/. Embryol. exp. Morph. Vol. 26, 2,pp. 181-193, 1971
Printed in Great Britain
A biochemical and ultrastructural study of RNA
in yolk platelets of Xenopus gastrulae
ROBERT O. KELLEY, 1 GEORGE S. NAKAI AND MARLENE
E. GUGANIG
From the Departments of Anatomy and Medicine,
The University of New Mexico
SUMMARY
Yolk platelets from Xenopus gastrulae were isolated in a sucrose-polyvinyl pyrrolidone
medium, washed, centrifuged four times, and portions of each pellet were prepared for
electron microscopy.
Electron microscopy revealed isolated platelets to be free of cytoplasmic contamination
with progressive disruption of the superficial layer after each washing.
Each washing and thefinalpellet were extracted with phenol and precipitated with ethanol.
Orcinol analysis indicated that 50-60 fig of RNA were present in yolk platelets isolated
from 1000 gastrulae.
Autoradiography of yolk platelets from cells incubated in [5-3H]uridine revealed label in
superficial and main body components after treatment with DNase but not after incubation in
RNase.
Acrylamide-gel electrophoresis suggests that yolk platelet RNA is of both high and low
molecular weight.
INTRODUCTION
The structure, chemical composition and role of yolk platelets during
amphibian embryogenesis has been a subject of active investigation and lively
controversy for several years. The ultrastructure of the yolk platelet was
revealed by Ward (1962) and Karasaki (1963) to consist of a main body component with a distinct crystalline matrix, a superficial layer surrounding this
main body, and a limiting membrane. Gross and Gilbert (1956) reviewed the
chemical composition of the platelet in amphibians; however, Lanzavecchia &
Le Coultre (1958) were among the first to propose that these structures contained
nucleic acids. Rounds & Flickinger (1958) suggested that nucleic acid found in
the 'yolk fraction' of mesodermal cells during primary induction participated
in early morphogenesis. However, in an effort to resolve and analyse yolk
platelet components, Wallace (1963 a) concluded that amphibian yolk platelets
in situ contained no RNA, but was uncertain of the content of DNA. Recently,
Baltus, Hanocq-Quertier & Brachet (1968) have reported double-stranded,
linear DNA in amphibian yolk platelets which they suggest plays a role in
1
Author's address: Departments of Anatomy and Medicine, The University of New
Mexico, School of Medicine, Albuquerque, New Mexico 87106, U.S.A.
182
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
synthesis of enzymes for breaking down platelets during development, if this
hypothesis is valid, then might yolk platelets contain RNA in addition to DNA?
We chose techniques of electron-microscopic autoradiography, phenol extraction
of RNA, and separation of RNA molecular species by means of sucrosegradient centrifugation and acrylamide-gel electrophoresis in an attempt to
answer this question.
MATERIALS AND METHODS
Fertilized eggs were obtained from the South African clawed frog, Xenopus
laevis, after the method of Gurdon (1967). Gastrulae (stage 11; Nieuwkoop &
Faber, 1956) were carefully divested of surrounding jelly and fertilization coat
in 2-0 % cysteine neutralized to pH 7-8 with NaOH and containing 0-2 % papain.
Electron microscopy
Entire embryos were preserved in a variety of fixatives (veronal acetate
buffered osmium tetroxide, phosphate buffered osmium tetroxide, phosphate
buffered formaldehyde, phosphate buffered glutaraldehyde, cacodylate buffered
glutaraldehyde, and veronal acetate buffered glutaraldehyde). Best preservation
was observed in specimens fixed with 1-75 % glutaraldehyde in 0 . 1 M phosphate
buffer (Polysciences, Inc.) for 1 h at 4 °C, postflxed with 2-0 % osmium tetroxide
in 0.1 M phosphate buffer at 4 °C for 1 h, rapidly dehydrated through an ethanol
series to propylene oxide and embedded in Epon 812. Thin sections (mounted
on uncoated grids) were stained for 1 h in saturated aqueous uranyl acetate at
35 °C, for 5 min in alkaline lead citrate at room temperature and examined
in an Hitachi HU-11C electron microscope.
Isolation of yolk platelets
To determine the presence of RNA chemically, yolk platelets were isolated
from gastrulae using modifications of the method of Wallace & Karasaki (1963).
The following procedures were performed°in a cold room (5 °C). Embryos were
washed with Niu-Twitty's (1953) solution containing penicillin (1000 i.u.) and
streptomycin (0-01 mg/ml) and were transferred to cold 0-25 M sucrose-5-0 %
(w/v) polyvinyl pyrrolidone (PVP). Gradients were prepared by placing 20 ml
of 1-0 M sucrose-5-0 % PVP in round-bottomed 35 ml Pyrex centrifuge tubes
(Sorvall rotor SS-34). One thousand embryos were gently homogenized in 60 ml
of the 0-25 M sucrose-5-0 % PVP medium, the grey homogenate then being
divided into four equal aliquots and carefully layered on to the surface of each
gradient.
Tubes were initially spun in a Sorvall RC2-B refrigerated centrifuge (0 °C)
for 10 min (F max = 600 g). This procedure produced a yellowish pellet of yolk
platelets, a thin superficial layer of contaminating pigment on top of the pellet,
and a grey supernatant. After decanting the supernatant (which was saved for
analysis), walls of the tubes were rinsed with 0-25 M sucrose-5-0 % PVP, the
RNA in yolk platelets
183
pellets resuspended by gentle manual shaking, and centrifuged again at 590 rev/
min for lOmin (F m a x = 500 g). This procedure was repeated four times, the
final pellet containing neither pigment nor contaminating particulate material
(for some experiments, final resuspension was accomplished by shaking in a
vortex mixer). Portions of each pellet after each spin were fixed for examination
in the electron microscope.
A utoradiography
To determine the localization of RNA in yolk platelets, gastrulae were
incubated in [5-3H]uridine (New England Nuclear Corp.; specific activity
13-12 Ci/mmole, in sterile aqueous medium) for 2 h, and were transferred into
unlabeled uridine (uracil riboside, Nutritional Biochemicals Corp.; 10 mg/ml
in sterile Niu-Twitty's solution) for 1 h. Some embryos were prepared for
electron-microscopic autoradiography after the method of Caro (1964), whereas
others were homogenized, their yolk platelets isolated as described, and the
pellets prepared for autoradiographic examination in the electron microscope.
Control specimens (intact embryos and platelet pellets) were fixed in 3:1
ethanol: acetic acid and embedded in paraplast. These were sectioned at 6 /*m,
mounted on gelatin-subbed slides, and subjected to one of the following
procedures: (1) 3 h in DNase (Worthington, x 2 crystallized, 0-5 mg/ml in
0-1 M phosphate buffer with 0-1 % phenol as preservative); (2) 3 h in RNase
(Worthington, 13 mg/ml in 0-1 M phosphate buffer with 0-1% phenol as
preservative); (3) 3 h in buffer and in water, both without enzyme; and (4) 1 NHC1 at 60 °C for 12 min. These slides were treated with cold 5-0 % trichloracetic
acid for 15 min, rinsed in 80 and 95 % ethanol and allowed to dry.
Random samples of silver grains over control sections were counted using a
grid (one square = 0-4 cm2) drawn on a transparent plastic sheet. All silver
grains over yolk platelets within 100 adjacent squares of the grid were counted.
Autoradiographs used for counting were at the same magnification.
Analytical procedures
Isolated yolk platelets were analysed by the following procedures:
Extraction of RNA
After isolation of yolk platelets, pellets were resuspended in 5-0 ml of saline
buffer (0-24 M NaCl, 0-01 M MgCl2, 0-01 M Tris, pH 5-0) to which was added
0-5 ml of 10% sodium dodecyl sulfate (SDS), 0-5 ml of 2-5% bentonite in
001 M sodium acetate, and 5-0 ml of water-saturated phenol containing 0-1 %
8-hydroxyquinoline (Cline, 1966). After manual shaking for 20 min at 0 °C,
phases were separated by centrifuging at 800 g for 10 min at 5 °C. The aqueous
layer was saved whereas the phenol layer was re-extracted with 5-0 ml saline
buffer by shaking for 5 min at 50 °C. The emulsion was chilled, centrifuged at
800 g for 10 min at 5 °C, the aqueous layer combined with the previous aqueous
portion and re-extracted twice with 5-0 ml phenol at 50 °C for 5 min. After final
184
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
extraction, the aqueous fraction was centrifuged at 15000 g for 30 min to remove
bentonite. One-tenth volume of 20 % sodium acetate and two volumes of 95 %
ethanol were added to the supernatant and the RNA was allowed to precipitate
overnight at - 20 °C. The precipitate was treated with DNase (Worthington,
13 mg/ml in 0-1 M phosphate buffer with 0-1 % phenol as preservative) prior to
sedimentation analysis and electrophoresis.
Orcinol colorimetric test
Ribonucleic acid in yolk platelet pellets was determined by standard orcinol
colorimetric analysis as described by Shatkin (1969).
Density-gradient separations of RNA
RNA was centrifuged at 12000 g for 30 min at 5 °C, the ethanol was decanted
and the precipitate dried by inverting the tube at 5 °C for 30 min. RNA was
dissolved in SDS buffer (0-1 M NaCl, 0 1 M EDTA, 0-01 M Tris-HCl, pH 7-4,
0-2 % SDS), layered on a 10-30 % linear sucrose gradient, and centrifuged in
a Spinco L2-65 ultracentrifuge (SW 65 rotor) for 2-5 h at 65000 rev/min
(300000 g) at 25 °C. The centrifuged gradients were displaced with a 40%
sucrose solution and continuously monitored at 254 nm through a Model D
Density Gradient Fractionator (Instrumentation Specialties Co., Inc., Lincoln,
Nebraska).
RNA fractionation on acrylamide gel
RNA was extracted with phenol, purified as described above, and subjected to
electrophoresis on an acrylamide gel according to Loening (1967). RNA from
Escherichia coli (23 S and 16S) were utilized as markers in control gels.
RESULTS
Electron microscopy
Yolk platelets are present in random pattern throughout cells of Xenopus
gastrulae (Fig. 1), the larger platelets (up to 50 /im in length) being present in
prospective entodermal regions. Most platelets exhibit three basic components
as described by Karasaki (1963): a limiting membrane, a superficial layer and a
main body component. However, platelets in presumptive neural ectoderm
appear to have lost superficial layers and limiting membranes by the midgastrula stage.
Isolated yolk platelets
Limiting membranes are generally lost during isolation procedures, whereas
superficial layers and crystalline inner matrices remain (Fig. 2). The superficial
layers consist of small electron-dense particles (50 A in diameter), larger, more
angular particles (150-250 A in diameter) and an amorphous background substance. The main body contains a highly structured crystalline matrix (for
review of fine structure in the main body component see Karasaki, 1963).
RNA in yolk platelets
Fig. 1. Portions of cells at mesoderm-ectoderm interface (stage 11). Note random
pattern of yolk platelets (yp). I, Lipid droplet; m, mitochondrion; p, cytoplasmic
particles, x 6000.
Fig. 2. Portion of isolated yolk platelet revealing crystalline main body component
(mbc) and superficial layer (s). Note presence of 150-250 A particles (arrows) in
periphery of superficial layer, x 82000.
185
186
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
RNA in yolk platelets
187
Pellets examined in the electron microscope after initial centrifugation revealed
presence of membranous material, lipid droplets and disrupted mitochondria
in addition to yolk platelets (Fig. 3). Superficial layers of the latter were fused
with neighbouring platelets, creating a continuum of yolk substance. After
resuspension and a second centrifugation, platelets regained their individuality,
losing contaminating materials (Fig. 4). Following a third centrifugation,
superficial layers began to separate from main body components (Fig. 5),
revealing particulate material in the supernatant. Main body components were
disrupted following suspension with a vortex mixer and a fourth centrifugation
(Fig. 6).
Autoradiography
Autoradiographs of gastrula cells cultured in [3H]uridine reveal silver grains
over both superficial and central components of yolk platelets (Fig. 7) in
addition to cytoplasmic particles, mitochondria and nuclei. In addition, label
remains in isolated platelets.
Table 1 illustrates the effect of acid hydrolysis and RNase on grain counts over
yolk platelets in situ. Thick sections of cells prepared for light-microscope
autoradiography have fewer silver grains after incubation in RNase and treatment with 1 N - H C I than when treated with DNase, phosphate buffer, and water
for similar periods of time at 38 °C.
Table 1. Numbers of silver grains over yolk platelets in ectodermal cells treated
with 1N-HCI, RNase, DNase, phosphate buffer without enzyme, and water
(Results represent the average of counts from at least ten light micrographs of
mid-gastrula ectoderm.)
RNase
38 °C
3h
DNase
38 °C
3h
Buffer
60 °C
12min
38 °C
3h
Water
38 °C
3h
63
91
274
297
311
1 N-HCI
Grains/grid
387 cm2
Fig. 3. Micrograph of yolk platelet pellet after initial centrifugation (Fmax = 600g).
I, Lipid droplet; m, mitochondrion (contracted, because the medium used for isolating
yolk platelets is hypertonic for mitochondria); cm, cytoplasmic membranes,
x 12000.
Fig. 4. Micrograph of yolk platelet pellet after resuspension and a second centrifugation. Note absence of cytoplasmic contamination, x 24000.
Fig. 5. Micrograph of yolk platelet pellet after gentle hand resuspension and a third
centrifugation revealing partial disruption of superficial layers, x 19000.
Fig. 6. Micrograph of yolk platelet pellet after resuspension by vortex mixer and a
fourth centrifugation revealing disruption of both main body and superficial
components, x 19000.
188
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
Fig. 7. Autoradiograph of ectodermal cell (stage 11) incubated in [3H]uridine for
1 h. x 44000.
Fig. 8. Autoradiograph of platelets isolated from mid-gastrula cells cultured in
[3H]uridine for 1 h. x 33000.
189
RNA in yolk platelets
10
20
Fraction number
Fig. 9. Sucrose density-gradient pattern of RNA from purified yolk platelet pellets.
Peak I at top of sucrose gradient represents contaminating sediments (nucleotides,
degraded DNA and protein, some low-molecular-weight RNA).
1-5 _
1-4 1-3 1-2 1-1 10 E 0-9 c
T
0-8 " 4 - 6 S
LO
(N 07
' A4-9S
f-\
" / S . 5-8 S
o 0-6
0-5
0-4
0-3
0-2
0-1 "Top
n
-
29 S
-
A
-
1
-
28 S
-
hi
J
18S
15SI20S
_
i
15
Bottom
Bottom
"Top
30
Fraction number
15
30
Fig. 10. RNA profile after acrylamide-gel electrophoresis of peaks 2 and 3 present
in Fig. 9. Diagram on left represents peak 2 (Fig. 9), whereas peak 3 is presented on
the right.
E M B 26
190
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
Analytical procedures
Orcinol analysis
Orcinol analysis (Shatkin, 1969) suggested that 50-60/tg of RNA were
present in yolk platelets isolated from 1000 Xenopus embryos (stage 11).
Sucrose-gradient centrifugation and acrylamide-gel electrophoresis
Optical-density measures of RNA from isolated pellets in sucrose-density
gradients revealed three distinct peaks (Fig. 9). The major peak (fraction 1) at
Fig. 11. Acrylamide-gel profile of phenol-extractable material from supernatant of
first centrifugation.
1-5 _ Wash 2
1-4
1-3
1-2
1-1
10 |09 -
O06
0-5
0-4
0-3 0-2
0-1 Top
0
_ Wash 3
I4S
/
30 S
27 SA
\
- \
|
15
Bottom ~ Top
30
Fraction number
15S
I
15
y\
Bottom
30
Fig. 12. Left: acrylamide-gel profile of phenol-extractable material from supernatant
of second centrifugation step in isolation procedure. Right: acrylamide-gel profile
of phenol extractable material from supernatant of third centrifugation step in
isolation procedure corresponding to disruption of superficial layers (Fig. 5).
RNA in yolk platelets
191
the top of the tubes represented contaminating sediments (nucleotides, degraded
DNA, and some protein) and low-molecular-weight RNA. Fractions 2 and 3
were isolated and electrophoresed on acrylamide gels, revealing several distinct
components (Fig. 10). Peaks corresponding to 23 S and 16 S RNA from E. coli in
control gels were noted and sedimentation values for peaks in experimental
profiles were computed from these data.
RNA extracted from washings obtained during platelet isolation procedures
yielded the following results. Fig. 11 represents RNA species separated by gel
electrophoresis from collected supernatants after a single centrifugation of
homogenate. Numerous peaks were present, representing high- and lowmolecular-weight RNA in both nucleus and cytoplasm of cells from stage 11
embryos. However, after resuspension and a second centrifugation of platelet
pellets, phenol extractions of supernatant did not provide optically active
material demonstrable in acrylamide gels (Fig. 12, left). Extractions of washings
from the third centrifugation, however (Fig. 12, right), yielded electrophoretic
patterns similar to those obtained from isolated yolk platelets. Low-molecularweight RNA as well as larger species were present. A 15 S species was present in
both third washings and isolated yolk platelet pellets. Analysis of washings from
a fourth resuspension and centrifugation revealed electrophoretic profiles similar
to those demonstrated after the third spin.
DISCUSSION
Questions concerning the presence and potential developmental role of nucleic
acids within amphibian yolk platelets are recurrent topics in numerous papers
(see Rounds & Flickinger, 1958; Yamada, 1961; Horn, 1962). Although the
existence of RNA in yolk platelets has been challenged by Wallace (1963 a, b)
and Ohno, Karasaki & Takata (1964), our results indicate that yolk platelets in
Xenopus gastrulae (stage 11) do possess small quantities of RNA (005-006 [i%\
embryo).
Of principal concern is the problem of contamination or artifact. Bacteria
would probably not contaminate an embryonic cell fraction to the extent of
contributing significant optical density to the RNA population. Furthermore,
bacteria can be easily removed from media. In addition, embryos were chemically
dejellied, which has been shown by Brown (1967) to decrease significantly
bacterial contamination. An additional potential contaminant is yolk phosphoprotein which has a molecular weight (30000) and phosphate content (8%)
similar to low molecular weight (4 S) RNA (Wallace, 1963a). Furthermore,
phosphoprotein can be extracted into the aqueous phase and precipitated with
ethanol. Fortunately removal of most of this contaminant is effected with
0-01 M MgCl2 in the initial homogenate (Brown, 1967).
Are the 150-250 A particles present in superficial layers of in vitro and in situ
platelets ribonucleoprotein (RNP)? And if so, are they normal components of
13-2
192
R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG
superficial layers or artifacts of the isolation procedure? Generally, particles
that are 150-250 A in diameter and either attached to membranes or free in the
cytoplasm of adult tissues are designated ribosomes (Palade, 1955), whereas
those larger in size (250-500 A in diameter) and free in the cytoplasm are
regarded as glycogen granules (Drochmans, 1962). It is thought then that larger
particles present in superficial layers are RNP because of their appearance and
affinity for aqueous uranyl acetate. In addition, the isolation procedure of
Wallace & Karasaki (1963) used in this study provides the investigator with
platelets which are reasonably free from contamination visible in the electron
microscope (Figs. 4-6). Furthermore, the absence of RNA in the supernatant
following a second resuspension and centrifugation (Fig. 12, left) and the
associated intact appearance of isolated platelets (Fig. 4) suggest that 150-250 A
particles are normal components of superficial layers and not free cytoplasmic
particles displaced by centrifugation.
Autoradiographs presented in this study of labeled (tritiated undine) yolk
platelets may provide morphological evidence for the presence of RNA in those
structures (Figs. 7, 8). Although nucleosides are not thought to be precursors of
nucleic acids in normal biosynthetic pathways, [3H]uridine has been used in a
variety of embryos and appears to be an efficient precursor of nucleic acids.
Bieliavsky & Tencer (1960) have demonstrated that during cleavage in amphibian
embryogenesis, labeled uridine is incorporated into DNA rather than RNA, but
the converse seems to be true during gastrulation. Since thick sections of cells,
prepared for light-microscopic autoradiography as controls, have fewer silver
grains over yolk platelets after incubation in RNase and 1 N - H C I than when
treated with either DNase, buffer or water, it is concluded that most [3H]uridine
is incorporated into RNA rather than DNA during this developmental period.
Failure to extract RNA from washings following a second resuspension and
centrifugation (Fig. 12, left) followed by an increase in RNA content in supernatants of successive spins (Fig. 13, right) may correlate with progressive disruption of main body components (Fig. 6), superficial layers (Figs. 5 and 6), and
the apparent release of RNP particles (Fig. 2) from the latter.
Hence, biochemical and autoradiographical evidence presented in this study
supports the hypothesis that yolk platelets in amphibian gastrulae contain RNA.
Acrylamide-gel profiles suggest that RNA species of high and low molecular
weight are present. Furthermore, the RNA content of isolated platelets can be
released to surrounding media during isolation procedures which supports
observations of Rounds & Flickinger (1958). The developmental significance of
yolk platelet RNA, however, remains uncertain.
Grateful acknowledgment is made to Professors A. J. Ladman and Leonard Napolitano
for helpful discussions and critical reading of the manuscript, to Mrs Margo G off for technical
assistance, and to the United States Public Health Service for facilities through CA-10694.
RNA in yolk platelets
193
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{Manuscript received 4 January 197T)