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Modification of the endoplasmic reticulum in some mammalian oocytes.

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Modification of the Endoplasmic Reticulum
in some Mammalian Oocytes ’
DANIEL SZOLLOSI
Department of Biological Structure, University of Washington,
Seattle, Washingfon
ABSTRACT
Oocytes of the hamster, rat, Mongolian gerbil and squirrel monkey
demonstrate a n unusual “twin” or multilaminated arrangement of the rough endoplasmic reticulum. Two or more cisternae appose laterally apparently after the ribosomes vanish from the adjacent surfaces. A central electron dense leaflet, composed of
filamentous components can be discerned between the apposed surfaces. Between two
cisternae of the membrane complex found in hamster oocytes, narrow connecting
pillars or walls also develop. The nuclear envelope frequently represents one cisternal
element in the membrane complex, at which place nuclear pores are lacking. In rat
oocytes the endoplasmic reticulum cisternae take on either a concentric form, apparently by end-to-end fusion of “twin” cisternal membranes, or form a spiral by several
turns of the same cisternum. No functional role could be suggested for the observed
specilization of the endoplasmic reticulum.
The ergastoplasm or granular endoplasmic reticulum consists of flattened
cisternal or vesicular cytoplasmic membranous elements. The cytoplasmic surfaces of these membranous elements are
studded with varying numbers of ribosomal
rosettes. The cisternae may be arranged
individually or, in cells actively engaged in
synthesis of proteins for export, they may
be found in parallel arrays. In the latter
case nearly their entire surface may be
covered by tightly packed ribosomal clusters. Communication between the cisternae
of the endoplasmic reticulum and the nuclear envelope has been convincingly demonstrated in a large number of different
cell types. Hence, the nuclear envelope has
been referred to by Porter (’61) as the perinuclear endoplasmic reticulum. (For detailed reviews on the endoplasmic reticulum and further references, the reader
should consult Porter, ’61; Fawcett,’63, ’66.)
In this study a morphologically unusual
“twin” or multiple layered cisternal arrangement of the rough endoplasmic reticulum is described which was encountered
regularly in oocytes of four mammals.
at pH 7.4. After a brief rinse in 0.15 M
solution of the appropriate buffer solution,
the tissues were post-osmicated in 1 or 2%
buffered osmium tetroxide. The small
pieces of tissue were then transferred directly to 70% alcohol and rinsed in several
changes of this solution before subsequent
dehydration in a series of SO%, 95% , and
absolute alcohol. After two changes of
propylene oxide, the tissues were passed
through a series of propylene oxide-Epon
mixtures in which the Epon concentration
was progressively increased. The tissue
segments were then embedded in Epon
(Luft, ’61). (A modified formula for Epon
stock solutions A and B was recently recommended by Luft, see Szollosi, ’67.)
Sections of 50-70 mcl thickness were cut
with a diamond knife on a Huxley ultramicrotome. The sections, stained with
saturated aqueous uranyl acetate and lead
citrate in sequence, were examined and
photographed in Elmiskop I or Philips 200
electron microscopes. Du Pont Orotho A
lithographic film on 0.007 inch Cronar
(polyester) base was used (Wood, ’60).
RESULTS
MATERIALS AND METHODS
Ovaries of 25-30 day old rats and hamsters, 7 day old Mongolian gerbils and adult
squirrel monkeys were fixed 30-45 minutes
in 2.5% glutaraldehyde and 1.5%acrolein
in 0.1 M cacodylate or phosphate buffers
ANAT.
REC., 158:
59-7.2.
An unusual “twin” granular endoplasmic
reticulum membrane complex within hamster oocytes is depicted in figures 1 and 2.
Two cisternae are arranged parallel in such
IThis study was supported by
USPHS grant HD-
01110 of the National Institutes of Health.
59
60
DANIEL SZOLLOSI
a manner that the apposing membranous
proflles remain separated by an average of
280-350 A. An amorphous material of
somewhat higher electron density than the
surrounding ground substance is visible between the apposed cisternal membrane
surfaces. Equidistant from the latter, a
central line of even higher electron density
is seen, In places, the cisternae are separated by a greater distance while two central lines are evident and the amount of
the amorphous material is also apparently
increased.
In certain regions, narrow pillars of 80120 A thickness extend between the apposing membrane surfaces. When such regions have been sectioned obliquely, it
seems that the pillars are perpendicular
sections of narrow wall or plate-like connections (figs. 1,3,4). In oblique sections
a further detail of the central dense line
can be resolved which seems to be composed of fine fibrillar elements (fig. 4).
The external or cytoplasmic surfaces of
the cisternae are densely studded with ribosomal clusters or rosettes.
In a few cases one component of the
“twin” endoplasmic reticulum complex was
found to be represented by both leaflets of
the nuclear envelope as shown in figure 5.
Nuclear pores are lacking in the regions of
membrane apposition but are present in
their immediate vicinity. A similar membrane complex has recently been depicted
in a study of the oocyte in a unilaminar
hamster follicle although its occurrence
was not further discussed or elucidated.
(Odor, ’65) .
Sections of Mongolian gerbil and squirrel monkey oocytes demonstrate several
laterally apposed cisternae of the endoplasmic reticulum forming stackes of cisternae basically similar to those described
above (fig. 6). In both of the latter cases
the nuclear envelope frequently represents
the innermost components of the parallel
membrane stacks (fig. 7). Nuclear pores
are usually lacking in the region of the
membrane appositions in these cases as in
the hamster. The cytoplasmic complexes
in the gerbil are several microns long and
in some instances two to four membrane
complexes can be found in the same thin
section. Although ribosomal clusters have
been seen occasionally on the external sur-
face of the membrane, their frequency is
lower than those seen in hamster oocytes
(figs. 6,7). When the cisternae were sectioned perpendicularly, a central dense
component became evident between the
apposing membrane surfaces, but this is
seldom distinct. The narrow wall-like supports, described above for the hamster, are
seemingly lacking altogether.
It was not recognized for some time that
similar membrane complexes are also present in different developmental stages of rat
ovarian oocytes. The difficulty in recognition resulted from the peculiar swirl-like
arrangement of the apposing endoplasmic
reticulum cisternae (fig. 8). Seemingly,
a cisternum is rolled up around a centrally placed cytoplasmic core to form the
“twin” membrane complex. The lumen of
the cisternum could be followed through
several turns. Observations of several adjacent sections have shown that the membrane complex is not spherically arranged.
Polysaccharide granules, ribosomes and
even small endoplasmic reticulum cisternae or vesicles bearing occasional ribosomes are entrapped in the central core
cytoplasm. The separation between the
adjacent cisternae is much larger than in
the other species discussed, and the amorphous material filling the cisternal space is
usually of higher electron density. The
dense material nearly suppresses the visibility of the dense line halfway between
the vicinal membrane surfaces. Some electron micrographs show self-enclosed, concentrically arranged membrane profiles
(fig. 9 ) . Such circular images could be
formed in thin sections if the membranes
of abutting cisternae actually fused forming cylindrical or cup-shaped configurations. Although observations to be discussed
later favor such an interpretation, other alternative explanations cannot be excluded.
The cisternal swirls found in some rat
oocytes suggest that the formation of these
structural specializations of the endoplasmic reticulum proceeds by apposition and
fusion of curved membrane systems. Figures 10, 11 and 12 demonstrate configurations which are consistent with this interpretation. Flattened or curved cisternae
seem to be clustered with a narrow but distinct cytoplasmic layer between them. If
the cytoplasm and ribosomes of apposing
UNUSUAL ER IN OOCYTES
61
Recently, similar membrane associations
were reported in dividing cells (Buck, ’61;
Dean, ’64; Porter, ’57; Enders, personal
communication; Szollosi, unpublished observations) as well as in different phases
of the life cycle in HeLa cells (Epstein,
’61). In the former cases the nuclear membrane was observed to be broken down and
the chromosomes were in different stages
of mitosis. The similarity of such membrane systems to those observed in mammalian oocytes may be important if the
cisternal associations represent some form
of temporary intracellular membrane reserve or if such structures serve a temporary inactivation of some membrane
bounded synthetic apparatus.
Superficially, the cisternal membrane
complex resembles annulate lamellae which
have been described in several cell types
but which are demonstrated with particular
DISCUSSION
clarity in the cytoplasm of several inverteMorphological observations do not allow brate eggs. The extensive differences, howany conclusions as to the function of this ever, make it rather unlikely that the morreported specialization of rough surfaced phological variants of the endoplasmic
endoplasmic reticulum complexes. Sugges- reticulum function similarly.
tions as to their participation or lack of
ACKNOWLEDGMENT
participation in protein synthesis either for
The author is indebted to Drs. N. B.
the rapidly growing ovum itself or for ex- Everett, D. E. Kelly and J. H. Luft for
port would be speculation. Synthetic activ- reading the manuscript carefully and for
ity of morphologically differentiated regions their helpful suggestions.
of the endoplasmic reticulum might, howLITERATURE CITED
ever, be responsible for accumulation of
Buck,
R.
C.
1961 Lamellae in the spindle of
specialized proteins of local importance.
mitotic cells of Walker 256 carcinoma. J. BioThey might, in this manner, express rephys. Biochem. Cytol., 11: 227-236.
gional functional specializations or segre- Deane, H. W. 1964 Some electron microscopic
observations on the lamina propria of the gut,
gation of oocyte cytoplasmic complexes.
with comments on the close association of
Alternatively, aggregation of membrane
macrophages, plasma cells and eosinophils.
systems could express a temporary inacAnat. Rec., 149: 453-474.
tivation of synthetic activity dependent on Epstein, M. A. 1961 Some unusual features of
fine structure observed in HeLa cells. J. Biomembrane attached polysomal complexes.
phys. Biochem. Cytol., 10: 153-162.
Furthermore, it is possible that the mem- Fawcett,
D. W. 1963 Structural and functional
brane complexes represent cytoplasmic
variations in the membranes of the cytoplasm.
In: Intracellular Membraneous Structure. S.
membrane conservation without any speSen0 and E. V. Cowdry, Editors. Japan Society
cialized immediate function, but in a form
of Cell Biology.
which could be called upon quickly without Fawcett,
D. W. 1966 In: The Cell, pp. 133-188,
high energy requirements for their own
W. B. Saunders Company, Philadelphia and
London.
synthesis.
No sign could be detected for a repeated Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem.
association of the cisternal complex with
Cytol., 9: 409.
any other cell organelle which would imply Odor, D. L. 1965 The ultrastructure of unia functional relationship as well. Rather,
laminar follicles of the hamster ovary. Am. J.
Anat., 11 6: 493-522.
any association seemed to be at random,
K. R. 1957 The submicroscopic morprobably expressing merely momentary Porter,
phology of protoplasm. Harvey Lectures, 51:
local proximity.
175-228.
cisternal surfaces are displaced in some
manner, the membranes could conceivably
come into close proximity. Several examples demonstrate that even in apparently well organized complexes some of the
cisternae are in end-to-end apposition and
have not actually fused. Frequently, a
small islet of cytoplasm has remained entrapped between regions of adjacent cisternae (fig. 8). A fusion of cisternae would
provide the simplest explanation for the
cisternal swirls and, particularly, for the
formation of the concentrically arranged
membrane system. Approximation of ribosome studded endoplasmic reticulum in
the oocyte cytoplasm can be observed frequently but close proximity alone does not
seem to be sufficient for the formation of
“twin” cisternal complexes.
62
DANIEL SZOLLOSI
1961 The ground substance: Observations from electron microscopy. I n : The Cell.
J. Brachet and A. E. Mirsky, Editors. Vol. 11,
Chap. 9. Academic Press, New York.
Szollosi, D. 1967 Fixation procedures of embryonal tissues for electron microscopy. In:
Methods i n Developmental Biology. F. H. Wilt
and N. K. Wessels, Editors, Thomas Y. Crowell
Company. In Press.
Wood, R. L. 1960 Considerations of photographic material for electron microscopy with
special reference to the use of sheet film.
Norelco Reptr., 7: 23.
Note Ad d ed in Proof
Thomas, J. A., E. Hollande, E. Delain 1967 Effet persistant de l’ergastoplasme et l’appareil de Golgi, in &TO. C. R. Acad. Sci., Paris, Serie D,
264: 134-137. The occurrence of assemblies of double lamellae of the
rough endoplasmic reticulum which seem very similar to the “twin” or
multilaminated rough endoplasmic reticulum report above. See figs. 16,
17 and 18 of Thomas et al. for comparison.
PLATE 1
EXPLANATION OF FIGURES
1-2
“Twin’’ ribosome studded endoplasmic reticulum cisternae are seen
in an oocyte from a hamster secondary follicle (fig. l), and in a
primary follicle (fig. 2), respcctively. The single central dense line,
two central lines and connecting pillars are demonstrated. Figure 1
x 87,500; figure 2 X 41,500.
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 1
63
PLATE 2
EXPLANATION OF FIGURES
64
3 4
A region of a hamster oocyte where the apposing cisternae were
sectioned more or less perpendicularly adjacent to a region where
it was obliquely cut. Ribosomes, central line and pillars are clearly
seen. In the obliquely sectioned portion the latter structures seem
to be plate-Like connections. In figure 4, the fibrillar substructure
of the central line becomes more clear in the obliquely sectioned
membrane portion (arrow). Figures 3 and 4 X 114,000.
5
The inner component of the “twin” endoplasmic reticulum cisternal
complex is represented by the nuclear envelope in a primary follicular oocyte of the hamster. X 39,500.
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 2
65
PLATE 3
EXPLANATION OF FIGURES
66
6a
Six flattened cisternae form one large membrane complex in the cytoplasm of the Mongolian gerbil primary oocyte. In this case the
cisternal complex is near the Golgi apparatus and a centriole. Occasionally a small cluster of ribosomes can be seen on the cytoplasmic
surface of the membrane complex. x 27,000.
6b
Higher magnification of
X 81,000.
a portion of three apposing cisternae.
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 3
67
PLATE 4
EXPLANATION O F FIGURE
7
68
Two “twin” cisternal complexes of the endoplasmic reticulum can be
seen in a Mongolian gerbil oocyte. In one case the nuclear envelope
represents one component of the membrane complex which is near the
terminal portion of a synaptinemal chromosome complex. X 43,500.
UNUSUAL ER IN OOCYTES
PLATE 4
Daniel Szollosi
69
PLATE 5
EXPLANATION OF FIGURES
8
In oocytes of rat secondary follicles spiral arrangements of the rough
endoplasmic reticulum is demonstrated. From the central cytoplasmic
core the folded cisternum can be traced through several turns (between arrows). Peripherally, cisternae of different sizes cluster around
the swirl-like portion of the cisternal complex. x 41,000.
9 A concentrical “twin” endoplasmic reticulum complex is found around
a central cytoplasmic core. Peripherally, several cisternae have further
aggregated and i n one case (arrow) has formed another “twin” membrane complex. The dense granule clusters are polysaccharide in
nature and are larger in diameter than the membrane associated
ribosomes. X 36,000.
70
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 5
71
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 6
EXPLANATION OF FIGURES
10-12
72
Cisternae of the rough endoplasmic reticulum demonstrate figures which were interpreted as
stages of formation either of the concentrically oriented membranes or of the membrane
swirls. Figure 10 X 34,000; figure 11 X 27,500; figure 12 X 24,500.
UNUSUAL ER IN OOCYTES
Daniel Szollosi
PLATE 7
73
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