(_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.