Read Ebook: The Kansas University science bulletin Vol. I No. 8 September 1902 by Various Editor

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PUBLISHED BY THE UNIVERSITY,

LAWRENCE, KAN.

Price of this number, 50 cents.

Entered at the post-office in Lawrence as second-class matter.

KANSAS UNIVERSITY SCIENCE BULLETIN.

THE SPERMATOCYTE DIVISIONS OF THE LOCUSTIDAE.

BY C. E. M'CLUNG.

Under the title, "A Peculiar Nuclear Element in the Male Reproductive Cells of Insects" , I published a preliminary account of the process characterizing the maturation divisions of the Locustidae. This was of a general character and served merely as a basis for a description of the accessory chromosome in these cells. It is my present intention to give a detailed history of the spermatocyte divisions occurring in this family, after the manner followed previously in considering corresponding stages in the Acrididae . Besides giving this account of processes, however, I shall be able to draw some comparisons between the two families. Eventually I hope to complete such a comparative study of all the Orthopteran families. Material for this larger investigation is now partially on hand, and is being added to as circumstances permit, so that it may be possible to carry through a study of the maturation stages in this order of insects within a few years.

The value of comparative cytological study was urged by Vom Rath , and its importance in relation to the accessory chromosome and the maturation mitoses received recognition in both my earlier papers . Recently Montgomery has added his influence to the movement.

For the fixation of material used in these studies, it has been found that the osmic acid mixtures of Flemming and Hermann are the most generally applicable and are productive of the best results. In connection with these, however, Gilson's acetonitric-sublimate mixture has been tried, and frequently affords an excellent fixation. Extensive shrinkage in the melted paraffin is sure to follow the use of sublimate mixtures unless celloidin is used to support the soft tissue. This double infiltration of celloidin, followed by paraffin, has been found the best method of securing clear and accurate figures, for, because of the lessened shrinkage, the elements are not crowded together and rendered indistinct. This circumstance is particularly fortunate in the case of the Locustid cells, where the nuclear elements are so numerous and crowded.

The stains employed are the iron-haematoxylin of Heidenhain and the safranin-gentian violet-orange combination of Flemming. For general purposes, nothing excels the haematoxylin stain, but it is frequently advantageous to trace the chemical changes undergone by the different cell elements in the process of mitosis, and the aniline stain above mentioned serves excellently for this. Kernschwarz has also been found a valuable stain for some purposes.

The terminology as outlined in a former paper will be followed in the present one.

The testes of the Locustidae 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.

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.

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.

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 . 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 . 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.

The main interest of these studies, however, attaches to the movements of the chromatin granules. As was suggested in an earlier paper , 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.

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 . 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.

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.

I have not yet found it possible to make a detailed study of the spermatogonia of the Locustids, as was done for the Acrididae 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.

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 .

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 . 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 . The accessory chromosome does not change its form and structure at this time . 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."

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 . 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 .

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.

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.

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 Sin?ty.

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.

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.

The literature relating to the spermatocytes of insects was reviewed at some length in my previous paper upon the history of these cells in the Acrididae . It is not my purpose to go over this same ground again except in so far as increased knowledge makes it necessary. More recent papers by Montgomery, Wilcox and others will, however, be discussed in detail. The policy previously announced, of restricting comparisons to results derived from insects, will again be adhered to. I believe that the main features of the maturation divisions are essentially the same in all insects, and I desire to see this belief either well established or overthrown. If it can be demonstrated that so large a class as the insects are characterized by a common process, it will be a firm basis upon which to conduct further comparative studies into more comprehensive groups. On the contrary, if it is shown that there is no type, even in the class, then it is useless to seek agreements between widely removed species.

A necessary basis for any comparative work is a common terminology. Confusion inevitably follows the loose application of names to the structures compared. This is perhaps unavoidable in the early stages of an investigation, but should be overcome as soon as possible. There is surely no reason for continuing uncertainty after terms have received general acceptance. Believing this, I feel called upon to repeat my criticisms of Montgomery's application of the well-accepted terms "prophase," "metaphase," "anaphase," and "telophase."

In reply to my previous objection directed against this part of his work, Montgomery acknowledges the validity of the criticism so far as it relates to the metaphase, but denies the application to the other phases, particularly to the anaphase. He alleges in support of his position that the introduction of an unusual condition, the "synapsis," makes it impossible to correlate strictly the stages of the germ-cells with those of ordinary divisions. Upon this point I must again disagree with him. It is impossible for any known modification of the prophase to change the essential character of the anaphase, so as to make it precede instead of follow the metaphase. This stage marks the movements of the chromosomes from the equatorial plate to the poles, and terminates when they are massed around the centrosomes. How can the "synapsis" in the least affect the duration or character of this process? It is apparent enough, I think, that Montgomery's subphases of the "anaphase" do not belong to this portion of the mitotic cycle at all, but are really portions of the telophase of the spermatogonia and prophase of the first spermatocyte. Further, it may be noted that, even were these subphases properly included in the anaphase, they would belong to the spermatogonia and not to the spermatocytes.

Montgomery's translocation of the terms makes the "synapsis" occur in the anaphase. This is manifestly an impossible condition of the chromatin at this time, and his figures show definitely enough that it is a prophase, or, at the earliest, a spermatogonial telophase, that witnesses the contraction of the chromatin. The objection urged in my earlier paper to the use of the term as a designation for the mere contracted condition of the chromatin cannot apply to Montgomery's latest use of it; for he here recognizes the justice of my contention that it was primarily designed to indicate the fusion of the spermatogonial chromosome to produce the chromosomes of the spermatocyte. He states this clearly in the following words: "Moore first gave the name 'synaptic phase' to that stage in the growth period of Elasmobranchs when the reduction in the number of chromosomes takes place. Accordingly, the criterion of the synapsis stage is, first of all, the combination of univalent chromosomes to form bivalent ones; whether the chromosomes are then densely grouped or not is of secondary importance."

The formation of the first spermatocyte chromosome gives us an insight into the later changes undergone by these elements such as cannot be obtained in any other way. The great importance attaching to this part of the spermatogonial process renders it desirable to exhaust every effort in obtaining a knowledge of the actual changes here taking place. This thought has been held constantly in mind during the progress of these investigations, and every point of resemblance or of difference between the various species studied has received careful attention. Despite variations in details, however, I must state that the essential features of the maturation divisions are the same in all species of the Orthoptera examined. It is true that as yet only two families, the Acrididae and the Locustidae, have been worked out in a detailed way, but the close agreement between these raises a strong presumption in favor of the general prevalence of the type. The processes of the two families have already been described in detail, but it will perhaps be well to call particular attention to some points worthy of mention.

The general appearance of the material derived from the two families is quite different in sections. Even the hastiest observation will show this. The spermatocytes of the Locustid testis are much smaller, denser and more deeply staining than those of the Acrididae. The relative quantity of chromatin is greater, so that it is possible by microscopical examination of a section to tell whether it was prepared from Locustid or Acridian material.

In connection with the transformation of the chromatin from the spermatogonial condition to that of the spermatocyte, we must take notice of that stage which is commonly denominated the "synapsis." The evidence afforded by the Orthopteran cells is entirely negative regarding this. In properly fixed material there is no distortion of the chromatin in the nucleus at any time. It would, if present, be particularly easy to observe, as was stated in my previous paper, for during the entire winter the spermatocytes exist in the spireme stage, and in a longitudinal section of a follicle all stages may be discerned. On the other hand, in poorly fixed or hastily prepared material the synapsis is present, and always in such a form as to indicate its artificial character. What is here said regarding the synapsis refer to the appearance commonly thus designated, but, as has already been stated, such an application of the term does not meet the spirit of the definition as intended by Moore . A fusion of the spermatogonial chromosomes of some sort must certainly occur, but that it is always marked by a unilateral massing of chromosomes, I deny.

I should like to emphasize the fact that the chromosomes in both the Orthopteran families studied have been carefully traced from their earlier appearance down to the time of their dissolution in the spermatid through such a gradual series of changes that there can be no reasonable doubt of the accuracy of the conclusion set forth in these papers. The Orthopteran material possesses one distinct advantage over the Hemipteran, in that the point of cross-division is always marked by the same sort of a protuberance as is to be distinguished in the early chromatin segments. When the two free ends of the element are brought around to form a closed ring, the last particle of doubt regarding the position of the planes of separation marked out for the two spermatocyte divisions is dispelled.

Paulmier advances the suggestion that in the double-V figures we may find a structure that will serve to reconcile the divergent accounts concerning the longitudinal and cross divisions of the tetrads. The only way in which this might be accomplished would be to suppose that each of the interspaces represents a longitudinal cleavage of the thread, the first being at right angles to the second. I have given this suggestion careful consideration, and find no evidence to support it. The double Vs are only of rare occurrence, the common element being a straight rod, in the center of which is a diamond-shaped clear spot representing the two planes of division laid out for the spermatocyte mitoses. If two longitudinal divisions occur, one must precede the other considerably and the resulting halves become mutually repulsive, so that they move apart and lie in one plane with only a slight connection at the point of final separation. Moreover, the second cleavage must begin at the opposite end of the segment and proceed in a reverse direction from the first. Not only this, but the first spermatocyte mitosis divides the elements along what is generally conceded to be the longitudinal split, and this must necessarily succeed the supposititious first longitudinal cleavage by some time. Without going into a consideration of these points, I may say that they suggest such deviation from normal processes that only extensive and accurate observations would make Paulmier's suggestion worthy of further consideration.

Wilcox claims the distinction of being the first and only investigator to doubt the hypothesis that longitudinal and cross divisions of the chromatic thread produce chromosomes of a different character. It is perhaps well that this is so, in view of the reasoning by which such a distinction is secured. Upon his own unconfirmed and disputed statement that there is no longitudinal division of the spireme, Wilcox presumes to disparage the accepted view of practically all cytologists. The constructive thought of the last two decades is summarily disposed of by this author in the following language: "The whole question, therefore, whether a certain division is longitudinal or transverse loses its practical significance, since the theoretical interpretation which has long been placed upon these divisions is shown to be impossible and absurd!" The showing alluded to consists in the statement that the chromosomes consist of an indefinite number of granules, which cannot be expected to arrange themselves in any order, and which, therefore, may be divided in any way without affecting the results.

Laying aside for a moment the question as to the occurrence of a longitudinal division, we may well inquire whether the belief that, "In view of this manner of the formation of the chromosomes , it seems absurd to assume that the separation of an individual chromosome by one plane could be quantitative while the separation by another plane was qualitative," is well founded. At the basis of such an assumption lies the implication that any definite arrangement of chromomeres is impossible; for if any definite order were possible, then the supposed argument against the longitudinal disposition of the chromomeres would be invalid.

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