as the nebenkern of the spermatid.

A marked difference between the second spermatocytes that contain the accessory chromosome and those which do not is observable. In the metaphase, the element, already longitudinally split in the prophase of the first spermatocyte, projects from the equatorial plate for some distance into the cytoplasm. It is very much larger than most of the other chromosomes, as may be seen in figure 28. It divides readily in metakinesis, and its chromatids travel to the poles with those of the other chromosomes, but, on account of their greater length, project downward from the mass (fig. 31). Here, as always, the accessory stubbornly maintains its independence, and can be seen extending out from the mass of other chromosomes at each end of the mother cell (fig. 32).

The division of the other class of second spermatocytes is, of course, unaccompanied by modifications due to the presence of the accessory chromosome. Aside from this, no difference between cells of the two classes is noticeable.

To summarize, we may say, that resulting from the division of each first spermatocyte are two second spermatocytes, one of which contains an accessory chromosome while the other does not. The second spermatocyte containing the accessory divides, and with it the accessory, so that each of the spermatids derived from it contains a chromatid from the accessory. The other second spermatocyte, not containing the accessory, also divides, producing two spermatids in which the accessory is absent. Thus half of the spermatids contain accessory chromosomes while the other half does not.

(e) Number of Chromosomes.

The enumeration of the chromatic elements, while a very important part of any study upon the nucleus, is unsatisfactory at the best. If there is any great number of chromosomes in the cell, it is impossible to secure a determination of it in a lateral view of the metaphase, because the elements overlie one another so as to render their distinction very uncertain. A polar view is much more desirable, but even here one is never certain that all the elements are represented, or that only entire chromosomes of one cell are present. The first of these contingencies arises from the fact that, in the event of a cell being cut in two, some of the chromosomes may drop out and not appear in the sections; or, if still on the slide, and in a small group, they may lie so close to a mass of chromosomes in another cell as to be confused with them. An excess in number may be found if a portion of the chromosomes have already divided in the equatorial plate, while the remainder are still united (cf. fig. 19), or if one or two from the fragment of another cell are in the neighborhood. All these embarrassments are increased when an independent structure like the accessory chromosome is present. These difficulties exist when the conditions are most favorable, i. e., when the chromosomes are arranged in the equatorial plate; they become practically insurmountable during any other stage of mitosis by the intertwining of the chromatic segments or by fusion of chromosomes in later stages.

Because of these considerations, I do not put implicit confidence in conclusions drawn from numerical relations when they involve the question of whether or not there is a difference of one chromosome between two cells. What I have to say, therefore, concerning the numbers of chromosomes in the different cell generations of the Locustid testis, I must state as my best judgment in the matter, based upon the most careful observations I could make upon cells showing the elements with the greatest clearness. While I regard them as in all probability correct, I do not rely so thoroughly upon them as I do upon observations of structural details, and have therefore based no conclusions upon numerical relations alone.

As is stated elsewhere, the number of chromosomes in the spermatogonia appears to be thirty-three. This was ascertained by selecting the clearest possible cases of the metaphase that could be found and drawing them under the camera lucida. Subsequent countings were made, and in most of the cells thirty-three chromosomes were found. An inspection of figure 1 will show that there is a characteristic arrangement of the chromatin bodies, the larger ones being on the outside of the group, the smaller within. Amongst the large ones, it was impossible to distinguish the accessory chromosome, but a lateral view of the anaphase shows it clearly. From the fact that it was a single element in the spermatogonia, it was to be expected that an uneven number of chromosomes would appear in this cell generation.

In the spermatocytes, as in the spermatogonia, the polar view of the metaphase was the stage selected for use in counting the chromatin elements. A large number of cases showed that sixteen and seventeen were the prevailing numbers. The smaller of these is easily accounted for when it is recalled that the accessory chromosome is at one pole of the spindle, and would very often lie in another section, where it would not be possible to be sure of its relations. I am convinced from these counts that seventeen is the reduced number in the first spermatocyte, sixteen of the elements being ordinary chromosomes, the other one being the accessory chromosome which has come over unaltered from the spermatogonia. This coincides with the theoretically expected number, deduced from the independently determined number of spermatogonial elements.

In view of the divergences found in insect spermatogenesis, the established theory that the reduced number of chromosomes is exactly half the normal or somatic number is not a strictly accurate one, for in this case the reduction is from thirty-three to seventeen. Similar instances may be found in the forms investigated by Montgomery and de Sinéty.

When we come to consider the second spermatocytes, spermatids, and spermatozoa, it is necessary to divide them into two classes, because of the unequal apportionment of the accessory chromosome consequent upon its remaining undivided in the first spermatocyte mitosis. There are formed, accordingly, two numerically equal classes of second spermatocytes—those containing sixteen chromosomes plus the accessory chromosome, and those with merely the sixteen chromosomes. The members of each of these classes divide and double their kind, forming spermatids marked as were the second spermatocytes—one class with seventeen chromatic elements, and the other with sixteen. From these, by the usual transformations, are derived the mature male elements, which are thus of two distinct kinds.

(f) Spermatids.

The limits set to this paper preclude anything more than passing mention of the spermatids. As stated above, cells at this stage of development are of two classes, depending upon the presence or absence of the accessory chromosome. The distinction thus set up continues to exist visibly far through the transformation stages of the spermatid, by reason of the persisting independence of the accessory chromosome. Of the dual nature of the spermatids I was very early convinced, because the accessory chromosome is so strikingly displayed by the nuclei in which it exists that it is impossible to overlook its absence in a large proportion of the cells. As to the certainty of this partial distribution in the transforming spermatozoa, I am rendered positive by the most careful and painstaking study. This is valuable corroboration of the observed fact that the accessory chromosome remains undivided in one of the spermatocyte mitoses.