The Kansas University Science Bulletin, Vol. I, No. 2, February, 1902

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Title: The Kansas University Science Bulletin, Vol. I, No. 2, February, 1902
Author: Baumgartner, W. J.
Language: English
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THE KANSAS UNIVERSITY SCIENCE BULLETIN.

(VOL. I, NO. 2—FEBRUARY, 1902.
Whole Series, Vol. XI, No. 2.)

CONTENTS:

SPERMATID TRANSFORMATIONS IN GRYLLUS ASSIMILIS,
WITH SPECIAL REFERENCE TO THE NEBENKERN.
—_W. J. Baumgartner._

PUBLISHED BY THE UNIVERSITY,

LAWRENCE, KAN.

Price of this number, 30 cents.

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

KANSAS UNIVERSITY SCIENCE BULLETIN.

VOL. I, NO. 2. FEBRUARY, 1902. {WHOLE SERIES,
{VOL. XI, NO. 2.

SPERMATID TRANSFORMATIONS

In Gryllus assimilis, with Special Reference to the Nebenkern.

(Thesis for the degree of Master of Arts.)

BY W. J. BAUMGARTNER.

With Plates II and III.

I.—METHODS.
II.—MATERIAL.
III.—OBSERVATIONS.
(_a_) NUCLEUS.
(_b_) Cytoplasmic Structures.
1. Nebenkern.
2. Acrosome.
3. Axial Filament.
4. Centrosome.
5. Cell Body.
(_c_) The Spermatozoon.
IV.—COMPARISON OF RESULTS.
V.—SUMMARY.

It is the object of these studies to follow carefully the spermatid
transformations, and, if possible, to get a complete series of
changes occurring therein. I do not pretend that I have as yet found
all the stages, but in the present paper will publish observations
made on _Gryllus assimilis_ pertaining chiefly to the nebenkern, of
which I have found some stages not previously described, as far as I
know. I shall first describe my own findings, and then compare them
with the results of others.

I. Methods.

While other fixing agents were used, Flemming’s chromo-acetic-osmic
mixture (strong) proved the best. Its fixation was satisfactory in
all but a few stages, where the chemical changes going on in the cell
are such that it seems difficult, probably impossible, to secure
good, clear results; at least, neither Gilson’s nor Zenker’s mixture
did any better on cells in these stages.

Heidenhain’s iron-hæmatoxylin stain gave the best results, although
Flemming’s three-color method was very satisfactory.

Most of the observations are made from sectioned material, but smear
preparations were used for advanced spermatozoa.

II. Material.

The first observations were made in January of the present year on
some material prepared by Professor McClung, to whom I wish here
to make most grateful acknowledgment for proposing the line of
investigation, and for many helpful suggestions during the progress
of the same. The testes were those of adult insects, and showed
mostly only mature or almost mature spermatozoa. Enough spermatids
were seen to indicate that their transformation into spermatozoa was
somewhat peculiar.

The material for the drawings was taken from specimens collected
under stones and boards on and near the University campus, at
Lawrence, Kan. Young nymphs of _Gryllus_ were caught as early as
March 1, but the cells of the testes were all in the spermatogonia
and spermatocyte stages. During the early part of May, after the
nymphs had passed their third molt, crickets were secured whose
testes showed all stages of development and transformation.

_Gryllus assimilis_, the common black field cricket, has paired
testes lying in the anterior dorsal part of the abdomen. These have
a whitish transparent appearance, which becomes duller in the adult,
showing sometimes a slight yellowish tinge. The shape of each is
that of a somewhat conically rounded body, not unlike a flattened
strawberry. Each testis consists of a central rachis about 3 mm.
long, from which extend a large number of curved follicles varying in
length from 1 to 3 mm.

The follicles are larger toward the blind end and taper toward
the rachis. Each follicle is divided into cysts, but more often
transversely than longitudinally, for frequently one cyst occupies
the whole follicle in cross-section. The cysts toward the rachis end
of the follicle are much longer and narrower than those of the blind
end. The cells are not very large and contain twenty-four chromosomes
in the spermatogonial generations; eleven and twelve were most
frequently found after the reduction.

The follicles of the cricket testis show the different cell
generations and the same relative arrangement of them as McClung
(’00) found in _Hippiscus_, and Sutton (’00) in _Brachystola_.
The spermatogonia are nearest the blind end of the follicles,
spermatocytes next, and the spermatids following the latter. The
cells of a cyst are not, as in _Anasa_ (Paulmier, ’99), “in the same
stage of development,” but only approximately so, for some cysts show
cells in the metaphase, while others have reached the telophase.
Successive cysts, as in _Brachystola_, do not show successive stages;
for, frequently, growing spermatocytes and late spermatids, or even
young spermatozoa, were observed side by side. The individual cells
in division pass through prophases, metaphases, anaphases, and
telophases—these terms being used according to their usually accepted
meanings. (See McClung, ’00.) In this paper I shall begin with the
cell when it has reached the telophase of the second spermatocyte
division; that is, just after the chromosomes have reached the poles
of the spindle.

III. Observations.

(_a_) THE NUCLEUS.

The chromatin in _Gryllus_ behaves very much as Henking (’91) has
described for _Pyrrhocoris_, and Paulmier (’99) for _Anasa_. At the
end of the anaphase the chromosomes are crowded together at the
poles of the spindle. (Fig. 1.) A clear space begins to be formed
around them, especially on the equatorial side. (Fig. 2.) They then
separate and are scattered somewhat evenly over the nuclear membrane
when it is formed, as seen in figs. 4, 5, and 6. Soon protuberances
appear on the surface of the individual chromosomes, making their
outlines irregular. At about this time they begin to show a granular
structure. The chromosomes break up more and more, and soon the
chromatin appears in irregularly arranged patches or blotches of
granules of various sizes. (Fig. 8.)

Meanwhile the nucleus has slowly increased in size. It reaches its
maximum, which is probably about treble its original diameter,
before the cell begins to elongate. The granules have been breaking
up into finer and finer pieces, until, a little after the cell has
reached its maximum, the chromatin has largely disappeared, or, to
be more exact, has lost its affinity for stains. (Fig. 16.) As the
cell begins to elongate, the nucleus goes to one end, namely, that
toward the periphery of the cyst. (Figs. 14 and 15.) The chromatin
again frequently appears in patches as the cell decreases in size.
(Figs. 33 and 36.) In many cells the patches never disappear. When it
has diminished to less than half its maximum diameter, the nucleus
begins to elongate, becoming elliptical, and finally forms the long,
tube-like head of the spermatozoon. (Figs. 43 and 45.)

I found here that the nucleus was hollow, or, better, that it
showed a clear space within. For a long time it contains the many
chromatin granules, as seen in figs. 39, 40, and 41; but at last the
walls seem to attract all these granular masses, and the center is
entirely clear, as in fig. 45. In fig. 44 are shown cross-sections
of spermatozoa heads, of the stage of fig. 43. In fig. 45 we have a
mature spermatozoon head.

The _accessory chromosome_ in _Gryllus_ lags behind during the
spermatogonial divisions, just as in _Xiphidium_ (McClung, ’99),
and retains its identity all through the growth period, in many
cases becoming quite large. At first it cannot be distinguished
in the spermatid, but soon its stronger stain and exemption from
disintegration make it apparent. (Fig. 4.) It is flattened against
the nuclear wall and, unlike Paulmier’s “small chromosome,” it
does not break up but remains intact, as Henking and McClung have
described. (Figs. 14, 18, 32, 37, and others.) It gets larger and
then smaller, and is finally lost in the rest of the chromatin, as
the latter condenses to form the spermatozoon head. I was not able
to see that it occupied one side of the elongating head, as McClung
describes it in _Xiphidium_. The darker stain and regular contour,
and sometimes its greater size, made it in most stages quite evident.

(_b_) CYTOPLASMIC STRUCTURES.

It is in the cytoplasmic structures that _Gryllus_ shows some things
that have not been described, so far as I know; yet I almost hesitate
to enter the discussion concerning the nebenkern, the mitosome, the
idiozome, the archoplasm, the attraction sphere, the acrosome, the
“mitochondrion körper” and other bodies which have been described in
the extra-nuclear parts of the germ-cell. But because I have been
unable to find in any of the papers a description of a structure
like the one in _Gryllus_, I shall proceed to describe the nebenkern
as I find it. I shall interpret the appearances as far as possible,
leaving to others, or to later efforts, the complete harmonizing of
this element with the structures of other species.

Before I proceed, I would add my protest against the name
“nebenkern,” as voiced by Calkins (’95), Erlanger (’96), Meves (’00),
and others. It is inapt and not at all descriptive or distinctive.
But since we have the word, and investigators will use it to mean
something, I think it would be best to restrict the word, as Calkins
and Erlanger suggest, to the remaining spindle fibers and connective
fibers which go to each spermatid and which have an important part
in forming the tail membrane. I shall retain the name for a further
reason, which St. George (’97) gives for first using it, “weil
sie die Frage nach der Herkunft dieses Gebildes offen lässt.” A
comparison of the literature will convince any one that the origin of
this element is still a much-disputed question.

1.—_Nebenkern._

There are, in _Gryllus_ spermatids, two quite permanent and prominent
extra-nuclear bodies. The larger of these I shall call _nebenkern_,
because I believe it originates from the spindle remains, and goes to
form the tail covering. Its behavior is as follows: At the beginning
of the telophase the fibers which had connected the centrosomes with
the chromosomes bulge out like barrel staves, as Henking (’91) has
described. These break loose from the chromosomes and centrosomes
as the clear space begins to form around the chromatin mass (fig.
2); but they are not separated into two rings, as in _Anasa_
and _Pyrrhocoris_. As the dividing cell membrane is formed, the
middle portions of the fibers are drawn together and the so-called
“mid-body” is formed. Some of the fibers unite, producing thicker
ones, as seen in figs. 1, 2, and 3. In the last-named figure some
of the central fibers seem to be still united with those of the
companion spermatids.

As the daughter-cell shortens and the nucleus takes a more central
position these fibers unite still more, shorten, bend together (figs.
4, 5, and 6), and finally assume the shape shown in fig. 7. I shall
call it for the present the “striated condition,” or simply “striated
nebenkern.” The shape resembles that of an egg with rather sharply
drawn-out ends. The surface is occupied at intervals with deeply
staining lines which look much like hoops. In most cases the long
axis of the nebenkern is at right angles to the axis of the spindle
from which it was formed, while the striæ, or dark lines, are in
planes parallel to it, as is seen in figs. 7 and 8_a_. In a few cases
the lines are parallel to the long axis of the nebenkern, as shown in
fig. 8_b_. Fig. 9 exhibits a partial end view of the nebenkern, while
fig. 26 represents a polar view in all planes.

The fibers in figs. 4 and 5 are not all in the same plane, giving
frequently, with changing focus, the impression that the lines are
the edges of plates. Even in figs. 7 and 8, the appearances shown
made me hesitate for some time before I dared say that the lines
represent striæ on the surface and not plates extending through the
body. But the end views (figs. 9 and 26) and the two drawings in fig.
19 show clearly that we are dealing with encircling hoops and not
dividing plates. Just how or why the fibers bend and assume the shape
of figs. 7 and 8 I am not able to say, but I am sure it is not an
artefact, as might be supposed, because I have found them occupying
whole cysts of several testes, and nearly all of them were as regular
and definite as fig. 7.[A]

I have found the nebenkern in its perfect condition (fig. 7) only in
material fixed in Flemming; although Zenker’s fixation showed some of
the breaking-up stages. This may be due partly to poorer fixation in
other mixtures and partly to chance, as not so many testes fixed in
other agents were examined, and the appearance evidently represents
a very brief stage. These fibers now begin to break up, and we get
figures that remind one of Meves’s “mitochondrion körper”; that is, a
darker staining center and radiating lines to the outer ring. (Fig.
11.) Sometimes stages a little later recall Paulmier’s “blackberry
stage.” (Figs. 12 and 25.) Fig. 19_a_ is a surface view and fig.
19_b_ an optical section of a nebenkern that is in the process of
breaking up. Figs. 20 and 21 have a part of one end cut off, while
fig. 23 is a polar view. Fig. 22 is probably a forming blackberry
stage.

All the above are brief stages, and we soon get the appearance of
figs. 13-16. Here we have the darkly staining central ball surrounded
by a clear space, enclosed by a ring, as Meves (’00) found it in
_Pygæra_. The central ball sometimes shows vacuoles (fig. 16),
but most frequently stains as one mass. In some cysts with cells
resembling fig. 16, it is difficult to distinguish between nucleus
and nebenkern, yet the latter’s stain is never as intense as that of
chromatin. The stages shown in figs. 13 and 14 persist for a long
time, and it is probable that this is the end of the telophase and
the beginning of the spermatid transformations. I have treated the
above more in detail and shown more drawings than its importance may
deserve, but it is in these stages that _Gryllus_ seems to differ
from other species, and I have tried to describe and illustrate the
appearances and changes fully.

As the cell elongates, the axial filament grows out, the nebenkern
approaches it, and a junction is effected in such a way that the
axial filament runs over the surface of the nebenkern. (Figs. 18,
27, 28, and 30.) At first the nebenkern is still almost round, but
it begins to elongate and the dark inner ball sends out a protrusion
to the nucleus, and sometimes back to the point of separation
between the external envelope and the axial filament. The outside
ring disappears and the dark mass moves down the axial filament.
(Fig. 31.) In many cases it breaks up into several small drops,
which appear at intervals on the axial filaments. (Figs. 34 and 36.)
In fig. 38 is seen such a hanging drop. In this way the nebenkern
material is distributed over the axial filament and forms a sheath
around it.

[A] Professor McClung, Mr. Sutton, and Mr. Blackman, of Kansas
University, and Doctor Child and Mr. Harper, of Chicago University,
have seen my preparations, and all confirm my statement that the
appearance is _not_ an artefact.

2.—_The Acrosome._

The second extra-nuclear body I shall call the acrosome, as it
forms the point of the spermatozoon head. I could not trace its
origin, because the fixation was not definite in some of the early
stages. Fig. 4 shows a small, darkly staining body, which may be a
centrosome, but more likely is the beginning of the acrosome, of
which figs. 7, 9 and 10 show developing stages, and figs. 11, 13 and
15 more advanced ones. Sometimes the central portion stains darkest
(figs. 11, 32, and 36), but more frequently there is a small clearer
space in the center surrounded by a ring or band of darker material.
(Figs. 10, 13, 27, and 30.) This ring usually stains darker on one
side, very often on the side toward the nucleus.

The position, which is, with few exceptions, in the angle between the
nucleus and the nebenkern, induces me to consider the differently
staining bodies as the same organ of the cell. For some time after
the nebenkern has disappeared the acrosome keeps its position;
then it approaches the nuclear wall, flattens against it, and
later wanders to the apical end, where it forms the tip of the
spermatozoon. (See figs. 32-36 and 39-42.) Fig. 16 shows the acrosome
back of the nebenkern. It is not an isolated example, still, I think,
it is an abnormality. Fig. 37 shows an apparent division of the
acrosome. I did not see enough instances to consider it a regular
occurrence.

3.—_The Axial Filament._

As the cell is elongating, the axial filament is seen apparently
growing out of the nucleus. I do not mean to say that it grows out
of the nuclear substance, but in almost every cell observed, where
the axial filament was incomplete, there was rather a large mass of
chromatin gathered at the place where the axial filament was attached
to the nucleus. It may be that the smaller one of the extra-nuclear
bodies in figs. 4, 7 and 9 is a centrosome which is passing to the
equatorial region of the nucleus, where it later develops the axial
filament. As already noted, the axial filament does not pass through
the nebenkern, but only over its surface. Fig. 29, _a_, _b_, and _c_,
shows different cross-sections through the elongating cell. In _b_
the axial filament was cut at an angle.

4.—_The Centrosome._

I have not followed the centrosome through its migrations. In fig.
2 one of the two bodies is the centrosome, but I did not trace its
changes farther.

5.—_Cell Body._

The cells, up to the stage of figs. 13 or 14, have been scattered
promiscuously through the cyst; but as the cell begins to elongate,
the one end becomes the anterior-nuclear end, and it shifts to the
periphery of the cyst. The central part of the cyst now shows the
so-called central lumen. As the lengthening goes on the heads of
the forming spermatozoa are all turned toward the distal end of the
cyst. The cyst becomes very narrow and long, apparently preparing
to contain the long, slender spermatozoa. Near the rachis the
spermatozoa seem much twisted, and I surmise that they turn so as to
have the head foremost when set free into the _vas deferens_. Fig. 42
may indicate some such behavior.

(_c_) THE SPERMATOZOON.

The mature spermatozoon consists: (1) of a sharp, spear-like
point; (2) a tube-like head about .02 mm. long, composed of a
densely staining outer wall and a clearer central cavity; and (3)
a filamentous tail about .5 mm. long. The point comes from the
acrosome, the head from the nucleus, and the tail from the axial
filament and nebenkern. While I do not deny the presence of a middle
piece, my observations do not warrant me in describing one, although
fig. 40 would suggest it.

IV. Comparison of Results.

Dr. R. von Erlanger (’97, 1, and ’97, 2) and Dr. F. Meves (’00) have
given excellent discussions of the literature on the nebenkern. I
cannot do better than direct the attention of other investigators
to them. Hence, I shall discuss only such authors and such points
as have special bearing on my own results. The spermatid changes in
the Gryllidæ have been studied, so far as I know, only by St. George
(’67) and by vom Rath (’92).

St. George, in his early paper, described the
transformation process in the “Hausgrille” along
with other insects. He found the “Nebenkörper,”
and saw that it took a part in forming the
envelope of the axial filament, a small particle
remaining near the nucleus and other drops of
it appearing at intervals on the tail. In his
paper on _Blatta_ (’86, 1) he accepted Bütchli’s
name, “nebenkern,” and traced its origin to the
spindle remains. In his paper on _Phratora_ (’86,
2) he takes up the description of the nebenkern
in the spermatocytes and spermatogonia. In the
spermatid, he describes the blackberry stage in
these words: “Während das andere (not nucleus)
Körperchen weniger lebhaft gefärbt als ein
kleiner Fadenknäuel mit unregelmässigem Contour
erscheint.”

There can be no true nebenkern in the spermatogonia and
spermatocytes, as St. George first used the word, and as I have
limited its application. But St. George found the object, traced its
origin and fate, and described several of the stages as they appear
in _Gryllus_.

Vom Rath (’92) found _Gryllotalpa_ very poor
material for following the spermatid changes.
However, he described an oval body which he found
varied in appearance, depending on the method of
fixation. He calls it the “nebenkern” and thinks
it goes to help form the tail.

The description is too incomplete for comparison. Judging from
_Gryllus_, I question its being very poor material.

Bütschli (’71) studied the spermatid
transformations in Acrididæ and Locustidæ along
with that of other animals. He saw the object
formerly described by St. George and named it
“nebenkern.” He saw it divide into halves,
elongate, and form the spermatozoon tail.

The dividing of the nebenkern into halves seems to be an appearance
quite common in insect spermatogenesis. I have myself seen it in
several genera of Acrididæ—_Hippiscus_, _Arphia_, _Melanoplus_, and
_Brachystola_; besides Bütschli, St. George, Henneguy, Platner,
Paulmier and others have described it.

Platner in his studies has given special attention
to the nebenkern. In his first paper on “Pulmonates”
(’85) he did not trace the origin of the nebenkern,
but described it as consisting of four to six rods
of different lengths and irregularly bent. These
were connected, forming an irregular polygon. In its
later stages he saw it with a mass of protoplasm pass
down along the primary tail—an early protrusion of
protoplasm. Finally it is lost.

In his succeeding paper (’86, 1) he studied the
“nebenkern” spindle remains—in the spermatogonia and
spermatocytes of pulmonates. In the spermatids, as in
the former generations of cells, the nebenkern grows out
of the nucleus, where it, with the chromatin, had formed
the spireme. It appears as a loop, which becomes larger,
twisted, and entangled, and finally breaks loose from
the nucleus. Later it goes to form the spiral covering
of the primary tail, changing it to the axial filament
and true tail.

In his next paper (’86, 2) he describes the changes
when the dividing of the protoplasm lags behind in the
spermatocyte divisions. His description agrees in so
many points with my own, that I shall quote his exact
words:

“Die Spindelfasern hingegen contrahiren sich mehr
und mehr nach dem Equator hin, wobei sie mit ein ander
verschmelzen und merkwürdiger Weise je weiter dieser
Verdichtungsprocess fort schreitet um so mehr an
Tinctions fähigkeit speciell gegenüber dem Hämatoxylin
gewinnen. Sie stellen jetzt zwei dreieckige oder
hakenförmige Gebilde dar, die mit der Spitze noch im
Equator zusammenhängen mit der breiten Seite sind sie
den zugehörigen Zellen zugewendet. Hier sind ihre
Grenzen undeutlicher, verwaschen und zeigen hier auch
noch häufig eine streifige Beschaffenheit, welche auf
ihren Ursprung hin weist. Zuweilen lassen sich einzelne
Fäden noch eine beträchtliche Strecke weit in das
Protoplasma hinein verfolgen, welches zwischen ihnen und
den sich ausbildenden Zellkernen liegt.”

The “häkenformige Gebilde” moves away from the
periphery, its sides elongate, break, and unite at
the nuclear end, thus form the polygonal nebenkern.
“Derselbe geht also in diesem Falle direckt aus den
Spindelfasern hervor.” When the protoplasm does divide a
similar process takes place. The spindle remains divide
at an equatorial line and each half forms a nebenkern,
and, as he says: “Also auch hier geht der Nebenkern
direckt aus den Spindelfasern hervor. Vielleicht geht
in den Spermatiden der Nebenkern überhaupt immer
aus den Spindelfasern hervor, in dem die langfädige
Verbindüngsbrücke, die ihn oft mit dem Kern verbindet,
sich wohl als ein noch einige zeit persisttirender Rest
der esteren deuten lässt.”

In his paper on _Limax_ (’89, 1) he followed the
nebenkern through all the divisions and thinks it a
constant organ of the cell. In the second part of the
paper on _Helix_ and _Paludina_ the nebenkern was
considered as formed from the remains of the spindlepole
and the centrosome. Later Platner (’89, 2) found the
nebenkern in the pancreas cells. In reports upon
_Pygæra_ and _Sphinx_ he changed the name nebenkern to
mitosome. The centrosome lies in front of the nucleus
and forms the point of the head. This he calls the
nebenkern. From the spindle remains arise two bodies. A
large, fibrous one from the equatorial end has a clear
space around it and the axial filament passes through
it. It soon elongates and forms the tail. This is the
large mitosome. The other is much smaller and arises
from the polar end of the spindle fibers. It takes its
place in the angle between the large mitosome and the
nucleus. Here it persists till the nucleus begins to
elongate, when it lengthens and surrounds the basal end
of the spermatozoon tail. This is the small mitosome.

Platner (’85) saw a true nebenkern. I have already criticized the
use of the word “nebenkern,” as the name for the spindle remains in
the spermatocytes. Platner himself later denied the nuclear origin
of the nebenkern. His results (’86, 2) agree with mine concerning
the uniting, converging, staining and bending of the spindle-fiber
remains and their passing to the tail. Again, Platner (’89, 1)
probably followed the centrosome and attraction sphere, as well
as the nebenkern, in the second part of the paper on _Helix_ and
_Paludina_. In the pancreas the body is a result of secretion, and
is not a nebenkern. In _Pygæra_ Platner’s large mitosome is the real
nebenkern, as shown by its fibrous structure, its surrounding clear
space and destiny. His small mitosome is what I have described as the
acrosome, and he is mistaken as to its final use. The body he called
centrosome in the nucleus is the persisting accessory chromosome.
Such would be my interpretation of his figures.

Henking (’91) has followed the spermatid changes
in _Pyrrhocoris_ quite carefully. He finds that the
fibers between the chromatin masses are separated into
peripheral fibers and central spindle fibers. The first,
a part of the second and the yolk mass, forms the
nebenkern, which passes down over the axial filament.
The rest of the central fibers form the mitosome. This
takes its position at the angle between the nebenkern
and the nucleus. On the surface of the nucleus it passes
to the anterior end, then back to its original position.
A piece now is constricted off and disappears, while the
rest, increasing its affinity for stains, again wanders
to the anterior pole of the nucleus, and becomes the
acrosome.

The large amount of yolk substance is a disturbing element in
_Pyrrhocoris_, but the nebenkern agrees with that of _Gryllus_ in
having the same origin and destiny, as does also the acrosome in fate
and position. Besides, there is much similarity in the stages, as
seen by comparing Henking’s fig. 63 with my fig. 25, and his fig. 85
with my fig. 32.

Wilcox (’95 and ’96) described the spermatid
metamorphosis in _Caloptenus_. The interzonal fibers, a
long, striated body composed very plainly of distinct
fibers, contracts longitudinally, and the corners round
themselves off, and this forms the nebenkern. It loses
its fibrous structure, comes close to the nucleus, and
then grows out into the axial filament. The centrosome
moves half way around the nucleus and lies between the
latter and the nebenkern. Later the centrosome becomes
the middle piece.

This description does not give much detail nor do the drawings show
the stages clearly. As far as given, the formation of the nebenkern
is the same as in _Gryllus_; but in _Gryllus_ the axial filament is
not formed from the nebenkern, as can be plainly seen from fig. 17.

Erlanger (’96), in a short paper, discusses the use
of the term “nebenkern,” and suggests limiting it as is
done in this paper. He opposes St. George’s opinion,
that the nebenkern comes from the cytomicrosomes. In
_Blatta_ the cytomicrosomes are preserved during the
whole process of mitosis, and have no connection with
the spindle fibers, but during the telophase they
collect in reduced numbers around the daughter nuclei.

In 1897 he called the collection of granules around
the centrosome, the centrodeutoplasm. He considers them
to be identical with St. George’s cytomicrosomes and the
archoplasm (or attraction sphere) of other writers. In
order to harmonize results, he suggests that, since the
centrosome sometimes wanders around the nucleus, the
centrodeutoplasm (or sphere) may unite with the spindle
remains in some cases to form one body, as shown by the
descriptions of Henking, Henneguy, Meves, and others.
He later (’97, 2) describes the so-called “sphere,” and
distinguishes between it and the true nebenkern.

I would strongly commend his excellent discussion of literature and
his careful comparison of the results of investigators. He has shown
clearly that the nebenkern comes from the spindle remains.

Calkins (’96) finds that the nebenkern comes from the spindle fibers
and is useless in the cell. But _Lumbricus_ is peculiar in having
the nebenkern simply disintegrate, for, in many cases, he admits
that the nebenkern has an important function. I do not have access
to Henneguy’s or Bolles-Lee’s or Toyama’s works, yet I should judge
from Erlanger’s and Meves’s criticism that all of these have the
nebenkern originate from the spindle remains, and Henneguy describes
it as having a “fibrillar appearance,” and Bolles-Lee as “fibrillar
structure.” Accordingly, I think that each of these has discovered
the correct origin of the structure, and I do not doubt that there
is, at least in the first two, a more or less direct change from the
spindle remains to the nebenkern.

Paulmier (’99) finds that in _Anasa_ the nebenkern
comes from the yolk mass and remains of the spindle
fibers. A part of this mass separates off, while the
whole is still in a confused condition, and forms the
acrosome. The nebenkern forms the tail sheath, while the
acrosome forms the point to the head.

A comparison of Paulmier’s fig. 42 with my fig. 4 suggests the
thought that they are the same stage, and his fibers are remains of
the spindle. His fig. 43, of course, agrees with my fig. 25; and fig.
43 may correspond to figs. 26 or 14, only that his stain is weaker.

Meves’s investigations are the most extensive of the recent ones on
the nebenkern. He has used _Paludina_ and _Pygæra_ as his objects.
The consideration of the mitochondrion in the spermatocytes I shall
pass over, as it is not within the bounds of this paper; yet I hope
to study the earlier generations of my material, and shall then
compare the results. In Meves’s description of _Paludina_, I find but
one point in which it agrees with _Gryllus_. The head of the young
spermatozoon in _Paludina_ has a clear space in the center filled
with nuclear fluid, which remains till the head begins to stretch. In
_Gryllus_ it is hollow until maturity.

In _Paludina_ the nebenkern in one kind of spermatids
is formed from threads made up of mitochondria—small,
round bodies identical with St. George’s cytomicrosomes
and Erlanger’s centrodeutoplasm. These threads change
to vesicles, which, reduced to four, closely surround
the centrosome as it lengthens into the middle piece.
At first they form a four-cleft cylinder, but finally
a single sheath. An idiozome and spindle remains are
seen in the spermatid. They persist for awhile, and
the former changes into the acrosome. In _Pygæra_
Meves finds two kinds of spermatids, distinguished
by a small difference in size. The larger forms the
typical spermatozoon. The spindle remains form a
“Spindelrest körper,” which is soon lost. The ends
of the mitochondrion chains form a ring of dark mass
surrounding a clear space. The ring is broken by
radiating clear spaces. These spaces collect and unite
into larger vacuoles, which surround the darker center.
Finally there is only a dark ball with a surrounding
clear space shut in by a ring. The centrosome with
attached axial filament fastens itself to the nucleus;
then both begin to grow longer. The axial filament
passes over the surface of the ring surrounding the
ball—the “mitochondrion körper” or nebenkern. This
body elongates a great deal then the darker mass puts
forth numerous threads which surround the axial filament.

As to _Pygæra_, the peculiarity is the complete agreement of his
“mitochondrion körper” and my nebenkern in appearance and behavior
for a part of the transformation and their complete disagreement in
the other part. The question with me is, “Do the two bodies whose
final stages are so similar originate so differently, or has one of
us mistaken the origin of the body?”

Since reading Meves’s paper I have carefully reexamined my material,
and I am positive that I am right as to the origin of the nebenkern;
but, on the other hand, I would not say that Meves is wrong in his
observations, as in doing so I should fall into the same error which
I think Meves himself has made. In his discussion of the literature
he has forced every description to agree with his ideas, or has
declared that the author has described some extra-nuclear organ as
a nebenkern which is _not_ a nebenkern. Thus by implication, if not
by direct statement, he says that a nebenkern never comes from the
spindle remains. With due respect to his ability and long experience
as an investigator, I must say that Meves is mistaken in this. St.
George himself traced the cytomicrosomes back to the spindle remains.
Besides, the many investigators whom I have cited above cannot be
mistaken as to the origin of the nebenkern. In my own material I am
positive that there is a direct passing of the interzonal fibers over
into the earlier stages of the nebenkern.

That the body which forms the tail covering does not come from the
spindle remains in all cases, I am willing to admit. Meves has cited
many investigators, especially on vertebrates, whose results favor
such an opinion. The spindle remains do not change into a nebenkern
even in all Arthropods, as Blackman (’01) finds no nebenkern, nor
anything in anywise resembling it, in _Scolopendra_.

From my study of the results of other investigators, it is evident
to me that there are at least two general methods for the formation
of the covering for the spermatozoon tail. One of these plans will
harmonize Meves’s mitochondrion körper, Erlanger’s centrodeutoplasm,
Heidenhain’s pseudo-chromosomes, and other similar structures. The
other will show that Platner’s large mitosome, Paulmier’s blackberry
stage and my striated nebenkern are only different stages of the
spindle remains changing into the tail covering.

V.—Summary.

1. The chromosomes of the second spermatocytes break up and the
chromatin becomes diffused all through the nucleus. Later the
chromatin collects in granules again and finally forms the walls of
the tube-like spermatozoon head.

2. The spindle fibers break loose as the clear space is formed around
the chromatin mass. They unite and contract, becoming fewer, thicker,
and shorter. These bend and form the “striated nebenkern.” The fibers
break up and sometimes show a blackberry appearance. Soon there is a
collection of darker material at the center, surrounded by a clear
space, which is shut off from the cytoplasm by a darkly staining
membrane which in sections appears as a ring. This stage persists
for some time; then the nebenkern moves against the axial filament,
elongates, loses the ring, and the dark mass passes down along the
axial filament. Often it appears in several small drops.

3. The axial filament does not come from the nebenkern nor from the
acrosome. It comes apparently from the nucleus; probably from the
centrosome closely attached to the nucleus. It never passes through
the nebenkern—only over its surface.

4. The acrosome occupies a position in the angle between the
nebenkern and the nucleus. It shows a central clear space surrounded
by a darker mass which stains more intensely on one side. Later
it passes to the front of the nucleus and forms the point of the
spermatozoon head.

5. The mature spermatozoon consists of a sharp point, a slender,
tube-like head filled with a clear fluid, and a long, thread-like
tail.

LABORATORY OF ZOOLOGY AND HISTOLOGY,
UNIVERSITY OF KANSAS.
September 27, 1901.

Bibliography.

1. Blackman, M. W., ’01: Spermatogenesis of the Myriapods. Kans.
Univ. Quart., vol. 10.

2. Bütschli, O., ’71, 1: Vorläufige Mittheilung über Bau und
Entwickelung, der Inseckten und Crustaceen. Zeitschr. f. wiss. Zool.,
Bd. 21.

3. —— ’71, 2: Nähere Mittheilung über die Entwickelung und den Bau
der Samenfäden der Inseckten. Zeitschr. f. wiss. Zool., Bd. 21.

4. Calkins, G. N., ’95: The Spermatogenesis of _Lumbricus_. Journ. of
Morph., vol. 11.

5. Erlanger, R., ’96, 1: Ueber den sogenannten Nebenkern in den
männlichen Geschlechtszellen der Inseckten. Zool. Anz., Bd. 19.

6. —— ’96, 2: Die Entwickelung der männlichen Geschlechtszellen.
Zool. Centralbl., Jahrg. 3.

7. —— ’97, 1: Ueber Spindelreste und den echten Nebenkern in den
Hodenzellen. Zool. Centralbl., Jahrg. 4.

8. —— ’97, 2: Ueber die sogenannten Sphäre in den männlichen
Geschlechtszellen. Zool. Centralbl., Jahrg. 4.

9. Heidenhain, M., ’00: Ueber die Centralkapseln und
Pseudochromosomen in den Samenzellen von _Proteus_, sowie über ihr
verhältniss zu den Idiozomen, Chondromiten und Archoplasmaschleifen.
Anat. Anz., Bd. 18.

10. Henking, H., ’91: Ueber Spermatogenese und deren Beziehung zur
Entwickelung bei _Pyrrhocoris apterus_. Zeitschr. f. wiss. Zool.,
Bd. 51.

11. McClung, C. E., ’99: A Peculiar Nuclear Element in the Male
Reproductive Cells of Insects. Zool. Bull., vol. 2.

12. —— ’00: The Spermatocyte Divisions of the Acrididæ. Kans. Univ.
Quart., vol. 9.

13. Meves, F., ’00: Ueber den von v. la Valette St. George endeckten
Nebenkern (Mitochondrion Körper) der Samenzellen. Arch. f. mikr.
Anat., Bd. 56.

14. Paulmier, F. C., ’99: The Spermatogenesis of _Anasa tristis_.
Journ. of Morph., vol. 15, supplement.

15. Platner, G., ’85: Ueber die Spermatogenese bei den Pulmonaten.
Arch. f. mikr. Anat., Bd. 25.

16. —— ’86, 1: Ueber die Enstehung des Nebenkerns und seine Beziehung
zur Kerntheilung. Arch. f. mikr. Anat., Bd. 26.

17. —— ’86, 2: Zur Bildung der Geschlechtsproduckte bei den
Pulmonaten. Arch. f. mikr. Anat., Bd. 26.

18. —— ’89, 1: Beiträge zur Kenntniss der Zelle und ihrer
Theilungserscheinungen, I und II. Arch. f. mikr. Anat., Bd. 33.

19. —— ’89, 2: Beiträge zur Kenntniss der Zelle und ihrer Theilung,
IV und V. Arch. f. mikr. Anat., Bd. 33.

20. Vom Rath, O., ’92: Zur Kenntniss der Spermatogenese von
_Gryllotalpa vulgaris_. Arch. f. mikr. Anat., Bd. 42.

21. St. George, v. la Valette, ’67: Ueber die Genese der Samen
Körper. Arch. f. mikr. Anat., Bd. 3.

22. —— ’86, 1: Spermatologische Beiträge, Zweite Mittheilung. Arch.
f. mikr. Anat., Bd. 27.

23. —— ’86, 2: Spermatologische Beiträge, Vierte Mittheilung. Arch.
f. mikr. Anat., Bd. 28.

24. —— ’87: Zelltheilung und Samenbildung bei _Forficula
auricularia_. Festschrift f. A. v. Kölliker.

25. —— ’97: Zur Samen- und Eibildung beim Seidenspinner (_Bombyx
mori_). Arch. f. mikr. Anat., Bd. 50.

[B]26. Sutton, W. S., ’00: Spermatogonial Divisions in _Brachystola
magna_. Kans. Univ. Quart., vol. 9.

27. Wilcox, E. V., ’95: Spermatogenesis of _Caloptenus femur-rubrum_
and _Cicada tibicen_. Bull. of Mus. of Comp. Zool. Harvard., vol. 27.

28. —— ’96: Further Studies on the Spermatogenesis of _Caloptenus
femur-rubrum_. Bull. of Mus. of Comp. Zool. Harvard., vol. 29.

[B] Nos. 12 and 26 were used when describing the material only and
not the spermatid transformations.

Explanation of Plate II.

All drawings were made by the author with the aid of a camera
lucida. All figures except 2, 3, 4 and 45 were made with a B. & L.
1/16 objective and one inch eye-piece, producing a magnification
of 1340 diameters. Figs. 2, 3, 4 and 45 were drawn with a Leitz
1/16 objective and a Zeiss compensating ocular No. 12, giving a
magnification of 2325 diameters. The drawings were not reduced in
the photo-mechanical reproduction.

FIG. 1. An early telophase, in which the fibers appear like barrel
staves. Some of the fibers have thickened.

FIG. 2. A later stage, in which the fibers are uniting, the clear
space is forming, and the chromosomes are beginning to separate.

FIG. 3. A daughter-cell, showing the chromosomes separating and the
fibers thickening.

FIG. 4. The chromosomes are scattered over the nuclear membrane; the
fibers appear as a few thick rods. The beginning of the acrosome is
seen. The accessory chromosome can be distinguished.

FIG. 5. Same as fig. 4, with some of the fibers curved and no
acrosome.

FIG. 6. Same as fig. 5, but with the acrosome visible.

FIG. 7. The chromosomes are granular; the fibers have bent and formed
the round “striated nebenkern.” The acrosome is present.

FIG. 8_a_. Same as fig. 7, but the acrosome is not present.

FIG. 8_b_. The fibers run parallel with the long axis of the
nebenkern.

FIG. 9. Here is shown a partial end view of the nebenkern. The
accessory chromosome and the acrosome are both prominent.

FIG. 10. The nebenkern shows some of the fibers on the under side. It
is beginning to break up.

FIG. 11. The nebenkern shows the beginning of the dark center; the
fibers extend from it to the ring. The acrosome has a peculiar
appearance.

FIG. 12. The fibers have broken up and the whole has assumed a
vesicular appearance, resembling a blackberry. The acrosome is
present.

FIG. 13. This shows a persisting spermatid stage. The chromatin
appears in patches. The nebenkern is in the ball-and-ring stage. The
acrosome shows its characteristic clear center surrounded by a ring
darker on one side. The two dark bodies on the nuclear surface are
probably artefacts.

FIG. 14. A stage a little later than 13. The dark central ball shows
some vacuoles. The cell is beginning to elongate.

FIG. 15. Same as fig. 14, with the ball denser.

FIG. 16. The nucleus has become almost clear. The acrosome is behind
the nebenkern.

FIG. 17. This shows the axial filament apparently growing out from
the nucleus. There is an aggregation of chromatin at its base.

FIG. 18. The axial filament passes over the nebenkern, which has
slightly elongated. The accessory chromosome is very plain.

FIG. 19. _a_ shows a surface view, and _b_ an optical section, of a
nebenkern which is in process of breaking up.

FIGS. 20 and 21 have part of the surface fibers cut off and show the
dark mass forming within.

FIG. 22. Shows a nebenkern passing from the blackberry stage into the
ring stage.

FIG. 23. This is an end view of the stage shown in figs. 20 and 21.

FIG. 24. The fainter lines are the fibers on the under side of the
nebenkern.

FIG. 25. The blackberry stage, showing the mass of vesicles.

FIG. 26. This represents a nebenkern of the stage shown in fig. 7,
drawn from a polar view, with adjusting focus.

[Illustration: PLATE II.]

Explanation of Plate III.

FIG. 27. This shows the nebenkern elongating. The axial filament
passes over its surface.

FIG. 28. A little later than fig. 27.

FIG. 29. Shows cross-sections of elongating nebenkern; _a_ shows
axial filament, nebenkern and acrosome; in _b_ the axial filament
is cut at an angle.

FIG. 30. A little later than fig. 28; the axial filament not so
plainly shown.

FIG. 31. The ring has disappeared, and the dark mass is passing down
along the filament. The acrosome is indistinct.

FIG. 32. The nebenkern has disappeared, but the acrosome has kept its
position.

FIG. 33. The acrosome has flattened against the nucleus. Some remains
of the nebenkern are seen.

FIG. 34. The acrosome has moved to the side of the nucleus. Some
nebenkern remains are shown.

FIG. 35. The acrosome is in front of the nucleus.

FIG. 36. A little later stage than fig. 35.

FIG. 37. Shows a divided acrosome. It is not a frequent appearance.

FIG. 38. The nebenkern appears as a hanging drop on the side of the
axial filament.

FIG. 39. The nucleus has condensed, the walls have thickened, and the
chromatin appears in granules. The acrosome is pointed.

FIG. 40. A little later stage than 39.

FIG. 41. A little later stage than 40.

FIG. 42. The head is stained intensely, while the acrosome is
lighter. Probably a middle piece indicated.

FIG. 43. The head shows the clear space.

FIG. 44. Cross-sections of the head, of the stage shown in fig. 43.

FIG. 45. The head and part of the tail of a mature spermatozoon.

[Illustration: PLATE III.]

*** End of this LibraryBlog Digital Book "The Kansas University Science Bulletin, Vol. I, No. 2, February, 1902" ***

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