353
The yolk nucleus of fish oocytes
By R. P. NAYYAR
(From the Department of Zoology, University of Delhi, Delhi 6, India)
With i plate (fig. 2)
Summary
A comparative study of the yolk nucleus in the oocytes from 8 species offisheshas been
carried out. The yolk nucleus appears as a mass of lipid granules and mitochondria
situated beside the nucleus. It becomes spherical, detaches itself from the nucleus,
and migrates to the peripheral region of the cytoplasm to lie just below the cell
membrane. During the migration, the lipid granules of the yolk nucleus grow and
move out of this organelle. When the oocyte is about 180/x in diameter, the yolk
nucleus breaks up and its components are dispersed in the cytoplasm. No differences
have been found in the morphology of the yolk nucleus in the species under investigation.
Introduction
T H E early oocytes of certain animals contain a mass of special character
situated beside the nucleus, to which the names of yolk nucleus, archoplasm,
centrosphere, and corps vitelline have been applied by different workers. This
cytoplasmic inclusion has been observed by many workers in spider oocytes
(Dyal and Nath, 1933; Krishna, 1953; Nath, Gupta, and Manocha, 1959;
Sareen, 1963). A similar structure has been reported in fish oocytes (Wheeler,
1924; Hibbard and Parat, 1928; Subramaniam, 1935; Narain, 1951; Chopra,
1958a, b, 1961; Sathyanesan, 1959; Stock, 1959, 1961), and in the oocytes of
reptiles (Srivastava, 1948; Dutta, 1950) and birds (Brambell, 1925; Das,
1931; Guraya, 1962).
Nath and others (1959) described the yolk nucleus of spiders as a 'dynamic
seat of lipid synthesis'. In fishes this inclusion is not so active as in spiders,
but its occurrence has been reported in the early oocytes of all the species of
fishes studied so far. Controversial reports exist regarding the origin, role, and
fate of the yolk nucleus (Wheeler, 1924; Dyal and Nath, 1933; Subramaniam,
1935; Sathyanesan, 1959; Stock, 1959, 1961; Chopra, 1961; Sareen, 1963).
With this-in mind, a comparative study of the yolk nucleus in 8 species of
teleosts has been carried out with improved histochemical techniques of
fixation and staining, and also by phase-contrast microscopy of the living
material.
Material and methods
The following species were studied:
Cyprinidae
Esomus dancricus (Ham.), Puntius conchonius Ham., Labeo dero (Ham.),
Chela bacaila (Ham.)
[Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 353-358, 1964.]
354
Nayyar—The yolk nucleus of fish oocytes
Heteropneustidae
Heteropneustes fossilis (Bloch)
Bagridae
Mystus seenghala (Sykes)
Channidae
Channa punctatus (Bloch)
Mastacembelidae
Rhynchobdella aculeata (Bloch).
Specimens of these species were collected from the River Jamuna and its
tributaries around Delhi during the months of April to August 1962. For
morphological studies, small pieces of the ovaries were fixed in Bouin's fluid
and in 10% formalin. Paraffin sections, 10 /x thick, were stained with
Heidenhain's haematoxylin.
Saturated solutions of Sudan black B in ethanol and in ethylene glycol were
used as general lipid colorants. For histochemical studies, the material was
processed by the techniques recommended by Baker (1946) for phospholipids
and by Cain (1947, 1948) for triglycerides; also by Ciaccio's post-chroming
technique as modified by Bradbury (1956) and the performic acid/ Schiff
reaction (Pearse, i960), to detect the degree of saturation of lipids. The
coupled tetrazonium reaction (CTZ) of Daneilli (Pearse, i960) was carried
out for the study of proteins. For carbohydrates, the periodic acid / Schiff
(PAS) reaction (Hotchkiss, 1948) was used, with various controls as recommended by Pearse (i960).
The methyl green / pyronin G (Brachet, 1953) and toluidine blue / methyl
green / orange G (Korson, 1951) methods were used, along with extraction
controls, for the demonstration of nucleic acids. Acid fuchsin was used for
mitochondria (Baker, 1956).
In addition, living oocytes were studied by phase-contrast microscopy.
Observations
The following description is based mainly on a study of the oocytes of
Heteropneustes fossilis. The general behaviour of the yolk nucleus is similar in
all the species under investigation, apart from minor differences in the size of
the oocyte at which the yolk nucleus appears. However, in all these fishes, the
activity of the yolk nucleus is confined to the stage when the oocyte has a
diameter between 50 and 200 /x.
The yolk nucleus first makes its appearance when the oocyte has a diameter
of about 50 /x, in the form of a concentration of granules round the nucleus
(figs. 1, A, B; 2, A). Under the phase-contrast microscope, granules of two
sizes can be distinguished. Histochemical tests reveal that the small granules
are mitochondria and the larger ones lipid globules. With the growth of the
oocyte, the concentration round the nucleus becomes more pronounced and
a conical cap is formed on one side of the nucleus (fig. 1, c to E). When the
oocyte measures 100 JU, in diameter, more mitochondria and lipid bodies
collect and the yolk nucleus now reveals two regions: an outer region of
Nayyar—The yolk nucleus of fish oocytes
355
diffuse lipids and mitochondria, and an inner or medullary region with an
aggregation of lipid bodies (fig. 1, F).
The yolk nucleus, after becoming spherical, starts migrating towards the
periphery of the oocyte (fig. 2, B, c). The lipid bodies of the central zone grow
and slowly move apart. Emerging from the yolk nucleus they become dispersed in the cytoplasm (fig. 2, E). This process stops as soon as the yolk
nucleus has reached the periphery and lies just below the egg membrane
yolk nucleus
nucleus
lipid body
FIG. 1. Camera lucida drawings of growing oocytes of Heteropneustes fossilis. (Formaldehydecalcium, post-chromed; frozen sections; Sudan black.) A, early oocyte, showing the concentration of lipid granules and mitochondria round the nucleus. B, the yolk nucleus has appeared
beside the nucleus in the form of a mass of lipid bodies and mitochondria. C, D, the yolk
nucleus has increased in size and formed a conical cap on one side of the nucleus. E, the yolk
nucleus has become oval, but still occupies a position beside the nucleus. F, later stage of
oocyte, showing the yolk nucleus during its migration towards the periphery. Note the increase
in size of the lipid bodies.
(fig. 2, D). Later, the yolk nucleus loses its spherical form and becomes
oblong or irregular in outline (fig. 2, D). When the oocyte is 180 fx, in diameter,
the yolk nucleus breaks up and its components disperse through the cytoplasm
and ultimately get mixed with other inclusions.
Living oocytes of diameter 130 to 150 /x were punctured and the contents
studied under the phase-contrast microscope. The yolk nucleus escaped from
the oocyte and could be clearly seen as an intact spherical mass of granules.
The colouring of formalin-fixed gelatin sections with Sudan black B
revealed two types of sudanophil granules in the yolk nucleus. Both types of
granules contain phospholipids (fig. 2, A, B, D). Triglycerides appear to be
absent. The larger lipid granules can be completely extracted with lipid
solvents such as chloroform / methanol. The application of Ciaccio's test as
modified by Bradbury (1956) and the PFAS test (Pearse, i960) reveals that the
phospholipids of the larger granules (lipid bodies) are unsaturated (fig. 2, E).
356
Nayyar—The yolk nucleus of fish oocytes
The smaller sudanophil granules colour red with the acid fuchsin techniques
for mitochondria.
In paraffin sections, the yolk nucleus contracts into a spherical mass and
becomes separated from the cytoplasm by a clear area. Staining with methyl
green / pyronin (Brachet, 1953) or by methyl green / toluidine blue / orange G
(Korson, 1951) indicates that the yolk nucleus consists largely of basiphil
material, rich in RNA. It gives a positive reaction to the coupled tetrazonium test, before and after benzoylation (Pearse, i960), which suggests
the presence of proteins containing tyrosine, tryptophane, and histidine. The
results given by the PAS test, with various controls, reveal the presence of
carbohydrates in the yolk nucleus, but no glycogen or acid mucopolysaccharide has been detected.
Discussion
Wilson (1925), Srivastava (1948), Chaudhry (1949), Chopra (1961), Guraya
(1962), and Sareen (1963) consider that the yolk nucleus is of cytoplasmic
origin, but Wheeler (1924), Hibbard and Parat (1928), Subramaniam (1935),
Sathyanesan (1959), and Stock (1959, 1961) believe it to be an organelle of
nuclear origin, which passes through the nuclear membrane to invade the
cytoplasm. The present observations support the cytoplasmic origin of the
yolk nucleus. The formation of the yolk nucleus has been studied in detail
from the initial stages of its appearance. At no stage of oocyte growth has any
intranuclear body been seen to pass out of the nucleus into the cytoplasm.
Chopra's (1961) attempt to follow the nuclear origin of the yolk nucleus in
the oocytes of Notomigonus chrysoleucas with tritium-labelled cytidine lends
support to this view.
The morphology of the yolk nucleus differs from one group of animals to
another. In spiders it is made of 4 concentric zones (Nath and others, 1959;
Sareen, 1963). Electron microscope studies have revealed that the cortex of
the spider yolk nucleus is formed of cisternae of the endoplasmic reticulum
intermingled with Golgi bodies and mitochondria, while the core is made up
of a spherical body, which has been called a 'capsulated body' (Sotelo and
Trujillo-Cenoz, 1957).
On the yolk nucleus of fishes the reports are conflicting. Sathyanesan (1959)
and Stock (1959, 1961) described the yolk nucleus as a spongy body in the
cytoplasm, possessing a large vacuole. With growth of the oocyte, according
to these workers, the yolk nucleus loses part of its staining capacity and moves
FIG. 2 (plate). A, photomicrograph of oocyte at the 80-ft stage, showing the yolk nucleus
lying beside the nucleus. Acid haematein.
B, oocyte at ioo-^i stage. The yolk nucleus has become an oval mass. Acid haematein.
c, oocyte at 125-/1* stage. The yolk nucleus is migrating towards the periphery of the oocyte.
Phase contrast.
D, oocyte at i6o-/i stage. The components of the yolk nucleus have started dispersing
through the cytoplasm. Acid haematein.
E, the yolk nucleus at the stage when the oocyte is 125 ft in diameter. Some of the lipid
bodies have moved out of the yolk nucleus. Ciaccio's method.
FIG.
2
R. P. NAYYAR
Nayyar—The yolk nucleus offish oocytes
357
towards the periphery, where it is reduced to a granule and ultimately merges
with the cytoplasm. Stock (1959, 1961), who only used material fixed in
Bouin and stained with haematoxylin, suggested that the morphological
characteristics of the yolk nucleus have some taxonomic significance among
fishes. He considered that a yolk nucleus with a granule in a vacuole was
characteristic of Cyprinids and Characids, while a non-granulated one was
characteristic of Silurids. The material used in the present investigation
includes both Cyprinids and Silurids, but in all these fishes, the yolk nucleus
first appears as a concentration of lipid bodies and mitochondria around the
nucleus, which later detaches itself from the nucleus, becomes spherical, and
moves to the peripheral region of the oocyte, where it becomes disorganized.
No significant difference in the yolk nucleus was observed between members
of the two families. Although Narain (1951) and Chopra (1961) observed tiny
neutral-red vacuoles in the fish yolk nucleus, large vacuoles of the type
reported by Sathyanesan (1959) and Stock (1959, 1961) were not reported by
the early workers on fish oogenesis. Such vacuolation, however, is sometimes
noticed in material fixed in Carnoy or Bouin.
In reptiles (Srivastava, 1948; Dutta, 1950) and birds (Brambell, 1925;
Das, 1931; Guraya, 1962), the yolk nucleus arises in the midst of a concentration of lipid bodies and mitochondria. According to Guraya (1962) the
yolk nucleus of bird oocytes is a homogeneous spherical mass of granules with
radiating projections. He believed that mitochondria and lipid bodies collect
in the vicinity of this structure and do not form a part of it. He further
suggested that the yolk nucleus of birds corresponds to the 'ergastoplasm' of
early workers (Haguenau, 1958). Guraya is the only worker to report the
reconstitution of the yolk nucleus after the yolk is fully formed. No structure
comparable to the yolk nucleus, however, has been observed in the fullygrown ova of fishes.
The present studies confirm the presence of mitochondria and lipid bodies
in the yolk nucleus of fishes. The yolk nucleus has also been found to be rich
in basiphil material, proteins, and carbohydrates. Similar results have been
obtained by Chopra (1961) in fishes, by Nath and others (1959) and Sareen
(1963) in spiders, and by Guraya (1962) in birds.
In spite of diverse views regarding the origin and morphology of the yolk
nucleus, the majority of workers believe it to be related in some way with yolk
formation and hence the term 'yolk nucleus' has been universally accepted. In
spiders, Nath and his colleagues (1959) and Sareen (1963) described the origin
of lipid bodies from the yolk nucleus in 4 cycles during oocyte growth.
Protein yolk has no relation with the yolk nucleus but arises independently in
the periphery of the oocyte. In fishes, Narain (1951) believed that the yolk
nucleus represents an area of concentration of Golgi bodies, mitochondria,
yolk bodies, and vacuoles in early oocytes.
In lizards, Srivastava (1948) reported the appearance of Golgi bodies
inside the yolk nucleus. Guraya (1962) suggested that in birds, the yolk
nucleus is the most important site of synthetic activity during oogenesis.
358
Nayyar—The yolk nucleus of fish oocytes
During the present studies it has been observed that although the lipid
bodies inside the yolk nucleus are the first to grow, the major increase in the
cytoplasmic lipids takes place long after the yolk nucleus has become disorganized. It seems probable that the yolk nucleus of fishes initiates the
synthesis of lipids. It is possible that components of yolk nucleus play some
role in further synthetic activities of the oocyte after the yolk nucleus itself
has lost its identity.
The author is grateful to Professor B. R. Seshachar for guidance and to
Dr. S. R. V. Rao for helpful criticism of the manuscript.
References
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Bradbury, S., 1956. Ibid., 97, 499.
Brambell, F. W. JR., 1925. J. roy. micr. Soc., 49, 17.
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Postscript.—While this paper was in press, a review appeared on the origin
and differentiation of cytoplasmic inclusions in the oocytes of Xenopus laevis
(Balinsky and Devis, 1963, Acta Embryol. Morphol. Exp., 6, 55). There is
much similarity between fish and amphibian oocytes as regards the origin,
morphology, and fate of the yolk nucleus. Balinsky and Devis concluded
that the yolk nucleus plays no part in the production of protein yolk platelets, its primary function being to act 'as a focus of reproduction of mitochondria'. They call this organelle simply 'Balbiani body'.