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Title: The Kansas University science bulletin, Vol. I, No. 8, September 1902
Editor: Various
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Language: English
Original publication: Lawrence: Kansas University, 1902
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*** START OF THE PROJECT GUTENBERG EBOOK THE KANSAS UNIVERSITY SCIENCE BULLETIN, VOL. I, NO. 8, SEPTEMBER 1902 ***
THE
KANSAS UNIVERSITY
SCIENCE BULLETIN.
Vol. I, No. 8—September 1902.
(Whole Series, Vol. XI, No. 8.)
CONTENTS:
THE SPERMATOCYTE DIVISIONS OF THE LOCUSTIDÆ, _C. E. McClung_.
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.
VOL. I, NO. 8. SEPTEMBER, 1902. {WHOLE SERIES,
{VOL. XI, NO. 8.
THE SPERMATOCYTE DIVISIONS OF THE LOCUSTIDÆ.
BY C. E. M’CLUNG.
With Plates VII, VIII, IX, and X.
I. INTRODUCTION.
II. METHODS.
III. NOMENCLATURE.
IV. OBSERVATIONS.
(a) General Structure and Form of the Testes.
(b) The Spermatogonia.
(c) The First Spermatocytes.
(d) The Second Spermatocytes.
(e) The Spermatids.
V. COMPARISONS AND CONCLUSIONS.
(a) Nomenclature.
(b) The Spermatocytes of the Acrididæ and the Locustidæ.
(c) Formation of the Tetrads.
(d) The Spermatocyte Divisions.
(e) The Accessory Chromosome.
(f) Individuality of the Chromosomes.
(g) Nucleoli.
(h) Rest Stage.
VI. SUMMARY.
VII. BIBLIOGRAPHY.
I. INTRODUCTION.
Under the title, “A Peculiar Nuclear Element in the Male Reproductive
Cells of Insects” (=16=), I published a preliminary account of the
process characterizing the maturation divisions of the Locustidæ. 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 Acrididæ (=17=). 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 (=25=),
and its importance in relation to the accessory chromosome and the
maturation mitoses received recognition in both my earlier papers (=16=,
=17=). Recently Montgomery (=15=) has added his influence to the
movement.
The observations upon which the present paper are based were originally
made upon _Xiphidium_. The cells in this genus are, unfortunately, small
in size, and much difficulty was experienced during the early work in
getting clear images. This embarrassment was further increased by the
large number of chromosomes and their habit of compact arrangement.
Later it was found that species of _Anabrus_, _Orchesticus_,
_Microcentra_ and _Scudderia_ have cells much better adapted for study,
and because of this they have been largely utilized. The account which
follows is therefore based upon a study of all these genera, and is
considered representative of the family. The accompanying figures are
principally from _Orchesticus_, since the number of stages represented
exceeded those in material derived from other genera. I am indebted to a
friend and former student, Mr. W. S. Sutton, now of Columbia University,
for a generous supply of _Orchesticus_ and _Anabrus_ testes from his
collections.
II. TECHNICAL METHODS.
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-hæmatoxylin of Heidenhain and the
safranin-gentian violet-orange combination of Flemming. For general
purposes, nothing excels the hæmatoxylin 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.
III. NOMENCLATURE.
The terminology as outlined in a former paper (=17=) will be followed in
the present one.
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.
V. COMPARISONS AND CONCLUSIONS.
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
Acrididæ (=17=). 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_) _Nomenclature._
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 himself seems to be rather uncertain of the position of his
“anaphase.” In the first paper, upon _Euchistus_ (=12=), it was put down
as the anaphase of the first spermatocyte; in his later paper (=14=),
upon _Peripatus_, it is recorded as the anaphase of the spermatogonia.
Still more confusing is his use of the “telophases,” for in the article
upon _Peripatus_ (=14=) it is, in the “Contents,” placed as a substage
of the spermatogonial anaphase, and in the body of the work, page 307,
as the telophase of the spermatocyte! Neither the anaphase nor the
telophase can, by any possible construction of their proper meanings, be
made to apply to the “growth period” of the germ cycle, as Montgomery
insists; they are the last stages of the “division period,” in reality.
The prophase of the first spermatocyte is the initial stage in the
constructive process marking the growth period.
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 (=17=) 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
(1895) 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.”
(_b_) _The Spermatocytes of the Locustidæ and Acrididæ._
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
Acrididæ and the Locustidæ, 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 Acrididæ. 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.
The transformation from the telophase of the last spermatogonial
division to the prophase of the first spermatocyte is marked by
practically the same changes in both families. It is to be observed,
however, that the derivation of the spireme from the disintegrating
chromosomes of the previous generation is not so clearly indicated in
the Locustid cells, and it was for this reason that in the examination
of _Xiphidium_ I was not able to determine certainly that the accessory
chromosome came over from the spermatogonia into the spermatocytes as a
formed element. Upon this point, as upon others, my later material is
clearer, and I was able to reconcile the appearances in the two
families. In both, unfortunately, it has been found impossible to
determine the exact origin of the first spermatocyte chromosomes.
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 (=20=). 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.
During the prophase the chromatin segments in the cells of _Orchesticus_
and other species of the Locustids are heavier, more granular and denser
than they are in _Hippiscus_. It is to be observed, also, that there is
a greater variation in the size of the elements. This fact is observable
from the earliest appearance of definite segments down through both the
spermatocyte mitoses. This disproportion may be such that one chromosome
will exceed another in the same cell by twenty or thirty times its
volume. We have here, as is pointed out in another place, a strong proof
concerning the individuality of the chromosomes, for in some species it
is possible to distinguish a particular chromosome in all the
spermatocytes. This is strikingly the case in _Anabrus_, where there is
always one chromosome very much larger than any of the others. It
exceeds in size even the accessory chromosome, and might be mistaken for
it were it not for the difference in form. It is, however, typically a
tetrad, and shows the four chromatids, while the accessory chromosome
exhibits the usual spermatogonial condition.
As was indicated under the head of “Observations,” the prophase tetrad
characteristic of _Anasa_ and _Hippiscus_ is again exemplified in the
Locustid cells. So close is the resemblance of the maturation
chromosomes of these various insect cells in their early stages, that I
now regard it as practically established that they are commonly present
in all insect spermatocytes. No more important evidence regarding
chromosome structure and behavior can be obtained than that afforded by
these elements. Particularly are the ring figures of value in the
determination of the sequence of the longitudinal and cross divisions,
and upon this point the material from the two families is equally
convincing and positive in demonstrating that the first spermatocyte
mitosis witnesses a separation along the longitudinal cleft of the
spireme thread.
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.
This diagnostic character seems to be lacking in the chromosomes of the
Hemiptera, and Paulmier, in his work on _Anasa_, depends for his
criteria of orientation upon the relative lengths of the chromosome
axes. Such a feature would be valueless in Orthopteran cells, because,
as has been shown, the chromatids move upon each other in such a way as
to exactly reverse the preexisting relation between the axes. How
applicable this observation may be to conditions in the Hemipteran
cells, I do not know; but, judging from the great resemblance of the
elements in the prophase, it would seem most reasonable to expect a
similarity of the divisions.
Paulmier (=22=) 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.
(_c_) _Formation of the Tetrads._
In my former paper I reviewed the results obtained by Montgomery upon
the Hemiptera, but further notice of his work will now be necessary,
since on almost every important point relating to chromosome structure
he has changed his opinion. His late extensive comparative study upon
the Hemipteran cells, as well as that upon _Peripatus_, will at the same
time receive consideration.
It appears from Montgomery’s account that at the point where the
Orthoptera are least valuable in demonstrating chromosomal relations the
Hemiptera and _Peripatus_ are most convincing. I refer here to the
derivation of the first spermatocyte chromosomes from the chromatin of
the spermatogonia. He claims to have observed the union by pairs of the
secondary spermatogonial chromosomes during the anaphase (his synapsis)
so clearly as to be positive of this fusion. I hope this may be
verified, for it offers a logical explanation of the process of
reduction, and is a confirmation of what has previously been assumed
true without sufficient basis in observed fact, as was suggested in my
paper on _Hippiscus_. This, if established, would also be a strong
support of the theory relating to the constancy of the chromosomes. If
this true synapsis is accomplished at this time, however, it must be
noted that it occurs during the last phase of the final spermatogonial
mitosis, and is not an act of the spermatocyte prophase. But as to the
exact location of this point no contention need be made, for it is
conceivable that the time of its occurrence might vary considerably
without affecting the essential nature of the process.
With regard to such an origin of the first spermatocyte chromosomes,
there is an important difference to be noted between the earlier and
later work of Montgomery, and one which he fails to mention. In his
paper (=12=) upon _Euchistus_ he states the matter as follows: “But in
the post synapsis we do not find seven chromosomes, the definitive
number present in the spermatocyte divisions, but a smaller number;
hence, in the synapsis the true (_i. e._, exactly half) reduction of the
chromosomes does not take place, but the number is reduced to less than
one-half.” This statement is based, he says, upon a most careful and
painstaking enumeration of the chromatic segments in a number of nuclei,
and is unhesitatingly declared correct.
In his later paper, on the contrary, he is just as positive that the
definitive reduction is here accomplished, for he says: “Since then I
have been able to demonstrate that this numerical reduction is effected
in the synapsis by the union into seven pairs of the fourteen
chromosomes, each of the seven bivalent chromosomes (pairs) being
composed of two univalent chromosomes joined end to end.” This statement
is made without adducing any specific proof, as was formerly done. By
what means we are to reconcile these diametrically opposite statements
Montgomery does not say. He, however, insists that he has always known
that the fusion by pairs takes place. How this was to be brought about
under his previous assumption that one of the fourteen spermatogonial
chromosomes became removed from participation in the usual processes of
the cell to form a “chromatin nucleolus,” he fails to state. Until the
confusion is cleared up by corroborative evidence on one side or the
other, a most important part of Montgomery’s work must still be regarded
as uncertain.
Despite his recognition of the fusion of the chromosomes in the synapsis
as the essential feature of this stage, Montgomery is insistent upon the
concentration of the chromatin as its distinguishing characteristic.
Regarding this he says: “McClung considers the appearance of the
synapsis stage as artefacts. It is hardly necessary to reply to this
criticism, since in all _Metazoa_ where the spermatogenesis has been
carefully examined, with the exception of certain _Amphibia_, the dense
massing of the chromosomes (?) in the synapsis stage has been shown to
be a perfectly normal phenomenon.”
Concerning two points in this statement I wish to take exception. First,
as was suggested in my previous paper (=17=), the term synapsis is
usually applied to a condition of the prophase in which the apparently
unsegmented spireme exists. It must be remembered that most
investigators consider that the reduction of the chromosomal number
takes place by the segmentation of a spireme into half the usual number
of segments. In the second place, I must resent the implication that the
work done in this laboratory is not “carefully” conducted. Many
“_Metazoa_” have been examined “carefully,” and in none has the
“synapsis” occurred when the material was well fixed and prepared. It
has, moreover, been found possible to produce the appearance at will.
One case of this kind is sufficient to raise the presumption that it may
not be normal even when constantly found in certain preparations. I have
not, however, absolutely denied the possibility of such an occurrence,
because it is conceivable that from the telophase of the preceding
division the massing of the chromosomes may persist during their
elongation. My contention is that the appearance is not a constant or
necessary condition in “all the _Metazoa_,” and this I have proven.
In rather striking contrast to the work of Montgomery, in which an
effort is made to formulate a typical process for the entire _Metazoa_
from the study of a single order, is that of Wilcox, wherein a general
denial of any apparent system in the maturation divisions of animals is
based practically upon the study of a single species. As was stated in
my former paper, I regard Wilcox entirely in error upon the vital point
of his theory of tetrad formation, not by “forced interpretation” of his
own views, but by an actual examination of the object upon which he
worked. There is no point upon which Orthopteran material affords more
indisputable evidence than upon the occurrence of the longitudinal
division of the chromatin thread in the early prophase. My statement
regarding Wilcox’s position on this subject was in no sense “misdirected
criticism,” but an actual statement of fact; it was not an attempt to
explain away “abundant and evident cases which cannot be made to fit
into the scheme,” but simply the presentation of proof that _one_ case
was wrongly interpreted.
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 (by the
aggregation of the chromomeres), 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.
The argument of Wilcox is therefore directed against order in general,
and not against order in any one particular, as he would have it appear.
For it must be admitted that if it is possible for the scattered
chromatic granules of the early prophase to arrange themselves at all
(and this even Wilcox does not deny), it is equally possible for them to
come together in a definite order. That they do this is amply evidenced
by the fact that later they appear in definite groups or chromosomes. It
is to be noted, moreover, that the later investigations tend to suggest
that the apparently unorganized chromatic granules in the first
spermatocyte prophase are really bound together and represent merely a
diffuse condition of the spermatogonial chromosomes.
Wilcox’s chief error, however, is not to be sought in speculative
theories, but rather in his faulty observations. He repeatedly denies
the occurrence of any longitudinal split in the chromatic thread of the
first spermatocyte prophase. That he is mistaken here I am thoroughly
convinced, both from a study of his own object and from investigations
upon many other species of the same family. At the present time, also,
practically every spermatologist is aligned in support of the view
denounced by Wilcox. For a while Wilcox had some backing, but most of
those who advocated only cross divisions of the thread have later been
able to demonstrate the longitudinal cleavage in better prepared
material.
There is general acceptance of the opinion that the chromomeres of the
last secondary spermatogonia appear in a linear arrangement to form what
is commonly known as the “spireme.” Wilcox declared that while in a very
fine condition this thread breaks across into segments, which unite by
pairs to form the chromosomes of the first spermatocyte. The great
majority of other investigators are unanimous in the opinion that this
fine thread, made up of granules, becomes double by the division of each
granule individually, thus producing a double thread. Thus it is that
the two halves of a longitudinally divided chromosome are made
equivalent, not by the sifting apart of preexisting granules, but by the
division of these after they are arranged in a linear series. It need
hardly be mentioned that the formation of the thread has here a reason
for existence which is entirely lacking according to Wilcox’s scheme.
This much space has been devoted to Wilcox’s statements, not because
they present any arguments against the generally accepted views of his
fellow workers, but because he represents a rapidly lessening minority
which is content to work in a very limited field and to resort for the
explanation of diverse results to the very convenient theory that great
differences may be expected in the normal processes of even closely
related forms. One needs only to glance at the work of all insect
spermatologists to see how closely the agreement now is upon the
important points of the process. This accordance of results Wilcox
notes, but interprets in his own way, which may be regarded as not
exactly complimentary to the skill and judgment of his colaborers. “It
is only necessary,” he says, “to refer to any recent publication on the
subject to find examples of this attempt to force the divergent
processes in different species to fit the same formula.” This is
certainly a very easy and convenient way to dispose of the accumulated
observations of the many careful investigators who have come to an
agreement upon the important questions under discussion, but I venture
to think will hardly satisfy any one except its sponsor.
After handing in this article for publication, I fortunately secured a
copy of the paper by R. de Sinéty (=37=) in which the spermatogenesis of
various Orthopteran species is described. I regret that the available
time is so short that I shall not be able to bestow upon this
contribution to insect spermatogenesis the attention it deserves, but I
shall try at least to consider the principal points wherein a difference
exists between the results of de Sinéty and of myself.
It is unfortunate that we have here a further complication of the
problem concerning the character of the two maturation divisions in
insects. At this time it had begun to appear as if there was every
possibility of insect spermatologists coming to an agreement with regard
to the maturation processes. Indeed, with the exception of Wilcox, who
occupies a unique and solitary position in the field, workers upon the
subject are committed to a belief in the occurrence of a cross and a
longitudinal division of the chromosomes in the spermatocyte mitoses.
The sole difference of opinion relates to the sequence of the divisions.
We have now to consider in connection with insects the remaining
possibility in tetrad formation—that of two longitudinal divisions—which
finds an advocate in de Sinéty.
Because of a thorough acquaintance with the forms upon which this author
has worked, I do not hesitate to say that he is entirely mistaken with
regard to the character of the second spermatocyte division. I am
convinced of this because of the fact that in the early period of my
work upon Orthopteran spermatogenesis I was inclined to place just such
an interpretation upon the phenomena encountered in the spermatocytes of
the Acrididæ as does de Sinéty. I soon became convinced, however, that I
was proceeding upon a wrong assumption, and abandoned it in favor of the
one which more extended observation taught me is correct. I hope to
demonstrate here the ground for my plain statement that de Sinéty is in
error upon the question of a double longitudinal division of the
chromatin thread during the formation of the tetrads in insect
spermatocytes.
It is fortunate that our author has properly appreciated the value of
the early prophase in the determination of the structure of the first
spermatocyte chromosomes, for we are here upon common ground, and need
only compare like stages in order to reach our conclusions. As will be
recalled, the statement is made elsewhere in this paper that the typical
chromosome of the first spermatocyte is an approximately straight rod,
split longitudinally, and again cleft in its middle by a second fissure
at right angles to the first. Such an element is represented in figures
15_a_, 17, D and E of my paper upon the Acrididæ, and in figures 7, 9,
11 and 38 of the present one. Although this is extremely common, and, as
the photomicrographs show, undeniably present, de Sinéty does not figure
it at all. The nearest approach to such a structure is found in figure
123_c_, where a cross with two nearly equal arms is represented. My
interpretation of this figure, based upon a great number of careful
observations, is that this represents merely an extension of the shorter
arms at the expense of the longer ones. In support of this, I have
stated that all intermediate stages between a rod with a mere
enlargement at the center and a cross with equal arms could be found.
How, according to de Sinéty’s conception of overlying free elements,
could these structures be explained?
It is not necessary, however, to have these gradations in order to
disprove the theory under discussion. One needs only to carefully
examine one of these crosses to be convinced that the two arms lie in
one plane where they intersect, and are not superimposed one upon the
other as de Sinéty shows in his figure 123. Our author clearly realizes
the importance of the cross, as may be judged by the following
quotation:
“La croix est de toutes ces figures celle dont la genèse peut le plus
facilement donner lieu à des interprétations en sens contraire.—C’est
précisément pour cette raison que nous croyons devoir l’étudier
spécialement au point de vue critique, persuadé que, cette figure une
fois rattachée à une théorie, les autres doivent en suivre le sort.”
It is unfortunate, therefore, that he was not able to trace the
formation of the element in its very early stages and through the
various modifications which connect it with the typical rod already
described.
As the simplest modification of this basic form, we find the one where
it is evident that the change consists merely in a flexure of the rod at
the weak spot in its center. Such forms are shown in figure 14 of my
former paper (=17=) and in figures 8, 9 and 11 of this one, but are not
illustrated by de Sinéty. It occasionally happens that in chromosomes of
this character the halves diverge widely at the center, producing the
double-Vs of Paulmier, as is represented in figure 14 of my paper upon
the Acrididæ (=17=) and in figure 8 of the present one. These structures
are not shown by de Sinéty and would be difficult to explain in
agreement with his conception of the tetrad.
I have consistently placed great reliance upon the frequent ring-shaped
chromosomes in determining the structure of the first spermatocyte
elements, and have no occasion to change my opinion of them since
examining the work of de Sinéty. This investigator joins issue with me
upon my interpretation of these structures, and states his attitude in
the following language:
“McClung fait grand fond, pour appuyer son interprétation, sur une
forme spéciale, la forme en anneau, qui pour lui dérive du bâtonnet
(a′ b′)/(a′ b′’), supposé placé transversalement sur le fuseau, inséré
par son milieu et incurvé en dehors jusqu’ à rapprochement et soudure
de ses extrémités.
“Le chromosome en anneau est en effet très fréquent chez les
acridiens; mais il nous a été possible d’en reconstituer l’histoire,
grâce à des détails qui ne semblent pas s’être rencontrés dans les
figures de McClung. On se souvient que nous avons établi les deux
points suivants en complet désaccord avec la théorie de l’auteur
américain:
“1. Les deux moitiés de l’anneau proviennent de la première division
longitudinale.
“2. L’insertion est terminale.”
With equal emphasis, I must deny that the enclosed space in the ring
represents any plane of division in the chromatin thread; and that the
insertion of the spindle fibers is at any place except at the center of
what would be the typical rod-shaped chromosome were the ring
straightened out. We encounter in de Sinéty’s interpretation of these
rings the very error against which I was careful to caution elsewhere in
this paper, _i. e._, _of regarding the points where the fibers are
attached as the crossed ends of a simple segment_. This mistake de
Sinéty has made, and has thereby vitiated all his conclusions concerning
the structure of the tetrads. It is not necessary to repeat here the
proof which I have brought forward in support of my views. No one, I am
sure, will find difficulty in reducing the various forms of chromosomes
found in the first spermatocytes to the type of a doubly split rod, in
which one plane of division is parallel to the long axis and the other
at right angles to it. The explanation offered by de Sinéty requires us
to conceive a doubly split rod in which one separating space may vary
indefinitely while the other is constant. There is here no common type,
but an infinitely variable one, which differs with every modification of
the interspace between the first pair of chromatids in each chromosome.
As a constructive basis for the foundation of his theory of a double
longitudinal division, de Sinéty uses particularly the chromosomes of
_Œdipoda_ (_Hippiscus_) _miniata_, represented in figures 129 and 130,
concerning which he says:
“Survient le phénomène exceptionnellement important de la seconde
division longitudinale; nous regardons comme un point capital dans
notre travail d’en mettre l’existence hors de doute et pour cela nous
désirons ne faire appel qu’à des images extrêmement claires. Nous
considérons comme telles les fig. 129 et 130 rapprochées l’une de
l’autre.
“Il est de toute évidence que le chromosome _a_, fig. 130, n’est que
le chromosome de même désignation, fig. 129, dont les deux anses
jumelles se sont clivées. De même, le chromosome en forme de boucle,
_c_, fig. 129, dont les deux branches représentent, comme nous l’avons
fait remarquer, deux anses jumelles, se retrouve avec un clivage très
évident en _d_, fig. 123. On pourrait faire les mêmes rapprochements
entre _b_, fig. 105, et _a_, fig. 107; ici, le clivage est moins
avancé, mais les granules sont nettement divisés.”
I am obliged to confess that I have never seen in other species of this
genus any appearances that would incline me to place an interpretation
upon them such as does our author upon these. I would venture to
suggest, on the contrary, that the chromosomes represented in figure 129
have not as yet demonstrated any division, but show merely irregular
spaces between chromosomes. At even an earlier stage (figs. 5, 37, and
38), I have shown the formation of the tetrads by means of simultaneous
cross and longitudinal divisions so clearly that presumed successive
divisions, as represented by de Sinéty, cannot be regarded as occurring.
Finally, I would emphasize the fact mentioned in connection with the
discussion of the cross-shaped chromosomes, that where the elements of
one of these compound chromosomes intersect _they lie in one plane, and
are not superimposed upon each other_, as de Sinéty’s theory demands and
as his figures represent. This was shown clearly in Paulmier’s figures
as well as in my own, and is even more clearly demonstrated, if
possible, in the very long, slender chromosomes of the myriapods, which
I have observed in Mr. Blackman’s preparations. This, and the continuity
of the chromatin in contiguous arms of the cross, is alone sufficient to
disprove de Sinéty’s theory, and, fortunately, is easily demonstrated.
This same fault of de Sinéty’s is encountered, in another form, in his
discussion of the ring figures. He asserts that the halves of the rings
are pulled past each other while they lie in the plane of the spindle
axis. Herein my observations fail entirely to agree with his. The rings
lie in the plane of the equator, and no elements of the mitotic figure
show a lateral displacement of the separating halves equal to the width
of the chromosome when viewed in this plane.
(_d_) _The Spermatocyte Divisions._
I approach a discussion of Montgomery’s conclusions regarding the form
of the chromosomes in the first spermatocyte, and the sequence of their
divisions, with considerable hesitation, because of the difficulty I
experience in appreciating his exact position. This is due, not to any
lack of positive statements on his part, but to the partial
contradictions that result from his frequent changes of opinion. The
most important statement in his first paper upon _Euchistus_ reads as
follows: “From the resting stage of the first spermatocyte to the
formation of the spermatid, there is absolutely no longitudinal division
of the chromosomes. I have studied hundreds of nuclei in these stages,
and at the first with a hope of finding a trace of such a process, but
observation shows that all divisions of the chromatin elements are
transverse divisions.”
This would certainly seem to be as strong a stand as one could take upon
the subject, but in later papers Montgomery assumes with equal assurance
the opposing position, which holds for a longitudinal division.
Regarding this he says: “During the synapsis stage the chromosomes
become split longitudinally, as was first shown by Paulmier (1898, 1899)
for _Anasa_—a process that I had overlooked (!) in my former paper
(1898).” Throughout his later investigations this hypothesis serves as
the basis of all his theories, and the careful longitudinal division of
the thread is assigned an important role in the maturation process. So
far as positive assertions to the contrary are concerned, a general
acceptance of the theoretical importance attaching to this act is to be
supposed.
Notwithstanding this, I find nowhere in his later writings any statement
that he abandons the conception formerly entertained regarding the
non-importance of the longitudinal cleavage. This attitude is indicated
in the following language: “If it can be proved that the mode of
division of a chromosome, _i. e._, the axis of the line of division, is
merely a function of its chromomeres, then it would be of no theoretical
value whether the division be longitudinal (equation) or transverse
(reduction). But it happens that the postulated difference forms one of
the main premises of Weismann’s theoretical superstructure. On account
of the differences observed in different objects in regard to the modes
of division of the chromosomes, it would appear that the differences
have no theoretical value, but that the halving of the mass of chromatin
is the process of importance—the standpoint taken by Hertwig.
“In the two reduction divisions the chromosomes may split by two
longitudinal divisions, by two transverse divisions, by one longitudinal
and one transverse division, or by one division (longitudinal or
transverse) preceded or followed by an elimination division. The facts
show already that there is no general uniformity in the mode of division
of the chromosomes in the reduction mitoses. The long line of
observations on different objects show this to be the case, and
demonstrates that the expected uniformity does not occur.”
Herein lies the essential conclusion of the work upon _Pentatoma_,
which, so far as a specific retraction is concerned, stands yet. If this
be abandoned, then the first work upon the chromatin structure of
_Pentatoma_ is practically discredited, for Montgomery has definitely
retreated from his positions concerning the absence of the “chromatin
nucleolus” in the spermatogonia, the non-occurrence of a longitudinal
cleft in the spireme thread, the lack of an equational division of the
chromatin in the spermatocyte, the origin of the “chromatin nucleolus,”
and the fragmentation of the “chromatin nucleolus.” In addition to these
specifically acknowledged errors, we may infer that Montgomery (=12=)
considers himself at fault in his views upon the production of
chromosomes from the “three to six chromatin loops” by breaking apart in
the prophase, and upon the occurrence of both longitudinal and cross
divisions of ordinary chromosomes in the same mitosis. The observations
recorded in his last paper (=15=) upon the production of the
spermatocyte chromosomes by the end-to-end union of those in the last
spermatogonial division warrant this assumption.
It follows from all this that we may practically disregard Montgomery’s
earlier work upon chromosomal structure and take his views as expressed
in the later papers (=14=, =15=) as representing his opinions upon the
subject. These later theories are largely the result of his
investigations upon _Peripatus_, but they seem to be carried over and
applied to the Hemiptera without essential modifications, and we may
regard this concept as applicable to the forms studied by him.
I called attention in my previous paper to the fact that, by many
investigators, the definitive form of the chromosome is used as the
basis for determining the direction and sequence of the chromosome
divisions. This fact and the danger attending the practice was partly
realized by Montgomery in his work upon _Euchistus_ (=12=), for he
devotes considerable space to a consideration of the prophase segments,
but in determining the character of the second spermatocyte division he
regards only the formed element. With respect to this he says: “And now
a fact may be determined which is of the greatest importance in
estimating the morphological value of the second division of the
chromosomes. While the latter are still parallel to the axis of the
spindle, there may be clearly seen in some cases a transverse
constriction on some of the chromosomes, so that they already acquire a
dumb-bell shape.” This constriction is not correlated with any similar
one on the prophase elements, and is here observed for the first time.
In his paper upon _Peripatus_, however, he definitely supports the
contention that it is only in the prophase of the first spermatocyte
that we can learn the construction of the chromosomes, for he says: “The
early stages in the prophase are of the greatest importance in
determining the exact constitution of the chromosomes of the first
maturation division.... Since, then, as has been shown in another
section of the present paper, the split of the univalent chromosome of
the second spermatocyte is a true longitudinal split, corresponding
perfectly in position with the longitudinal split of the early prophase,
it follows that the univalent chromosome does not become turned upon its
axis to take its place on the equator of the spindle.” Orientation is in
both spermatocytes based, accordingly, upon planes determined in the
prophase. Upon this point Paulmier and Montgomery, as students of
Hemipteran spermatogenesis, are now agreed, and their results correspond
with observations made upon Orthopteran cells.
It is upon the sequence of divisions in the spermatocyte that
differences now exist between these investigators and myself. In my
previous paper I took occasion to elaborate the proof in support of my
position regarding the early occurrences of the longitudinal division in
the Orthopteran spermatocytes. Montgomery follows Paulmier in ascribing
the reduction division to the first spermatocyte, and takes no account
of my results upon _Hippiscus_. The objections that I previously urged
against Paulmier’s conclusions apply equally well to Montgomery’s. Until
the chromosomes are traced in a more detailed way through the prophase
to the metaphase, I shall consider the presumption against the
occurrence of the cross-division in the first spermatocyte mitosis. In
this I believe that I am justified by the definite proof of my position
brought forward in the work upon _Hippiscus_. Here, it may be recalled,
I observed and photographed in the same mitosis all stages of movement
by the chromatids along the plane of the longitudinal split. In
addition, I was able to locate definitely the position of the future
cross-division in the ring figures, so that it is impossible to mistake
the character of the first division in them. These two proofs I consider
incontrovertible so far as they apply to the Orthopteran families
studied.
Paulmier judged the planes of the division by the relative lengths of
the chromosome axes, but, as I pointed out, this is not conclusive
unless it can be shown that they have not shifted, as it is possible for
them to do, during the prophase. The value of the ring figure, which is
formed at such an early stage that it would be impossible for the
shifting of the axis to occur, is here evident.
Montgomery finds these rings in _Peripatus_, and realizes the importance
of their evidence in determining the planes of division, but places his
conclusions upon a much more insecure footing than those founded upon
the Orthopteran cells, because of the criterion used in determining
which point represents the junction of the paired chromosomes. The
diagnostic feature he uses is the linin connection persisting between
the “central ends” of the chromosome, which holds them together until
the “distal fibers” connect with the centrosomes and cause the rupture
of the “central” fiber. Since the whole of his elaborate theory
regarding the continuance of the linin spireme is practically a
theoretical conception with little basis in observed fact, the value of
such proof cannot compare with that furnished by the definitely formed
chromosomes themselves in the Orthopteran cells.
In view of all these facts, I think it must still be held an open
question as to which is the reduction and which the equation division in
the Hemipteran spermatocytes, although it is not to be doubted that the
probability of the first spermatocyte being witness of the reduction
division is much increased when thus interpreted by two independent
observers.
(_e_) _The Accessory Chromosome._
I have already, in another paper (=19=), taken up a comparative study of
the accessory chromosome in different insect spermatocytes, and shall
not be obliged, for that reason, to enter into a very lengthy discussion
of the subject here. The great interest attaching to this structure,
however, compels me to consider the work that has been done since the
manuscript of the earlier article was sent in for publication. This
review will concern, very largely, the investigations of Montgomery upon
a considerable number of Hemipteran species, which are set forth in his
paper under the pretentious title “A Study of the Chromosomes in the
Germ Cells of Metazoa.”
In his first work upon _Euchistus_, Montgomery describes a cell element
under the name “chromatin nucleolus” which corresponded so closely to my
accessory chromosome that I concluded the two structures were identical.
These similarities were, the origin from a spermatogonial chromosome,
the integrity and constancy of staining power and position during the
spermatocyte prophase, and participation in the division act during
metakinesis of a spermatocyte.
Among the numerous changes of opinion recorded by Montgomery in his
latest work, there are several relating to his “chromatin nucleolus”
that materially alter the aspect of the question. Perhaps the most
important of these concerns the origin of the element. I was some time
in determining that the accessory chromosome is a spermatogonial
chromosome which divides in the spermatogonia with the other chromatin
elements and comes over into the first spermatocyte as a formed
structure. The work of Sutton upon the early history of the element in
_Brachystola_, however, was convincing in this respect and confirmed me
in the opinion I had already formed. I therefore gave Montgomery the
credit for this discovery, and set it down as strong confirmation of the
assumption that we were dealing with similar structures in the two
orders of insects.
Upon this point Montgomery now completely reverses himself, and declares
that his “chromatin nucleolus” is not a spermatogonial chromosome, but
may be noted in the earlier generations as a nucleolar structure, which,
however, divides in metakinesis. The most important feature to be noted
in this connection is the fact that the structure does not exist as a
simple element, but is observed as a number of granules, and that this
number varies considerably in different species. These granules fuse
during the “synapsis stage,” as do the chromosomes, to produce in the
spermatocyte half the number of “chromatin nucleoli” that were present
in the spermatogonia. In this respect the “chromatin nucleolus” differs
radically from the accessory chromosome, which has the same valence in
both cell generations. The indefinite number and insignificant size of
Montgomery’s structures are other characters that point to extensive
differences between them and the accessory chromosome.
In his work upon _Peripatus_, Montgomery states that in restudying his
preparations of _Euchistus_ he observes a continuous linin spireme which
involves the “chromatin nucleolus” as well as the chromosomes. Here,
again, there is a difference between the Hemipteran element and the
accessory chromosome; for the latter is entirely free from linin
connections in the prophase and is usually surrounded by a hyaloplasmic
investment.
According to Montgomery, also, his “chromatin nucleolus” usually takes
part in both spermatocyte mitoses. In this respect there exists an
essential difference between his element and that found in the
Orthoptera, for, after extended and most critical studies, I have become
convinced that only one division takes place in the spermatocytes. In
those cases where Montgomery admits but a single division, it is stated
to occur in the first spermatocyte, while in the Orthoptera the
accessory chromosome remains undivided here and is halved in the second
spermatocyte.
If, therefore, Montgomery’s recent observations are correct, it must
follow, I think, that his “chromatin nucleolus” and the accessory
chromosome are different structures. I am free to admit, however, that
his statements are far from convincing. So much dependence is placed
upon the numerical relationships of elements that are admittedly very
minute, and so little corroborative proof is given, that I entertain
serious doubts as to the accuracy of the observations. In this
connection I would suggest a comparison between the figures of the
“chromatin nucleolus” in the first paper upon _Euchistus_ (figs. 55–68)
(=12=) and those in the last one (figs. 1–17) (=15=). The showing here
made would alone be sufficient to raise a question as to the nature of
the “chromatin nucleolus,” and until further evidence is forthcoming the
character of the peculiarly modified chromosomes in the spermatocyte of
the Hemiptera must remain in doubt.
Aside from definite retractions that Montgomery has made regarding his
earlier views on the character of the “chromatin nucleolus,” there are
noticeable different attitudes toward it in his earlier and later works.
Thus, in his lecture at Woods Holl (=13=_a_), we find the following:
“These remarkable ‘nucleolar’ structures which stain like chromatin have
been observed by numerous writers, but as yet no satisfactory
description has been given of their mode of origin. They have been
observed by me in spermatocytes of various insects, in hypodermal and
other cells of _Carpocopsa_, and in follicle cells of the testicles of
_Plethodon_ and _Mus_.” At this early stage of Montgomery’s
investigations it is apparent that he views his “chromatin nucleolus”
primarily as a nucleolus with chromatic origin and characters, but the
fact is equally apparent that he now regards it primarily as a
“chromosome” with nucleolar attributes. This is made evident in his
recent definition, which reads: “The chromatin nucleoli are
morphologically chromosomes, undergoing division in mitosis like the
other chromosomes, but differing from them in the rest stage by
preserving a definite (usually rounded) form.”
What has here been said regarding the “chromatin nucleolus” applies to
those structures in _Euchistus_ and other Hemiptera to which Montgomery
has given the name without qualification. According to his definition,
however, there is present in the cells of _Protenor_ and other species
another form, the “chromosome x.” Not only by inference is this
classification operative, but by direct statement we learn that
Montgomery regards this element as a member of the class of bodies which
he calls “chromatin nucleoli.” In speaking of _Protenor_ chromosomes, he
says: “This is the only case in the Hemiptera where one chromosome
becomes differentiated into a ‘chromatin nucleolus’ for the first time
in the spermatocyte generation.”
The noteworthy thing about this “chromosome x” is the fact that in every
essential detail it corresponds to the accessory chromosome of the
Orthoptera. It is a spermatogonial chromosome that comes over intact
into the spermatocyte; it retains its form and staining power unchanged
through the prophase of the spermatocyte; it divides in only one of the
spermatocyte mitoses; and is a large and conspicuous element of the cell
at all times.
This “chromosome x” agrees just as closely in its description to the
accessory chromosome as do the ordinary ones of the two orders, and, if
Montgomery’s account is correct, there would seem to be no reason for
doubting their identity. In two respects, however, there are differences
between these structures. First, it is to be noted that the “chromosome
x” divides in the first spermatocyte, while the accessory chromosome
undergoes separation in the second spermatocyte. Should Montgomery’s
observations prove correct, it would yet indicate no fundamental
difference in the character of the element, for the result is the same
whether division takes place in the first or second mitosis. In either
event, one-half the spermatozoa are provided with the odd chromosome
while the remaining half are not.
The second point of difference would seem to be a more serious one.
Montgomery states that during the spermatogonial mitosis the “chromosome
x” regularly divides as do all the other chromosomes, _i. e._,
longitudinally. In the spermatocyte mitosis, however, the element is
broken across, and the longitudinal split, which is apparent in the
early stages, disappears and is not utilized in division. We have here
the remarkable occurrence of a chromosome entirely unchanged in its
structure, but merely differing in its surroundings, which, instead of
dividing along the plane marked out for it, as it has done in all
preceding mitoses, breaks across after it is a formed element. An
occurrence of this kind, so different from the usual method of division,
would require strong proof to establish it, and this, in my opinion,
Montgomery has not brought forward.
A criticism of the degeneration theory as advocated by Paulmier and
Montgomery has already been given (=17=), so that it would not be
necessary to consider it here except in so far as it has been modified
since its promulgation. As a rule, Montgomery refers to his “chromatin
nucleoli” throughout his late paper (=15=) as degenerating chromosomes,
but in discussing their function specifically he makes important changes
in this conception. These are stated as follows: “When we find,
accordingly, the mutual apposition of them (true nucleoli) to chromatin
nucleoli, it would be permissible to conclude that the chromatin
nucleoli are chromosomes which are especially concerned with nucleolar
metabolism. And this, I think, would be the correct interpretation. The
chromatin nucleoli are in that sense degenerate that they no longer
behave like the other chromosomes in the rest stages, but they would be
specialized for a metabolic function; and from this point of view they
would certainly seem to be much more than degenerate organs.”
It is difficult to comment upon a contradictory statement like this;
but, fortunately, it is not necessary to do so, since it carries with it
its own refutation. The conception of a chromosome specialized in the
direction of increased metabolic activity as being in the process of
disappearing from the species can hardly be regarded seriously.
Taking everything into consideration, it may be said that Montgomery’s
work upon the Hemiptera has left the subject in a very disturbed
condition, and any prospect of a complete agreement between the
accessory chromosome of the Orthoptera and the “chromatin nucleolus” of
the Hemiptera is made more remote than was previously the case. This, I
think, is largely due to the inferior character of the Hemipteran
material, which has lead to misconception of phenomena that are clearly
marked in Orthopteran cells.
It is gratifying to note that the recent work of de Sinéty (=37=)
practically corroborates the conclusions herein set forth regarding the
history of the accessory chromosome. Aside from failure to observe the
important spireme condition of this element in the first spermatocyte
prophase, de Sinéty describes the same series of processes with scarcely
an exception. His summary contains the following account of the
accessory chromosome:
“Le ‘chromosome accessoire,’ découvert par McClung chez _Xiphidium
fasciatum_, se retrouve chez les locustiens que nous avons étudiés.
Chez _Orphania_, il se divise dans les spermatogonies en deux masses
volumineuses et allongés, que l’on reconnait dans les nucléoles,
également volumineux et allongés, des spermatocytes de premier ordre
en prophase. A la métaphase de la première cinèse, on le trouve situé
excentriquement et plus près de l’un des pôles; _il va tout entier a
l’une des cellules-filles_. Dans celle-ci, il se divise comme un
chromosome ordinaire, d’où il suit que _sur quatre spermatides formant
la descendance d’un spermatocyte, deux se trouvent privilegiees_. Par
ce partage inégal, non réalise dans _Xiphidium fasciatum_, d’après
McClung, le chromosome spécial d’Orphania rappelle celui des
hémiptères.”
A like series of processes is recognized in the Phasmids.
As is elsewhere explained in this paper, the occurrence of two divisions
of the accessory chromosome in _Xiphidium_, which was mentioned as a
possible occurrence in my preliminary paper, is shown not to take place.
While it is much more difficult to demonstrate the undivided condition
of the accessory chromosome in one of the spermatocyte mitoses of
_Xiphidium_ than it is in the cells of _Orchesticus_, _Anabrus_,
_Scudderia_, and _Microcentrum_, I am convinced that it does not differ
from the other Locustids in this respect.
We may therefore feel assured that our knowledge of the morphological
character of the accessory chromosome in the Orthoptera is fairly well
established. This gives us a good base from which to conduct further
comparative studies into other groups, and it is to be hoped that our
knowledge of this element will rapidly increase.
Unfortunately, de Sinéty has chosen to add another name to the already
overburdened list of synonyms, and “chromosome spécial” now takes its
place in the literature of insect spermatogenesis. The reason for adding
this name—
“Il reçu successivement leg noms de ‘accessory chromosome’ (McClung),
‘small chromosome’ (Paulmier), ‘chromatin nucleolus’ (?), ‘chromosome
_x_’ (Montgomery). Nous avons préféré éviter ces appellations, qui
semblent toutes supposer une signification qui n’a jamais été définie
ou s’appuyer sur des caractères plus ou moins secondaires, pour
adopter un nom indifférent, celui de ‘chromosome spécial,’ nous
conformant à l’idée de Wilson, pour qui c’est un ‘extra chromosome,’”
would seem to be at least insufficient, since “accessory chromosome” can
scarcely be regarded as implying any more primary or secondary function
than can “chromosome spécial.”
(_f_) _Individuality of the Chromosomes._
In each of my preceding papers I took the opportunity to point out the
fact that, even were the accessory chromosome of no other value, it
would certainly be worthy of study for the light it throws upon the
question of the individuality of the chromosomes. On this point
Montgomery has much to say in his late paper (=15=). I think it cannot
be questioned that we have here indisputable proof that at least one
chromosome may be identified through all the cell generations of the
testis. While this does not prove that chromosomes are persisting and
independent structures, it does evidence the fact that they may be, and
greatly strengthens the hypothesis that they are.
In addition to the evidence here offered by the accessory chromosome,
there must be noted that derived from a study of spermatocytes in which
there is always present one ordinary chromosome that greatly exceeds the
others in size. Such a condition is found in the cells of _Anabrus_. The
disproportion in size of the elements is here so striking that it would
be impossible to fail in distinguishing the giant chromosome. In each of
the spermatocytes of _Anabrus_ there are therefore two chromosomes which
are plainly recognizable. It may be observed further that the remaining
chromosomes are quite different in size, and it may be possible within
reasonable limits of certainty to pick out one or more other chromosomes
in each cell. Unless this could be done for each element, however, it
would not definitely prove that all the chromosomes are distinct and
recognizable structures. The actual recognition of two elements in each
cell of the same generation and its ancestors or descendants in other
generations goes far, however, to render probable the individuality of
each chromosome.
Beyond this point studies upon the Orthopteran cells will not permit me
to go; but Montgomery has been fortunate enough to find in _Peripatus_
an object in which he considers it possible to demonstrate the
continuity of the chromosomes from one generation to another, and their
fusion by pairs in the early history of the spermatocyte to bring about
the reduced number. This is, in the main, a logical conclusion to my own
work, and I am therefore bound to regard his results as probably
correct. While doing this, however, I recognize that the absolute proof
he brings forward in support of his hypothesis is very slight. I
consider any deductions based upon observations of linin structures as
very insecure, and it is upon these that Montgomery principally relies
to demonstrate his theory. Further observations upon the behavior of the
chromosomes between the spermatogonia and the spermatocytes in objects
favorable for study will be awaited with interest. In the meantime it
must be conceded that the work upon insect spermatogenesis has at least
lent strong support to the theory of the individuality of the
chromosomes in general and has definitely shown that there is such a
thing in some instances.
(_g_) _Nucleoli._
Considerable importance is attached by some investigators to the nuclear
structures, properly called plasmasomes, that occur in the
spermatocytes. It is probable that there are marked differences between
the cells of various species in regard to the occurrence of these
bodies, for in the Orthoptera they either do not appear at all, or, if
present, they are minute and inconspicuous. This fact would tend to
disprove any theory which would attach a fundamental importance to these
structures, such as is conceived for the chromatin. The Orthopteran
cells do not allow any observations which would add to our positive
knowledge of the nucleoli, and I include this brief statement merely for
the negative value it may possess.
(_h_) _Rest Stage._
In his first paper upon _Euchistus_, Montgomery assigns an important and
conspicuous place to the “rest stage” among his numerous subphases
preceding the first spermatocyte mitosis. As a result of his later
comparative work upon the Hemiptera, however, we learn that in certain
families no trace of such a condition of diffusion on the part of the
chromatin is observable, from which we conclude that “accordingly such a
stage would appear to have no broad significance.” It has already been
announced that nothing like a rest stage intervenes between the
spermatogonia and spermatocytes of the Orthoptera, and the work of most
investigators would tend to indicate that it is the exception rather
than the rule. In those cases where such a condition of the nucleus
exists, it would seem to be true that nothing more unusual than an
excessive diffusion of the spermatogonial chromosomes occurs, and this
is of hardly sufficient importance to receive a special designation.
The existence of a rest stage between the first and second spermatocytes
is also negatived by the conditions found in the Orthopteran cells. The
formation of chromosomes in the prophase of the first spermatocyte that
are already prepared for two divisions would _a priori_ render
improbable the intervention of a rest stage here; and the actual
observed persistence of the chromosomes, as such, through the telophase
of the first spermatocyte and through the modified prophase of the
second spermatocyte gives actual proof in support of the view that
commonly prevails regarding the suppression of the second spermatocyte
rest stage.
Observations upon numerous species tend to show that the behavior of the
chromatin during the period between the two spermatocyte mitoses varies
considerably with the species and even within the species itself. The
amount of diffusion would, in some measure, seem to be related to the
form of the chromosomes and to vary correspondingly in those individuals
where the chromosomes are of diverse forms. Thus, where the elements of
the second spermatocyte metaphase appear as short double rods, the
amount of diffusion is slight, and the individual chromosomes may be
distinguished throughout the telophase of the first spermatocyte; but in
those cases where the members of the mitotic figure are much elongated
the diffusion is more extensive and the distinction between elements is
made difficult or impossible. Since these two conditions may prevail in
the same testis, it is probably only a question as to the extent of
elongation on the part of each chromosome. In those cases where the
elements become very much extended the appearance of the resting
condition would be simulated closely, while, on the contrary,
chromosomes consisting of spherical or short cylindrical chromatids
would never give a suggestion of such a stage. In this we may find, I
think, an explanation for those cases in which a rest stage is described
as occurring between the spermatocyte generations.
VI. SUMMARY.
1. The secondary spermatogonia are much reduced in size at the end of
their divisions and the cytoplasm is very small in amount. The
rod-shaped chromosomes number thirty-three, and, of these, one is to be
distinguished from its fellows by greater size and slower division.
2. From the substance of the disintegrated spermatogonial chromosomes,
the tetrads of the first spermatocytes are formed. It was impossible to
determine the relation of the elements of the two generations, but the
changes are rapid and there is no intervening resting condition of the
nucleus.
3. It could not be determined whether or not the spireme is continuous.
A longitudinal split appears very early, and shortly after the chromatin
segments may be seen. These soon betray at their centers an indication
of the cross-division, producing crosses with arms that may vary
considerably in relative lengths. No reason was found for considering
both divisions longitudinal.
4. The typical element is granular and more or less rod-shaped, with the
longitudinal division merely indicated by a narrow line, and with but
slight elongation of the chromatids along the plane of the
cross-division. Various modifications of this occur, by which the
longitudinal cleft is much increased in width at the center, the
cross-arms are greatly extended, or approximation of the ends of the rod
brought about, producing a ring.
5. The definitive chromosomes of the metaphase are produced by a
concentration of the prophase elements, whereby they become shorter,
heavier, and entirely homogeneous in structure. Distinct lines of
division between the chromatids are not visible, but the tetrad
character of the elements is readily established by observing the steps
in their formation.
6. The accessory chromosome early becomes distinguishable because of its
peripheral position and strong tendency to stain with safranin, while
the remaining chromatin takes the gentian violet by Flemming’s
three-color method. At first it appears as a homogeneous plate, but
later this is seen to be a closely coiled thread. As the chromatin
segments shorten and broaden to form the chromosomes of the mitotic
figure, this thread also grows shorter and heavier until it forms an
element of essentially the same character as that of the spermatogonial
chromosome from which it was derived.
7. Upon the establishment of the mitotic figure, the chromosomes arrange
themselves in the equatorial plate with their longer axis perpendicular
to the spindle axis. Division of the elements is not synchronous, so
that all stages of the chromatid movements may be observed in one
nucleus. By this means it is possible to determine that separation of
the chromosomes takes place along the plane which marked the
longitudinal division of the prophase thread in such a way that the
chromatids show no clear interspaces. The individual chromosome near the
end of its division has the same form as that with which it started,
except for the difference that the chromatids are now in contact for the
greater part of their length along the plane of their cross-division. As
the daughter chromosomes separate, this line of division comes into
evidence through the springing apart of the two chromatids now composing
each chromosome. The result is the formation of two V-shaped chromosomes
with mantle fibers attached to their apices. The accessory chromosome
does not participate in this division, but passes unchanged to one pole
of the spindle.
8. By reason of the action of the accessory chromosome in the first
spermatocyte mitosis, there are produced two numerically equal classes
of second spermatocytes—(_a_) those containing sixteen dyad chromosomes
and an undivided accessory chromosome, and (_b_) those with merely the
sixteen dyad elements. In both cases the mitotic figure quickly reforms
without an intervening rest stage in which the chromosomes lose their
identity. There is a loosening up of the chromomeres in all the elements
except the accessory chromosome, so that they have a structure and
staining reaction similar to that of the first spermatocyte chromosomes
just before they enter the metaphase. The dyads of the first
spermatocyte telophase, and of the succeeding and greatly abbreviated
second spermatocyte prophase, are quite as definite structures as are
the chromosomes of the first spermatocyte prophase.
9. All the chromosomes of the second spermatocyte are paired structures
and divide in a similar way. The spindle is small and weak as compared
with that of the first spermatocyte, and the chromosomes arrange
themselves radially on its periphery in such a way that the pairs lie in
the plane of the spindle axis with their joined ends inward. The space
between the chromatids represents the line of cross-division observable
in the prophase segments of the first spermatocyte, and their separation
accordingly represents a reduction division. The accessory chromosome,
on the contrary, divides along the plane marking the longitudinal cleft
of the spermatogonial spireme.
10. From each first spermatocyte there are formed, by two divisions,
four spermatids, of which two are distinguished from the remaining pair
by the possession of an extra chromosome in addition to the
number—sixteen—common to them all. Both classes undergo a like series of
transformations by which they become mature spermatozoa. These are
necessarily of two kinds; and it is believed that those containing the
accessory chromosome, in the act of fertilizing the egg, determine that
the germ-cells of the embryo shall be sexually male, or like themselves,
while those from which it is absent are unable to impress their sex upon
the egg and assist in producing female embryos.
VII. BIBLIOGRAPHY.
=1=. Atkinson, Geo. F., 1899. Studies on Reduction in Plants, Bot. Gaz.,
vol. 28.
=2=. Baumgartner, W. J., 1902. The Spermatid Transformations of _Gryllus
assimilis_, etc., Kans. Univ. Sci. Bull., vol. 1, No. 1.
=3=. Brauer, A., 1893. Zur Kenntniss der Spermatogenese von _Ascaris
megalocephala_, Arch. mikr. Anat., vol 42.
=4=. Carnoy, 1895. La Cytodiérese chez les Arthropodes, La Cellule, vol.
1.
=5=. Cholodkovsky, N., 1894. Zur Frage über die Anfangsstadien der
Spermatogenese bei den Insecten, Zool. Anz., vol. 17.
=6=. Griffin, B. B., 1899. Studies on Maturation, Fertilization and
Cleavage of _Thalassema_ and _Zirphæa_, Jr. Morph., vol. 15.
=7=. Häcker, Valetin, 1897. Ueber weitere Uebereinstimmungen zwischen
den Fortpflianzungsvorgängen der Tiere und Pflanzen, Biol. Cent., vol.
17.
=8=. Henking, H., 1890. Untersuchungen über die ersten
Entwicklungsvorgänge in den Eiern der Insecten. 2. Ueber Spermatogenese
und deren Beziehung zur Eientwicklung bei _Pyrrhocoris apterus_ M., Z.
wiss. Zool., vol. 51.
=9=. Henking, H., 1892, idem. 3. Specielles und Allgemeines, _ibid._,
vol. 54.
=10=. Ischikawa, 1891. Studies of Reproductive Elements.
Spermatogenesis, Ovogenesis and Fertilization in _Diaptomus_ sp., J.
Coll. Sc. Imp. Univ. Japan, vol. 5.
=11=. Kingsbury, B. F., 1902. The Spermatogenesis of _Desmognathus
fusca_, Am. Jour. Anat., vol. 1, No. 2.
=12=. Montgomery, T. H., jr., 1898. The Spermatogenesis in _Pentatoma_
up to the Formation of the Spermatid, Zool. Jahrb., vol. 12.
=13=. Montgomery, T. H., jr., 1898. Chromatin Reduction in the
Hemiptera: a Correction, Zool. Anz., vol. 22.
=13=_a_. Montgomery, T. H., jr., 1898. Observations on Various Nucleolar
Structures of the Cell, Biol. Lect. of the Woods Holl Laboratory, 1898.
=14=. Montgomery, T. H., jr., 1900. The Spermatogenesis of _Peripatus
balfouri_ up to the Formation of the Spermatid, Zool. Jahrb., vol. 14.
=15=. Montgomery, T. H., jr., 1901. A Study of the Germ Cells of
Metazoa, Trans. Amer. Phil. Soc., vol. 20.
=16=. McClung, C. E., 1899. A Peculiar Nuclear Element in the Male
Reproductive Cells of Insects, Zool. Bull., vol. 2.
=17=. McClung, C. E., 1900. The Spermatocyte Divisions of the Acrididæ,
Kans. Univ. Quart., vol. 9, No. 1.
=18=. McClung, C. E., 1901. Notes on the Accessory Chromosome, Anat.
Anz., Bd. 20, Nos. 8, 9.
=19=. McClung, C. E., 1902. The Accessory Chromosome—Sex Determinant?
Biol. Bull., vol. 3, Nos. 1, 2.
=20=. Moore, J. E. S., 1895. On the Structural Changes in the
Reproductive Cells during the Spermatogenesis of Elasmobranchs, Quart.
J. Micr. Sc., vol. 38.
=21=. Paulmier, F. C., 1898. Chromatin Reduction in the Hemiptera, Anat.
Anz., vol. 14.
=22=. Paulmier, F. C., 1899. The Spermatogenesis of _Anasa tristis_, Jr.
Morph., vol. 15.
=23=. Platner, G., 1886. Die Karyokinese bei den Lepidopteran als
Grundlage für eine Theorie der Zelltheilung, Internat. Monatsschr. Anat.
Histol., vol. 3.
=24=. Rath, O. vom, 1891. Ueber die Reduction der Chromatischen Elemente
in der Samenbildung von _Gryllotalpa_, Ber. Naturf. Ges. Freiburg, vol.
6.
=25=. Rath, O. vom, 1892. Zur Kenntniss der Spermatogenese von
_Gryllotalpa vulgaris_ Latr., Arch. Mikr. Anat., vol. 40.
=26=. Rath, O. vom, 1895. Neue Beiträge zur Frage der Chromatinreduction
in der Samen und Eireife, _ibid._, vol. 4.
=27=. Sabatier, A., 1890. De la Spermatogénese chez les Locustides,
Comptes Rend., vol. 111.
=28=. Sutton, W. S., 1900. The Spermatogonial Divisions of _Brachystola
magna_, Kans. Univ. Quart., vol. 9, No. 2.
=29=. Toyama, K., 1894. On the Spermatogenesis of the Silkworm, Bull.
Coll. Agric. Imp. Univ. Japan, vol. 2.
=30=. V. la Valette St. George, 1897. Zur Samen-und Eibildung beim
Seidenspinner (_Bombyx mori_), Arch. mikr. Anat., vol. 50.
=31=. Wagner, J., 1896. Beiträge zur Kenntniss der Spermatogenese bei
den Spinnen, Arb. Nat. Ges. St. Petersburg, vol. 26.
=32=. Wilcox, E. V., 1895. Spermatogenesis of _Caloptenus femur-rubrum_
and _Cicada tibicen_, Bull. Mus. Comp. Zool. Harvard Univ., vol. 27.
=33=. Wilcox, E. V., 1896. Further Studies on the Spermatogenesis of
_Caloptenus femur-rubrum_, _ibid._, vol. 29.
=34=. Wilcox, E. V., 1897. Chromatic Tetrads, Anat. Anz., vol. 14.
=35=. Wilcox, E. V., 1901. Longitudinal and Transverse Divisions of
Chromosomes, Anat. Anz., Bd. 19, No. 13.
=36=. Wilson, E. B., 1900. The Cell in Development and Inheritance. New
York; 2d ed.
=37=. de Sinéty, R., 1901. Recherches sur la Biologie et l’Anatomie des
Phasmes, La Cellule, Tome 19, 1er fascicule.
DESCRIPTION OF FIGURES.
Drawings were made with a _camera lucida_, the optical combination being
a 1–16 B. & L. objective and a Watson “Holoscopic” ocular No. 7. Details
were studied with a Zeiss 2-mm. apochromat, N. A. 1.30. As reduced in
reproduction, an enlargement of 1500 diameters exists. Photomicrographs,
excepting those of figures 37 and 38, were made by the use of the arc
light and horizontal camera. The exceptions represent illumination by
ordinary diffuse daylight. In all cases the lenses used were the Zeiss 2
mm., N. A. 1.30 objective and projection oculars. A Watson
“Parachromatic” oil-immersion condenser of 1.30 N. A. was employed to
illuminate the objects. In use it was stopped down to between .75 N. A.
and 1.0 N. A.
Explanation of Plate VII.
FIG. 1. Pole view of spermatogonial metaphase, showing the thirty-three
chromosomes. It will be observed that the chromosomes are of unequal
sizes, and that the large ones arrange themselves in a circle on the
outside of the figure.
FIG. 2. Very young spermatocyte. The chromatin derived from the breaking
down of the spermatogonial chromosomes in a diffuse condition, with no
trace of a linear arrangement. The accessory chromosome _x_ on the
periphery of the nucleus, darkly staining and homogeneous.
FIG. 3. Early stage in the formation of the spireme. In the cytoplasm
the remains of the spermatogonial spindle. The cell has entered upon the
growth period.
FIG. 4. A later stage in the spireme formation. The accessory chromosome
larger and more flattened. A surface view shows it as an apparently
fenestrated plate. The remains of the two spermatogonial spindles still
persisting.
FIG. 5. First appearance of definite chromosomes. One shown entire with
longitudinal and cross-divisions marked. The accessory chromosome is
here seen to be in a spireme condition.
FIG. 6. Condition of the chromosomes after further contraction of the
early segments. As here shown, they are more granular than is usually
the case.
FIG. 7. Common types of the prophase chromosomes.
FIG. 8. A cell in which one of the chromosomes has its halves widely
separated along the longitudinal division, forming Paulmier’s double-V
figure.
FIG. 9. In this cell may be seen the variation in form and size of the
early spermatocyte chromosomes.
FIG. 10. Two cells of the late prophase, with the chromosomes at almost
the extreme degree of concentration.
FIG. 11. Chromosomes of cells in the stage shown in figure 10. These
represent the different types of rings, crosses, etc., commonly observed
in first spermatocytes just before the formation of the mitotic figure.
FIG. 12. Different forms assumed by the accessory chromosome in the
prophase of the first spermatocytes of _Xiphidium_.
FIG. 13. Metaphase of the first spermatocyte. The accessory chromosome
is seen at one pole of the spindle, to which it has moved before the
separation of the chromatids of the remaining chromosomes.
FIG. 14. Another cell in about the same stage as that represented in the
preceding figure.
FIG. 15. A first spermatocyte metaphase in which the accessory
chromosome has not as yet moved to the pole of the spindle. This is
uncommon in _Orchesticus_, but frequent in _Anabrus_.
FIG. 16. Pole view of a first spermatocyte metaphase, showing seventeen
chromosomes. The variation in size of the elements, so marked in the
spermatogonia, is even more pronounced here. This is a cell similar to
that of figure 15, in which the accessory chromosome lies in the
equatorial plate.
[Illustration: Spermatocytes]
Explanation of Plate VIII.
FIG. 17. Two cells in metaphase—a pole view of one and an oblique view
of the other. The accessory chromosome does not show in the former, the
cell being such a one as is represented in figures 14 and 15.
FIG. 18. Pole view of another cell, showing but sixteen chromosomes.
FIG. 19. Early anaphase of the first spermatocyte, with the accessory
chromosome already at one pole.
FIG. 20. Mid-anaphase, with the giant chromosome still undivided.
FIG. 21. Later anaphase, in which the accessory chromosome is seen at
the lower pole. This figure shows, also, the character and extent of the
intercellular material.
FIG. 22. Later anaphase. The accessory chromosome at the upper pole. An
undivided chromosome lying between the groups of daughter chromosomes.
FIG. 23. About the stage of figure 22, but the lagging chromosome has
divided.
FIG. 24. Very late anaphase. Here, again, the lagging chromosome is
divided.
FIG. 25. Pole view of first spermatocyte telophase, showing the
accessory chromosome at one side of the daughter chromosomes.
FIG. 26. Pole view of a cell in the same stage as that represented in
figure 25. Here, however, the accessory chromosome is not present.
FIG. 27. Lateral view of telophase, with the accessory chromosome in the
lower daughter-cell.
FIG. 28. Fragment of second spermatocyte, showing the chromosomes in
metaphase. The relative sizes of the accessory chromosome and the
remaining chromosomes is well shown.
FIG. 29. Metaphase of a second spermatocyte, in which the accessory
chromosome is not present.
FIG. 30. Anaphase of second spermatocyte, in which there is no accessory
chromosome.
FIG. 31. Anaphase of second spermatocyte, where the accessory chromosome
is present—_x_^1 and _x_^2.
FIG. 32. Telophase of the same class of second spermatocytes. The
accessory chromosome extends out from the mass of chromosomes at each
pole—_x_^1 and _x_^2.
FIG. 33. Telophase of the class of second spermatocytes from which the
accessory chromosome is absent.
[Illustration: Spermatocytes]
Explanation of Plate IX.
FIG. 34. Photomicrograph of early spireme stage of first spermatocyte,
showing peripheral position of the accessory chromosome _x_. At the
left, secondary spermatogonia, last generation. × 1300.
FIG. 35. A late prophase, showing accessory chromosome _x_, and spindle
remains _s_ (_cf._ figs. 3 and 4). × 1300.
FIG. 36. Coarse spireme of first spermatocyte. × 1300.
FIG. 37. Prophase, with chromosomes in the form of long segments. At
_a_, the cell drawn in figure 9. In the cyst at the left are
spermatocytes in a later stage, with the chromosomes homogeneous. ×
1000.
FIG. 38. Prophase with segments divided longitudinally and across. At
_a_ is one shown _en face_. Accessory chromosome at x. × 1000.
FIG. 39. Metaphase and anaphase of first spermatocyte. The accessory
chromosome _x_ at one pole of the spindle. Lagging chromosome at _c_. ×
1300.
[Illustration: Spermatocytes]
Explanation of Plate X.
FIG. 40. Anaphase of first spermatocyte. Accessory chromosome _x_ at one
pole. The form of chromosome in the anaphase well shown. The lagging
chromosome _c_ seen in two cells. × 1300.
FIG. 41. Anaphase of the first spermatocyte, showing the longitudinally
divided condition of the accessory chromosome _x_ in the cell near the
center. Compare with the accessory chromosome in the metaphase of second
spermatocyte, figure 43. × 1300.
FIG. 42. Second spermatocyte in metaphase. In most of the cells the
focus is upon the ends of the chromosomes, but in one a side view is
obtainable. Compare with the chromosome of the upper cell in figure 40.
No accessory chromosome in most of the cells in focus. × 1300.
FIG. 43. Second spermatocyte metaphase and spermatids. Note the relative
sizes of the accessory chromosome and the other chromosomes. In the
spermatids the accessory chromosome has taken its place on the periphery
of the nucleus in the same way that it does in the prophase of the first
spermatocyte. × 1300.
FIG. 44. Anaphase of the second spermatocyte, showing the accessory
chromosome _x_ separated. Other cells in metaphase. × 1300.
FIG. 45. Telophase of the second spermatocyte. Two daughter-cells with
persisting spindle between, showing the accessory chromosome _x_ in
each. Other nuclei in focus show no accessory chromosomes. × 1300.
[Illustration: Spermatocytes]
------------------------------------------------------------------------
TRANSCRIBER’S NOTES
1. Silently corrected obvious typographical errors and variations in
spelling.
2. Retained archaic, non-standard, and uncertain spellings as printed.
3. Enclosed italics font in _underscores_.
4. Enclosed bold font in =equals=.
5. Denoted superscripts by a caret before a single superscript
character.
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