T E E GERM-CELLS OF GRANTIA UOMPRESYA
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261
The Germ.Cells. Fertilization. and Early Development of Grantia ($'con)
conywessa *. By J . BRONTG
GATENBY.B.A., B.Sc., D.Phil., 8enior
Deiny. Magdaleii College. Oxford ; Senior Assistant in Zoology. Lecturer
in Cy tologg. University College. London (Conimiinicated by Prof . E 8
.
aOODRICH.
..
F.R.S., sec.L.8.)
(PLATEB19-23; and 4 Text-figures.)
[Read 11th December. 1919.1
.
CONTENTS
1. Introductory ........................................................
2. General Statement of the Problems investigated ......................
3. Previous Work ....................................................
4. Material and Nethods ..............................................
6 Genertd Account of the Development of a Sycon, up to the Formation of the
Free-swimming Amphiblastula Larva ................................
6 The Structure of the Amphiblastula Larva ..............................
7 The Minute Cytology of the Cfastral Epithelial or Collar Cull of Grantia ....
8. Amoeboid Cell-elements of Gruntia ...................................
9 The Question of the Origin of the Germ-cells ............................
10. How Collar-cells become Amoeboid Cells of the Mesoglea . . . . . . . . . . . . . . . .
11 Formation of Oogonia in d f u from Collar-cdls ..........................
12. Position of Germ-cells in Gruntiu conzpressu ............................
13. Spermatogenesis in Grantin compressa ..................................
14. General Description of the lnclusions of the Cytoplasm in Oogeuesiv. . . . . . . .
16. Ectoplasmic and Jhdoplnsmic Regions in the Egg ......................
16. The Problem of the Polarity of the Oocpte, the Ovum, and the Embryo . . . .
17 Preliminary Outline of Events lotiding to the Union of Sperm nud Egg . . . .
18 Evidence as to the True Identity of the Sperm-carrying Cell . . . . . . . . . . . . . .
19. Behaviour of Sperm after Entry into Collar-cell ........................
80. Changes in the Collar-cell brought about by the Entry of the Spermatozoon .
21. The Fate of the Sperm-carrying Cell after the Sperm pasees into the Oocyte .
22. Behaviour of Sperm and Sperm-bearing Cell explicable as a Chemotactic
Phenomenon ......................................................
23. The Entry of the Spermatozoon into the Egg and the Effect on both Gametes.
24 . The Fate of the Middle-piece or Sperm Macromitosome ("X benkern ") during
Fertilization ......................................................
36. The Mitochondria in the Development of Grantia compress@ ..............
36. Differentiation of the Nuclei of the Blastula-wall heralding the Formation of
TwoDefiniteTissues ..............................................
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* [The materials and part of the reagents used in this research were purchased by a
Government Resenrcli Great of the Royal Society, for whicli I express 'my grateful
thanks.]
&I"JOURN.-ZOOLOGY,
VOL XXXIV
20
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262
DR. J. B. GATENBP ON THE GERM-(IELLS ANT)
27. DI~CESSION
.......................................................
( a ) Organ-formin Substances? ....................................
b) Chromidia an! Mitochondria .................................
...............................................
considered as a Mesogled Smoebocyte ? , .
(e) Germ-cells and Sex in Sponges ................................
(f)Srmatogeneaia in Grantin. ...................................
(y) T e Colln.r-cells as the Dominant and most Charncteristic Timue of the
Sponge ..................................................
(a) FinalRemnrks ..............................................
28. SEMWARY.
.........................................................
29. BIBLIOQRAPHY..
....................................................
DEBCRIPTION
OF THE PLATRE,.
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[c)
Fertilization
( d ) The Sperm-carrying Cell
Page
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1. Int roiluct owj.
THE following investigation into the gametogenesis, fertilization, and early
development of a common British sponge, Grantia compressa, constitutes the
opening of a new field of research in sponge-embryology, for the new
inethods of cytology have been used with fruitful results, ant1 for the first
time.
Quite recently Professor Arthur Dendy has published a papor in the
‘ Quarterly Journal of Microscopical Science’, on “ The Gametogenesis of
Grantia conzpressa,” in which he has described many interesting facts.
Only such problems as those not treated by Professor Dendy have been dealt
with here : for a long description of the nuclear phenomena in the germ-cell
cycle of a Sycon, Jorgensen’s paper (8a) may be consulted.
In this paper I atn able to announce a remarkable discovery in the early
stilges of conjugation of the gametes in Grantia compressa and Sycon eiliatum.
This work was partly carried out a t Oxford in Professor Sherrington’s
laboratory, but niainly at Universi t8yCollege, London.
Some lime was also spent a t King’s College, Strand, London, in going
through some of Professor Dendy’s preparations. I t is a pleasure for me to
acknowledge the great help I have derived from various discussions with
Professor Dendy, who has carritid out such valuable invebtigations on tlie
Porifera. Professor Dendy has followed with much interest this applicution
of the modern technique to sponge-enibryology.
2 . General Statement of the Prollenas iilaustigated.
It is a well-known fact that the amphiblttstuln larvz of the Syconidae are
formed irtainly of two sorts bf cells-flagellilted and granular. According to
previous investigations, the cytoplasm in the two sorts of cells is of different
quality; it was to examine this phenomenon that the present work was
undertaken. I t was necessary to see whether, during cleavage, there was a
special segregaiion in any blastonieres of granules or organ-forming substances such as occurs in Aacidia (1). I n addition, it mas planned to study
the fate of the middle-piece of the spermatozoon dpring fertilization. As is
EARLY DEVELOPMENT OF.GRANTIA COMPRESBA.
263
now well known, some workers claim that the middle-piece of the animal
sperm breaks up during fertilization, and growing i u the egg-cytoplasm
takes part i n the phenomenon of amphiinixis.
En pussunt, I examined the various statements with regard to the origin
of the germ-cells in sponges. Dendy (2), in his liite paper, supports
Haeckel’s view that the germ-cells are foriiied from metamorphosed collarcells, a view which is strenuously opposed by the Neo-Weismannians. As
the work progressed, I found that two well-known apongologists, Jorgensen
and GBrich, had made an extraordinary misiiitcrprctation of tlis early stages
in fertilization. This paper gives the first account of the peculiar fertilization of the sponge and of the spermatogenesis of a marine sponge.
3. Previozrs Work.
There is no previous work on Grantia (Sycon) executed by any observer
acquainted with modern interpretation as to the bodies in the cell. There
are, however, several papers by earlier authors which itre important ; I refer
to those of Giirich (6), Jijrgensen (8a), Maas (lo), and Dendy (2). J a n
Hirschler (8) has examined the collar-cells of Xpongillu, while various other
authors have written papers on the development and IarvE. The papers of
Garich and Jorgensen are reviewed by Dendy (2), and will only he mentioned
in the text when neceesary.
Maas (10) in Sycundra ruphanus describes the maturation and fertilization.
The sperm penetrates the egg before the formation of the second polar body.
With regard to the entry of the sperm, l e says : “ Der Sperimkerii zeigt
sich beim Eiiitritt hestehend aus einer einzigen dichten Chrematinmasse von
Bohnenform und einem dahinter liegenden stark lichtbrechenden Kiirper,
beides umgeben van Zone verdichteten Plasmas mit sehr intensiver radijirer
Strahlung.” The “ stark lichtbrechenden Kiirper ” is the ‘‘ (~hromidium”
of JSrgensen. The two pronuclei come to lie in the central region of the
ovum, chromosomes appear in each, and tho first segmentation-spindle is
formed. J a n Hirschler (8) describes briefly the Golgi body in the flagellated
cells of Spongilla. It lies beneath the collar, is spherical, and resembles that
of the central capsule (idiozome) in many metazoan germ-cells. This author
also notes sniall granules of mitochondria1 nature. H e describes no stapes
in gametogenesis or development. Dendy (2), in a late paper, describes the
gametogenesis of Gruntiu compressa. H e adopts Haeclrel’s (7) idea that
collar-cells can metamorphose into germ-cells. He describes oogenesis in
detail, deriving oogonia From collar-cells, “ which accumulate reserve material,
enlarge, withdraw their collars and flagella, become amwhoid and wander
into the mesoglea, re-entering the chambers before dividing mitotically into
oogonia of the second generation.” Dendy finds ‘‘ chromidin ” in the oocyte
cytoplasm, wliich lie thinks are formed by :i“ process of exteiisive chroiiiidiuni20*
264
DR. J. B. aATENBY ON THE GERM-UELLS AND
forniation by extrusion of chroinatin from the nucleolus into the cytoplasm.”
Dendy’s “ chrornidia ” are the mitochondria ; so far as I can niake out, Dendy
does not describe or figure triie yolk, nor has any other author done so.
Dendy shows that during oogenesis young oocytes engulf entire cells,
which are brought to thein hy special carrier (nurse) cells. This process is
very peculiar, and, so far as I can remember, unique in the Animal Kingdom.
Dondy also describes a reiriarliable process in which the nucleolus becomes
squeezed out to form lunips, which are extruded into the cytoplasm, and
from these Innips he coiisiders the chromidia are formed.
I n his plate 24, figure 52, he gives a drawing of wh:it we now know to be
the fertilization. Dendy tacitly accepted JSrgensen’s interpretation of these
stages, but did not make a special study of developillelit later than the fullgrown oocyte.
Dendy’s account of the origin of germ-cells from collar-cells has nut met
with any sort of generul ncceptmce, principally because ISngli*h zoologists
have allowed themselves to be influenced by Weismann’a germ-plasm theory
in an uncompromising form. More wlll be said of this matter Inter on. I t
is tilerely necessary to add that tho other view about the origin of the gernicells in sponges derives thein from waiidering amoeboid cells of the iiiesogleii, and the wandericg amceboid cells lire themselves derived during early
larval life froin posterior griinular cells or archeocytes (Minchin, 9).
Both JSrgensen and Dendg describe n process whereby a part of one of
the pronuclei occasionally beooines sapurated, and lies in the cytoplasni as
an accessory separate nucleus, or <‘ karyornere.” Such a phenomenon does
not happen, according to my own observiition, in healthy cells, and I consider
that the “ karyomeres ” are possibly acceesory sperni-pronuelei, and that
such a cell as drawn by Dendy in his pl. 26, fig. 95, is a case OF polyspermy.
Subsequently to the writing of the above, I have seen many of Professor
Dendy’s preparations showing young oocytes engulfing other cells (see (2)
pl. 24.figs. 50 & 51), and have found similar stages in my own slides. I
accept fully this pitrt of ProEessor Dendy’s account, and draw attention here
to the extraordinary character of this phenomenon. The oocyte of &ll71tilc
in a veritable phagocyte, and markedly amceboid ant1 peripatetic.
4. Material and Methods.
The sponges used for this research were procured from the Plymouth
Biological Station during the months of July and August, 1919 ; the species
used was Grantia compreesa, sometimes known as Sycon compwssum. Special
fixatives with directions for use were sent to Plymouth, and the specimens
were preserved there. I have to thank Dr. Allen, F.R.S., for seeing that
my directions were carried out. For soiiie reason or other many of the
pieces of sponge showed signs of having died of asphyxiation, and, though
BAHLP Ul?JELOPMENT OF GRANTIA CONPRESSA.
265
the methods wed are the best known to modern cytology, I cannot say that
my material was, as a whole, completelg satisfactorg ; I can onlyassuine that
soine of the sponges were not transferred to the fixatives quickly enough after
their collection. For this reason I have been somewhat handicappod, and
have had a certain measure of difficulty in working out part of my results ;
nevertheless, among the large numbers of slides prepared some perfect
examples were procured.
The methods which gave the best results, and which future workers may
use with confidence, are as follows : (1) Clliampy-Kull ; (2) Hermann-or
Fleiiiming-without acetic acid followed by two or three days’ mordnnting in
3 per cent. K2Cr2O7j (3) Kopscli’s original method. For these techniques
see my late paper (4). I n addition, I tried the method of Cajal aud used
glycogen techniques. I had access to Professor E. S. Goodrich’s foriiiolFlemming material oE Sycon ciliatum, and at IL late stage of this work to
Professor Dendy’s slides. I also had at my disposal the sponge collections
at Oxford, and a t University College, London. I have therefore based my
remarks and figures on a very extensive colleotion. On inj Plates the
collar-cells are drawn from some very successful Kopsch preparations.
5. General Account oj*the Uerelopment oj’ a Sycogi, up to the Formation of tlie
Free-switnming Amnphiblastula Larva.
The ovum undergoes total and regular cleavage, resulting i n tlie forniiltion
of a hollow blastula lying beneath the collar epitheliutn, PI. 21. figs. 19, 20,
22, and 21.
For some time the walls of the blastula are formed of cells, which are
suboqual in size and general appearance (PI. 21. fig. 21).
Subsoquently one hemisphere of the blastula, generally that which lies
iuimedihtely beneath the nearest gastral cavity, and touching the collar
epithelium, becomes modified to form a regular epithelium of deep cells,
with small dense nuclei like those of the choanocytes ; flagella soon appear
on these cells.
The other hemisphere, generally that which lies away from the epithelium
of the gastral cavity, and in contact with the mesoglea, becoinee changed
very little, its cells dividing less rapidly and reinuining clenrer and larger.
Examined fresh, much pigmented yolk is seen to repose i n the former
(flagellated) cells, while such granules of yolk are less evident) in the latter
(granular) cells. According to the late Professor Mincliin (Q), the freeswimming larva contains three cell-elements, “ columnar flagellated cells at
the anterior pole, rounded non-flagellated cells :It tlie posterior pole, and :I
central inass of granular amoebocgtes.” In all probability the latter are
derived from the flagellated cells by a process of‘ i m m i g d i o n inwards into
the reduced blnstocael.
266
DR. J. B. GATENBY ON THE QERM-UELLS AND
6 . Tile Structure qf the Amnphiblastulu Larva.
The amphiblastula larvas of Grantia contpressa develop under the
flagellated collar-epithelium, and are partially or wholly surrounded by a
capsule of nutrient squainous cells. I n PI. 23. figs: 28 and 32 the capsule
is marked LGC ; a t first I believed that the capsule was really part of the
larva-that is embryonic tissue-but Professor Dendy kindly pointed out its
true nature ; this partly investing tnembrane is formed of inaternul cells,
and when the larva breaks forth from the sponge, the capsule Is left behind.
The subject of ihis nutrient capsule will be dealt with below.
When the larva, after having become free, is extimined alive, it will be
noted that the histocytes or flagellated cells, while being clear a t their outer
extremities, contain in their inner ends abutting against the blastoccel, a
large number of brownish-yellow yolk-spheres : in other species of spongelarvs, the colour of the yolk may be much brighter. In the fresh, the
archeocytes or posterior granular cell8 are found to contain fewer yolkspheres, or at least they are not grouped in such a way as to forin a
conspicuous coloured band as in the flagellated cells.
Now if the sponge, is fixed in Clhanipy's chrome-osmium mixture, and
treated as elsewhere described (4) by the (Ihampy-Kull method, in the
embryos true yolk is greenish-brown, mitochondria are red, and nuclear
substance is bluish-green. Moreover, by the Flemming-wi thout-acetic
acid and Iron Esematoxylin method, the yolk is greenish-brown, the
initoohondria are black. By Altmrtnn's method, yolk is greenish-brown and
mitochondria red.
As is well known, if one fixes the sponge in highly coagulative fluids such
as alcohol, corrosive acetic or Bouin's fluid, the posterior granular cells
become more chromophile and granular on staining than the flagellated
hy stocy tes.
Nevertheless, I cannot say that this more marked granulation in the
posterior cells is traceable to a greater content in the latter of either
initochondrial or yolk spheres. On the contrary, niost of the granular celle
contain fewer grauules of yolk or mitochondria than the histocytes. This
can be seen on examining PI. 23.figs. 28 and 32.
Some amphihlastulas embryos prepared in alcohol and stained in carmine
showed the posterior or lower cells much denser and chrornophile than the
flagellated cells, while yolk and mitochondria wew absent or, at the best,
hardly observable. I trace the difference in quality of the granular and
flagellated cells not to their contained granules, but to certain differences in
the ground-cytoplasm, which in the granular cells is coarser and more
ahromophile on fixation in alcohol-acetic-corrosive liquids.
The cytoplasni of the granular cells is extremely oxyphile, nuclei are
rimpliopliile ;inti con t,iiiri :I true 1il:isinosonie ; it] iodine the granular cells go
EARLY DEVELOPMENT OF GRANTIA COMPRESSA.
267
much darker than the flagellated cells. The nuclei of the flagellated cells
are basophile and the cytoplasm only faintly oxyphile. The mitochondria of
the amphiblastula larva are not pigmented i n the fresh state. I n P1. 23.
fig. 31 is drawn a t a higher magnification a posterior granular cell. I n this
there are both yolli and mitochondrial spheres (Y and M) and also probably
Golgi elements (GX) ; the nuclei of these cells are most characteristic, being
large, pale, and with an open reticulum. By the chrome-osmium method of
fixation, the ground-cytoplasm of these cells is quite smooth and fine,
The minute structure, of the flagellated cell is interesting, for the yolkgranules are aggregated into a special group beneath the nucleus and at the
inner pole of tho cell, as in P1. 23.fig. 33, Y ; the mitochondrial granules
also lie nearly in the same region. o n the periphery of the flagellated cells
are found certain eriiginatic granules whose histochemical reactions are
unlike those of either yolk or mitochondria ; these fine granules are inarked
OG in PI. 23. fig. 33.
The nucleus is formed of a dense basophil nialrix in which a nucleolus is
visible. On the outer side of the nucleus is u coarse granule from which
originates the flagellum. This coarse granule is apparetl tly the Golgi
apparatus, which surrounds the centrosome (see GA in P1. 23. fig, 33).
Lying inside the sin:tll blilstocel are found a few stnall ameboid cells, which
have a nucleus like that of the flagellated cell ; the cytoplasin of these cells is
craininsd with mitochondrja; in P1. 23. figs. 28 and 32 such cells are inarked
GAMYand in P1. 23. fig. 30 one of these cells is drawn at a higher power.
I conld not find any yolk-granules in these calls-only mitochondria.
Apart froni these three cell-constituents, the sponge larva, while still i n
situ within the maternal tissues, is found to be invested by a cell-capsule, as
in P1. 23. fig. 32 a t LGC' ; these cells are seen to contain a large nuniber OE
mitochondria of somewhat irregular shape ; i n imperfect preparations this
layer early peels off froin the posterior granular cells. I n P1. 23.fig. 29 one
of these capsule-cells is drawn a t a higher power. They contain mainly
mitochondria, but no certain y olk-spheres.
So far as I cnn understand, the capsule around the Grantia larva is formed
just after the period of differsi~t~iation
of the flagellated hemisphere, froin
ameboid elements of the mesoglea. The matter, however, is far froin heing
settled. There is on0 point to which I must draw attention : tlie maternal
nutrient capsule often simply surrounds the cells of tlie posterior pole of the
larva as in P1. 23. fig. 32, but in other cases the maternal cells partly tend
to penetrate between aiid well into the granular posterior cells 11s in
PI. 23. fig. 28 at LGC'. This niatter will be further investigated by me.
268
DR. J. B. GATENBY ON THE GERM-OELLS AND
7 . The Minute Cytology Ofthe Gastral Epithelial or
Collar Cell of Grantia.
Hirschler (8) was the first to show the presence of a typical Golgi
apparatus in the collar cell of a sponge (S'ongilla)
. U ~ ~ f o r t ~ ~ n this
i ~ t eable
ly
observer did not push his work very far, and we do not know in S~~ongilla
in what way the Golgi appar&us is related to the centrosome or flagellum.
The technique indicated for work on the Golgi apparatus of sponges is that
of Kopsch (4) ; pieces of sponge are left for two weeks in 2 per cent. OsO,
and then washed, dehydrated, and embedded in hard wax. The sections are
cut as thinly as possible tind may be treated in turpentine to clear them
somewhat-five minutes is long enough for thin sections. I n Grantia such
preparations show the Golgi apparatus in the collar-cells in the same position
as indicated by Hirschler for Sponyilla. PI. 19. figs. 1, 2, 3, and 4 are drawn
from preparations of collar-cells, to show their minute structure. I n PI. 19.
fig. 3 is a group of cells fixed in Hermann-without-acetic acid, mordanted
in K,Cr,O, and stained in either Iron Eamatoxylin or Altniann. The
granules contained in these cells are, I believe, the true mitochondria.
Hirschler (8) figures such fine mitochondria1 granules in Spongilla, and I
think there can be no doubt as to the presence of mitochondria in the flagellated cells of GrBantia.
I n P1. 19. fig. 2 is drawn a Kopsch preparation of Grantia cornpressa to
show the Golgi apparatus; as in Simu$la, it is a spherical or irregular
object lying on the periphery of the cell and staining dark brown or black
in osinic acid. In these cells the other cytoplasmic granules become light
brown. P1. 19.fig. 4 is a liigh-power drawing of a collar-cell treated by
Kopsch. The Golgi apparatus is seen to be formed of a central archoplasmic
sphere (AS), surrounded by a rind of Golgi apparatus substance (GA) ;
this preparation was differentiated in turpentine, while the apparatus in
PI. 19.tig. 1 A & B was left untreated.
I n many cases, such as in PI. 19.fig. 1 A, it can be shown that the flagellum
passes into the apparatus, but in otheis, such as in PI. 19. fig. 1 B, the
centrosome is distinct from the archoplasin and lies on the nucleus. I think
that in most examples the cerrtrosome lies inside the Golgi-cum-archoplasm
apparatus, and the filament issues from the latter. I n P1. 19. fig. 4, below
the apparatus at C: ?, lay a granule which is possibly the centrosome. Now
besides the mitochondria, which in some cases may he mucli finer than
depicted in PI. 19.fig. 3, there are other much larger granules marked Y in
PI. 19.figs. 1 and 4. These are difficult to distinguish from the initochondriii,
and they may stain black by prolonged immersion in Iron Hsematoxylin ;
by staining in Altmann they are not tinged, and then can be distinguished
from the mitochondria. I n Cajal's silver nitratu method the granules may
EARLY DEVELOPMENT OF GRANTIA UOMPBICSSA.
269
go intensely brown or black.
Such large apparently non-mitochondria1
granules are possibly yollry bodiecl partly formed of a dense proteid basis,
whence their histochemical behaviour.
Presumably these nulrient granules are tlie direct descendants of those
yolk-spheres which one finds in the histocytes or flagellated cells of the
amphiblastula larva (PI, 23.fig. 28,Y,and PI. 23.fig. 32, Y). Nevertheless,
such large nutrient granules of the flagellated collar-cell of Grantia are not
quite liko tlie yolk-spheres of the eiiibryonic histocyte ; the former granule
has a heavier proteid basis, which takes crystal violet and haematoxylin, as
well its occasional impregnatioii in silver nitrate. At one stage of this work
I did not feel sure that the nutrient granule and the mitochondriuni of the
collar-cell could be distinguished from each other, but some of my later
preparations appeared conclusive. Probably the most convincing evidence
is acquired by exaniining two sets of preparations, one by the Kopsch,
the other by the Fleniming-without-acetic method (4). I n the foriner,
yolk or nutrient bodies are markedly demonstrated, ill tlie latter such bodies
are not properly shou n, while mitochondria stain densely.
8. Ammboid Cell-elements of Grantia.
Besides the ordinary flagellated cells in situ lining the gastral cavities, one
finds many wandering mesogleal cells whose origin can be traced to the
flagellated cells, as depicted in P1. 22. fig. 24 (see also PI. 19. fig. 7).
Such amoeboid cells are the commonest elements of the mesoglea and very
probably give rise to both spermatozoa and ova. I n addition to the ordinary
amoeboid cells, whose cytoplasmic inclusions much resemble those of the
collar-cells, one occasionally finds small cells loaded with mitochondria.
These granular cells are very distinctive, and examples of them itre seen at
GAM in P1. 21.fig. 21 and PI. 22. fig. 23. Such cells contain in their
oytoplasm a large number of mitochondria1 granules, and very little of any
nudrient yolk-spheres. They are probably formed from the histocytes
during late larval and early pupal life, as shown in P1. 23. fig. 28, GAM,
and in P1. 23,fig. 30. I do not intend here to enter into the questions
surrounding other elements of the sponge, but the two kinds of cells abovementioned are the only sorts of ameboid cells which have been met with by
me, and which could give rise to gametes.
That the germ-cells may arise from the ordinary wandering mesogleal
cell (Pl. 19. fig. 7) is very probable, but the smaller and more densely
granular cells are less common-so rare, in fact, that one could not believe
that they provide a large quantity of germ-cells, if any at all.
270
DR. 3. B. GATENBY ON THE GERM-CELLS ANI)
9. The Question of the 0rigi.n of the Germ-cells.
That ova and spermatozoa ultimately arose from collrired cells was the
conclusion of Haeckel, but Scliultze and others claimed that they arose frotii
wandering ameboid cells of the niesoglea, which they supposed were
unrelated to the collar-cells. Poldjaeff and Gorich also consider that germcells ariso from wandering mesogleal cells, which are not connected with
the gastral epithelium. Jorgensen likewise derives liis germ-cells froin
the so-called mesoderm (mesogleal) cells, either resting stellate connectivetissue cells, or ammbocj.tes. Dendy, as before mentioned, in 1915 publ~shed
a paper in which he disinterred Haeckcl’s “ heresy,” and courageously stated
that in his opinion collar-cells (lid metamorphose into gerni-cells. I n the
present paper I a111finally obliged to follow Dendy’s unpopular view, simply
because I find myself unable to overlook directly confirmatory evidence,
derived from examination of my O W I I and other workers’ sections. It is niy
opinion that not only do collar-cells migrate into the mesoglea and subsequently become germ-cells, but also that collar-cells in situ may met:imorphose
into oogonia. Schultxe, Poldjaeff, Jiirgensen, and others may be correct in
considering the germ-cells as originating from inesogleal ameboid cells, but
I claim to have shown herc that collar-cells are the inuin source of the
above-mentioned amoeboid cells, so that in the long run Haeckel and Dendy
are correct.
10. How Collar-cells becowe Bnmboid Cells of the Mesoylea.
I n PI. 22. fig. 24 I have drawn by means of the camera lucida a part of
a sponge showing the iiiigration of collar-cells in to the mesoglea. The cells
marked a 1, a 2 , and a 3 nre three stages in the process ; in P1. 19. figs. 7
and 8 are two later stnges showing the inatamorphosis of an arnceboid cell
into an oogonium. The cells drawn in P1. 22. fig. 24 can only be doing one
of two thinge-either they are migrating into the inesoglea or they. are
migrating from the mesoglea to the gastral cavity. That they are not doing
the latter seems indicuted by the fact that just ’near the figure a 2 in PI. 22.
fig. 24 are gaps in the epithelium showing that cells have left their place, and
the cell marked by the figure a 3 is opposite one of these gaps. When the
collar-cells begin to migrate inwards they lose their collur and flagellum
and become amceboid. As Deiidy and Carter showed, collar-cells teased
out alive often begin to thrust out pseudopodia. M m y amoeboid cells of
the inesoglea contain similar cytoplasmic inclusions to those of the ordinary
collar-cells, but later their mitochondria often becoine more marked, as in
PI. 19.fig. 7. Most of the pieces of sponge I have sectioned exhibit appearances such as drawn in fig. 84 of Plate 22, and I am led to believe that this
behaviour of collar-cells is of constant occurreiice during the life of the
sponge individual.
EARLY DEI'ELOPMEKT OF GHANTIA COIPREBSA.
271
11. J'ornmtion of Ooyoniu in situ from Collar-cells.
I n P1. 22. figs. 23, 25, and 26, I have drawn with the camera lucida parts
of the gastral epithelium to show the metamorphosis in situ of collar-cells
into oogonia. The first stage is in P1. 22. fig. 26 ; tit PC, a collnr-cell has
sunk below or become partly covered by its immediate neighhours. That
this cell, which is larger than ordinary collar-cells, is really a lately metamorphosed gastral epithelial cell is indicated, firstly because its nucleus resembles
that of the collar-cell, and secondly because its cytoplasmic inclusions are
ciasely similar to those oE its neighbours. I n P1. 22. fig. 85, the cell marked
OG has a cjtoplasin just like that of the collar-cell except that its size is
greater ; the nucleus of this cell is like the nucleus of the collar-cell, except
for the size of the karyosoine. I n PI. 22. fig. 23 at PC: and OG are two
stages which are similar to those already described. The cell OG is now an
oogoniuni, and is about at the same stage as that drawn in P1. 19. fig. 8.
I n PI. 22. fig. 27 at OTE is a still later stage.
The figures 23-26 of Plate 22 were drawn carefully with the camera
lucida, from the edge of a sponge where a large number of young oocytes
wore found. All stages up to the formation of larger oocytes were present,
and I believe there can be no doubt that such a cell aa that in P1. 22.
fig. 25 is a stage i n oogenesis, and is derived from a collar-cell.
12. Positicm of Germ-cells in Grantia compressa.
I n P1.19. fig. 5 is part of a sponge showing an egg (OTE) on the left
and a grciup of spermatids (SPT) on the right. These lie beneath the collarcells, and tend to abut into the gastral cavity (GCAV). Jijrgensen (8a)
mentions that oogonial divisions may take place in the gastral cavity
beneath the collared epithelium, but more often in the mesoglea. The
growing oocyte always occupies a position beneath the basementmembrane
of the collar-epithelium, and I believe that it may come to lie in this position,
in inany cases by a simple sinking inwards as shown in P1. 22. figs. 25 and 26.
Between the stages drtiwn in the latter figures and in P1. 22. fig. 27, OTE,
Jorgenseii finds oogouial divisions aud the prophases of the first maturakion
division. These latter have occurred in PI. 22. fig. 27, OTE, which is well
advanced in growth. Jiirgensen would possibly interpret the cells inarked
PC and OGA in PI. 22. figs. 23, 25, and 26, as secondary oogonia which had
inigrated froin the mesogle:~to thoir present position ; this I would dispute,
for I find all stages intermediate between suclr cells tind the collar-epithelium
cell. The migrations of the oogonia are very peculiar, and I cannot claiin
that this account is a t all exhaustive. The problem is one which will need
special study and a larger amount of iiiaterial than at my disposal ; the main
point is to note that collar-cells do not always rcainain as such, but may grow
and nietainorpl~oseinto otlier elements.
272
DR. J. B. GATENBY OH'THE QERM-CELLS AKD
13. Spermatogenesis in Grantia compressa.
About twenty-five pieces of sponge were sectioned by me, and of these
only one contained any obvious stages of spermatogenesis. The spermelements of Grantia consist of sinall groups oE cells lying in the interstitia
between the gastral chambers. Each group lies enclosed in a special
Camerducida drawing of part of sponge showing oacytes (OTE) and nests of spermatids
(SPT) with tails. Compare with P1. 19. figa. 5 and 6 A. The entire substauce of
this sponge contained patches of spermatids nnd isolated growing oocytes ; it WAS R
lightly protogynous hermaphrodite, as some of the oocytes were being fertilized.
capsule of niesoglesl cells, the whole arrangement being unlike anything
described by previous authors.
It is a moot point as to whether each group of sperm-cells should he
called a testis ; I think that the cells are too much scattered to form a
distinct gonad (text-fig. 1).
EARLY DEVELOPMENT OF ORANTIA COMPRESSA.
273
I n P1. 19. fig. 5 is depicted a part of a hermaphrodite individual
showing one gastral cavitry, on the left an oocyte about to be fertilized, and
on the right a nest of spermatids (SPT), e n c l o d by their nurse-cells (NSC).
The liitter are pale cells, cubical, and contain faint granulations ; they are
not ciliated and the epithelium they form tnay be partly interrupted by
absence of cells a t any one side.
The spermatids lie inside the cavity of the nurse-cell chamber (SPA), and
their tails can easily be seen.
I n my material I wits in addition able’ to recognise larger cells also
enclosed by a nurse-cell chamber-these
I believe to be spermatocytes, a8
they are of the expected size.
These aggregations of sperm-cells probably arise a t a certain period of the
year from aiiiceboid elements of the mesogle:L, and the nurse-chamber would
be formed subsequent to the inception of spermatogenesis.
14. General Desrv*iption of the Inclusions of the Cytoplasm in Ooyenesis.
The young oocyte of Grantia contains the usual three categories of
cytoplnsmic granules or bodies, i. e. yolk-spheres, mitochondria, and Golgi
apparatus. I n PI. 19. fig. 2 is a young oocyte containing three Golgi elements
(GAO), with yolk and mitochondria stained yellowish. T h e apparatus is
like that of the flagellated cells, being formed of an inner protoplasniic
(nrchoplasmic) area, surrounded by a peripheral rind of Golgi material.
My Kopsch preparations were not suitable to study the Golgi apparatus in
all stages, because those which were successful did not contain oocytes older
than that drawn in P1. 19. fig. 2.
T h e mitochondria I worked out fully : they are the “chroniiClia~’of
JargonFen. Without clouding this Iwesent section with controversy with
regard to the origin of the germ-cells, it can he stated that those cells (in
fitct, all cells of the sponge) from which the gametes nltimatsly arise
colltain mitochontlria in their earliest stages.
There can then be no
question as to the origin of the sponge mitochondria Crom chromatin.
The mitochondria in the youllg oocyte (1’1. 19. fig. 8) niiiy be extreniely
large and coarse, and they lie in the endoplauniic region. As the oocyte
grows larger, the mitochondria become more numerous and generally smaller
a t first, but later they may grow very large and dense. As soon as the
oocyte has grown so large that the crowding of the granules is less marked,
vaclloles begin to appear in the endoplasm.
The yolk in the Grantia coinpressa oocyte cannot be deirionrtrated properly
by short fixation in chrome-osmium, as is suitable for mitochondria. The
best method for demonstrating yolk is the unmodified Kopsch (7).
I n P1. 19. fig, 9 is an oocyte just before fertilization, showing the mitochondria (black) and the very fine numerous yolk-granules as lightly stained
bodies. The yolk-bodies of the egg of Grantia are so delicate that only
274
DR. J. B. GATENBY ON THE GERM-CELLS AND
prolonged osmication enables them properly to withstand contact with alcohol
and xylol in the subsequent embedding and staining. I have not hitherto
met with such delicate pollr, though that o f the niollusc eggsis often ycry
easily destroyed. No other author has described the true yolk of spongeeggs.
While the yolk granules are beautifully fine nnd regulnr in cize and shape,
the mitochondria are often extreoiely ’coarse, and of irregular shape, and
may occasionally be abnormally large (Pl. 20. fig. 13, MI.
I n the unfertilized and unmatured oocyte, the yolk granules and niitochondria are evenly distributed in the endoplasm, iis depicted in P1. 19.
fig. 9, and, as will be seen, are subsequently evenly sorted out among the
blastomeres during cleavage of the ovum.
15. Ectoplasmic and Endoplasmic Regions in the Egg.
Even in the youngest oocytes one may notice that a t an early stage a clear
ectoplasmic zone becomes differentiated from an inner or endoplusiiiic zone.
T h e ectoplasmic zone (PI. 19. figs. 5 and 9, P1. 20. figs. 10-13, P1. 21.
figs. 18-20) contains few or no vacuolee, is amooth, and is often drub\ n out in
the form of blunt pseudopodia or filamentous dendriform threads. T h e
inner or endoplasmic zone is vacuoliited completely and has a fine frothy
appearance ; it is i n this region that the cytoplasmic inclusions lie, granules
in the ectoplasm of the ooojte being rare or never found. Occasionally, in
preparations fixed in inixtares containing nlcohol or acetic acid, the vacuoles
collapse, and the egg comes to have a curious radintion of fibres tiround the
nucleus (see Jiirgensen’s figures, 88). Eggs treated with silver nitiate
solution show the endoplasin browner thon the ectoplasm, and in eonie cases
the difference in qualit,y of ectoplasm nncl endoplasni is as iiiarked as that
shown in P1. 19. fig. 5, END, ECT. In favourirble cases not only young
oocytes, but arnehocFtes, may he seen to posRess ectoplasmic t i n d endoplasmic zones (PI. 19. figs. 7 atid 8). As in the case of the older oocyte the
cytoplasmic inclusions lie in the endoplasm, while the pseudopodia consist
inainlp of ectoplasmic material.
During development of the egg, all the blastorneres i n most cases come l o
have an equal portion ot’tlie ectoplasm.
The ectoplasin can be traced through cleavage up to the formation of Q
blastula (PI. 21. fig. 21), but it soon either bocomes absorbed or is invaded
by endoplasmic substance. I n PI. 21. fig. 22, a young blastula is shown,
which still has distinctly marked ectoplasinic regions. In R rare number of
cases, it was found that in young blastulse the cells of one side had less
ectoplasm than those elsewhere, but in no example could I show that this
inequality had any relationship to the iormation of the flagellated and 1lOFlflagellated parts of the aniphiblastula.
EARLY DEVELOPMENT OF GRANTIA COMPRESSA.
16. The Problem of the Polarity of the Oocyte, the
275
Ovum, and the Embryo.
This important problem is yery difficult to nnderstand properly, because
the granules in the Grantia egg are evenly disposed, and one cannot identif'y
a vegetative or animal pole by this means. I n fact, one is obliged to
consider that the egg of Grarctia is a primitive amceboid body, without
visible signs of a polarity sucli as one findn in the amphibian egg (PI. 19.
fig. 9).
I n most cases the egg of Grantia lies beneath the gastral epithelium in
such a way as to be compressed into an oval form, a s shown in P1. 19. fig. 9 ;
the nucleus itself is rarely quite spherical, being oval in the vatlie direction
as the egg. While some oocytes at this stage are quite spherical, and their
nuclei likewise, it is true that by far the majority of Grantia oocytes possess
this form of bilateral polarity. While the oocyte nucleus is most commonly
oval or elliptical, the pronuclei are often quite spherical. Thongh it is
difficult to conie to a conclusion, I fear that the above observations on the
gross polarity of the oocyte are of only small value.
The ectoplusm, too, lies around the entire periphery of the egg, and lends
no clue to the solving of this probletn. Polar bodies are most often given
o f towards tho gastral side of the egg, but I know of one case where a
polar body was passed out on that side of the egg lying towards the mesoglea.
I do not consider that inoch significance can be attached to this.
I have been forced to the conclusion that, apart from the bilateral polarity
caused by pressure, the oocyte shows no permanent signs of definitc polarity.
Now during fertilization the sperm alwriys, in my experience, enters a t
that side OE the egg nearest the gastral cavity (Pi. 19. fig. 9, 8),arid in by
far the greater number of examples the penetration of the sperm causes a
partial flow of cytoplasmic graiiules to that side of tLe oocyte on which
the sperm entered (PI. 20. fig. 13). The egg thus comes to have a definite
polarity temporarily conferred by the entry of the sperm. Tho question
then arises as to whether the place of entry of the spermatozoon marks
the animal pole, and whether it is the animal pole which later becomes the
flagellated hemisphere of the blastula P
There are several established facts which we may notice at once: The
flagellated half of the amphiblastula larva almost R ~ \ ays
I
becomes formed
froin that hemisphere of the undifferentiated blastula itbutting against the
gastral liiyer ; the sperm always enters on this mine side, m d tlw polar
bodies are most often given off on this side ; in the differentiating blastula
(PI. 22. fig. 27), the line of demarcation between future flagellate cells ant1
future granular cells is sharply distinguishable.
I may say, nevertheless, that I am aware that the above evidence is
insufficient fully to eettle the question of the polarity. One may, however,
follow out, this line of' reasoning to its fullest extent,. though it should be
276
DR. e l . B. GATENBY ON T H E GERM-CELLS AND
noticed that the constant position of entry of the sperm may only be due
to the fact that the gastral side is the nearest path which the sperm could
take to the egg.
The fertilized egg soon loses th:it temporary polarity conferred by the
disturbing influence of the spermatozoon, and soon the granules so disarranged flow back and become evenly disposed (Pl. 21. figs. 18 und 19).
No methods used bg me succeeded in demonstrating any differences in
cells of the two halves of the stirly blastula (Pl. 21. fig 21), and it should be
mentioned that in several cases it seemed to me that the future flagellate
hemisphere, instead of being turned towards the nearest gastral cavity, wafl
turned away from it, u s drawn in Pl. 22. fig. 27, where I presume the
spermatozoon entered near the letters SPX.
The conclusion which seems most likely to be correct is that the oocyte
lies with its animal pole towards the gastral cavity, and that the sperm
enters at this pole ; one map also suppose that the anim:il pole gives rise
to the flagellated cells or histocytes, while the vegetative hemisphere gives
rise tn the granular cells. I t may be noted, too, that the oocyte cytoplasin
shows, 011 a microscopic:~lbasis, no signs of regional differuntiation.
I believe that while the latter remark is correct, it may be true that the
ground-plasma of the oocyte is more or less perfectly differentiated into
two sorts of subgtances, which may be the forerunners of the two different
categories of cells of the amphiblastula larva
17. Preliminary Outline of Events leading to-the Union of Sperm and R9.q.
T h e following account of tho stages leading to the union of sperm and
egg is derived only partly from assumption, but mainly from actual observation of microscopical preparations.
The spermatozoa are carried into the gastral cavities, presumably being
borne along lry the inhalent currents. Pas-ing to that region of the gastral
epithelial carpet overlying a ripe oocyte, one spermatozoon enters a collarcell (Pl. 19. fig. 9). After the entry of the sperm into the choanooyte or
collar-cell, the l a t k r loses its collar and flagelluni, becomes ~ m c e l ~ o i and
d,
gradu:rlly passes through the baseineiit-meoibrane of the collar-cell epithelium
(PI. 20. fig. 14), and penetrating all obstacles finally comes to lie on what I
have tentatively presumed to be the animal pole of the egg : generally the
sperm-carrying collar-cell makes n special depression on the surface of the
oocyte. Protoplasmic continuity is estuhlished between oocy te and spermcarrying cell, and the spermatozoon is passively borne into the egg by
nieans of ptotoplasmic currents (1’1. 20. fig. 12).
I n short, this account would mean to say that the differentiated flagellated
collar-cell niny become modified, in a short space of time, into an amoeboid
non-flagelhted cell, whose functioir is to carry the spermatozoon to the egg.
EARLY DEVELOPMENT OF GRANTIA UOMPRESSA.
277
I consider that I have found all stages confirniing this peculiar happening,
not only iii Grantia, but also in p u t in Sycon ciliatznn. Jijrgensen's
figures (8a), misinterpreted, show that the same process takes place in
Sycandra rapJi anu s
.
18. Ebidence as to the True Identity of the Sperm-carrying Cell.
In by far the greater number of' cases, the earliest stages of fertilization
that I have found are those at a time when the sperm-carrying cell has
reached the egg (PI. 20. figs. 11and 14). I n many such cases it is quite clear
that the nucleus of the sperm-carryin! cell has a somewhat clearer substratum, and may be bigger than that of the collai-cell. Did one know only
these special cases, one might be led to consider that the sperm-carrying cell
was some sort of aincebocyte from the mesoglea. Nevertheless, I have concluded definitely that the sperm-carrying cell is, or was, a collar-cell, and
for the following reasons :1. In certain few but definite examples the ripe spermatozoon has been
found in a colhr epithelial cell, which reposed in place alongside its fellows
(text-fig. 1, cainera lucida).
2. In B very large number of examples I found spermatozoa in cells in the
position indicated in text-fig. 3-that is to say, still in the epithelium, but
more or less covered by its neighbours.
3. I n a very large number of cases, I believe the majority, the nucleus
and the cytoplasmic inclusions of the sperm-carrying cell exactly resembled
those of the collar-cell or choanocyte, while the size of the former approximated closely to that of the latter (see page 279).
4. The position of the point of entry of the sperm-carrying cell into tissue
of the sponge, on its journey to the oocyte, is remarkably constant (compare
text-fig. 4) ; yet when one examines ripe oocytes before the advent of the
spermatozoon, no special cells can be found in the epithelium at the region
where the sperm-hearing cells later appear.
These reasons lead me to believe that the sperm-carrying cell is a modified
choanokyte ; I consider that any one of the statements given in the above
four paragraphs is sufficient to indicate the true nature of the sperm-carrying
cell, and all the facts together conditute a clear and indisputable proof.
The occasional slight change in the density of the nuclear substratum
(]inin) I consider explicable under the assumption that the presence of the
spermatozoon exerts an influence leading to this change, and it is important
to note that this change does not always occur.
The matter is further discussed on page 283.
19. Brhaviour ?f' Sperm after Etttry into Collar-cell.
I n my own preparations I nevor found the last stages in spermatogenesis,
SO that it is impossible for me to say whether the nucleus of the sperm is
altered by its entry into the collar-call. I n PI. 19.fig. 5 A, I have drawn
LINN. J0URN.-ZOOLOGY,
VOL. XXXIV.
21
Letters CHC=collar-cell of gastral epithelium. ECT and END=ectoplnamic and endoplasmic regions of oocyte. M =mitochondria. SP= spermatozoon.
Figs. 2 and 3.-TWO etag6s in the fertilization of Qrantia ; in fig. 2 the s ermatozoon (Sp)
ha8 just entered a collar-cell ; in fig.3 the collar-cell has lost its cofiar and is sinking
below its follows. Subsequent stages rire drawn on PI. 20. figs. 12, 16, and 17.
Fig. 4.-Part of an oocyte Rnd the covering-wall of collar-cells (CHC). The black dots
represent the comparative ositions of twenty-five spermatozoa in that number of
cases examined in the stage rawn in fig.3. In all I have over fifty oocytrs in stages
at or between Text-figs. 2 & 3 and P1. 20. fig. 11,
i
279
EARLY DEVELOPMENT OF QRANTIA COMPRESSA.
the oldest spermatids I could find, and in PI. 19, fig. 6 I have drawn the
sperm possibly just after it has entered the collar-cell. The tail of this sperm
has been added to my drawing. From what evidence I gained by examining
a number of ripe sperms lying in collar-cells, I believe that the only
change undergone by the sperm in this position is the loss of its tail or axial
filament. The nucleus of the sperm, after entry into the collar-cell, might
swell a little, but I have no evidence that such is the case". It is only
when the sperm is drawn into the egg that the former breaks into parts,
and the nucleus begins to swell. The collar-cell has no such effect on the
sperm : this is a very interesting point.
GSrich, however, in Spongilla draws the sperm quite differently from that
of Grantia, and if the free sperm of the latter resembles that of Spongilla it
is certain that some change comes over the Grantia sperm after entry into
the collar-cells. GSrich's account I do not trust in details.
20. Changes in the Collar-cell brought ubout 6y the R71try ofthe
S'ermat ozoon.
While most sperm-carrying cells a t the stages drawn in text-fig. 3 closely
resemble the collar-cell, it is true that by the time the former reach the
oocyte some undoubted changes have taken place, especially within the
nucleus.
The example drawn by Professor Dendy (2) on P1. 26. fig. 52 of his paper
is n fairly typical case, such as I have occasionally noted in my own
material ; a similar example is drawn on P1. 20. fig. 16 of this paper.
I find that a good dual of variation in the general appearance of the nuclei
of the sperin-carrying cell call be noted, and some nuclei are exactly similar
in size, shape, and staining-power to the collar-cell nucleus, while others
lack especially that dense substratum peculiar to collar-cell nuclei. I consider
that this partial clearing-up of the dense nuclear substratum is caused by the
prolonged presence of the sperm, and is in some way connected with the
amceboid phenomenon of the nietamorphosed collar-cell.
I n addition to this occasional change, all sperm-bearing collar-cells lose
their collar and flagellum, and become amceboid. As would be expected,
most sperm-bearing cells are a little larger than the unmodified collar-cell :
this is due to the presence in them of the sperm.
21. The Fate qf the SpeTm-carrying Cell after the Sperm passes into
the Oocyte.
The sperm-carrying cell lying upon the surface of the oocyte is a most
conspicuous object, yet in the cleavage-stages subject to fertilization this cell
could not any longer be made out. Having examined a number of early
* I have lately procured evidence that the sponge Rperm is filiform and flagellate, so R
great change comes over it nfter entry into the collar-cell (see Journ. Roy. Dficr. SUC.1920).
21*
280
DR. J. B. GATENBY ON THE GERM-UELLS AND
cleavage-stages I have come to the conclusion that the sperm-carrying cell
either wanders awny after the spermatozoon has passed out of its cyt,oplasin,
or in some way effnce4 itself by degenerating or shrinking in size. I found
only one case suggestiiig that the sperin-carrying cell subsequently degenerates : in P1. 21. fig. 18, the cell at SCC is obviously unhealthy. A t present
I ain led to consider that the sperm-carrying cell subsequently wanders away
through the mesoglea.
22. Beliasioiir oj' Sperm and Sperm-bearing Cell explicable as a
Clhemotactic Phenomenon.
I n text-fig. 4 is drawn diagrammatically a part of an oocyte and the neighbouring row of collnr-cells. Twenty-five oocytes were exanlined a t the stage
ot' fertilization drawn in text-fig. 3, and the position of the sperm was
indicated in the diagram in fig. 4 by a black dot. This diegram, therefore,
gives a graphic representation of the fact that tlie swiiniiiing spermatozoon
is definitely attrmted in some way towards the egg. If this were not so, one
would not find the sperm almost invariiibly entering cells neighbouring the
egg. I have not found sperms in collar-cells further away froin the egg
than those drawn on the extreme left and right in fig. 4.
Chemotaxis as an explanation of the attraction of any sperm hy any egg
has heen abandoned to a great extent by zoologists, but surely here is a
definite case of attraction between egg and sperm ? The egg of the sponge
seems able to give forth some substance which causes the sperm to swim to
that region of the gastral epithelial carpet which just overlies an oocyte. I
cannot think of any other explanation which accounts for text-fig. 4 so
satisfactorily.
23. Tlie /:'ntry of the Spermatozoon into the Egij and the Efect on
both Gametes.
The sperm is drawn into the unmatured oocyte prolmbly by synchronous
flowing movements of the protoplasm of both sperm-carrying cell and the
oocyte. That the sperm itself has no iininediate part in the process seeiiis
indicated by the fact that it is able to pass into the egg with its iiucleus
forwards or its middle-piece forwards (Pl. 20. fig. 11) or sideways. There
is undoubtedly a complete protoplasmic continuity established between the
cytoplasni of the carrier-cell and that of the oocyto ; here the vitelline
menibrane of the egg is interrupted as shown in PI. 20. figs. 12 and 14.
When the sperm has passed into the egg, the first change noticed in it is
the breakiug-down of the sperin-cell wall (PI. 20. fig. 16). The acrosome
(AC) becomes faint, and i s just disappearing in the last figure. The sperm
to plasm becomes furred a t its edges where it touche3 tlie egg-cytoplasm ;
soon a flowing movement, apparently initiated by the entry of the sperm,
tends to disturb the arrangement of mitochondria, which partly pass to that
hemisphere of the oocyte at which the sperm entered (PI. 20. figs. 10, 12,13).
281
EARLY DEVELOPMEKT OF GRANTlA COMPREFSA.
Vacuoles collect near the region of t,he sperm, and the path of the latter
into the egg becomes inarlted by an area formed of more liquid su1)stance
(PI. 20. figs. 13 and 17). The fine yolk-spheres Beep closely around this area,
but do not invade it, as is shown in PI. 20. fig. 13. Subsequent to the entry
of the sperm, the oocyte undergoes maturation giving off' two polar bodies.
24. The Fate of the Xiiddle-piece or Spevnt SIauromitosonw
d i i r i i y J'eidlizatioli.
(ir
Nebeiikern ")
I n Ascaris and Phalliisia it has been shown that the mitochondria1 substance
in the middle-piece of tho spermatozoon does not degenerate during fertilization, but retains its faculty for growth and divisiou and apparently takes
some ininor par,t in the phenomenon of fertilization (14).
With Champy-&ill technique the middle-piece of the Graufin sperm
stains a bright red, while the egg-cytoplasm is a greenish or golden-grey
colour ; it is, nevertheless, a somewhat difficult matter t o ascertain exactly
what beconies of this body during fertilization. Between the stages of
P1. 20. fig. 12 and fig, 13, the niiddle.piecc in its forni of a bun-slial"I bead
completely disappears. I n PI. 20. fig. 1 7 , IMAM, i t is quite clear that the
middle-piece is breaking up into a cloud of sinall grannles which adhere to
the growing inale pronacleus like the tail of n comet. I n subsequent stages
I never found any granules which I could identify for certain as having
originated Erom the ~n:icroinito~oine,
but in P1. 20. fig. 13, at x, are certain
grains which might be the remains of the original middle-piece. While I
do not wish to deny that there may be activity of the male iiiitochondria
subsequent to the stage of P1. 20. fig. 17, I consider that sucli is unlikely.
The evidence either way is not complete enough to allow me to express :t
definite opinion.
25. The Mitochondria in the Development of Grantia conipressa.
Tn certain Ascidi:i i t is established that the mitochondria beconie unevenly
segregated into different blastomeres, the niyoplasiii being formed inainly
from these granules. The two regions of the spongo auiphiblastula do not
contain granules in equal quantity, and it i s interesting to asccrtain wliether
in Graiitia there is any special segregation of cytop1:ismic granulations into
special blastomeres.
Subsequent to fertilization in Gruntiu ( * o t n p e s 8 4thc ~nitocliondrinbeconie
niore or less evenly epread out in the endoplasnl (Pl. 21. fig. 11), and
during cleavage rho distribution of the mitochondria between the blastonieres
is equal, as shown in PI. 21. figs. 19, 20, 21, and 22.
I n P1. 21. fig. 22, the blastula is found to hc formed of cells, all of wlrich
contitin approximately equal numbers of mitochondria1 grains. It. is only
niuch later during the differentiation of the two main cell-layers in the
282
DIt. J. B. GATENBY ON THE GERM-CELLS AND
embryo that any differentiation of granules appears, and these differences
seem due to slow growth and not to any segregation during cleavage. It
is apparent that pari passu with the cleavage of the egg into smaller and
smaller blastomeres, the mitochondria either shrink considerably in size or
break up into smaller fragments (compare the size of the granules in PI. 21.
fig. 1 9 and fig. 22). It is not true that the mitochondria (chroniidia of
Jiirgensen) disappear in the late blastuh-stage, as was thought to be
the case (8 a).
26. Uiferentiation of the Xuclei of tlie Blastula-wall heralding the
Formation oj' Two Dejinite lfssues.
On PI. 21.fig. 21 I have drawn a Grantia blastula at a stage when all
the cells appear alike ; the nuclei and the inclusions of the cytoplasm of'
these cells seeined to me to be similar throughout the entire blastula. On
P1. 22. fig. 27 is a blastula fixed a t the period when the nuclei of the future
flagellated hemisphere have begun to be easily distinguishable ; the earliest
signs of differentiation of the future flagellated cells is constituted by a
tendency for the cells of one hemisphere to divide more rapidly, and to become
more regularly arranged : the peculiar denseness of the nuclei of this layer
is a subsequent and rapid differentiation, In P1. 22. fig. 87 the granules in
the cytoplasm are not different in either region, and it seems certain that
nuclear differentiation precedes or instigates special cytoplasmic activity.
Soon after the stage depicted in P1. 22. fig. 27, the character of the yolkspheres becomes altered ; this is indicated by the fact that the yolk-spheres
of the embryonic tissues are more persistent and less easily destroyed than
those of the egg or earlier blastula.
Owing to lack of suitable material, there were several interesting problems
which I did not examine, but it is hoped to investigate these a t some later
period.
27. DISCUSSION.
(a) Organ-fornting Substances ?
I n this study the behaviour of yolk and mitochondria has been followed
out in the development of the sponge Grantia conzpressa. The introduction
of specific and selective staining and fixing methods has enabled me to
identify mitochondria, yolk, and Golyi apparatus. The sponge falls into
the group of animal forms vhich have been shown to possess the two
protoplasmic inclusions.
The main result of this research, in so far as it touches on the mitochondria,
has been to show that they pass through developinent without losing their
individuality, and that they are distributed evenly during cleavage to the
blastomeres. No sort of segregation of materials in special blastomeres could
be detected with the methods used by me, and I have come to the conclusioii
that such differentiation of cell-layers and materials as one finds in the
EARLY DEVELOPMENT OF GRANTIA CONPRESSA.
283
amphiblastula larva is of gradual growth in the stages between the end of
cleavage and the final stages of tho forination of the free-swiniining larva.
I could not find any evidence for such segregation of iiiaterials as described
by Uonltlin (1) in Cyritliia, and I have come to the conclusion that, so far
as fixed tilaterial goes, no visible organ-forming areas can be demonstrated
in the egg of Graiitia coinpressa. I t is possible, however, that delicate easily
destroyed pigments may be present in the fresh ripe egg, and these inight
mark out ground-protoplasmic areas not to be distinguished in fixed material.
Of this I have no evidence.
(b) Cliromidia and Mitochondria.
eJijrgensen and Dendy both describe the mitochondria under the name
‘‘ chromidia,JJ implying that they have a true chroinatinic nature and are
related to the nucleus.
If the egg is treated by Champy-Kull’s method the mitochondria are red
(fuchsin) and the nucleus is bluish. In the Bensley-Cowdry stain the mitochondria are red (fuchsin) and the nucleus is green (methyl-green). In
Clarnoy’s fluid the mitochondria are dissolved away and the nucleus remains.
The mitochondria of Grantia are not “ chrornidia ” in the proper sense of the
word, nor are they derived from the nucleus at any period of oogenesis *. All
embryonic cells contain mitochondria, and there is no question of the latter
appearing froin the nucleus at any stage of the oogenesis of egg-cells derived
from the amoeboid or other elements of the iiiesoglea or collar-epithelium.
I n a late paper on Apanteles I described true cytoplasmic chroiiiidia in the
egg of this form ; such chroniidia stain like chromatin, and ultimately
metamorphose into secondary nuclei. I n the Hyrnenopterous egg the
secondary nuclei (chromidia) and the mitochondria are easily distinguishable
and can he stained in different coloiirs.
( c ) Fertilization.
I n this paper I have given the first correct interpretation of the peculiar
stages already described by Giirich, tJorgensen, and Dendy. I t has been
demonstrated beyond a doubt that the spermatozoon of Grantia is brought to
the oocyte by a carrier-cell. On page 277 I have given my reasons for supposing that the sperm-carrying cell is really a collar-cell which has lost its
collar and flagellutn, and become ameboid.
Should subsequent work confirm this description (and I hive little doubt
that there has been no mistake) this will constitute one of the most remarkable occurrences in the fertilization of any aniinal.
It is rumarkable that the collar-cell teniporarily containing the sperniatozoon
should desert its position in the &astral epithelium and wander into the
* I now abandon this view and admit Uendy’s description of uucleolar extriisions to be
correct. This matter is treated at leugth in my new paper i n the Juurn. Boy. Micr. Soc.
1920.
284
DR.
3. B. GATENBY ON THE GERM-OELLS AND
mesogleal tissue. That the sperm is the instigntor of this unusual behaviour
seeins certain, and appaiwitly any collar-cell may transform into a spermQoodrich’s sinall series of
carrying cell. Froin exaininat~ionof Dr. E.
S’con ciliaturn slides, I believe that in this forin also the collar-crll acts as a
sperm-carrier, and JGrgensen’s account of similar stages in Syca?~di*u
raplmnrs,
all misinterpreted, lead me to consider that the process described by me f o r
Grantia compressu will apply to other forms of sponges.
It is evident, nu Professor Deiidy has hiniself pointed out to me, that two
distinct processes have been confused. The young oocyte is fed by a process
of phagocytosis. It is definitely establishcd by Profebsor Dendy that, certain
large amceboid elements of the mesoglea carry other cell-t.leiiients to the
young sponge-oocyte, and the latter engulfs the food-matter so offered. ’l’he
cell-elements which bring such cell-food to the growing oocyte are urililie
the cell which later brings the sperinatozoon to the oocyte ; iiioreover, this
process of feeding only takes place in sinall oocytes. After the latter have
grown to half thcir ultimate size, ingestion of other cells is rarely found to
occur, and, moreover, ingestion of other cells by the growing oocyte takes
place on any part of its surface, whereas the process of fertilization occurs
on one defiilite side of the oocyte. There can be little doubt that not only is
the process of phagocytosis i n the oocyto different from the stages leading
to fertilization, but also that the cells engnged in the two processes are not so
alike as to lead one to believe that they are derived from the same source.
The cells engaged in bringing food-niatter to the young oocpte are nearly
always much bigger than those which bring the sperm to tho egg, and the
cell-contents of the two cells are not alike.
s.
(d) l’lie Spernt-carryitig Cell considered as a Mesoyleal A ~ n a d m y t e?
The sperm-carrying cell has beeu assumed by me to he a modified choanocyte or collar-cell. There may be critics of this view who would prefer to
take the other alternative, and look upon this peculiar cell as an amoebocyle
from the niesoglea : according to such critics, the ainwbouyte would be much
more likely to behave in the inanner of the sperm-carrying cells ; moreover,
it is true tliat some of the latter have nuclei rather unlike those of the collarcells, and similar to the true mesogleal amcebocytes.
I n the first place, oiie never finds aincebocytes near unfertilized but ripe
oocytes in the position in which the sperm-carrying cell later appears (textfigs. 3 and 4). Secondly, I have never found spermatozoa in ammbocyte
elements of the niesoglea, and I have found several cases where sperms lie
within what 1preduine to be choanocytes (text-fig. 2, SP). Thirdly, there
is the fact that the position of the sperm-carrying cell is reinarkably constant
(text-fig. 4): now, if nie?ogleal amcebocytes carry the sperin to the e g i , why
is this position so constant ? Presuming that sperms swim through into the
EARLY DEVELOPMENT OF GRANTIA COMPHESSA.
285
inesoglea and get into amcebocytes, we are then forced to believe that each
spertn-bearing arnabocyte, whatever it5 primary position, alway8 carries the
sperm to one side of the egg ; there would then undoubtedly be cases where
the amabocyte would have to perEorm a comparatively long journey with the
opposite pole of the egg as its goal. Agninst this amcebocyte view there is a
fourth reapon : cells carrying sperms can never to n i j lrno\vledge be found
on the short “ side ” of the egg, as marked by the .;tar on the right of textfig. 3. Had the spermbearing cell been a rnesogleal amcehocyte I would
surely have found one with a sperm, in positions other than the gastral side
of the oocyte (test-fig. 4).
Reference to Professor Dendy’s pl. 24. figs. 50 and 51, and comparison with
fig. 52, will show that the cell which carries food to the oocyte is not only
bigger than that which carries the sperni, but has different cytoplasmic
contents. I n Prof. Dendy’s fig. 52 the sperni-carrjing cell (NO) is about
the same size :is a collar-cell.
To explain my text-fig. 4 satisfactorily on the basis of the aincebocyte
view, one would need to assume that there was a row of waiting amcebocytes
ready in the position in which the sperm is later found. No author who has
drawn sponge-oocytes has figured such anicebocytes in this region, and I
have never seen an aincebocyte waiting in this part of‘the gastral epithelium.
To assuine that the sperm pushes its wily into the niesoglea till it finds a n
amcebocyte to carry it to the egg is preposterous, for if the sperm can push
its way, “ under its own power,’’ into the niesoglea, there \I ould be no need
for another cell to carry it to the egg.
I think it will be obvious to all that the sperm does penetrate into a collarcell, cannot itself pass through to the oocyte, but is carried by the collar-cell,
which, during the process, may occasionally become somewhat altered in
appearance.
(e) Germ-cells atid Sex
itt
Sponges.
My observittions lead nie to consider that all Gmiitia individuals are
probably potential hermaphrodites, but. that the occurrence of sponges with
sperm-stages is somewhat rare. Tn only one case out of Iwenty-five that I
know WV;ISthe sponge a positive herniaplirodite, and in all the other examples
I examine I only oogenesis stages were discovered. 1iini led to consider that
Grantia conapressa is either ii siinultaneous herinaphroclite, or female, both
sorts of individuals being found. The sponges which I found to contain
oogenesis stages alone might have been either 1)rotandrous or protogynous
hennaphroditw, but this s e e m rather iniproLable, for at the time I fixed ntp
material the bulk of the oocytes were ripe and being fertilized, while there
was a general absence of spenn-stages, except in one individual, which
contained both spermatids aud oocytes being fertilized.
286
DR. J. B. OATENBP ON THE GERM-CELLS AND
One small point I may mention with regard to any possibility of selffertilization in Grantia : the sole individual in which I found unripe
spermatids also contained oocytes just beini fertilized, showing that the
sperms must have conie from some other sponge, or otherwise one would
have found later stages in spermatogenesis,
As a niatter of fact, the whole question of sex in sponges is far from being
definitely settled, for I doubt whether certain previous observers of marine
sponges have really identified the true sperm-stages.
(f) Spermatogenesis in Grantia.
Haeckel in his ‘ Kal.kschwamme ’ figures small ‘‘ sperni-balls ” just under
the gastral epithelium, much in the same position :IS in Dendy’s figures.
Neither Haeckel nor Dendy have produced convincing evidence as to their
‘(sperm-balls.”
Subsequently to my writing the account of Gvantia sperinatogenedis given
on page 272, I had the opportunity, througli Professor Uendy’s kindness,
of seeing not only his own ‘‘ sperin ” stages, but also thosd of Dr. PolPjaeff.
I n figs, S6-93 of Professor Dendy’s plate 46 are drawn what this observer
describes :is (‘sperm-morulae,” with spermatozoon lieads ; he has :dso
described the derivation of these i n o r u h from other cells which he believed
to be sperinatogonia and spermatocytes. On pl. 26. fig. !I4 he gives figures
from Dr. Polejacff’s slides. After cure fully reading Dendy’s account, and
having seen his preparations, I have little hesitatioii in claiming that what
he bas described are certainly not sperm-stages of tiny sort ; I believe that
these bodies he describes are parasites or inquilinus of some kind, probably
of plant nature. Dr. PolBjaeff’s sperni-balls are, I consider, iiothing inore
than some protozoan, while the granules inside the cell are not individual
cells, but discrete cytoplasmid aggregations. Moreover, neither Dendy nor
Polejaeff figure tails on their spermatozoa, while I find tails on my
spermatids *.
I believe that there is no doubt that the cells I have described are true
stages of spermatogenesis, and I sectioned twenty-five sponges before I
found them. I wish to emphasize thiR fact, for other observers have found
the same scarcity of spermatogenesis stages in sponges, and this is one
reason why two observers (and probably three) identified either parasites or
inquilines as spermstages.
Wilhelm Giirich in Spongilla $uviatilis describes what are undouhtedly
stages in spermatogenesis ;his description of the formation of the spermatozoon from the spermatid is quite detailed, but he mentions neither mitochondria
nor Golgi apparatus. I n SpongiZEa the sperm-stages lie h i d e a wall of
nurse-cells (“ Cystenzellen”), somewhat as in Gmntia. I n Sycandra raplianus
* At the meeting of the Society, after I had read this paper, Prof. Dendy, i a reply,
abandoned his previous views with regard to his ‘‘ sperm-morula,” and stated thctt in two
other spoiiges he had foiuud stages like those which I have described in this paper.
EARLY DEVELOPMEKT O F GRANTIA COMPRESSA.
287
he describes the formation of the I‘ Spermatocyste ” by means of cell which
approitches the spermatogonium (“Spermazelle”), and surrounds it completely; lie figures and describes the sperni-cell dividing inside the cyst-cell.
If Garich’s account of the formation of the nutrient-cell capsule is correct,
it would mean that in Chantia probably all the cells in P1. 19. fig. 5 , marked
NSC, would have originated from a single nurse-cell. I doubt this, but
am unprepared at present either finally to reject or adopt Gijrich’s account.
In Gorich’s spermatid and spermatozoon there is no nucleolus.
(g) The Collar-cells as the Donainant and most Characteristic Tissue
of the Sponge.
Dendy (2) remarks, I‘ The one constant and Characteristic feature about
sponge histology is, of course, the collared cell, and that is only constiint i n
the sense that its typical form is that which possesses a collar and a flagellum.
l h e sponge is, after all, not very much more highly advanced in organisation
than a colony of choano-flagellate Protozoa.” This paragraph aptly describes
the views, a t which I have myself arrived by my independent observation.
The collar-cell is the dominant cell in the sponge, and I feel that Haeckel
was correct in tracing the origination of germ-cells from collar-cells.
Collar-cells are continually migrating into, and reinforcing, thG mesoglea
of the growing sponge : the collar-cell is really very little differentiated
except for the flagellum and collar, which, we are now quite sure, can readily
be withdrawn.
The collar-cell then become6 i i n active amoeboid cellindividual, whose subsequent fate possibly inay be to metamorphose into a
germ-cell.
One is obliged to remark that in the discussion on the origin of germcells-not only in sponges, but in other animals-there has been far too much
reliance set upon theoretical conceptions, and not enough on simple observation. It does not aufice for critics to state that they “ do not think that
collar-cells can metamorpliose into germ-cells ” ; they must bring forward
something more definite before one can give their views adequate attention.
So far as I am able to understand, the critics of Haeckel’s view as to
the origin of sponge germ-cells base their assumptions on the illogical
and unnecessary extension of Weismann’s views to sponge eihbryology.
According to these observers, gorm-cells are supposed to be derived from
non-differentiated embryonic elements, and the collar-cell, having becoiiie
differentiated, is debarred from entering the charmed circle o€ cells which
alone can hecome germ-cells.
Speaking for myseif, I am quite unable to see why a. collar-cell cannot
differentiate, especially when one remembers that an embryonic cell can
become a collar-cell. I f the stimulus can be pro&led to make a cell undergo
certain changes in one direction, why should not otlier stimuli Cause the
changed cell to pass back along its former path ? Tlic collar-cell is not a
highly differentiated cell, such as u metazoan ganglion or gland-cell.
‘ 1
288
DR. J. B. GATENBY ON THE GERM-UELLS AND
(h) E’inal Remarks.
Professor Dendy has given me permission .to state that he accepts my
explanatioii that what he described in his plate 21. fig. 52, as feeding of the
oocyte by nurse-cells, is really a stage in fertilization.
The question of the true nature of the sperm-carrying cell is less well
established, hut Professor Dendy considers that in all probability my
interpretation will be found to be the correct one.
The problem of the spermatogenesis is a moot point, hut I believe that
few will doubt that what I have described in text-fig. 1and P1.19. figs. 5 and
3 A are the true stages in G~antia. Haeckel, Pokjaeff, and Dendy have
failed to bring forward convincing evidence as to the spermatogenesis, and
no cytologist acquainted with these questions would identify the “ spermballs” of these workers as authentic stages in the formation of spermatozoa
in any animal.
Attention is drawn to the reinarkable facts which other authors and I
have established with reference to sponge gametogenesis :1. The young oocyte bodily engulfs large cells and feeds at their expense.
2. The oocyte and ovum are both amceboid and capable of moving
through the mesoglea.
3. Spermatozoa are developed in special nurse-chambers, possibly formed
of mesogleal cells.
4. The spermatozoa are carried to the oocytes by special carrier-cells,
which are very probably collar-cells.
5. The larva is nourished by a speoial nutrient chamber foriried of
mesogleal cells.
6. The pupal stages comprise many peculiar events, which lead to the
formation of the sponge.
Probably in no other order does the gametogenesis and early development
preseat so many extraordinary peculiarities.
This paper merely touches on a few of these peculiarities, and I hope in
subsequent work further to pursue the subject.
28. SUMMARY.
Choanocyte or Collar- Cell.
1. The nuclear background contains a fairly dense basophil substance,
which is either a dense linin network or a chroiiiophil karyoplasm-more
probably the latter (Pl. 19. fig. 1).
2. The collar of the choanocyte has two thickenings, an inner and an
outer ; the latter one is very well marked ;both thickenings are iiiwe easily
seen in the partly withdrawn collar (Pl. 19. fig. 1, B).
3. The collar consists of delicate protoplasm which is best preserved by
289
EARLY DEVELOPMENT OF GRANTIA UOMPRESSA.
prolonged osinication ; the collar-cell is often drawn out into two regions, an
outer ci neck” and an inner “ base,” containing all the granules and the
nucleus (P1.19. fig. 4) ; the Golgi apparatus and centrosome generally lie
in the c 6 neck,” other granules in the “ base.”
4. Col1:ir-cells contain three cell-elements other than the nucleus : the
Golgi apparatns (and centrosome), the mitochondrih, and metaplasmic storegranules.
5. The mitochondria are irregular, but fine, and lie around the nucleus
(PI. 19. fig. 1, M,fig. 3, M.)
6. The metnplnsmic or yolky store granules are much larger than the
mitochondria, but lie in the same region. (Pl. 19. figs. 1and 4,Y).
7. The Golgi apparatus lies in the “neclc” of the bottle-shaped cell,
either upon the nuclcus (Pl. 19. fig. 1, A) or separately (Pl. 19. fig. 1, B).
8. I n certain dcfinite cases the centroeoine is seen to lie inside the Golgi
appnratris (Pl. 19. fig. 1, A) and the flagellum originates from tlie ccntrosome.
I n other cases the centrosome appeared sepnrate (Pl. 19. fig. 4, C 3).
I
Oogenesis.
1. The young oocyte contains the three cytoplasmic inclusions : yolk
granules, Golgi apparatus, and mitochondria (PI. 19. figs. 2 and 9).
2. The oocyte consists of two well-m;rrlred regions : a peripheral smooth
ectoplasm and an internal frothy endoplasm, in which all the cytoplasmic
granules lie (PI. 19. fig. 9).
3. The Golgi apparatus consists of isolated multiple elements formed of
several rods arranged in angular formation, much resembling that of the
mollusc oocyte (Pl. 19. fig. 2, GAO).
4. The mitochondria are fairly large, often irregular bodies, lying in the
endoplasm (PI. 19. fig. 9, M). I n the ripe oocyte they are oftea abnormally
large, and are never tery numerous. The mitochondria constitute the
‘ 6 chromidia ” of JSrgensen and Dendy.
I n their histochemical reactions
they differ from chromatin.
5. Yolk-spheres are very fine and delicate, entirely filling the endoplasm
and lying in the trabeculE between the endoplasmic vacuoles. The yolk is
only properly preserved by Kopsch’s methotl. Yolk-granules are formed
in and by the ground-cytoplasm, and have no connection with the mitochondria (Pl. 19. fig. 9 etc., Y).
6 . The egg is amceboid and ectoplasmic pseudopodia are common (PI. 19.
fig. 9,ECT).
7. The full-grown oocyte is oval or elliptical in shape, as is the nucleus,
but tlie granule? and yolk are evenly disposed, and there is no sign of a
definite vegetative or animal pole.
8. No organ-forming arms, or visible aggregations of cytoplasmic materials,
was made out.
2 90
DR, J. B. GATINBY ON THE GERM-OELLS AND
.
Spermatogenesis *
1. The spermatozoon is probably a (flagellated) ovoid body, containing a
bun-shaped nucleus. The spermatid is flagellate, so we know that the
spermatozoon must be flagellated, though one was never found outside a
sperm-carrying cell. I n PI. 19. fig. G is drawn a sperm from a spermcarrying cell, the tail being added from observation of spernintids. The
sperm has an acrosome in front, which lies on the edge of the cell, but which
is not pointed. The nucleus of the spermatozoon is bun-shaped, reticulate,
and contains a nucleolus (lsaryosome) ; the mitochondria1 apparatus is in the
form of R bun-shaped fuchsinophile, macromitosome (" Nebenkern "), or
middle-piece. The centrosome lies partly between the nucleus and macromitosome, but more in the latter than in the fornier (Pl. 19. fig. 6).
2. Spermatogenesis takes place inside a definite capsule of pale, cubical,
granular, non-flagellated mesogleal cells. The sperm-stiiges are aggregnted into little patches here and there throughout the entire area OF
the hermaphrodite sponge ; these patches lie beneath the flagellated
gastral epithelium, and stain more darkly than their surroundings. Both
spermatooytes and spermatids have been identified ; the latter are flagellated
(text-fig. 1 and PI. 19. fig. 5 A).
3. The number of spermatids in one patch is generally above fifty.
Fertilization (1st P a r t ) .
1. The ripe sperm, after having entered into the gnstral cavities, is carried
from the collar-epithelium to the oocyte by means of a carrier-cell. The
sperm lies inside the carrier-cell, which hears its passenger to the oocyte.
2. The carrier-cell is assumed to be a modified collar-cell or choanocyte,
for the following reasons :(a) There are two cases in which I have found a sperm inside a collarcell (text-fig. 2, PI. 20. fig. 10).
(b) There are a large number of' cases in which I have found a sperm
inside a cell which closely resembles a collar-cell, except for absence
of collar and flagellum, and which lies still within the collarepithelium in the position drawn in text-fig. 3.
(F) There are about fifty cases in which I found the spermatozoon
contained within a cell which always lay in the region indicated by
the black dots in text-fig. 4. No sperm-carrying cells or spermatozoa
were found in any part of the mesoglea or at the short sides of
the egg.
( d ) I have never fgund, nor has any other author figured, mesogleal
amcehocytes lying near unfertilized oocytes, in the invariable
position in which the sperm-carrying cell later appears-only
'
collar-cells are found there ; the gastral cavities were not found to
contain sperm-carrying cells-the collar-cells alone can be the
source of the sperm-carrying cells.
* See foot-note on page 279,
EARLY DEVELOPMENT OF GRANTIA COMPRESBA.
291
3. The entry of the spermatozoon into the collar-cell does not cause the
former to break up into its constituent parts.
4. The entry of the sperm into the collar-cell or choanocyte causes the
latter to lose its collar and flagellum. Subsequently the nucleus of the
collar-cell may lose that characteristic smooth chromophil background, and
become more open and reticulate-this does not alwnys occur. The spermcontaining chonnocyte sinks below its fellows in t8heepithelinm (text-fig. 3),
paRses through the hasement-membrane of the choanocyte epithelium, and
comes to rest on that surface of the oocyte nearest the collar-epithelium.
Occasionally, the sperm-bearing cell makes a hollow in the egg, into which
it fits (Pl. 20. fig. 11).
5. The wall of the sperm-carrying cell and of the oocyte vitelline membrane
come into direct contact ; they become interrupted, protoplasmic continuity
is established, and the spermatozoon flows passively into the egg (Pl. 20.
figs. 11, 12, 14, and 16).
6. The sperm may enter the egg frontways, backways, or sideways (Pl. 20.
figs. 11 and 12).
7. The sperm does not appear to nndergo the special rotation noted in
other forms.
8. The sperm-ciirrying cell, after the sperm has passed away, does not
immediately change, for it can be found during early cleavage. Later on it
becomes increasingly difficult to find this cell, and one is led to believe that
it subsequently wanders away from the scene of its former activity. I n one
case I thought the sperm-carrying cell liad begun to degenerate.
9. From the fact that the spermatozoa are always found inn circumscribed
area of the fliigellittecl cavities, just where the eggs lie, it has been concluded
that this is a definite example of' chemotaxis (text-fig. 4).
FeertiEization (2nd Part).
10. The spermatozoon, after entry into the egg, first of all loses its cellwall, which degenerates.
11. The acrosome next becomes chromophobe, while the edges of the
sperm-cytoplasm tend to spread into the surrounding egg-cytoplasm, being
for some time recognizable by its coarse stringy appearance, but later it can
no longer be noticed (Pl. 20. figs. 16 and 17).
12. The. sperm nucleus now begins to grow, and other nucleoli put in an
appearance (Bl. 20. fig. 13). The nucleus becomes spherical.
13. The sperm-middle-piece or macromitosome breaks up into a cloud of
fine granules, which adhere to the growing male-pronucleus like the tail of a
comet (Pl. 20. fig. 17, MAM). I n a later stage these granules could not be
identified.
1.4. Soon after the entry of the sperm, one finds that in the majority of
oocytes there is n flowing towards the region of entry of many of the
292
DR.
.r.
B. GATENBT ON THE GERM-CELLS A N D
mitochondria1 spheres, so that the hemisphere away froin the gastral layer is
partly bereft of niitochondria (PI. 20. figs. 10 and 13).
15. Tlie path of entry of the sperm is marked by an area formed of‘ a
chromophobe vacuolated mbstance, probably iatra vitarn of a liquid nature.
16. The oocyte nucleus breaks up aiid forms two polar bodies during these
changes in the spermatozoon (Pl. 20. fig. 13, PB),and finally the male and
female pronuclei grow to the same large size (PI. 21. fig. 18).
Cleavage and Developnzent.
1. During cleavage and early development the yolk and mitochondria1
granules are sub-equally sorted out among the blastomeres (Pl. 21. figs. 19,
20, 22, and 21).
2. During the histogenesis of the two main tissues of the sponge-larva
(Pl. 23. fig, 28, FC! B GC), these mitochondria and yolk-grains (and probably
the Golgi eleinen ts), though sorted out previously into subequal quantities
between the blastoineres, later become more or less numerous, or specially
modified or grouped in special regions, as the tissues develop and differentiate.
3. Such differeritiatiori is not, so far as the granules themselves are concerned, in any way traceable to a special segregation during cleavage.
4. The fixing and staining reactions of the yolk-spheres of the egg are
different from those of the amphiblastula larva, for duriiig differentiation or
histogenesis the yolk-spheres become denser i i n d less delicate, especlially in
the fl:tgellatetl cells. The same applies, though less well, in the case of the
mitochondria.
The A iyhillast it 1a Car aa.
1. The larva is form6d of three sorts of cells : anterior flagellated histocytes, posterior “ granular ” arclieocy tes, and inner amoebocgtes. All these
originate and differentiate from a unihninar blastula formed of similar cells
(PI. 21.fig. 21). For larvm see P1. 23. figs. 28 and 32.
2. The larva in I:ttm stages of its formation becomes partly or wholly
surrounded ky :L layer of squainous inesogleal (inaternal) cells, which are
lriiirked hy their containing peculiar irregular mitochondria. This nutrielit
capsule is best developed near the posterior “ grunular-cell ” pole of the
larva, and often interrupted on the flagellated hemisphere (Pi. 23. figs. 28
and 32).
3. In many cases these maternal cells penetrate and lie among the posterior
‘‘granular cells” (PI. 23.fig. 28, LGO) ; when the 1arv:r breaks away f’rorn
the sponge the nutrient capsule is left behind.
4. The histology of the tliree cell-elements of the 1arv:i is as follows :The flagellated lieinisphere is formed of flagellated cells, marked especially
in the live animal by the possession at their posterior or inner poleof a dense
group of yolk-spheres which :we pigmented (brownish). There is mixed up
EARLY DEVELOPMENT OF GRANTIA COMPRESSA.
293
with these yolk-granules, but more towards the posterior or inner pole, a
number of mitochondria. At the outer or anterior pole ofthe cell lie a group
of special granules doubtfully identified a$ of true yollry nature. The nucleus
is small, extremely hasophil, and contains a lraryosome. Lying on the
nucleus is a distinctly marked capsular body, from which the flagellum
arises ; this body is thought to he the GFolgi apparatus (Pl. 23. fig. 33).
The large centrally situated “granular cells” (GC in P1.23. figs. 28 and 32)
are formed of :inucleus which stains palelg, and contains a coarse karyosome
anti a cytoplasm in which lies a number of granuleq, some mitochondria,
others yolk, and certain others (GX in P1.23. fig. 31) possibly Golgi apparatus.
I n most cases these large granular ccBlls contain fewer yollr-spheres than the
flagellated cells arid about the same number of niitochondria.
5. The smaller inner granular amceboid cells (or mesenchyme) are
apparently derived by an i n w d immigration from the flagellated cells, and
are distinguished by the fact that t1ie.v are crammed with mitochondria1
granules and possess very little, if any, yolk. In Grantia conzpressu the
amphiblastula, :it the period drawn in P1. 23. fig. 28, generally contains only
two or three such cells (PI. 23. fig. 30).
University College, London,
October 30,1919.
29. BIBLIOGRAPHY.
1. C~ONKLIN, E. G.-“ The Orientation and Cell-Lineage of the Ascidian
Egg.” Journ. Acad. Philadelphia, vol. xiii., 1905.
2. D E N DY, A.-‘‘ G:inietogcnesis of Grantia compressa.” Quart. Journ.
of Micro. Science, vol. lx., 1915.
3. - “ The Pseudo-gastrula Stage: in the Devvlopmen t of Calcareous
Sponges.” Proceed. Roy. SOC. Victori:i (Australia), 1898.
4. GATENBY,J. BRONT&-“ The Modern Technique of Cytology.” Quart.
Journ. c t f Micro. Science, no. 63, 1919.
‘‘ The Identification of Intracellular Bodies.” Journ. Royal Micr.
5.
Society, 1919.
6. G ~ R I OW.-“
R,
Zur Kenntnis der Spermatogenese bei den Poriferen und
(lo3lrnteraten nebst Benierlrungen iiber die Ovogenese tler Ersteren.”
Zeit. f. wiss. Zool., Bd. lxxvi.
7. HAECKEL,
E.-“ Ueber die sexuelle Fortpflanzung und das naturliche
System der Schwlimme.” Jenaische Zeit. f. Med. u. Naturti iss.,
Bd. vi.
8. H~KSCHLBII,
JAN.--“ Ueber Plasmastrukturen in den Tunicaten-, Spongien-, und Protozoenzellen.” Antlt. Anz. vol. xlvii., 1914-15.
8a. JSRGENSEN, MAx.--“BeitrLge
xur Kenntnis Eibildung Reifung, Uef r u o l ~ t u n g ,u u d Furcliung bei Schwiimmen (Syconen).” Arcli. fur
Zellfor., Bd. iv., 1910.
9. MINCHIW,
A.-Lankester’s
‘ Treatise on Zoology,’ Part 11.
LINN. JOURN,-ZOOLOUY,
VOL. XXXIV.
22
-
294
DR. J. B. OATENBY ON THE UERM-CELLS AND
Anat.
10. MAAS, O.-" Ueber R e i h n g und Befruchtnng bei Spongien."
Anz., Bd. xvi., 1900.
11. MACBRIDE,
E. W
Test-Book of Embryology, P a r t I." Macmillan,
London, 1914.
12. MORGAN,
T. H.--' Regeneration.' Macmillan Company, N.Y., 1901.
13. ROBINSON,
M.--"The
Division of the Collar-Cells of' the Calcarea
Heteroccela." Quart. Journ. of Micro. Science, vol. lvii., 1920.
14. Q-ATENBY, J. BRONTE.--"
The Gametogenesis and Early Development of
Limnaa stagnalis, etc." Quart. Journ. of Micro. Science, vol. lxiii.,
1919.
.-"
DESCRIPTION OF T H E PLATES.
EXPLANATION
OF LETTERINQ.
a l , a2,a3=stages in passage of collar-cell
into mesoglea.
AC =acrosome.
AS= archoplasnlic sphere.
BN=basement niembrane of collar
epithelium.
C=centrosome.
COL= collar of collar-cell.
ECT=ectoplasmic zone of oocyte or
-
egg.
ENl)=endoplnsmic zone of oocyte.
FC=future histocytes or collar-cells
of larva.
FL =flagellum.
FPN =fern816 pronnclens.
GA=Gol@;iapparatus.
GAO=Golgi apparatus of oocyte.
OAM=grmular (initochondria) ammbocyte.
GC = granular cells of larva.
GCAV=@;astralcavity of sponge.
GCX= future grannlar(8nd lower cells?)
of larva.
GE = gastral or collar-cell epithelium.
GX= large granules possibly of Golgi
nature.
IT= inner thickening of collar.
LaC=lower granular cells of larva, as
distinguished from inner granular cells.
M =mitochondrium.
M &NG= mitochondria and nutrient (yolk)
granules.
MAM=macromitoeome ((-'Nebenkern ")
or middle-piece of eperm.
MES=mesoglenl cells.
MPN=male pronucleus.
N=nncleus.
NC€I=nucleus of collar-cell (choanocyte)
NIbdrawn-out neck" of collar-cell
formed of hyaline protoplasm.
NSC=nurse-cells associated with spermutogenesis.
OG=outer granules of flagellated cell
(possibly yolk of some sort).
OGA =oogonium (young oocyte) formed
in situ from collar-cell.
OT =outer thickening of collar.
OTE =oocyte.
PB =polar body.
PC=first stages of nietamorphosis of
collar-cell into germ-cell.
PEZthickening of protoplasm on pa&
pliery of collar-cell, inside the
collar.
S=spmn.
SCC =sperm-carrying cell (metamorphosed collar-cell).
SN =sperm-nucleus.
SPA=space around, forming spermatozoa or nurse-cavity.
SPT =spermatids.
VAC =vncuolated region of cytoplasm
caused by entry of s erm and
flow of material to t i e region
of entry.
s=supposed remains of macromitosome (MAM).
Y =yolk.sphere or nutrient metaplasniic granule.
.
With regard to fixation and staining, the following abbrevintions have been used :-
F.W.A,= Flemming's fluid without acetic acid ; H.W..k.=Herruann's fluid without acetic
EARLY DEVELOPMENT OF QRANTIA COMPRESSA.
295.
acid ; K.=Kopsch ; I.H. =Heidenhain’s Iron Alum Hamatoxylin j Ch.K. =Champy-Kull ;
Alt.=Altmann’s stain.
All figures have been drawn with camera lucida by means of a & semi-apochromatic
oil immersion and compensating eye-pieces. Figs. 2, 3, 5 a, 6, 7, 8, 9, 11-22, and 99-31,
and 33 were drawn with an 18 compensnting eye-piece. Rgs. 6 and 23-28 and 32 with an
8 compensating eye-piece, Figs. 1 and 4 were not drawn to scale. Pig. 10 was drawn
with a 12 compensating eye-piece.
The Plates have been reduced, but a scale is given a t the side of Plate 21, which
applies to all figs. drawn with an 18 compensating eye-piece. Scale to fig. 10 is alongside
the drawing. Scale to all the figs, on Plate 22 is given at the left side, excepting to
figs. 29-31 and 33, whose scale is found on the right.
While P1. 19. figs. 1,4, and 9 are drawn with the camera lncida, they are n combination,
of both Kopsch, Chanipy-Hull, and Iron Alum Hamatoxylin preparations. The same
applies to P1. 20. figs, 13, 14, and P1.21. lip. 18, 22. Every care has been taken, however,
to see that the strictest accuracy has been preserved, corresponding stages in Champy-Kull
or E.W.A. and I.H. being united with those in Kopsch preparations. All the other
figures are drawn from one preparation only. In each case the urrow points to the nearest
gastral cavity (compare 1’1. 19. fig. 5 to illustrate this).
PLATE19.
Fig. 1 A & B. Two collar-cells or choanocgtes drawn and combined from two preparations,
one by Kopsch’s method (fig. 2), the other by chrome-osmium and Iron
Ilsmatoxylin. The smaller granules are mitochondria, the larger yolk-spheres
(reserve granules). I n fig. 1 A, the nucleus lies on the outer part of the cell, in
fig. 1 B in the middle; in each the Golgi apparatus is shown at QA ; in fig. 1A
the flagellum comes out of the latter, in fig. 1 I3 it passes through or to one side
of the Golgi apparatus, and reaches to the ceutrosome, which lies on the nucleus.
The collar in fig. 1 B is partly retracted and shows an outer (OT) and an inner
thickening (IT) ; in fig. 1 A the collar is stretched out and the thickenings fire
not so marked. On the periphery of the cell, at PE, is a denser region of the
protoplasm.
K, and I. H.
Fig. 2. Row of choanocytes and a young oocyte by Kopsch’s method.
Fig. 3. Same, fixed in Hermann-without-acetic acid and stained in Iron Hamatoxylin.
Fig. 4. Choanocyte by Kopsch’s method, to show characteristic neck to cell (NK). This
consists of hyaline protoplasm. The cfolgi apparatus (GA) is very clearly
marked, and consists of archoplasm (AS) surrounded by a peripheral layer of
darkly staining matter (GA).
Fig. 5. Part of a hermaphrodite sponge which contained ripe oocytes and ripening
epermatids. The latter (SPT) lie in a special cavity (SPA), lined by aonflagellate cells (NSC) without collars. These cells are probably for!ned in the
meroglea. At GCAV is the gastral cavity lined by choanocytes (GE), and at
OTE is the oocyte showing ectoplasniic (ECT) and endoplaamic (END) regions ;
this oocyte is just about to be fertilized (S). Note the taila (axial filaments) of
the spermatids.
Fig. 8 A. A group of three spermatids drawn at a high power showing nucleus with karyosome, the centrosome, and the axial filament. The mitochondria1 substance was
washed away. Cajal.
Fig. 6. Spermatozoon drawn from an example which has passed into a collrtr-cell as i n
fig. 9, S. The tail has been added from observation of ripening spermatids, and
this figure is thought to represent the ripe unchanged sperm of Ginnliu comprersn.
At AC is the acrosome. Ch.K. (See footnote on page 279.)
296
DR. S. B. OATENBY ON THE GERM-CELLS AND
Fig. 7. Ordinary nmceboid cell of the mesoglea of ffrantiu; i t lies in the nieaogled fibrous
tissue (MES). A t M are its mitochondria. There is a distinct ectoplasm.
Ch.K., 1.13.
Fig. 8. An oogonium of the mesoglea; the urrowa point to the nearest gastrd cavities.
Ch.K.
Pig. 9. Full-grown oocyte of Cfrantia with nearest accompanying choanocytes, in one of
which (at S) a sperm has entered. This oocyte shows the typiral endoplasmic
region with the grltnulnr inclusioiis and the ectoplasmic part; on the left is a
pseudopodium. A t M the mitochondria, and a t Y the yolk-granules, are evenly
distributed; no animal or vegetative pole could be identified by reason of unequal
distribution of the cell-granules.
Ch.K., I.H., & K.
PLATE
20.
Fig. 10. Oocyte at lower magnification to preceding, showing large sperm just after entry.
The cell-granules tend to approach the region of entry of the sperm.
Figs. 11%, 14. Gaetral side of oocyte showing sperm-carrying cell lying upon the former ;
in both sperm-carrying cells the nucleus is flattened on one side owing to the
pressure of the contained sperm ; in tig. 11 the sperm will enter middle-piece
forwards, in fig. 14 middle-piece backwards. The ectoplasm in the region of
entry of the sperm is interrupted. In fig.11 yolk is not shown. Ch.K.
Fig. 13. Sperm just entering, acroaome first. Shows protoplasmic continuity between
sperm-carrying cell and oocyte. Ch.K.
Fig. 13. Later stnge, polar body at PB, and female pronucleus growing (PPN). Spermpath marked by pale chromophobe area (VAC). Note large size of mitochondria
(M). Macromitosome of sperm disappeared, but may be represented by granules
at x. Cli.K., I.H., k K.
Fig. 14. See description t o fig, 11. Ch.K., I.H., st K.
Fig. 16. Flattened sperm-bearing call, which was compressed by egg, and so looks abnormally large. Its nucleus is of the open type. Ch.K.
Fig. 16. Sperm just after entry ; its cell-wall has disappeared, macromitosome still complete,
and sperm-cytoplasm furred at edges but still distinguishable from egg-cytoplasm by its greater chromophility and stringiness. Acrosome pale. Nucleus
nearly tjpherical and has grown. Sperm-carrying cell at SCC, with open
nucleus.
Fig. 16. Stage in fertilization lifter that in fig. 12. The sperm cell-wall has disappeared and
its stringy cytoplasm is still distinct from that of the egg. The acrosome (AC)
is becoming faint. Ch.K.
Fig. 17. Later stage just after breaking up of sperin middle-piece or macromitosome (MAM).
This preparation was fixed by Champy-Kull and stained by Heidenhain’s Iron
Alum Hsmatoxylin. Note that nuclei of sperm-carrying cell (SCC) and those
of ahoanocytes (NCH) stain dike. Ch.K., I.H.
PLATB
21.
Fig. 18. The two pronuclei side by side prior to last atage of fertilization. Note even disposal of inclusions (M& Y). The sperm-carrying cell (SUC) is degenerating in
’
this cue. Yolk added from another preparation at a siuilar rtage. Ch.K. &K.
Pig. 19. Two-cell stage, showing even disposal of mitochondria. Upper blastomere not cut
across middle. Ectoplaem evenly divided. Ch.K., I.H.
Fig. 20. Four-cell stcage,showing continued subequal distribution of the mitochondria and
of ectoplasm. . H. W.A., 1.H.
G atenby.
LINN.SOC.JOURN.ZOOL.VOL~.XXXW,PL.~~.
J.B.G .deL
Huth,London.
GRANTIA ( S Y C O N ) COMPAESSA
G at enby.
Huth, L o n don.
J.B.G. dal
GRANTIA ( S Y C O N ) COMPRESSA.
G atenby.
Huth,London
J.B.G.dal
G R A N T I A ( S Y C O N ) COMPRESSA
EARLY DEVELOPMENT OF GRANTIA COMPRESSA.
297
Fig. 21. Blaatula just before differentiation of flagellnted hemisphere (see P1. 22. flg. 27).
The fine mitochondria, still visible but staining less well, lie around the nuclei.
The yolk is not shown. At GAM is a granultLr ameboid cell. Judging from
the contour of this blastula, the future flagellated hemisphere will differentiate
on the right, and away from the ueitrest collar-cell. Ch.K., I.H.
Fig. 22. Younger blasttilit showing even segregation in each blastomere of yolk-spheres and
mitochondria. The ectoplasm is slightly larger in quantity on the right than on
the left. The yolk-spheres have been added from another preparation prepared
by Kopsch’s method. Ch.K., I.H., & K.
PLATE22.
Figs. 23, 2G, SZ 26. Parts of flagellated’gestrnl epithelium, Bhowing at PC and OGA two
stages in met:nnorphosis of collar-cell i n t o oogonium. Ch.K., 1.11.
Fig. 24. €’art of flagellated g w t r d epithelium showing immigration inwards of collar-cells,
al, a 2 , S: a3, into the mesoglea. Ch.II., I.H.
Fig. 27. Differentiating amphiblestula, showing future flngellnted rells (PC) and future
lower and granular rells (GCX). At OTE is a young oocyte in growth-jtage.
PLATE23.
Figs. 28 B 32, Two arnphiblastula larvce, for description of which see page 266. Ch.K.,
I.H..and Ch.K., Alt.
Fig. 29. Maternal nutrient capsule-cell (LGC in fig. 28), showing irregular and numerous
mitochondria (M). Ch.IL, 1.H.
Fig. 30. Inner ameboid granular cell (GHM in fig. 28) showing dense nuuierous mitochondria.
Fig. 31. Granular cell (GCi n fig. 28) showing scattered yolk-spheres (Y), mitochondria (M),
and what are possibly Golgi elements a t GX. Ch.K., I.H.
Fig. 32. (See legend to fig. 28.)
Fig. 33. Flzgullated cell or hiatocyte showiug four cytoplasmic elements. Mitochondria
(M), yolk (y),Golgi apparntus (GA ?), and outer “yolk ” granules a t OG.