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THE GERM-CELLS AHD THE PROCESS OF FERTILIZATION

In tha
CRAB MEHIPPE MERGEHARIA

Raymond Binford.

A DISSERTATION

Submitted to the Board of University Studies
of the Johns Hopkins University in conformity
with the Requirements for the Degree of Doc-
tor of Philosophy.

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THE GERM-CEILS AUD THE PROCESS 05* FERTILIZATION

in the
CRAB IvIEUIPPE IvIERCElTARIA.

Contents.

Page.

I . Introduction "b-c

II. Spermat ogene si s :

1. The testis 1

2. Methods 1

3. The testicular tuhules 2-4

4. General statement of spermatogenesis 4-5

5. The spermatogonial mitoses 5-6

6. Maturation mitoses 6-9

7. The transformation of the spermatid into

the sperm 9-14

8. The sperm 14-15

9. Sperms in the deferent duct ..15

lu. Discussion 15-17

III. Copulation 17-18

lY. Spawning hahits 18-19

V. The reproductive organs of the female 19-21

VI. The "behavior of the sperms:

1. Methods of study 21-22

2. Changes in the nuclear cup 23

3 . Change s in the capsule 23-25

4. Changes in the central "body 25-27

6. The dynamics of eversion 27-32

6. The effect of reagents on everted sperms. .. .32-33

VII. The entrance of the sperm 33-36

VIII. Fertilization 36-39

iZ. Discussion 39-42

Z. Summary 42-44

IlITRODUCTIOU.

In spite of the extensive researches iat© the spermato-
genesis of the Decapods, the use of the peculiar structures
found in the sperms of these animals is still an unsolved
prol)lem. This is due to the fact that the entrance of the
sperm into the egg has never "been reported. While studying
the hat its and structure of Menippe mercenaria, a large
edible crah found along the southern part of the Atlantic
coast of the United. States, I had the good fortune to obtain
material which shows the essential features of this process.
In order to show which parts of the seminal cell are involved
in the process of fertilization, the genesis of the sperm
and the formation of the pronucleus in the fertilization of
the egg, as well as the entrance of the sperm, are here de-
scribed. The history of the male cell from its origin in
the epithelium of the wall of a tubule of the testis to its
association with the female nucleus in the center of the egg,
is here presented.

The study of the life-history of Menippe mercenaria,
which led to the discoveries presented in this paper, was
undertaken at the suggestion of Prof. E. A. Andrews, and at
every step in the progress of this work I have received his
kind advice and helpful criticism. I am also greatly indebt-
ed to the Hon. Geo. M. Bowers, United States Commissioner of
Fish and Fisheries, for the privilege of working in the
Marine Biological Laboratory at Beaufort, II. C., and for the

lilDeral help extended to me in carrying on ray researches there.
My thanks are also due to Mr. H. D. Aller, Director of the
Laboratory, for his ready cooperation in placing at my dis-
posal the conveniences necessary for carrying forward my
work.

SPERMATOGENESIS.

1. The Testis.

The testis of Menlppe Is a large paired organ lying Just
underneath the dorsal wall of the carapace. The Inner ends
of the right and left portions lie close together Just anter-
ior to the heart and from here diverge anteriorly and later-
ally to the outer edge of the carapace. It is composed of
relatively long and complexly folded tubules which vary in
diameter from 0.14 to 0.33 mm. The deferent duct, one on
each side, leads from the testis to the base of the last
thoracic leg. It is extensively convoluted so as to form
two large masses, one lateral to the posterior part of the
testis and the other beneath the posterior part of the heart.
The deferent duct is lined with a layer of columnar epithelium
which secrets the substance that forms the walls of the
spermatophores .

2. Methods.

Pieces of the testis, obtained by cutting across the
organ, were fixed in Worcester's fluid. This is a saturated
solution of sublimate in 10^ formalin. Other fixing fluids
were used but did not give as satisfactory results. The sec-
tions were cut Iji to lOjx thick. The stains used were thionln
and eosin, safranin and LichtgrUn, iron-haeraatoxylln, and
Delafleld's haematoxylln.

&»ltt

3. The TeBtiottlar Tatules.

The walls of the tubules of the testis are thin and con-
tain flattened nuclei. Figures 1, 2, 3 and 4 are drawings of
transverse sections of the tuhules and show different stages
In the development of the seminal elements. In Pig. 4 the
tubule is seen to be made up of three regions: The smallest
one which bordars the more sharply curved side at the bottom
of the drawing contains mature sperms. Next to this and fill-
ing the central region is a space filled with sperms nearly-
mature. The third region, which forms the crescent shaped por-
tion on the tipper side, contains spermatocytes in the early
propase of the first maturation division. There are more or
less definite layers of epithelial cells between the different
regions of the tubule. The outer wall and sometimes these
inner partitions which border the regions containing mature
sperms, become thick and columnar in structure (Pigs. 3 and
4).

Hot only do the seminal elements in these separate paral-
lel cavities of the tubule differ in th« stages of their devel-
opment but in the same cavity the elements at one end of the
tubule are further along in their development than those at
the other end. Thus In one end of a cavity the cells may be
In the early prophase of the division of the spermatocytes
of the first order while in the other end they have reached
the spermatid stage. All the stages in the transformation
of a spermatid into a sperm may be found in passing from one
end of a tubule to the other.

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At the center of the upper border of Fig. 4, p.s., there
is one cell with a large nucleus. This is one of a single
row of cells along the side of the tubule which may be called
the prlDBtry sperraatogonial cells since they, by division,
give rise to a new lot of spermatogonia. Hear the top of Fig.

3, p«s., we find a similar cell. The cells forming the cres-
cent-shaped region are In this case not so far advanced as in J*'ig.

4. Figure 1 represents a tubule the largest portion of which
is filled with spermatids which have already entered upon their
transformation into sperms. In the upper portion of the draw-
ing we have an early stage in the foimation of a new batch of
spermatogonia. There are four large spermatogonial nuclei
surrounded by many epithelial nuclei and a considerable amount
of cytoplasm. Delicate cell walls cutting O'.t the cytoplasm
that belongs to each spermatogonial cell can sometimes be made
out at this stage. A later stage in the multiplication of
these cells is shown in Fig. 2. A large nucleus, p.s,, near
the middle of the convex border of the spermatogonial mass,
doubtless marks the position of the row of primary spermatogon-
ial cells which will persist unmodified to form, at a later
period another lot of spermatogonia. The largest cavity of
this tubule contains spermatids well advanced in their trans-
formation into sperms. They fill the cavity but only a por-
tion of those seen in the cross-section are represented in the
drawing. In Fig. 3 the mass of spermatogonial nuclei is still
further enlarged. Indeed most of thorn have probably reached
the spermatocyte stage. The spermatocytes in the early prophase
of the first maturation division are shown in Fig. 4. By put-

Tiidfdi «T'

ting these observations together we may determine the approxi-
mate order of events In the genesis of the sperms.

4. A General Statement of Spermatogenesis*

There persists along one side of the tuhnle a single row
of cells with large nuclei, the division of which give rise to
the spermatogonia. The latter multiply irregularly to form a
large mass which in transverse section has the shape of a
crescent. At first cell walls can "be made out, hut later the
nuclei seem to lie in an undivided mass of cytoplasm. Grad-
ually the division of these nuclei ceases and a spireme is
formed within each of them. The division up to this time has
taken place without the formation of any spireme structure.
The appearance of the latter is the first indication that the
cells have reached the spermatocyte stage. After the spireme
has been formed the nuclei pass into synapsis which lasts for
a comparatively long time, so that all the nuoell in a con-
siderable portion of a tubule will be found in this stage at
the same time. After synapsis cell walls are formed in the
cytoplasm which persist up to the anaphase of the first ma-
turation division. In the nucleus the chromosomes are form-
ed and the maturation division follow one another in quick
succession. They begin at one end of the tubule and pass along
it like a wave so that the spindle-figures are found in only
a small section of the tubule at any given time. While these
events are taking place within the tubule the cells in the
wall of the latter multiply so that the wall becomes consider-
ably thickened. The primary spermatogonia! cells also divide

♦jLJTflxr;

to start a new group of spermatogonia. Between these cells
and the spermatocytes there Is always a layer of epithelial
cells which persist to form the partitions "between the two
successive hatches of seminal elements. The mass of sperma-
togonlal nuclei remains small until the spermatids are well
advanced In their transformation into sperms.

As the mass of spermatogonia Increases the developing
sperms are crowded more and more to one side of the tuhule.
These sperms reach their mature state hefore the second batch
enter synapsis. The epithelial cells surrounding the mature
sperms, we may suppose, secret a fluid which together with the
increasing mass of spermatogonia press the mature sperms out
of the tubule. This process is not completed, however, before
a third batch is formed or even a fourth started.

5. Spermatogonial Mitoses.

In the resting spermatogonia! nucleus the chromatin is
arranged in a loose net-work with enlargements at various
places tPig. 5). This net, for the most part, lies just in-
side the nuclear membrane, the central part of the nucleus
containing almost no staining material. The behavior of the
chromatin during the prophase of Bltosis is as follows; the
knots of chromatin become enlarged and more regular in out-
line while the connecting threads become smaller and disap-
pear. The chromatin finally assumes the form of a large num-
ber of paired spheres (Pig. 6i. An effort was made to count
these spheres and numbers were obtained as follows: 61, 55,
67, 58, 6E, 62, 68 and 80. One may not however place very

c©yit

muoh dependence in these numbers for soiirie of the spheres are
always somewhat aggregated In one or two places so that they
oan not be definitely distinguished. These chromosomes at
first lie in the outer part of the nucleus just inside the
nuclear membrane, but are later massed in the center, from
which condition they move to their positions in the equitorial
plate. Figure 7 is an optical section of the nucleus showing
the peripheral arrangement of the chromosomes, m the meta-
phase and anaphase of the mitosis the members of each pair
are separated from each other and pass to opposite poles of
the spindle iPig. 8>. These divisions of the spermatogonial
nuclei do not occur simultaneously throughout the mass, but
singly here and there amongst the nuclei. The spermatogonia
become smaller as they become more numerous.

6. Maturation Mitoses.

Finally the spermatogonial divisions cease and the nuclei
prepare for the reduction divisions. The quantity of chromatin
seems to increase and the spireme makes its appearance. At
first it is very long and slender and complexly folded all
through the nucleus. The iron-haematoxylin stain can be con-
trolled so that the spireme has the appearance of a brown
thread with granules distributed irregularly along it iFlg.9).
The diameter of the granules is slightly greater than that of
the thread between the granules. The spireme now becomes
shorter and thicker and is finally massed at one side of the
nucleus in the condition of synapsis (Fig. 10). This stage
persists for a comparatively long period. The spermatocytes

enter sTnapsls Irregularly, in a sort of one -at-a- time fashion
but they tarry here tmtll all of the cells In the greater part
of the tuhule have reached this stage, then the nuclei of a
given portion all proceed to the open spireme stage, shown in
Pigs. 11 and IE. These figures show only the chromatin which
lies on the side from which the nucleus was observed. The
chromatic material is again arranged in the peripheral portion
of the nucleus and is segregated Into the chromosomes which
become somewhat massed In the center of the nucleus. The
spindle next makes its appearance (Fig. 13 J and the chromosomes
are drawn into the equatorial plate (Pig. 14).

The mitotic figure represented in Pig. 15 shows the pos-
sibility of a tri-polar division. Such a condition may have
been brought about by the formation of one of the spindles of
the second division before that of the first division was com-
pleted. There is a small portion of the chromatin of this
nucleus that is not Involved in the mitotic figure. This
portion is shown at £., in Pig. 15a which Is a drawing of
what was seen at a lower level than that shown in JTig. 16.

The chromosomes in these nuclei are so small and so close-
ly crowded together it is very difficult to determine their
structure or their number. In one preparation, however, I
obtained a ring-shaped appearance of the chromosomes lPig.l6).
These forms were seen in the equatorial plate and also before
the chromosomes had been arranged in the plate. In most of
the preparations the chromosomes appear as mere granules. It
may be that the ring shaped forms were produced by the fixing

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reagent, which may have caused a ewelllng of the chromosomes.
This result was not always obtained however hy the same re-
agent .

when de staining Is carried so far as to remove all the
stain from the cytoplasm and the achromatic figure, the equa-
torial plate may be shown to have a structure like that repre-
sented in Pig. 16a. This was drawn from a section cut from
the edge of the plate. Here it appears that the chromosoines
are stretched as they are pulled apart. Strands of chromatin
pulled out between the separating groups of chromosomes may
be seen In the later stages of the anaphase. By more exten-
sive destainlng we may obtain what appears to be only the
cores of the chromosomes as shown in Pig. 16b.

In Pigs. 17 to 22 various stages in the anaphase are rep-
resented. The interzonal fibers and the mid-body axe very
distinct In Pigs. 19 to 21.

The second mitotic division follows very soon after the
first. The chromosomes become somewhat separated and are
then drawn together again Into the equatorial plate ready
for the second division (Pigs. 23 to 25). Plgure 26 shows
the beginning, and Pig. 27, the end of the anaphase. Here
again the Interzonal fibers and the mid-body are distinctly
seen and a portion of the cytoplasm is definitely associated
with each daughter nucleus. The nucleus of the spermatid
Is now organized and persists in a sort of resting condition
for a comparatively long time. The centrosome may also be
distinguished for a considerable time but later I was unable

to recognize it (Figs. 28 to 32). A clear space surrounding
the nucleus Is also seen in these figures. The spermatid as
it appears In Pig. 32 rests for a conslderahle period hefore
any change towards the formation of the sperm is observed. The
boundaries "between the cytoplasm of the different cells dis-
appear and the nuclei come to lie in a sort of Plasmodium.

7. The transformation of the spermatid into the sperm.

In serial sections of a single tuhule we may trace every
stage in the transformation of the spermatid into the sperm,
and since the two ends of the series are in opposite ends of
the tubule and the intermediate stages lie in serial order
between these ends we may use the position of a seminal ele-
ment in the tubule as a criterion for determining its relative
stage in the course of development. The first evident step in
the transformation of the spermatid is the appearance of
vacuoles in the cytoplasm next to the nucleus. These are at
first small but by coalescing they soon form a large, clear
vacuole on one side of the nucleus (Figs. 3J5 to 38). Some-
times it appears that the vacuole may have arisen by the nu-
cleus settling to one side of the clear space surrounding it
in Pig* 31. The nuclei, each with its accompanying vacuole,
now lie in a common mass of cytoplasm. In the further develop-
ment of these cells there are three parts that must be con-
stantly borne in mind, viz., the nucleus, the vacuole (here-
after called the capsule) and the cytoplasm. We shall take
up certain stages in the differentiation of these three

10

parts, and consider their relation to each other.

In Figs. 37 to 41 the shape of the nucleus may he some-
T?hat modified by strains In the cytoplasm or by the crowding
of the elements in the tubule. In these drawings there is
no evidence of a granular or reticular structure, although
such structure was made out in some preparations which were
destained to a greater degree. In Fig. 37 It may be observed
that the outer layer of the nucleus stains more densely than
the inner portion. The nucleus in Fig. 38 contains a vacuole
which does not take the stain. The cytoplasm surrounding the
nucleus and capsule in Figs. 37 to 39 Is nearly uniform in
appearance, with probably a tendency to be a little more deep-
ly stained near the nucleus. In Figs. 40 and 41 there is a
concentration of a portion of the cytoplasm on one side of the
capsule and bordering the nucleus. This is finely alveolar
and stains more deeply than the rest of the cytoplasm. It
may be that this patch of cytoplasm is seen in an earlier
stage In Fig. 36c. This portion of the cytoplasm crowds in
between the nucleus and the capsule (Fig. 42). About this
time the capsule begins to take a brownish color when stain-
ed with iron-haematoxylin.

The otigin and development of this portion of cytoplasm
which appears on the side of the capsule and nucleus and
wedges in between them is a striking feature In the develop-
ment of the sperm. Its behavior is well brought out in Figs.
45-48. In Fig. 45 we see this substance slipped in like a
wedge between the nucleus and the capsule with a clear space
between it and the nucleus. If the spermatid shown in this

11

figure were rotated to the right through 90** so as to hrlng
the outer surface of the wedge of cytoplasm on the side to-
ward the observer we would have the appearance presented In
?lg. 46. If we should turn this through 180° so as to throw
the wedge on the opposite side from the observer the spermatid
would appear as in Pig* 47 where just the tips of the orescent
shaped wedge are seen. The tips of this crescent progress on
so'ound the capsule along the boundary line between the nucleus
and the capsule. At the same time the thick side of the wedge
Is reduced and the material Is distributed equally around this
border-line to form a complete ring, which viewed from any
lateral direction, has the appearance shown In Fig. 48. At
first the substance of the wedge Is finely alveolar In appear-
ance but by the time the ring Is completed It seems to be uni-
form throughout and Is stained black with Iron-haematoxylln.
It seems to be identical with the mitochondrial substance de-
scribed by Koltzoff '06.

After the mitochondrial ring is completed, the nucleus
becomes widely separated from it and the capsule (Figs. 50
to 52). This however Is not always the case. In two prepara-
tions from which Figs. 33 to 35 and 37 to 43 were drawn, the
nucleus remained fitted closely down on the capsule as shown
in Fig. 43. As the two different conditions were obtained
with the same fixing fluid it is hardly probable that the
difference was caused by the fixing. The nucleus at this time
looses the last trace of any granular or reticular structure
and becomes uniform ih its staining reactions, and somewhat
reduced in size.

12

About the time the mitochondrial mass begins to slip in
between the nucleus and the capsule, one or two deeply stain-
ing granules appear on the border line between the nucleus
and the capsule (Figs. 44 to 48). Koltzoff '06 in his re-
searches on the spermatogenesis in Galathea squamifera has
identified these granules with the centrosome. In my prepara-
tions of Menippe meroenaria I am able to distinguish the centro-
some for some time after the second maturation division (Pigs.
28 to 3E), but in the later resting period of the spermatid
and in the stages during the origin of the capsule, I am unable
to distinguish any granule that can with any certainty be iden-
tified with the centrosome » I shall call the structure develop-
ed from this granule, the central body. I am unable to follow
the development of two distinct granules although two could
some times be clearly distinguished as shown in ?ig. 44. Prob-
ably only the outer one is concerned in the development which
is here presented.

This outer granule elongates (Fig. 47) and becones tubular
(Figs. 50 to 56). There soon appears at the outer end a vesicle
which Increases in size as the central body elongates. yiThile
the vesicle is still small there appears in its outer wall a
flattened granule which Is usually seen to be connected with
the end of the central body by means of a fine strand as though
it might have been derived from this body. As the central
body Increases in length and the vesicle enlarges its outer
wall approximates the outer wall of the capsule. The deeply
staining substance in the outer wall of the vesicle now becomes
connected with the wall of the capsule. (Pig. 56 to 58). A sec-

ibX'

13

ond vesicle now forms. (Pig. 59). These two vesicles "become
transformed Into a tu"bule containing the central body. This
tuhule will hereafter be spoken of, as the inner tubule . At
its outer end a ring of darkly staining substance is found (Pig.
60). This seems to have* been derived from the central body. At
least a study of Pigs. 54 to 60 may well suggest such an inter-
pretation. The central body finally becomes reduced in diameter
and appears to be a solid rod. It is not stained by thionin,
nor by safranin,but is readily stained with iron-haematoxylin.

The inner tubule is stained green with safranin coimter
stained with Lichtgriin; blue with thionin counter stained with
eosin; and black with iron-haematoxylin.

During this whole period the content of the capsule shows
an increasing affinity for chromatin stains. It is colored
brown with iron-haematoxylin. In some series a sort of ring
shaped cloud appears in the capsular contents. At first it is
near the outer wall but gradually it contracts towards the ves-
icle at the end of the central body and finally settles in the
wall of the tubule when that structure takes its final form.
With Delafield's haematoxylin the contents of the capsule is
readily stained and with safranin it takes a dull red color.
In the early stages of development the content of the capsule
is stained green when the preparation is treated with the
safranin and Llchtgrun combination, but in the later stages
the green is masked by the red. In stages represented in
Pigs. 53 to 55 a sort of foam or alveolar structure can some-
times be observed in this substance.

While the capsule and the structures within it are assum-

bevxret

3Ti?9qq

14

Ing their mature form the nncleus has heoome less densely
stained and settles down upon the capsule like a cap. (Pigs.
62 to 59). It becomes thin In the center so that Its final
shape Is that of a cup with a rounded, thin hottom and a
thickened rim. This thickened border fits upon the mltoohron-
drlal ring so that In the mature sperm It is not possible to
distinguish It from that ring.

Protoplasmic rays or pseudopodla develop out from the
rlm of the cup. I have been unable to determine whether they
arise from the mitochondrial substance or from the' nucleus.

8. The Sperm.

We may now consider the structure of the mature sperm.
Figure 61 Is a drawing of a sperm taken from the seminal recep-
tacle of the female and killed in the vapor of osmlc acid then
stained with gold chloride after treatment with formic acid.
We observe the nuclear cup, n.c. from which the pseudopodla,
ps. arise. Inside the cup Is the spherical capsule, c. within
which there is the capsular oavlty, c.c; and the inner tubule,
l.t. with its cavity divided Into the inner tubular cavity,
l.t.c. ; and the outer tubular cavity, o.o. Running through
the Inner tubular cavity and through the wall of the inner end
of the tubule to the bottom of the capsule we see the central
body, c.b. Figure 62 was drawn from a live sperm in A% ENO3,
and Pigs. 63 to 65 are from sperms mounted in the serum of
the crab' s blood. Movements of the blood have bent the pseudo-
podla of these sperms. Otherwise they have more nearly the
natural shape and proportions than those shown in Pigs. 61 and

10

3 ' dfit'

15

62. The diameter of the capsule of these sperms is about
3.8^ and the pseud opodia are some times as much as 7^ long*

9. Sperms in the deferent duct.

The mature sperms pass from the tuhules of the testis in-
to the deferent duct. The latter is a long extensively fold-
ed tuhe lined with glandular epithelium. The sperms form a
common mass when they enter this tube but the secretion form-
ed by its lining flows in amongst them and separates them in-
to groups. The secretion surrounding each group then hardens
and so forms a membrane, so that finally there are an immense
number of capsules containing the sperms. These capsules are
known as spermatophores. In this condition the sperms are
transferred to the seminal receptacle of the female crab.

10. Summary and discussion.

In this study of spermatogenesis in Menippe mercenaria
the principal points brought out are as follows:

1. There is a single row of cells which persists on one
side of the testicular tubule and gives rise to successive
hatches of spermatozoa.

2. The spermatogonia divide without the formation of a
spireme. The chromatin simply aggregates into chromosomes
which are then gathered into an equatorial plate.

3. The maturation divisions follow one another quickly.
They are proceeded by spireme formation and a long period of
synapsis.

.flc..*-ei-v^f

16

4. There also seems to be a relatively long resting
stage after the nucleus of the spermatid Is formed hefore the
transformation Into the sperm heglns.

6. In the transformation of the spermatid, three struc-
tures must he considered, viz., the nucleus, the capsule and
the mitochondrial ring.

^. The nucleus becomes uniform In consistency, reduced
In size and cup-shaped.

7^» The capsule arises In the cytoplasm as a clear
vacuole which may be stained V7lth Llohtgrun. Its content is
gradually changed to have a greater affinity for chromatin.

8,. Prom a granule on the proximal side of the capsule
the central body develops Into the capsule. At the distal
end of this body a vesicle arises, which is changed into the
inner tubule.

9. The mitochondrial substance is segregated from the
cytoplasm and deposited as a ring between the nucleus and
the capsule.

Some of the theoretical questions connected with the
development and structure of the sperms of the decapods will
be taken up at the end of this article. At this point I wish
to say that the above description Is in agreement with the
principal observations made by Grobben '78, Gllson '86, Saba-
tler '93, Brandes '97 and Koltzoff '06. These authors have
all seen the same general structures and transformations.
They all describe a nucleus which during development Is modi-
fied in its staining reactions, reduced in size and often
flattened or otherwise changed in its shape. They do not dis-

17

agree as to which part of the cell is the nucleus. They like-
wise desorihe a vesicle which arises in the cytoplasm either
against the nucleus or close to it, and mention the substance
of cytoplasmic origin which appears hetween the nucleus and
the vesicle. Most of them see a structure like the central
body and describe the inner tubule. There are many variations
in the detail of the development of these last two structures
and different species seem to differ widely in this respect.
There is mxxch disagreement concerning the destiny of the nu-
cleus and the origin and nature of the substance in the cap-
sule. These points of disagreement do not however affect the
statements I have made concerning the structure of the mature
sperm. It is with this structure that we have to do in the
further course of the present investigation.

III. Copulation.

We come now to the question of the transfer of the sperm-
atophores from the body of the male to that of the female,
from the deferent duct to the seminal receptacle. We there-
fore turn our attention from the sperm itself to some of the
habits of these crabs. Menippe mercenaria lives in crevices
under or between the rocks or in burrows which it digs in
the mud along the shore a little below low water line. Usual-
ly one crab is found in each burrow, but occasionally, and
even frequently in the month of August, a male crab will be
found guarding a hole in which there is a female. Sometimes
the female thus found has a soft shell. If its shell is

18

hard It molts within a few days after helng hronght Into cap-
tivity. On Aug. 17, a female with a soft shell and a male
crab which had been taken from the same hole about noon were
placed together In a compartment of a floating cage. At
5:45 P.M. they were observed to be copulating. On being dis-
turbed they separated. Their behavior was then observed
while copulation was resumed. The most significant point
with regard to this behavior was the apparent care with which
the male acted in order to inflict no injury upon the soft,
delicate shell of the female.

During copulation the spermatophores are transferred from
the deferent duct to the portion of the seminal receptacle
that is lined with chitin, where they are deposited in a very
compact mass. Here they remain until the next spawning of
eggs. Only a portion of the sperms are used for the fertili-
zation of any one batch of eggs. One crab kept by itself in
a compartment of a floating cage for 69 days during the summer
of 1911, spawned six times sind apparently all of the eggs
in the six different batches of 500,000 to 1,000,000 eggs each,
were fertilized and developed normally.

IV. Spawning Habits.

The spawning habits and the development of this crab will
be discussed In a later paper. Here we shall present only
such points as are necessary in order to make it clear how the
stages in the entrance of the sperm and fertilization are ob-
tained.

jfilti^oX'

19

When a female is ready to lay a batch of eggs she assumes
an upright position and holcis the abdomen out from her body
so that it and the exopods of the abdominal appendages form
a basket into which the eggs are run. They there become at-
tached to the hairs of the andopods of the appendages and
pass through the embryonic stages of their development, which
requires from nine to thirteen days. The eggs then hatch and
the larvae escape. The female then cleans off the egg-shells
and their stalks from the hairs of the pleopods and after one
day to three weeks she spawns again. Eight days is a very
common length for the period between the hatching of one batch
of eggs and the spawning of the next. With these facts in
mind I made a large floating cage with fifty compartments and
collected a large number of females with eggs and placed one
in each compartment. After tjfie eggs of several of these had
hatched so that there were some fifteen crabs without eggs I
kept these under almost constant observation, day and night.
When one assumed the position ready for spawning it was natur-
ally supposed to contain eggs that were mature if they were
not already fertilized. Before describing the process of
fertilization we should consider briefly the structure of the
genital organs of the female.

V. TH3 RfiPRODUGTIVE ORGMS OF THE FEMALE.

Figure 181 Is a diagramatic representation of the ovary
and one seminal receptacle and oviduct of this crab. The
ovary is an H shaped tube, the lumen of which opens directly

80

into the seminal receptacle at a point a little posterior to
the cross connection of the H. The eggs are produced in the
wall of this tuhe and when mature are set free in the lumen.

The seminal receptacle is composed of two parts, a
glandular portion (Pigs. 121 and 122, g.) into which the ovary
opens and a portion lined with chitin (Figs. 121 and 122, c.)
from which the oviduct leads to the third segment of the
sternum. The speraatophores are stored in the latter division.
The cavities of the two portions communicate through a large
opening (Fig. 121,0.) in the chitinous lining. Just before
the crab molts the glandular portion secretes a mass of
gelatinous material which greatly distends it (Fig. 122) and
the spermatophores are "by some means transferred to this part
of the receptacle where they lie in the mass of jelly. This
prevents them from "being lost at the time of molting when the
chitinous lining is shed. Whether they are returned to this
part of the receptacle after the molting has not been deter-
mined. The glandular part of the receptacle is rapidly re-
duced after the shell is shed, but I do not know what becomes
of the secretion. During spawning the glandular portion is
very much contracted (Fig. 121) so that it is little more than
a tube connecting the ovary with the chitinous receptacle.
There is one possibility which may be mentioned here and that
is that the glandular receptacle may secret a semi-fluid sub-
stance and then by contracting force the sperms Intoithe lumen
of the ovary Just before spawning begins. Asni I shall show
later the sperms are transferred to the ovary. This however
is only a conjecture as to the method of the transfer. The

i":s '. 97orf

ibt^e^s

21

only time at which the receptacle is known to "be actively
secreting a suhstance is Just "before molting and it may simply
be a device for retaining the spermatophores at the molting
period.

If a crah that has Just hegun to lay its eggs is opened
the lumen of the ovary and the oviduct will he found to he
full of eggs. Some eggs were taken from the lumen of the
ovary with a sterilized plpet and placed in filtered sea-
water. Since these developed into emhryoes it is evident
that fertilization takes place in the ovary. Sections were
made of eggs taken from the lumen of the ovary and from the
oviduct smd from these the phenomena of fertilization were
observed, hut we shall return to this later.

VI. THE BEHAVIOR OP THE SPERMS.

The sperms of this crah sure so very minute, the eggs so
relatively large and opaque, and the conditions for sperm en-
trance so difficult to reproduce on the microscopic slide,
I did not see the living sperm enter the egg. It is easy
however to interpret the structures seen in sections of eggs
taken at spawning time, after one has observed the behavior
of the spermatozoa under certain experimental conditions. We
shall proceed therefore to a description of this behavior.

1. Methods of Study.

Koltzoff '06, by his careful analysis of the effects on
the sperm of solutions differing in osmotic pressure, has

22

Cleared up many of the mysteries of the decapod sperm. Accord-
ing to his researches the sperms maintain their normal form
in solutions of salts having the same osmotic pressure as
sea-water. He also found that 5'^ KNO3. 2.8^ HaCl, 4.25^ UalTOg,
18.5^ MgS04, 1% glycerine and 25.66/S sugar solutions are
isotonic with sea-water. Solutions of these salts at a lower
concentration cause a deformation of the sperms.

For my studies solutions of KHO3, UaCl and BiaH03 were
used. The sperms taken from the seminal receptacle and placed
in solutions of these salts isotonic with sea-water would re-
main many days without perceptahle change. When they were
placed in weaker solutions of these salts transformations oc-
curred. In studying these changes I proceeded as follows:
Sperms from the seminal receptacle were placed in the serum
of the crab's blood or in the isotonic solutions of KNO3,
HaCl, or HaHOg. In these solutions they were transferred to
the slide, covered and examined under the high power of the
microscope. Then, by placing a weaker solution of one of the
salts at the edge of the cover-glass and allowing it to dif-
fuse underneath, a slow change in the form of the sperm was
obtained. This change was thus followed in detail. It is to
these changes that we will now turn our attention.

By referring to Pig. 61 we may again call to our minds
the normal condition of the mature sperm which consists of a
chltinous capsule, set in a protoplasmic cup. The capsule
contains a tubule with an inner and outer cavity and running
through the inner cavity of the tubule is the central body,
the proximal end of which rests on the wall of the capsule.

Bnime

23

E. Changes In the nuclear cup*

When solutions with a lower osmotic concentration than
sea-water come in contact with the nuclear or protoplasmic cup
it becomes thicker and the pseudopodia &re withdrawn so that
the outline of the sperm, viewed from the top or bottom of
the cup, is circular instead of star-shaped. The disappear-
ance of the pseudopodia proceeds by a swelling at the base
while the outer portion tapers very gradually to an extremely
fine point. Compare Fig* 62 with 63 and 65. As the base
widens out still father the rays are reduced to a very fine
thread which either breaks off or is contracted into the body.
When the pseudopodia break loose from their attachment the
whole sperm is apt to move suddenly and then be borne away if
there are any currents in the containing fluid. This sudden
movement probably results from some of the pseudopodia break-
ing loose slightly before the others. This rather than the
explosion of the capsule may be the explanation of the "spring-
ing of the sperm" discussed by Kolteoff '06. This rounding
up of the protoplasmic portion of the sperm is apt to be com-
pleted before any change takes place In the capsule. Some-
times however the capsule may be completely changed before
the disappearance of the pseudopodia. Probably In rapid ex-
plosion the two take place simultaneously.

8. Changes in the capsule.

For the Interpretation of the entrance of the sperm into
the egg the transformation of the capsule is much more import-

24

ant than the changes in the protoplasmic cup. We shall there-
fore follow the capsular changes very carefully. The first
change is the out pushing of the outer cavity of the inner
tubule. CoBjpare Pig. 61 with 67. Here it is evident that
the wall of the outer cavity of the inner tuhule has been
everted, while the wall of the inner cavity (Pig. 61 » i.t.o.)
has been stretched. It is difficult to see Just what change
has taken place at this time in the central body. In some
instances it appeared that it had been lengthed, and in some
specimens I thought the end of it could be seen at the summit
of the out pushed portion. It may be that the lengthening
of this body is the force that turns this distal cavity inside
out.

In the next step of the capsular inversion the thick cov-
ering of the out pushed part shown in Pig. 67 becomes turned
out lateraly so as to form a collar (Pig. 68, r.) and the inner
tubule becomes father everted. The collar formed at this
stage persists unchanged throughout all the further modifica-
tions of the capsule. The central body may now become great-
ly increased in length so that it projects beyond the out
turned part of the inner tubule (Pig. 69, c.b. also Pigs.
70 to 72). Prom this stage on to the completion of the ever-
sion there is little further Increase in the length of this
axis. The everted portion of the inner tubule however swells
out more and more (Pigs. 77 to 79). The transition from the
condition shown in Pigs. 71 and 76 to that in Pigs. 77 to 79
is brought about by a further everslon of the inner tubule.
The part of the inner tubule involved in this second definite

25

everslon Is probably marked by the funnel-Bhaped portion In
Pig. 76. The portion of the everted wall, derived from the
part of the tubnle turned by this second everslon is Indicat-
ed by the granule g. Pig. 77. At this stage there Is another
pause while the out turned part continues to swell.

Finally the tension becomes so great that another portion
of the inner tubule is everted and as it turns the wall of the
capsule is also turned through the collar formed in the early
stage of the process of everslon. This last everslon is
shown halfway completed in Pig. 80, and the completed process
In Pig. 81. In the latter figure the central body stands on
the apex of the everslon and the inverted capsule inv.c. is
above the collar r. In dilute solutions of the salts used,
the protoplasmic portion which contains the nucleus and mi-
tochrondrlfld substance swells up to a spherical body as shown
in Pig. 82. Often one finds on the slides, bodies like the
one represented in Pig. 83. It is evident that these are ex-
ploded sperms from which the everted inner tubule has dis-
appeared leaving the central body, c.b.; the inverted capsule,
Inv.v. ; the collar, r; and the shrunken nuclear cup, n.c.

4. Changes in the central body.

We shall now return to a more complete consideration of
the behavior of the central body and the part that it plays
in the explosion of the sperm. These sperms are so very small
it is difficult in many cases to distinguish the central body,
especially in the live unstained material. Some significant
facts however have been observed. As is shown in Pig. 61,

26

the central body Is oomposed of two distinct parts, the distal
part within the cavity of the inner tuhule and a proximal part
connecting the inner end of the tubule with the wall of the
capsule. Whether the central body projects Into the outer
cavity of the inner tubule or ends against the shelf separat-
ing the inner and outer cavities of the tubule was not definite-
ly determined but the latter seems to be the case.

In Pig. 66, which was drawn from a sperm in the tubule
of the testis that was fixed in Worcester's fluid and stained
in iron haematoxylin, the central body projects through the
apex of the capsule. This condition may have been brought
about by an elongation of the central body or by a shrinking
of the capsule. In either case it indicates that the central
body is more or less rigid. One should notice also that the
fixing fluid has caused a shrinking of the nuclear cup so that
it is now more like a saucer than a cup.

In Pigs. 69 to 72 which were drawn from living sperms the
central body projects beyond the everted tubule like a rigid
rod and one gets the impression that its elongation may have
had something to do with the stretching of the tubule and the
lengthening of that axis of the sperm. The idea that the
central body is somewhat rigid is further supported by its ap-
pearance in PigB, 73 and 74, where it stands out above the
everted tubule. The same condition is produced in Pigs. 81
and 82 » Probably the strongest evidence in favor of the rigid-
ity of this structure is found in Pig. 75, where in lengthen-
ing it has pushed backwards through the wall of the cep sule
and pushed the nuclear cup away from the wall of the capsule.

27

There are some Indications that the central "body is not
firm hut a plastlo, semiflnld euhstance. This Is supported hy
the fact that It sometimes glides out through the Inner tubule
at stages such as that shown In Pig. 76 and adheres to the
surface of the everted tuhule In one or more amorphous masses
(Pigs. 77,g.» 79 and 82). This condition may have heen brought
about by a degeneration of the body as a result of keeping
the sperms in the serum of the blood or in salt solutions.
Sometimes in unexploded sperms the central body adheres to one
side of the tubule instead of standing in the center, and it
may be that it was only in such cases as this that it adhered
to the everted wall of the tubule.

5. Dynamics of Ever si on.

We may now consider the forces Involved in the turning
of the tubule and capsule inside out. We may divide this in-
quiry Into two questions: (l) what are the external conditions
necessary to initiate and csirry on the process? (2) What eire
the internal conditions that respond to the external ones and
determine the nature of the process?

As stated above a decrease In the osmotic pressure of the
medium in which the sperms lie, will oause the everslon. Un-
exploded sperms taken from blood serum and placed in 5% KlOg
do not explode; placed in 3% to A% ZSOg they take the forms
shown in Pigs. 67 to 70; in 2?5 to 3^ KHO3 the forms in Pigs.
70 to 72 and 77 are obtained; in 1.5^ to 1^ KNO3 the everslon
proceeds to the stages shown in Pigs. 77 to 82. Like results

28

were obtained by treating the sperms with dilutions of 2.8^
HaCl or 4.25^ NaHOg. Hot all the individuals are equally af-
fected by these solutions. Many of the sperms retain the un-
exploded conditions of the capsule for a long time in a Z%
MO3 solutions, and often none of them attain to the stage
represented in ?lg. 82 when treated with 1% KMO3.

Sperms kept for several days in 2.8?^ HaCl exploded when
transferred to 4.25^ I^aHO^. Here we had an explosion when
the sperms were transferred from a solution of one salt to
that of another with equal osmotic pressure. Presh sperms
do not explode when placed in 4.25^ SaHO^; therefore the
sperms must have been changed by the HaCl, or the presence
of these two salts must have had an effect that neither had
when acting alone. To determine the factors here acting will
require further experimentation.

Some of the sperms explode whenever they are transferred
to a slide and covered with a cover-glass. What may be the
cause of such explosions was not determined. Koltzoff found
that mechanical pressure would cause the explosion of the
sperms of some decapods. I failed to produce any explosion
by pressing on the cover-glass of a preparation containing
them. Koltzoff '06 made extensive researches to find some
specific stimulus that would cause a certain definite explosion
which he believed to be the normal one but failed to find
such a stimulus. It appears however that a careful investi-
gation of the conditions which initiate the process followed
up by an analysis of the conditions which may Increase the

'.VHJ

29

pressure within the capsular cavity (Fig. 61 c.o.) would throw
valuable light on this subject. My researches have been con-
cerned with the exact changes that occur in the sperm rather
than with the conditions that cause the changes.

The second question, that Is the one concerning the in-
ternal conditions which determine the response of the sperm
to the external conditions, may now be considered. What is
there In the sperm that may react to a decrease of the osmotic
pressure of the solution which surrotmds it? An examination
of Figs. 68 to 82 clearly shows that it is the capsular cavity
that increases in size. It must therefore contain a substance
which is Isotonic with sea-water and with the blood of the
crab and which absorbs water when placed in any solution which
is of a lower concentration. This water is doubtless taken
in through the wall of the inner tubule which seems to be semi-
permeable, while the outer wall of the capsule is probably
impervious.

Another striking feature of the explosion is the remark-
able extensibility of the wall of the inner tubule which is
everted to form the wall of a structure many times larger
than the capsule. The central body must also be considered
as one of the structures taking a part in the explosion of the
capsule. We have therefore three changing structures, a swell-
ing mass, a stretching membrane, and an elOEgating body, each
of which take a part in determining the form of the Inversion.
To these must be added two structures which do not change and
are resistant in their nature. These are the wall of the

ntti-}

.Trrt-M'jtT

30

capsule and the collar surrounding the hole In the capsule,
through which the tubule Is everted. Let us now follow the
interaction of the forces Involved in the behavior of these
changing structures. For this purpose we shall divide the
explosion into four stages.

Stage 1. The eversion of the outer cavity of the tuhule
(Pig. 67). Two forces probably take part in this, the pres-
sure in the capsular cavity and the elongation of the central
body.

Stage 2. The elongation of the everted outer cavity (Pigs.
68 to 71 and 76). This results in the formation of the collar
(Pig. 68,r.). Here again two forces may be involved, the
swelling of the material in the capsular cavity and the fur-
ther elongation of the central body which stretches the por-
tion of the inner tubule which bounds the inner tubular cav-
ity. The fact that the everted portion is sometimes longer
in the axis through which the central body passes indicates
that this body may be exerting an out-pushing force. If this
is the case, we have here an elastic body that has become
active by being released from compression, that is the central
body elongates like a coiled spring. This action is fully
discussed by Koltzoff . The pressure in the capsular cavity
is sometimes showa by the squeezing of the central body out
through the outer end of the tubule when it has lost its re-
sistant properties.

Stage 3. The second eversion of the inner tubule (Pigs.
77 to 79). Prom the condition shown in Pigs. 71 and 76, the
increasing pressure in the capsular cavity causes the wall

31

of the everted tutule to swell up to the form shown in Pig. 72.
Finally the pressure becomes so great that the ring which
formed the division "between the inner and outer tuhular cavi-
ties (Fig. 61, i.t.c. and o.c.) gives away and a part of the
tubule bounding the inner tubular cavity becomes everted.
Portions of the central body often adhere to the wall of the
tubule and are carried outward and so mark the extent of this
second eversion (Fig. 77, g. ) .

Stage 4« The third eversion of the tubule , accompanied
by the inversion of the capsule (Figs. 80 to 82). The in-
ternal pressure continues to increase as is shown by the
bulging out of the walls of the everted portion (Fips. 78 and
79). This brings a strain upon the axis in which the tubule
and central body lie. This tends to stretch these structures
as is shown by the incurving of the apical wall of the everted
portion in Figs. 78 and 79. This causes the base of the evert-
ed part to press on the sides of the capsule. This pressure
on the sides of the capsule together with an up-pulling along
the line of the central body results in turning the capsule
through the collar when the last section of the tubule is
everted. In Fig. 82 the portions of the everted wall con-
tributed by the second, thlrci and fourth stages of the ever-
sion are probably Indicated by the granules g^ and g^. Thus
we see that the whole transformation may be explained by the
increase of pressure in the capsular cavity together with
tensions along the line of the inner tubule and the central
body*.

T«C

32

Efforts were made to reverse this process ty placing the
sperms In very concentrated solutions of the salts used. The
only effect of this treatment was a shrlnkihg of the everted
portion, which would again swell up and the process of eversion
continue when dilute solutions were again used. After the
explosion had reached the stage presented in Pig. 8E the only
part affected by concentrated solutions was the protoplasmic
portion at the bottom. It is also true that this is the only
part that takes methylene blue, methyl green or thronin
stains when these are applied to the living sperms. It was
rather surprising that the contents of the everted capsule
were not stained by these stains. Sometimes a few granules
can be seen in this cavity.

Figures 73, 74, 84 and 85 were made from sperms that had
been kept in a 5% KUO3 solution for 51 days. We observe here
that partial explosion had taken place. Those shown in Figs.
84 and 85 had reached the stage corresponding to Fig. 70.
When these were treated with a solution of lower concentration
the eversion continued but the wall of the part already evert-
ed seemed to be hardened so that it made an elongated collar
through which the further inversion took place.

6. The effect of reagents on the everted sperms.

After trying several fixing reagents it was found that
for imbedding and cutting the crab's eggs Morgan's fluid gave
decidedly the best results. This fluid is a saturated solu-
tion of picric acid in ^0% alcohol, to 100 c.c. of which 2 o.c.
of H2SO4 are added. Sperms in various stages of the process

;jiii:nf tsdcfT'

33

of inversion and those In the normal mature condition were
motinted under the microsoope and killed with this fluid in
order to determine its effect upon them. A considerable
amount of shrinking was observed, hut no decided change in
the relationship of the parts seemed to take place. Figures
86 and 87 were drawn from sperms exploded in distilled water,
fixed in Morgan's fluid, stained in thionin and eosin, dehy-
drated In alcohols, cleared in xylol and mounted in balsam.
The general relations of the parts is the same as in Figs. 81
and 82. The space between the everted tubule and the invert-
ed capsule seems to have shrunk relatively more than the in-
verted capsule. Having now observed the behavior of the
sperms under experimental conditions we may proceed to our
observations concerning the entrance of the sperm and the
process of fertilization.

711. THE ENTRAHCE OF THE SPERM.

Eggs were taken from the lumen of the ovary just after
the crab began to spawn, and were fixed in Morgan's fluid,
imbedded in paraffin and sectioned. A microscopic examina-
tion of these eggs showed the sperm in the act of entering
the egg. The best stain for the study of these sections is
thionin, for it stains the chromosomes in the mitotic figure
of the nucleus of the egg and the nuclear cup of the sperm a
deep blue. It stains the everted portion of the sperm faint-
ly and the food material, cytoplasm and egg-shell are unstain-
ed or only faintly stained. This treatment makes it possible

34

to find these minute structures in the relatively immense egg.
After one has heGorne familiar with these structures and their
position on the egg it is possible to find them quite readily
with other stains such as Delafield's haematoxylin, iron-
haematoxylin, or safranin and Liohtgrun.

The relation of the sperms to eggs taken from the lumen
of the ovary is shown in Pigs. 88 to 92. Sperms in the same
condition are also found In eggs taken from the oviduct as
is shown in Pigs. 9Z and 94. How when these figures are com-
pared with Pigs. 96 and 87, which have been treated with the
same fixing reagents, it is evident that it is the everted
portion of the sperm that has gone through the shell of the
egg. The nuclear cup (n.c. Pigs. 91 and 92) is on the out-
side of the shell. The everted tuhule forms a vesicle with-
in which one sees the Inverted capsule (inv.c. Pig. 91). Hera-
after I shall call this everted tuhule and capsule, the sperm-
vesicle. At the inner-end of this sperm-vesicle the ejected
central hody may he seen (Pigs. 88, 91 to 94 c.b.). That the
part which remains outside is the nuclear cup with its radiat-
ing pseudopodla can he more clearly seen hy a surface view of
the structure as it lies upon the eg^. Such a view is present-
ed in Pig. 95. Furthermore the staining reactions are in ac-
cord with those observed in the mat:ire sperm and the artificial-
ly exploded ones and are as follows:

) the part outside of the shell-blue.
Thionin and eosin (

) the part inside of the shell--red.

„ (part outside of shell--red
Safranin and Liohtgrun )

(part inside, green mixed with red.

35

( part outside of shell, tlack.
Iron-Haematoxylin )

( part inside, 'brown except the central "body
) which is hlaok.

Koltzoff '06 claimed that the sperms. In certain decapods
settled on the egg with the nuclear cup towards the egg and the
capsule pointed away from the egg. He was also of the opinion
that the rehound from the explosion of the capsule was suffi-
cient to drive the nucleus Into the egg. On eggs taken from
the ovary of Menippe mercenaria, I found a few sperms attach-
ed as shown in Fig. 96 with the nuclear cup next to the shell
of the egg. That the eversion of the capsule does not force
the nucleus through the shell in this case is shown in Pig.
97, where a sperm has exploded with the nuclear cup against
the egg. So far as my ohservations go there is no evidence
whatever that the eversion of the capsule causes any sudden
movement of the sperm hody as a whole.

The niimber of sperms that have pierced the shells of the
eggs is much greater for eggs taken from the oviduct than for
those taken from the lumen of the ovary. The ntimher was count-
ed In a few eggs that had Just heen spawned and the number
per egg was as follows: 28,44,52,52,54, 71, 73 and in one ex-
ceptional case 679.

So far there seems to "be no doubt as to the behavior of
the sperm in entering the egg, but we may ask; Is this the
final stage in the entrance of the sperm? Is not the nuclear
cup drawn through the shell at a later stage? If it is not
what becomes of it?

That the nuclear cup does not enter the egg^ but falls
off is shown in Figs. 98 to 101. Here we see that the nuclear

.3,^$.:^

36

cup has moved away from the egg-shell and that a strauid of sorae
substance hy which It was prohably attached to the hotton of
the capsule is drawn out with it. Sometimes the nuclear cup
breaks loose from the strand and leaves it projecting through
the shell into the capsule (Pig. 100). In eggs taken from
the oviduct or from the pleopeds Just after spawning, large
numbers of sperm-vesicles are found sticking to the inside
of the shells after the nuclear cup has fallen off (Pig. 101).
It is very clear then that in most cases at least the nuclear
cup does not enter the egg* But, does it thus fall off from
the particular sperm that fertilizes the egg^ or only from
those which have failed to perform the work of fertilization?
This question can best be answered by a further study of the
events of fertilization.

VIII. PERTILIZATIOBT.

In eggs taken from the oviduct or just after leaving it,
many sperm-vesicles may be found lying on the edge of the
cytoplasm as shown in Pigs. 102 and 103, while one is found
down in the cytoplasm (Pigs. 104-108). Here it is evident
that the movement of the cytoplasm has carried the vesicle
below the surface. Before the sperm-vesicle enters there is
a layer of cytoplasm Just inside of the egg-shell. The rest
of the egg is filled with spherules of food material in the
interspaces of which the cytoplasm extends from the peripheral
layer, by fine strands, all through the egg. The fact, that
the sperules of food move apart and a small mass of cytoplasm

:c?

37

acoompanles the sperm-vesicle into the egg» Is "best explained
"by supposing that the first vesicle that comes in contact
with the cytoplasm initiates a flowing movement of the latter
along the inner surface of the shell, from all sides towards
the newly entered vesicle. The cytoplasm moving thus along
the inner surface of the shell towards one point would he de-
flected in towards the center of the Qggt and would tend to
carry the vesicle in with it. We may suppose further that
when the cytoplasm has once responded to such a stimulus its
physiological state is so changed that it will not respond
to another. As a result only one vesicle becomes imbedded
In the cytoplasm of the egg where it is to be treuasformed in-
to the male pronucleus.

The vitelline membrane (Pig. 104, v.) is formed just after
the entrance of the sperm-vesicle into the cytoplasm and
the vesicles that failed to enter lie between it and the
shell of the egg» The first polar body (Pig. 114) which is
cast off while the eggs are passing through the oviduct is
also found between the vitelline membreme and the shell.

The first step in the transformation of the sperm-vesi-
cle into the male pronucleus is a thickening of its lateral
walls. This may be observed in Pigs, 105 to 111. Accompany-
ing this there is an increase of affinity for the stains used
(thionln and Delafield's haematoxylin) . Uext there seams to
be an extrusion of the old capsular wall which, if we recall
the method of the eversion of the sperm, we know, forms the
inner lining of the sperm-vesicle. The discarding of this
capsular wall was not clearly made out, bnt Pigs. 109 and 110

38

indicate that suoh a change takes place. In eggs taken soon
after spwaning the sperm-vesicle has increased considerably
in size and the wall has taken on a vesicular appearance.
This is shown in Figs. 110 eoid 111 which show the sperm-ve si-
de in two different aspects. The cavity of the vesicle
gradually disappears leaving only a notch on one side (Pigs.
108, 109, 110 and 112). Figure 112 was about an hour old.
In eggs two hours old the vesicle has taken on the appearance
of an ordinary nucleus containing granules of chromatin (Fig.
116). At this time it has gone about one fourth of the dis-
tance from the circumference to the center of the egg (Fig. 117)

We will now turn our attention to the egg-nucleus. When
the eggs are set free in the lumen of the ovary Just before
spawning begins, the spindle for the first maturation division
is already formed and its long axis is parallel with the sur-
face of the egg (Fig. 113). As the egg passes through the
oviduct the spindle turns to a position perpendicular to the
surface of the egg and the first polar body is cut off. This
is shown in Fig. 114 which is from em egg that has Just pass-
ed out of the oviduct. Efforts were made to count the chrom-
osomes just after this division. Twenty-five to twenty-eight
were counted. Thus the double number would be fifty to fifty-
six. They are however so small and placed so closely together
that it is difficult to distinguish them accurately.

Between one and one and a half hours after spawning the
second maturation division takes place. The second polar
body is apparently not cut off, but remains in the egg where
it degenerates and is absorbed by the cytoplasm.

39
The female pronucleus Is now formed and proceeds to the
center of the egg where It meets the male pronucleus. Figures
116 and 116 are drawn from nuclei fixed at 2 hours and 15 min-
utes after spawning. At this time it is not possible to tell
which is the male and which the female pronucleus tmless the
slightly concave side of the one shown in Pig. 116 indicates
that it is related to the nucleus shown in Fig. 112. In this
case it would he the male pronucleus. The nuclei have grown
rapidly and continue to do so until they reach the center of
the ogg. Their contents are finely granular. These granules
increase in size as the nuclei hecome larger. Figures 118 and
119 show the position of the nuclei four hours after spawning
and Fig. 120 (from en agg six hours old) shows them lying side
by side in the center of the egg. They have become elongated
and many times larger than they were when first formed. From
the above description of fertilization it is evident that the
nuclear cup takes no part in the formation of the male pro-
nucleus for the latter is derived from the sperm-vesicle which
is the Inverted capsule.

This completes the description of the structure and be-
havior of the male cells in the stone-crabe. We have here some-
thing unique in the method by which the sperm enters the egg
and something exceptional in the phenomena of fertilization.
These observations raise several theoretical questions, some
of which we will now briefly consider.

IX. Discussion.
We have here a case in which an Infolded vacuole that
arose in the cytoplasm is everted through the shell of the
egg and fertilizes it. How may such an event be brought into

40

harmony with the existing theories ooncernlng the chromoaomes?
In all other cases of fertilization the nucleus with its
chromosomes or at least Its chromatin is considered the essen-
tial thing; the bearer of the paternal qualities to the egg.
The part that they play in the theories concerning heredity
is too Important and useful to be lightly discarded. But,
granting all that is claimed for the chromosomes, we are never-
theless face to face with the fact that in most cases they
disappear during the telophase and are reformed in the next
prophase of cell division. Between the miotic divisions they
can be followed from one spindle to the next and in some other
oases some Investigators have claimed to have been able to ob-
serve the continuity of Individual chromosomes from one divi-
sion of the cell to the next one. But these are exceptions.
The problem of the origin of the chromosome is a real problem.
For many reasons, in our final analysis, we must go back of
the chromosome. So without attacking the proposition that
chromosomes are the means for distributing the hereditary
elements at the time of division, we may take up the question
of the origin of the chromosome before division.

The phenomena described in this paper force us to con-
sider this question if we are to bring the facts concerning
fertilization in this crab, in line with existing theories.
Does any of the chromatin from the nucleus of the spermatid
enter the egg"? We have shown that it is the substance in
the wall or in the cavity of the capsule that enters and
fertilizes the egg' Now is there any evidence that the chrom-
atic substance in the nucleus is transferred to the capsule

snT

ei-ff

41

during spermatogenesis f

Gro'b'ben '78 claimed that the capsule Is derived from the
nucleus of the spermatid. He described a change In the con-
sistency and a reduction in size of the nucleus which occurred
simultaneously with the development of the capsule. He seem-
ed to he of the opinion that the nuclear material was trans-
ferred hy diffusion from the nucleus to the capsule.

Herrmann '90 suggests that when one follows the parallel
transformations of the capsule and the nucleus, one gets the
impression that there Is a sort of migration of the chromatic
suhstance from the nucleus to the capsule.

Brandos '97 found two substances in the nucleus of the
spermatid. One was stained blue with methylene blue, the
other red with acid fuchsln. The latter settles to one side
of the nucleus and then passes out into the cytoplasm.

The later workers Koltzoff '06 and Spltschakoff '09 de-
*^ scribe no such d process.

In my own investigations I have noted a decrease in af-
finity for chromatic stains, and in the size of the nucleus.
The capsule on the other hand showed an increasing affinity
for iron-haematoxylin and safranin. These facts suggest a
transfer of nuclear material.

Finally if we may accept the views of Stauffacher '10
and Dersohan 'll, that basichromatin is derived from oxy-
chromatin, the former being deposited from the latter, we may
postulate a theory for the explanation of the phenomena of
fertilization in this crab. I do not claim that the facts

42

establish the theory. They only suggest it. Some of the
"b as i chromatin in the nucleus of the spermatid is dissolved
by the oxychrooatin and transferred to the capsule. After
the capsule is everted into the egg and has entered the cyto-
plasm of the latter, the b^sichromatin is redeposited and
thus the granular structure of the male pronucleus appears.
It may be possible to explain the number of chromosomes that
appear by supposing that there are a certain number of dif-
ferent kinds of molecules which are deposited out of the
oxyohromatin and that these have such an affinity for each
other that they are aggregated into a definite number of
groups or they may be of such a structural nature than they
can fall only into certain groups. Of course, I claim for
this only that it Is a possible explanation of phenomena
which are apparently not In accord with the conception of an
individual continuity of the chromosomes.

Z. SUMMARY.

1. The seminal elements in Menlppe mercenaria arise
from a single row of primary spermatagonlal cells which per-
sist along one side of the testicular tubule.

2. The tubule is divided into three or four regions by
longitudinal partitions composed of epithelial cells. The
seminal elements in the division next to the row of spermato-
gonlal cells, are younger than those in any other division.
The region on the opposite side contains mature sperms. The
seminal elements In one end of a given division are further

43

along in their development than those In the other end.

g. The spermatogonia! nuclei lie in a oommon cytoplasmic
mass and multiply irregularly without the formation of a
spireme. A spireme and synapsis occur in connection with the
first miotic division. The second miotic division follows
soon after the first.

4. In the mature sperm, the protoplasmic portion, con-
taining the nucleus, is cup- shaped. Prom the rim of the cup
peeudopodia project out like the rays of a star. There Is
a capsule half imhedded in the cup. An inturned tubule is
connected with an opening in the distal portion of the cap-
sular wall and a rod-like central hody arises from the prox-
imal side of the capsule and projects into the inner tuhule.

5. In the transformation of the spermatid, the nucleus
becomes uniform in consistency, reduced in size and cup-shaped.
A mitochrondrial ring is formed between the nucleus and the
capsule. The capsule arises as a vacuole in the cytoplasm. In
the course of its development it shows an increasing affinity
for nuclear steins.

6. The central body develops from a granule that appears
on the proximal side of the capsule. The inner tubule is
formed from two vesicles that arise at the distal end of the
central body.

7. Hypotonic solutions of various salts and possibly
other stimuli cause a lengthening of the central body, an
eversion of the inner tubule and an inversion of the wall
of the capsule.

8. When the sperms come in contact with the egg under

?er t-Xi

44

normal conditions, the capsule is usually applied to the shell
of the egg and the nuclear cup is directed away from the egg.
In this position eversion takes place and the ejected central
hody, the inner tubule and the capsule with its contents are
thus turned through the shell into the egg.

9. The nuclear cup is left on the outside of the egg
and it soon falls off.

10. The wall of the capsule together with its everted
contents, which we now call the sperm- vesicle, sinks into
the cytoplams of the egg^ where it is enlarged and transform-
ed into the male pronucleus.

11. The contents of the capsule may he derived from
the nucleus of the spermatid and is prohahly oxychromatin
which deposits hasichromatin after it enters the egg and so
gives rise to the chromosomes in the male pronucleus.

(TITO

45

LITERATURE CITED.

Brandes, G. '97, Die Spermatozoen der Dekapoden. Sitzungsbe-
richte d.K.P. Akad. d. Wlssen, Berlin, 1R97.

II
Derschan, M. '11, ITber KermbruGken nnd Kernsubstance in pflanz-

lichen Zellen. Arch. f. Zellforseh. Bd.7.

Gllson, Gr. '86, Etude comparee de la spermatogenese chez les
arthropodes. La Cellule, Tome II.

Grobben, C. '78, Baitrage zur Keuntniss der mazmllchen

Geschlechtsorgane der Dekapoden. Arbeit. a. d.
Zool. Inst. d. Univ. Wien. Bd. i.

Herrmann, G.'90, Botes sur la structure et le developpement
des spermatozoides chez les Decapodes. Bull.
Sc. de la Prance et de la Belgique. Vol. XZII.

Koltzoff, U.K. '06, Studlen uber die Gestalt der Zelle . I. Un-

tersuchimgen uber die Spermlen der Decapod eh,
als Einleitung das Problem der Zellengestalt.
Arch.f. mikros. Anat. Bd. 67.

Sabatler, A. '93, De la Spermatogenese chez les Crustaces
Decapodes. Travaux d.l'Inst d. Zool. de
Montpellier et Sta. ilar. Gette. 3er.9. Mem.
No. 3.

Spitschakoff , Th. Spermohistogenese bei Gariden. Archiv.f .
Zellforschung-. III.

Stanffacher, Hch. Beitrage zur Zenntniss der Zernstrukturen.
Zeitschrift f. wissench. Zoologue XGV. Bd.

46

BXPLAUATION OP THK i<'IGURES.

Fig. 1. Transverse section of a testicular tubule of a
small crat. Fixed In Petrunkewitsch* s fluid. Z600 Spermatogonia
at the top* Sperms at the "bottom, spermatids between.

Fig. 2. Transverse section of testicular tubule, p.s.,
primary spermatogonlal cell. The mass of spermatogonia larger
than in Pig. 1.

Fig. 3. Transverse section of testicular tubule showing
larger mass of spermatogonia, p.s., primary spermatogonlal
cell.

Fig. 4. Transverse section of testicular tubule showing
a large mass of spermatocytes in synapsis, p.s., primary
spermatogonlal cell.

Figures 5-llS (except 7u-72) were all drawn with the
camera lucida and a Zeiss 1.5 m.m. apochromatic objective and
a compensating ocular (either a No. 6 or XJo. 8). Then the
drawings were enlarged so that in the plates there is a mag-
nification of 3000 diameters. In making Pigs. 7u-72 the
camera lucida was not used.

Fig. 5. Resting phase of a spermatogonlal nucleus.

Fig. 6« Prophase of a spermatogonlal nucleus showing
the paired chromosomes which were seen in the upper one-half
of the nucleus.

Fig. 7. Optical section df a spermatogonlal nucleus
showing the peripheral arrangement of the paired chromosomes
in the prophase.

Fig. 8» The mitotic figure in a spermatogonlal division.

Fig. 9. Early prophase of the first miotic division of
the spermatocyte .

Fig. 10. SytapslB in the first miotic division of a
spermatocyte.

JTlgs. 11 and 12. The stage following synapsis showing
the spireme loosened up and separating into chromosomes.

Fig. 13. First miotic division, stage Jxlst preceding
the formation of the equatorial plate.

Fig. 14. Equatorial plate and spindle in first miotic
division.

Fig. 15. A tripolar division of the nucleus of a sperma-

47

tocyte of the first order. 15a is a drawing of the same
nucleus made from a lower plain and showing a portion e. of
the chromatin which was not included in the equatorial plate.

Pig. 16. Chromosomes found In the equatorial plate of
the first miotic division.

Figs. 16a and 16h. Portions of the first miotic figure
in the metaphase, showing the chromosomes.

Figs. 17 and 18. Two stages in the anaphase of the
first miotsis.

Figs. 19-22. Show different stages and variations in the
early telophase of the first miotic division. Interzonal
flhers are shown stretching hetween the masses of chromatin
and the mld-hody apparently forming a hand around the flhers.

Figs. 23-25. The second miotic division: Stages in the
formation of the equatorial plate.

Fig. 26. The metaphase of the second miotic division.

Fig. 27. Telophase of second miotic division, showing
Intersonal flhers and the mid-body.

Fig. 28. Later telophase. The centrosome is still vis-
ible here and is still attached hy flhers to the nucleus. A
clear area also surrounds the nucleus.

gigs. 29-52. Different stages In the formation of a
reticulate nucleus in the spermatid. A black granule, the
centrosome may be seen and a more or less complete zone free
from granules around the nucleus.

Figs. 55-56. Spermatids with vacuoles in the cytoplasm,
c.a. mass of finely granular cytoplasm which may be the mlto-
chrondria.

Fig. 57. Spermatid with vacuole or capsule and nucleus.
The periphery of nucleus more densely stained than center.

Fig. 58. Spermatid showing a small clear vacuole in the
nucleus.

Figs. 59-42. Later stages in the transformation of the
spermatids mt. mitochondrial substance.

Pig. 45. Spermatid showing two sides of a dark ring,
d. in the capsule; mt. mitochondria, also the central body
on the nucleus at the bottom of the capsule.

Fig. 44. Spermatid showing two granules on the border
line between the nucleus and capsule.

48

Figa. 45-47 » Spermatid from different view points show-
ing the mitochondria, mt. and a granule. In "bottom of cap-
sule.

Plg» 48« Spermatid with mitochondrial ring completed.

Pigs. 49-60. Spermatids showing stages In the develop-
ment of the central "body and the Inner tuhule within the cap-
sule. A clear space appears for a time "between the capsule
and the nucleus.

Pig. 61. Mature sperm, fixed In vapor of osmic acid and
stained In gold chloride preparation, n.c, nucleus cup; p.s.,
pseudopodla; c, wall of the capsule; c.c, cavity of the
capsule; l.t., inner tubule; l.t.c. Inner cavity of the
Inner tuhule; c.b., central body; o.c. outer cavity of the
inner tubule.

Pig. 52. Sperm viewed from the top, mounted In 4% M0„.

Fig. 63. Sperm mounted In the serum of the crab's blood.

Pig. 64. Side view of sperm In the serum of the blood.

Pig. 65. Sperm in serum with pseudopodla all turned to
one side by currents In the serum.

Pig. 66. Sperm in testicular tubule. Fixed in Worcester's
fluid. Stained In Iron-haematoxylln, central body projecting
from the top.

Pig. 67. A sperm treated with 3% EIO.'^, fixed with Morgan's
fluid and stained with Delafleld's haematoxylln.

Fig. 68. A sperm treated with 3% KtlOs, fixed with Mor-
gan's fluid and stained with eosln.

Figs. 69-85. Were all drawn from living sperms that had
been treated with hjrpotonie solutions of salts, and show
various stages in the eversion of the capsule, r.. the collar;
c.b., central body; Inv.c. Inverted capsule; g, g-^ and g^,
pieces of the central body on the everted wall of the inner
tubule.

Figs. 86-87. Sperms that were exploded in distilled water
and fixed in Morgan's fluid then stained with thlonln and
eosln.

Figs. 88 to 92. F.verted sperms extending through the shell
of the egg, from the lumen of the ovary; fixed in Morgan's
fluid. In Fig. 89 the shell of the egg Is badly warped, c.b.
ejected central body; Inv.c, Inverted capsule; n.c, nuclear
cup; r., collar of the capsule.

49

Figs. 93 and 94 » Everted sperms in the shells of eggs from
the oviduct, c.h., central hody ejected; r. , collar.

Fig. 95. The portion of the sperm v/hlch remains on the
outside of the egg seen from the hottom of the nuclear cup.

Pig. 96. Sperm on the shell of the egg with nuclear cup
next to the shell.

Pig. 97. A sperm that has exploded with nucleus next to
the egg.

Figs. 98-111. Portions of eggs with sperms, taken from
the oviduct or soon after leaving It.

Figs. 98 and 99. Show the nuclear cup falling away from
the egg and pulling a strand of some suhstance out with it.

Fig. 100. Sperm-vesicle with the strand from which the
nuclear cup has hroken away, projecting through the shell
into the vesicle.

Pig. 101. Sperm-vesicle just inside the shell.

Pigs. 102 and 103. Sperm-vesicles lying on the cytoplasm.

Fig. 104. Sperm-vesicle that has just entered the cyto-
plasm, v., vitelline memhrane.

Pigs. 105-108. Sperm-vesicles down in the cytoplasm.

Pig. 109. Sperm-vesicle with capsular wall projecting
out of it.

Pig. 110. Sperm-vesicle seen from one side; shows vesi-
cular structure.

Pig. 111. Sperm-vesicle seen from top-vesicular struc-
ture.

Pig. 112. Sperm-vesicle in egg 1 hour, 15 minutes old.
This may now "be called the male pronucleus.

Pig. 113. 'Rgg from the lumen of the ovary showing the
spindle of the first miotic division.

Fig. 114. First polar body of an egg Just spawned.

Pigs. 115 and 116. Two pronuclei found in one egg Z
\ and 15 minutes old.

Fig. 117. Egg 2 hours 15 minutes old, h., transverse sec-

50

tlon of a hair of the pleopod with the shell of the egg wrap-
ed part of the way around it; n., pronucleus, prohahly the
male; v., vitelline memhrane. X425.

Figs. 118 and 119 » Egg 4 hours old showing the pronuclei.

Fig« 120. Egg 6 hours old showing the pronuclei side hy
side in the center of the egg.

IlKi

121. Diagram of the ovary, the seminal receptacle
The latter turned out to one side so as to

and oviduct.

hring it in the same plane with the rest of the ovary; c,
portion of the seminal receptacle lined with chitin; g.,
glandular portion of the seminal receptacle; o., opening
between the glandular and chitinous portion of the recept-
acle; od., oviduct.

Fig. 122. Seminal receptacle at the time of Molting,
c, chitinous portion; g. , glandular portion.

51

VITA.

I was Ijorn near Carthage, Rush County, Indiana, July
15, 1676, and am the son of Josiah and Margaret F. Binford.
My family moved to western Kansas in 1885. I graduated from
the Haviland Academy In 1896. Then I taught a district
school one year, studied at warlham College, Richmond, Ind.
two years, and again taught a district school at Haviland,
Kansas for one year and the next year returned to Sarlham
College where I received the B.3. degree in 1901. Since
1901 I have held the position of Professor of Biology at
Guilford College, U.C. During the summer quarters of 19u2
and 1903, and for five quarters in 1905- '06 I studied in the
University of Chicago, devoting my attention principally to
the subject of botany, and received the degree of S.M. from
that institution.

During the academic year of 1907- '08 I devoted my time
to zoological studies at Johns Hopkins University. I was
scientific assistant at the U.S. Marine Biological Labora-
tory at Beaufort, H.G. during the four summers; 1908, '09, '10
and '11. At this laboratory I worked on the life histories
of several crabs, though primarily investigating that of
Menippe raercenaria.

In the fall of 1910 I returned to the Johns Hopkins Uni-
versity where the past two years have been spent in working
up the foregoing dissertation and in other zoological studies.

Raymond Binford.
Baltimore, Md. May 1, 1912.

i*».;

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