475
The histochemistry and fine structure of the accessory
nuclei in the oocyte of Bombus terrestris
By C. R. HOPKINS
(From the Zoology Department, University College of Swansea, Singleton Park, Swansea)
With 2 plates (figs, i and 2)
Summary
The accessory nuclei are present throughout vitellogenesis in the peripheral ooplasm;
they probably originate from the oocyte nucleus. Structurally they resemble nuclei
and in addition their electron-dense inclusions contain RNA, possibly of nucleolar
origin. They do not, however, contain chromosomal material.
During development they increase in size and multiply by equal division or by a
form of terminal budding.
They do not become transformed into albuminous yolk spheres but are probably
concerned with the control of albuminous yolk synthesis at the periphery of the oocyte.
They remain after the termination of yolk synthesis and are associated with the
formation of the vitelline membrane.
Introduction
ACCESSORY nuclei were first described by Blochmann in 1886 as structures like
nuclei occurring during vitellogenesis in the developing hymenopteran oocyte.
Many publications have been concerned with these structures, among them
those of Will (1884), Korschelt (1886), Gross (1903), Loyez (1908), and Palm
(1948), who investigated their role in the developing oocyte of Bombus sp.
Although the accessory nuclei have been said to resemble nuclei, they have
been shown by Bauer (1933), Mukerji (1930), and Cruikshank (1964) to be
Feulgen-negative.
Similar structures have been described in the oocytes of other insect orders,
but there has been little agreement concerning their origin, role, or ultimate
fate. They have been variously described as arising from the germinal vesicle
(Blochmann, 1886; Marshall, 1907), the trophocyte follicle (Gross, 1903), or
the follicular epithelial cells (Brunelli, 1904), or from granules in the ooplasm
contributed by either the oocyte (Hegner 1915; Buchner, 1918), the trophocytes (Gresson, 1930; Palm, 1948), or the follicular epithelial cells (Loyez,
1908; Peacock and Gresson, 1928). Marshall (1907) suggested that the role
of the accessory nuclei may be to change the trophocyte material passing
through the nutritive pore into a usable form. The more usual view, however,
is that the accessory nuclei are precursor bodies producing the albuminous
yolk spheres (Loyez, 1908; Gresson, 1930; Palm, 1948). Recently Cruikshank
(1964) suggested that in Anagaster the accessory nuclei may be involved in the
production of the vitelline membrane.
[Quart. J. micr. Sci., Vol. 105, pt. 4, pp. 475-80, 1964.]
476
Hopkins—Accessory nuclei in oocyte of Bombm
The previous study of these bodies had been mainly restricted to standard
histological methods. The present work was carried out to reinvestigate their
nature by histochemical and electron-microscopical techniques.
Materials and methods
Ovaries of Bombus terrestris were dissected from laying queens or from
workers that had been isolated and fed on sucrose for two weeks to promote
ovarian development. For electron microscope work individual oocyte
follicles were fixed for 1 h at o° C in 1 % osmium tetroxide buffered with phosphate at pH 7-8 and with the addition of 4-5% of sucrose. After rapid dehydration in a series of cold ethanols the tissue was embedded in araldite (Luft,
1961). Sections with grey interference colours were cut with a Huxley
microtome and mounted on untreated grids. The sections were stained for
5 min. with lead citrate (Reynolds, 1963) before being viewed with an AEI
EM6 electron microscope.
For histochemical procedures ovaries were fixed in a variety of fixatives
depending upon the technique to be used, dehydrated in ethanol, embedded
in paraffin, and sectioned at a thickness of 5 fi. Material fixed in Carnoy's
6:3:1 fluid was used for nucleic acid techniques. Oocytes fixed in Helly's
fluid were useful for demonstrating the form and distribution of nucleoli.
The Feulgen technique (Feulgen and Rossenbeck, 1924), with nonhydrolysed
controls, was employed for the demonstration of DNA. Pyronin/methyl green
(Brachet, 1942) and gallocyanin (de Boer and Sarnaker, 1956) were used
according to the instructions of Pearse (i960). Control sections were pretreated with ribonuclease (0-5 gm/ml, glass distilled water) or N.HC1 (6 min.
at 6o° C). These methods revealed the distribution of DNA and RNA.
Mercuric bromophenol blue (Bonhag modification, 1955), and ninhydrinSchiff (Yasuma and Itchikawa, 1953) were used with Bouin-fixed material
as general protein tests. The pH 8-i fast green method of Alfert and Gerschwind (1953) was employed with Carnoy-fixed sections to test for nucleic and
cytoplasmic histones. Baker's modification (1947) of the Sakaguchi reaction
was used as a test for argenine.
The periodic acid / Schiff (PAS) technique was used as a general test for
carbohydrates (Hotchkiss, 1948) and lipids were demonstrated by the Sudan
black B procedure (Pearse, i960), with gelatin sections cut on the freezing
microtome.
Observations
During vitellogenesis the accessory nuclei are confined to the peripheral
regions of the ooplasm. Initially they vary between 2 and 4 /J, in diameter
(fig. 1, B), but when albuminous yolk begins to be synthesized, they increase
in size and become ellipsoidal, with a maximum diameter of 15 /x. With the
electron microscope they can be seen to have a double limiting membrane,
provided with numerous annuli (fig. 1, A, c). These structures are more
obvious during the later stages of yolk formation. The limiting membrane of
the accessory nucleus is coloured intensely by Sudan black B.
Hopkins—Accessory nuclei in oocyte of Bombus
477
Inside the accessory nuclei is an electron-translucent matrix which contains two kinds of inclusions (fig. 1, A). Numerous small (200 m/x) electrondense bodies are distributed throughout it; these show little variation in form
and are present throughout vitellogenesis. There are also some larger electrondense spherical bodies, approximately 3 /x in diameter; they are often less
dense internally. These are few before vitellogenesis begins, but the number
rises to about 10 or more in each accessory nucleus during the synthesis of
albuminous yolk. The larger inclusions are Feulgen-negative but RNApositive. Failure to react with the fast green at pH 8-i implies the absence of
nucleohistones. They react positively with general protein procedures, but
consistent results were not obtained with the Sakaguchi reaction. The
accessory nucleus is at all stages PAS-negative, but the inclusions and the
limiting membrane alike stain intensely with Sudan black B.
Electron microscope observation has shown that the accessory nuclei are
present in the peripheral ooplasm during the germarial phase of development.
At this stage the trophocyte cells show little indication of secretory activity
and there is no evidence of the transfer of material from the trophocyte
follicle to the oocyte. The follicular cells are dividing during the germarial
phases and show no evidence of giving rise to accessory nuclei or their precursors (fig. 1, B).
The oocyte nucleus at this time is Feulgen-negative, but the nucleolus is
large and lobate; there are also numerous small RNA-positive bodies present
in the nucleoplasm. There are accessory nuclei in the perinuclear ooplasm
throughout development. The structure of their limiting membranes appears
exactly like that of the oocyte nucleus and their inclusions are similar in form
and staining reactions to those distributed in the peripheral nucleoplasm.
Although light-microscopic observation has indicated that the accessory
nuclei arise from the germinal vesicle by a blebbing process at the nuclear
membrane, it has not been possible to confirm these observations by electron
microscopy.
Throughout oocyte development the accessory nuclei increase in number by
division. During the earlier stages of vitellogenesis the products of division
are equal in size, but during the synthesis of albuminous yolk, and especially
during the formation of the vitelline membrane, division is unequal and small
terminal buds are formed by constriction. The daughter accessory nuclei
arising from such divisions are usually 3 to 5 \x, in diameter and contain from
3 to 5 electron-dense, RNA-positive inclusions (fig. 2, c). Loyez (1908) and
Palm (1948) have suggested that accessory nuclei become transformed into
albuminous yolk spheres during the later stages of vitellogenesis. With most
histological staining procedures the more peripheral albuminous yolk spheres
often appear to be transitional between the accessory nuclei and the yolk
spheres. These structures vary in their distribution with the fixative used,
however, and consist of a protein-carbohydrate complex similar to yolk
spheres, and do not contain RNA. If the albuminous yolk spheres are liberated into sodium chloride solution more dilute than 0-4 M, they gradually
478
Hopkins—Accessory nuclei in oocyte of Bombus
become granular and eventually dissolve. These observations are in agreement with those of Telfer (1961) on saturniid moths. Telfer suggested that
changes in the form of albuminous yolk spheres are caused by certain protein
elements of the sphere becoming insoluble in media of low ionic concentration. If precautions are taken against such changes, the albuminous spheres
appear in fixed sections as homogeneous structures (fig. 1, A; 2, c). There is
no evidence from electron microscope observations for the transformation of
accessory nuclei into yolk spheres.
During the formation of albuminous yolk spheres the limiting membranes of
the accessory nuclei are often irregular in outline (fig. 2, B). The protuberances so formed measure from 0-3 to 0-5 p. and are not associated with the
budding process described above. Possibly the irregularity of the membrane
during this phase is associated with an increase in its surface area and secretory
activity.
When the synthesis of albuminous yolk is complete and the protein uptake
has ceased at the ooplasma membrane, the vitelline membrane is formed in
the intercellular space between the oocyte and follicular cells (Hopkins and
King, in press). At this time a rearrangement of the peripheral ooplasm occurs
(fig. 2, A); mitochondria and small ooplasmic particles become concentrated
centripetally, leaving the most peripheral ooplasm a clearly-defined region
lacking cytoplasmic inclusions.
The accessory nuclei lie adjacent to the peripheral ooplasmic region. Their
inclusions are often concentrated towards the oocyte periphery and there is
some indication that electron-dense material is transferred across the limiting
membrane through the annuli towards the oocyte plasma membrane. The
accessory nuclei disappear after vitelline membrane formation and cannot be
identified during the later stages of oocyte development.
Discussion
Although it has not been possible to determine the origin of the accessory
nuclei with certainty, it is unlikely that they arise during the early phases of
oocyte development from either the trophocytes or the follicular epithelial
FIG. 1 (plate), A, electron micrograph of peripheral ooplasm during an early phase of
albuminous yolk synthesis. Each of the two accessory nuclei (an) contains 2 or 3 larger ribonucleoprotein bodies (ri) and numerous smaller inclusions (1). Formed albuminous yolk
spheres (ays) and yolk sphere precursors (ysp) are shown, probably arising as a result of the
pinocytotic activity at the interface between the oocyte and the follicle cell (fc).
B, electron micrograph of the peripheral ooplasm during an earlier germarial phase of
development. The ooplasm lacks yolk spheres but contains numerous mitochondria (m).
Accessory nuclei (an) are present in the peripheral ooplasm. fc, follicle cell.
c, electron micrograph of the oocyte periphery after the earlier phases of yolk synthesis
shown in A. The accessory nucleus (an) contains ribonucleoprotein bodies (ri) and smaller
inclusions; there are annuli (arrows) in its limiting membrane. The microvilli of the follicle
cell (fcm) and of the oocyte (oom) protrude into the intercellular space (ics), which lies between
the follicle cells (fc) and the oocyte. Albuminous yolk sphere precursors (ysp) adjoin regions
where there are signs of pinocytotic activity. The insert shows the annuli of the membrane
that limits the accessory nucleus, in transverse section.
FIG. I
C. R. HOPKINS
FIG.
2
C. R. H O P K I N S
Hopkins—Accessory nuclei in oocyte of Bombus
479
cells. Their probable site of origin appears to be at the limiting membrane of
the germinal vesicle. There is no evidence that they arise in the ooplasm
from any~organelle or from nuclear material transferred to the ooplasm from
the germinal vesicle. If such a process occurs rapidly and only during the
early stages of development in a laying Bombus queen, it would be extremely
difficult to detect. Autoradiographic procedures with C14 adenine were used,
but the slow rate of egg production of Bombus necessitates long periods of
incubation. This resulted in heavy labelling throughout the ovariole, preventing the identification of newly formed accessory nuclei.
The Feulgen-negative condition of the accessory nuclei has been previously
recorded by Bauer (1933), Mukerji (1930), and Cruikshank (1964), and
although the germinal vesicle is also Feulgen-negative during yolk synthesis,
it is known to contain DNA (Brachet, 1957). The absence of nucleohistones
and the mode of amitotic division indicates that the accessory nuclei do not
contain chromosomal elements. They do, however, contain RNA, which is
probably of nuclear origin.
The peripheral ooplasm, throughout which the accessory nuclei are distributed, contains large numbers of mitochondria, a few lamellar stacks, and
dense concentrations of free ribosomes. Recent work suggests that in the
insect oocyte protein from the haemolymph (Telfer 1961; Bier, 1962; Ramamurty, 1963) is taken up pinocytotically at the plasma membrane of the
oocyte (Roth and Porter, 1962; Kessel and Beams, 1963; Anderson, 1962,
1964), and contributes towards the large albuminous yolk spheres (Telfer,
1961). Evidence indicates that a similar process occurs in the developing
Bombus oocyte during yolk synthesis (Hopkins, 1964). The distribution
throughout such a region of large numbers of bodies resembling nuclei,
containing RNA possibly derived from the oocyte nucleus, and appearing in
maximum numbers at about the same time as the peak of albuminous yolk
synthesis, suggests that they may in some way control this process, even if
they are not direct precursors of deuteroplasmic bodies.
It seems likely that the accessory nuclei play a similar role during the
formation of the vitelline membrane, to which the oocyte is believed to make a
significant contribution (Hopkins and King, in press). The accessory nuclei
described above closely resemble the 'heavy bodies' occurring in the sea urchin
oocyte, as described by Afzelius (195b, 1957). The heavy bodies similarly
FIG. 2 (plate). A, electron micrograph of peripheral ooplasm showing accessory nucleus
(an) close to the vitelline membrane (vm). The pinocytotic activity seems to have terminated.
The peripheral positions of both the accessory nuclei and the large inclusion (n) are characteristic, a, annuli; oo, ooplasm; oom, oocyte microvilli; vm, vitelline membrane.
B, electron micrograph showing accessory nuclei (an) and albuminous yolk spheres (ays)
in the peripheral ooplasm at a stage slightly earlier than that shown in A. This is characterized by the folding of the membrane (arrows) and the presence of a bud (b). oo, ooplasm;
ri, ribonucleoprotein body.
C, a light micrograph showing the oocyte periphery at the beginning of vitelline membrane
formation. The formation of terminal buds (b) with inclusions is indicated, an, accessory
nuclei; ays, albuminous yolk spheres;/c, follicle cell. 5 /J. paraffin section stained with Heidenhain's haematoxylin.
480
Hopkins—Accessory nuclei in oocyte of Bombus
contain RNA, are bounded by a membrane similar to that of the nucleus, and
are believed to arise from the oocyte nucleus. Afzelius (1956, 1957) does not,
however, describe the function or fate of these structures.
I wish to acknowledge with gratitude the helpful guidance and supervision
given throughout this work by Dr. P. E. King. I am indebted to Professor
E. W. Knight-Jones in whose Department the work was carried out, and to
the D.S.I.R. for a Research Studentship during the tenure of which this
investigation was made.
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