J. Embryo/, e.xp. Morph. Vol. 21, 2, pp. 243-54, April 1%9
Printed in Great Britain
243
The yolk of Holostean fishes
By WINCENTY K1LARSKI 1 AND ZYGMUNT GRODZINSKI 1
From the Department of Comparative Anatomy, JagelIonian University,
Krakow
Eggs of some archaic fishes, such as Polypterus, sturgeon or the holosteans,
resemble amphibian eggs. Not only has the similarity been found in the amount
of yolk (mesolecithal types of eggs), which indicates the manner of egg cleavage
(holoblastic) in these vertebrates, but also in the morphological composition of
yolk. Fat droplets, yolk platelets and an unidentified fluid in which they are
suspended, in fishes sometimes called ichthulin, are three main components of
yolk in these eggs.
Yolk platelets have been an object of interest to various observers because of
their unique structure (Hwan Sun, 1962; Ward, 1962; Karasaki, 1963) and the
problems of their formation (Lanzavecchia, 1960; Ward, 1962) and breakdown
when being utilized (Karasaki & Komoda, 1959; Hwan Sun, 1962; Jurand &
Selman, 1964). Most information concerning their structure and physicochemical properties has, however, been acquired by observations of amphibian
yolk platelets. When in 1946 Holtfreter described the behaviour of yolk platelets
of the frog (Rana pipiens) at different osmotic concentrations he found that yolk
platelets are marked by birefringence and that during intracellular digestion
they can split into smaller discs, which still show birefringence. A similar effect
can be achieved by treating yolk platelets with weak acids or alkali (Gross &
Gilbert, 1956; Ringle & Gross, 1962), the smaller discs thus obtained still
having the analogous properties of anisotropic substances. These observations
made it possible to demonstrate that amphibian yolk platelets are composed of a
substance having a laminar structure. Observations were made using the electron microscope by Karasaki & Komoda (1958), who described the hexagonal
crystalline structure limited to the core of the platelet, while the superficial
portion surrounding the core has a granulo-fibrous structure enclosed by a
semi-permeable membrane.
Yolk platelets of the fishes mentioned at the beginning of this paper behave
similarly to the amphibian ones, which they also resemble in appearance
(Grodzinski, 1958, 1963, 1968). The authors studied yolk platelets of the eggs of
1
Authors' address: Department of Comparative Anatomy, ul. Krupnicza 50, Krackow,
Poland.
244
W. KILARSKI & Z. GRODZlfiSKI
Amia and Lepisosteus (Holostei) to determine to what degree the ultrastructure
of these platelets resembles that of the amphibian yolk platelets.
MATERIALS AND METHODS
Mature ovarian eggs of two species of fishes, the gar-pike (Lepisosteus osseus)
and bowfin (Amia calva), caught in Wisconsin in the United States, were studied.
The eggs were obtained from females in the month of May, in the middle of the
spawning season. They were quite ripe; their size (2-2-2-5 mm in diameter) and
appearance corresponded to the description of freshly layed eggs of Amia and
Lepisosteus (Dean, 1895; Whitman & Eydeshymer, 1897). Eggs were fixed whole
or crushed and the yolk mass, flowing out of them, was fixed. Parallel fixatives
applied were 4 % formalin neutralized with magnesium carbonate, cacodylatebuffered 5 % glutaraldehyde and 2 % aqueous solution of osmium tetroxide
with 002 M calcium chloride added. The eggs were left in the aldehyde fixatives
for 24 h at 4 °C. In the osmium tetroxide solution they were fixed for 4 h at 4 °C.
After fixation all the preparations were dehydrated through a graded series
of ethanol and then kept in 96 % alcohol for several weeks. The material
collected was fixed and partly dehydrated in the Institute of Biophysics, University
of Chicago, and sent in 96 % alcohol to Poland. The dehydrated material was
embedded in Epon 812 (Luft, 1961) and polymerized at 60 °C. Ultra-thin sections
were cut with a diamond knife on a Porter-Blum microtome. The sections, silver
in colour, were picked up on copper grids coated with carbon film and 'stained'
in uranyl acetate and lead citrate (Reynolds, 1963). They were examined with a
Japanese Electron Optics Co. electron microscope, type JEM-5Y. Measurements
were taken using an eyepiece micrometer to an accuracy of 0-1 mm on positives
enlarged 150000 times.
RESULTS
Light microscope observations
In the yolk of Holostean fishes one can distinguish fat droplets and yolk
platelets in the light microscope. The latter behave characteristically in NaCl
solutions of different osmolarity and react with some supravital dyes (Grodzinski, 1963, 1968).
The yolk platelets of both species investigated, as a rule, are elliptical. Their
dimensions vary within wide limits. The long axis of the platelet of Amia
measures 5-6 /*, of Lepisosteus 3-22 JLI. In the yolk of the latter fish some yolk
platelets are rectangular in shape and 20-25 JLL long (Fig. la).
The homogeneous appearance of the yolk platelets changes after a few
minutes in a hypertonic (1 M) solution. A borderline appears between the
transparent sheath and greyish core (Fig. \b). Shortly after, the core disintegrates into extremely fine granules, which start rapid whirling movements. At
the same time the sheath contracts and rounds up, forming a rectangular disc
(Fig. \c). In isotonic or slightly hypotonic solutions (012—018 M) the sheath
r
Holostean fish yolk
245
10//
1a
:*•
1c
f
• r
1b
Fig. I. A single yolk platelet from an oocyte of Amia in 018 M-NaCl solution (a),
and in 1 M-NaCl solution (b, c). A single yolk platelet from an oocyte of Lepisosteus
(cl) in 1 M-NaCl solution, x 1000.
1d
246
W. KILARSKI & Z. GRODZINSKI
moves apart from the core and forms two ear-like protrusions in Lepisosteus,
whereas in Amia it keeps its previous outlines (Fig. \d).
The supravital basic dyes, such as neutral red, colour the core. The acid
ones, e.g. trypan red, do not enter the platelet. The sheath exhibits therefore two
qualities: semipermeability and elasticity. The core is of a stiff structure, but
may break up into fine granules.
Electron microscope observations
Yolk platelets are composed of three readily distinguishable components.
The central portion of the platelet is a structurally homogeneous dense oval core.
Stained with lead or uranium ions, the platelet core increases in density. Another
component of the yolk platelet is the layer surrounding the core, which differs
from the core in its lower density and granular structure (Figs. 2, 3). The whole
is enclosed by the third component, a single membrane (Fig. 3).
The seemingly homogeneous structure of the core of the yolk platelet observed
under high magnification reveals distinct zonal differences in density. The core
consists of a less dense central part and a periphereal layer of a greater density,
giving it the shape of a regular oval (Fig. 3). Besides this difference in density the
two zones differ also in the organization of the macromolecules of which they are
built.
The cores of most yolk platelets observed showed a compact granular structure with no special regular organization. The central part differed from the
cortical part only in density. However, in some yolk platelets we observed
regular striations (Fig. 4), consisting of light and dark bands. The striations of
the core were at varying angles in individual platelets and no regularity in
relation to the long axis of the yolk platelet could be observed. The measurement of the light and dark bands was very difficult, for the boundary between
these bands, being blurred, was hard to define precisely. Another difficulty was
more fundamental: measurements were being made on bands magnified
1500000 times, which appeared to be built of globules connected by blurred
narrowings. These narrowings, however, seem to be optical artifacts caused by
the resolving power being insufficient to separate individual particles arranged
linearly, which under low magnification look like a band. This is true of both
dark and light bands. An attempt was therefore made to measure the diameters
of these particles. In the case of light particles the diameter ranged between 68
and 70 A, and the narrowing between two such particles measured about 47 A.
Similar values were obtained for dark particles and their narrowings. The cores
of a few yolk platelets looked as if they had two systems of striation, intersecting
each other at an angle of 47° (Fig. 5). This image could, however, be changed by
shifting the focus at the section to the rear, in which case only one system of
bands was seen, as in Fig. 4. The value for the angle (47°) does not seem to be
constant, since various angles of intersection were observed in particular platelets. In some of the gar-pike platelets examined the linearly arranged particles
Hoiostean fish yolk
Fig. 2. Components of yolk of an oocyte of Amia. The yolk platelets (YP) vary in
size and shape. Among the yolk platelets are very dense fat droplets (LP). x 1700.
247
248
W. KILARSKI & Z. G R O D Z I N S K I
Fig. 3. A single yolk platelet from an oocyte of Amia. The core has two components,
the central one occupying the greater part of the platelet and the peripheral cortex
of somewhat greater density. The core is surrounded by the superficiail layer (SL),
limited in turn by the membrane (Mb), x 6000.
Holostean fish yolk
249
gradually changed their arrangement into a hexagonal one at the opposite
margin of the platelet (Fig. 6). In this arrangement we observed a hexagonal
figure built of light particles about 47 A thick, the sides of this figure being 68 A
long. If dark particles were assumed to be the starting point, the figure obtained
was also hexagonal, but this time it was built of dark particles, 68 A thick and
arranged round a central particle. In such a figure all the dark particles were
47 A apart.
Cortical layer of the core
The cortical layer of the core differs from the central part in density. Surrounding the central part, it surrounds the polygonal outlines of this part so as to
give it the shape of an oval (Fig. 3). The structure of the cortex is distinguished
by an irregular arrangement of granules or short fibrils, which vary in density
and are very closely packed (Fig. 7). A gradual disorganization of the regular
structure of the central part can be observed at its boundary with the cortex,
which might suggest that the cortical layer is a portion of the central part, the
particles of which are not arranged in a crystalline manner. The measurements
of dark and light particles of the cortex also agree with those in the central
part, which may also indicate their identity. In the zone bordering upon the
superficial layer the granulo-fibroid structure of the cortex of the core is less
dense and has irregular outlines. No membrane separating the core from the
superficial layer was observed (Fig. 7).
Superficial layer
The superficial layer surrounding the core of a platelet varies in thickness; in
some platelets it may even appear to vanish at one pole and increase its thickness
by many times at the opposite one (Fig. 2). This suggests the possibility of displacement of the core within the superficial layer, or may be due to the fact that
the section plane runs tangentially to the long axis of the platelet. The density
of the superficial layer is considerably less than that of the core. This layer has a
loose organization and it is made up of fine granules and fibrils, about 130 A
thick, arranged irregularly. The organization observed here seems to have been
brought about by the coagulating action of the fixatives (Fig. 2, 3). The superficial layer of the yolk platelet, and consequently the whole platelet, is surrounded by a single membrane 180-200 A thick. In our preparations this
membrane is very badly preserved. Poor fixation is indicated here by lack of
continuity or by vesiculation of the membrane. Yolk platelets derived from
crushed preparations and those kept in alcohol too long were completely devoid
of the membrane and, occasionally, of the superficial layer.
DISCUSSION
The yolk of phylogenetically newer fishes (mainly teleosts) is organized
in the form of lipoprotein spheres (without a crystalline core) like the yolk of
250
W. KILARSKI & Z. G R O D Z I N S K I
Fig. 4. Fragment of the central part of the core of a yolk platelet of Amia. The distinct striation consists of dark and light bands alternating regularly at intervals of
65 and 47 A. x 128000.
Fig. 5. Fragment of the central part of the core in a yolk platelet of Amia. The central part looks as if there were two systems of striation intersecting at an angle of 47°.
This apparent image is formed due to the hexagonal arrangement of dark (dense)
particles, x 128000.
Holostean fish yolk
251
Fig. 6. Fragment of the core in a yolk platelet of Lapisosteus. The linear arrangement of particles at the lower edge of the platelet passes gradually into the hexagonal
system at the upper edge, x 168000.
Fig. 7. Fragment of the core of an Amia yolk platelet. It shows the border area between the central part and the cortex, this last zone having a distinctly denser structure than the central part. The crystalline arrangement of particles in the central part
passes gradually into the structural disorder of particles of the cortex (arrows),
x 168000.
17
J E EM 2 1
252
W. KILARSKI & Z. GRODZINSKI
birds or reptiles (Grodzinski, 1939, 1949, 1951, 1954, 1956). This type of yolk
might be called 'bird type'. The phylogenetically older groups of fishes (sharks,
sturgeons or holosteans) have yolk organized in the form of platelets (with a
crystalline core) like amphibians. Therefore we propose to call this type of yolk
organization the 'amphibian type'.
The yolk platelets of a holostean consist of three main components: the
crystalline core, the surrounding superficial layer and the single membrane,
which encloses the whole platelet. This organizational pattern can also be seen
in another group of vertebrates, amphibians, e.g. Triturus, Diemictylus, Rana
and Bufo (Ward, 1962; Karasaki & Komoda, 1963). Our attention was concentrated mainly on one element of the platelet, the core. The central part of
the core, composed of two types of substances varying in density and arranged
alternately, shows a clear crystalline structure, shown by its periodic striation.
However, this would raise doubts whether our images of yolk platelets are
images of actual molecular architecture or whether they are effects of the diffraction of electrons by this architecture. Karasaki (1963) rejects the diffractive
interpretation of the striation on the assumption that the particles which make
up the central part of the core of an amphibian yolk platelet are sufficiently
large and can be resolved with the electron microscope. Karasaki had at his
disposal the results of chemical analyses of amphibian yolk (Wallace, quoted
by Karasaki, 1963), which allowed the rough determination of the size of particles
on the basis of their molecular weight. No data concerning the chemical
composition of holostean yolk have been published as yet. Nevertheless, great similarities in molecular organization of yolk platelets between
holosteans and amphibians have provided grounds for us to compare
these structures and state that there is an analogy between them in the size
of particles of which they are built. Consequently we would suggest that
the striations observed in the central part of the core of the holostean yolk
platelet is an image of the actual molecular structure and not a diffraction
effect.
The values obtained by us for the widths of the light and dark bands differ
from those given by the authors who worked on amphibians (Karaski &
Komoda, 1959, 1963; Ward, 1962; Jurand & Selman, 1964), but the differences
do not seem to be significant. Under high magnification and at better resolution
the uniform bands split into regularly arranged spherical particles, whose diameter corresponds more or less exactly to the width of the band. If one manages
to find yolk platelets sectioned at different angles, then the particles making up
the band are arranged into parallelograms, rhomboids, or, in the case of the
greatest inclination of the platelet, a hexagonal pattern. On the basis of these
observations we may venture the conclusion that the particles are arranged in
long hexagonal 'pillars'.
Our observations showed that the density of the particles can be further increased by the use of uranium or lead salts. This phenomenon is due to the
Holostean fish yolk
253
specific adsorption of these ions on phosphate particles present in protein and
lipid molecules in the yolk platelets.
A small difference in density and lack of crystalline structure distinguish the
central part of the core from the thin cortical layer. Lack of a sharp demarcation line between these zones and the existence of some continuation between
the regularly organized molecules of the central part and the molecules of the
cortex scattered in a disorderly manner permit the supposition that we are
concerned here with the same components. The measurements of the cortical
particles agree with those of the particles of the central part of the core.
The core of the yolk platelet is surrounded by the superficial layer and the
whole is enclosed by the membrane, which structurally resembles the membranes
making up other organelles of the oocyte. The presence of this membrane,
though sometimes called into question (Ringle & Gross, 1962) is confirmed by
some physico-chemical properties of holostean yolk platelets as well as by the
morphological evidence. Such phenomena as the swelling or shrinking of the
superficial layer in NaCl solutions of different osmolarity, the penetration of
alkaline dyes and the blocking of acid dyes can be explained only by the presence
of a semi-permeable membrane (Grodzinski, 1968). The poor state of preservation of the membrane or sometimes even its absence in some of the platelets
observed by us is due to the poor conditions of fixation or dehydration of the
material.
SUMMARY
The yolk of holostean fishes {Lepisosteus and Amid) consists of fat droplets
and yolk platelets. This type of yolk can be called the 'amphibian type'. The
yolk platelets are composed of three main components: a crystalline core, a
superficial layer surrounding it and a membrane, which encloses the whole
platelet. The platelet core shows distinct dark and light periodic bands alternating at regular intervals of 65 and 47 A. Yolk platelets sectioned at various
angles reveal corresponding changes in the arrangement of bands. High magnification and good resolution made it possible to show that the bands are made
up of dark and light spherical particles, 65 and 47 A in diameter. These particles
are arranged hexagonally in the core of the platelet.
RESUME
Le vitellus des Poissons Holosteens
Le vitellus des Holosteens {Lepisosteus et ^m/a)consiste en goutteletteslipidique
et en plaquettes vitellines. Les auteurs ont nomme ce type de vitellus le 'type
amphibien'. Les plaquettes vitellines sont formees de trois composants principaux: un noyau cristallin, une couche superficielle l'entourant et une membrane
qui entoure la plaquettes entiere. Le noyau de la plaquette presente des bandes
periodiques distinctes, sombres et claires, alternant a intervalles reguliers de
65 et 47 A. Des plaquettes vitelline sectionnees selon des angles varies revelent
17-2
254
W. KILARSKT & Z. GRODZINSKI
des modifications correlatives dans la disposition des bandes. Un fort grossissement et une bonne resolution ont permis de constater que les bandes sont formees de particules spheriques sombres et claires, de 65 et 45 A de diametre.
Ces particules sont disposees en hexagones dans le noyau de la plaquette.
We are grateful to Professor W. Bloom, from the Department of Biophysics, University of
Chicago, for his personal and financial help in supplying us with the material for investigations, and J. Bigaj for his skilful technical assistance.
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(Manuscript received 16 May 1968—revised 30 June 1968)