J. Cell Set. 7,5-13(1970)
Printed in Great Britain
THE CONCENTRATIONS OF WATER, SODIUM
AND POTASSIUM IN THE NUCLEUS AND
CYTOPLASM OF AMPHIBIAN OOCYTES
T. J. CENTURY
Department of Biology, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, U.S.A.
AND I. R. FENICHEL AND S. B. HOROWITZ
Laboratory of Cellular Biophysics, Albert Einstein Medical Center,
Philadelphia, Pennsylvania 19141, U.S A.
SUMMARY
Techniques are described for the isolation of nuclei and cytoplasm from frozen amphibian
oocytes at — 40 °C and their analysis for water, sodium, and potassium contents. Nuclei of
the 4 species studied contain 74-85% water while cytoplasm is only 35-50% water. Nuclei
contain 7-17/tequiv. Na/ml H 2 O and 123-129 /tequiv. K/ml H , O ; cytoplasm contains
50-84/iequiv. Na/ml H 2 O and 81-94/iequiv. K/ml H,O. Mechanisms suggested to explain
the observed nucleo-cytoplasmic asymmetries are the selective sequestering of sodium in the
cytoplasm and a fraction of cytoplasmic water which does not act as solvent. Asymmetric
cation transport by the nuclear membrane is not indicated.
INTRODUCTION
Chemical analysis of cell nuclei usually involves physical separation of the nucleus
from the rest of the cell. When cell integrity is disrupted, in vivo transport barriers
may be extensively modified, and diffusion, especially of small solutes, may lead to
redistribution of materials between the nucleus and the cytoplasm. This problem can
be largely averted if the cell is frozen rapidly and kept at low temperatures throughout
the entire process of nuclear isolation. This paper describes a technique in which
amphibian oocytes are frozen in Freon-12 at —155 °C and nuclei and pieces of cytoplasm are isolated by free-hand microdissection at —40 °C. Results of dry weight, Na,
and K analyses of the isolated cell components are given and discussed.
MATERIALS
Oocytes
The oocytes of 2 species of salamanders and 2 frogs were studied. The salamanders were
Eurycea b. bulineata, collected in the Perkiomen Creek drainage, Montgomery County,
Pennsylvania in November 1968, and Desmognathus o. ochrophaeus, from the Pine Creek
drainage, Tioga County, Pennsylvania in June 1969. Frogs (Rana pipiens and Rana catesbtana)
were obtained from a New Jersey dealer. Mature oocytes from healthy females were used in
this study. Typical oocyte diameters and weights were: R. pipiens, 1 6 mm, 265 mg; R. catesinana, 1 3 mm, 1-27 mg; Eurycea, 1-7 mm, 312 mg, Desmognathus, 1 8 mm, 3-50 mg.
6
T. J. Century, I. R. Fenichel and S. B. Horowitz
Apparatus
The apparatus used in the experiments is shown in Fig. i. It is designed to allow free-hand
microdissection of the oocyte at low magnification (20 x ) on a low-temperature stage (— 40 °C)
in a dry atmosphere (to prevent water condensation on the stage and on the materials to be
analysed). An airtight box (c) with glove ports (g) is built around a dissecting microscope (a) so
that the microscope can be focused from inside the box. A rubber boot (d) provides a seal
between the arm of the microscope and the box; this boot is airtight but flexible enough to
accommodate movements of the arm when the microscope is focused. The base of the microscope is bolted directly to the floor of the box.
Fig. 1. Dry box with microscope and Dewar flask in place; drying train is shown
schematically. Rubber gloves which fit glove ports are not shown.
A 4-in (io-2-cm) diameter hole in the floor of the box accommodates the mouth of a small
Dewar flask (1); a rubber gasket (h) provides an airtight seal between the box and the Dewar
flask. A rotatable cold stage (/) is mounted inside the Dewar flask. In this way the stage may be
viewed with the microscope through a double-paned glass (e) in the roof of the box. A lamp (b)
illuminates the stage through this same double-paned glass.
The drying train consists of two parallel stages, shown schematically in Fig. 1. The first
stage is a cold trap (!) immersed in dry ice-methanol. The second stage is a P,O 6 trap (m) consisting
of concentric funnels; a Pern dish containing a small amount of PjOn (n) is sealed to the mouth of
the outer funnel so that air flowing through the inner funnel passes over the P a O 6 before
leaving the trap. Air is pumped through the system by a small closed-circuit pump (j), and a
deflated, large balloon (k) accommodates the increase in volume of the system due to sublimation of the dry ice in the Dewar flask (1). With valves (q) and (r) open (o and p closed), air is
circulated through the cold trap until there is no further increase in the amount of ice deposited
in the trap (about 1 h). Valves (o) and (p) are then opened (q and r closed) and the air is further
dried by passage through the P a O 6 trap. The air continues to be pumped through the P,O 6 trap
Na and K in the nucleus and cytoplasm of oocytes
7
during the course of each experiment. This drying system does not entirely eliminate condensation of water vapour on the cold working area, but reduces it to a level such that only occasional
light brushing is needed to keep the working area frost-free. The small amount of water vapour
which condenses in the Parafilm sample containers (see below) is monitored by using appropriate controls (see section on Controls below).
Cold dissection stage
An exploded view of the cold stage is shown in Fig. 2. An aluminium stage (/) rotates in an
aluminium base (y) fixed in a 500-ml Dewar flask (») packed with powdered dry ice. The side of
the rotating stage is grooved to receive a spring-clip (x) so that different positions on the top of
the stage may be fixed in the field of view of the microscope. The stage is rotated by turning the
inverted U-tube (v), the arms of which fit into 2 holes in the top of the stage. Other holes in the
top of the stage accommodate a mounting with frozen oocytes (w), a mounting for cleaning
isolated nuclei (s), and a o-5-ml polyethylene Beem capsule (t) containing 2 small tared Parafilm
envelopes (;/).
1 cm
Fig. 2. Exploded view of cold dissection stage and its components.
See text for detail.
The oocyte mounting (w) is made of stainless steel and its face contains a number of small
depressions each large enough to accommodate the bottom fourth of an oocyte. When frozen
into one of these depressions, an oocyte remains firmly in place throughout the dissection
procedure.
The mounting for final cleaning of the nucleus (s) consists of a piece of stainless steel rod
with a hole, 500 /(m in diameter by 500 fim deep, drilled into one end. The nucleus is held in
this hole while it is being cleaned.
Envelopes (K) are made by folding an 8 x 4 mm piece of Parafilm in half and pressuresealing the sides, giving 8 4 x 4 mm envelope.
8
T. J. Century, I. R. Fenichel and S. B. Horowitz
METHODS
Preparation of oocytes for microdissection
Whole ovaries or pieces of ovary are removed from the abdominal cavity of the female, placed
into ice-cold amphibian Ringer's solution, and divided into loose aggregates of 5-10 oocytes
by carefully cutting the ovarian wall. Enough oocytes to perform one complete experiment
are transferred to the face of the oocyte mounting (w) and plunged into liquid Freon-12 at
— 155 °C, giving a single layer of frozen oocytes. These mounted oocytes may be stored in
liquid nitrogen for later use.
The oocyte mounting (w), with frozen oocytes, is inserted into the cold stage along with the
mounting for final cleaning of the nucleus (s) and the capsule (i). The mouth of the Dewar
flask is then inserted into the rubber gasket in the floor of the dry box and the cold stage is
rotated to bring the oocytes into the field of view of the microscope.
Isolation of nuclei and pieces of cytoplasm
Using carefully cleaned microtools, the surface and cortex of an oocyte are scraped away until
the nucleus becomes visible. The nucleus is cleaned and semi-isolated as well as possible
while still in place in the cytoplasm; it is then broken free of the cytoplasm and transferred with
a small (no. 000) camel's-hair brush to the small hole m mounting (s) of Fig. 2. The cold stage is
rotated until mounting (1) is fixed in the field of view of the microscope. The nucleus is then
held in place with an insulated microneedle while the remaining cytoplasm is carefully scraped
off with a small scalpel. Nuclear purity is determined visually by the distinct differences in
colour and consistency between the nucleus and the cytoplasm.
The cold stage is then rotated so that the field of view includes both mounting (s) and the
Beem capsule (t), which is opened to give access to the Parafilm envelopes. The clean, frozen
nucleus is then transferred with a small brush from mounting (j) to the envelope (u). The Beem
capsule is closed, the cold stage rotated back to the oocyte mounting and the operation is repeated. Pieces of cytoplasm are chipped out of the remaining piece of oocyte and placed in
envelopes in the same way as the nuclei, except that no cleaning is necessary.
We have found it convenient to isolate 25 to 30 nuclei at a sitting (about 5 h), and these are
divided into 3 groups of 7-10 nuclei each for analysis.
Determination of wet and dry weights
When a group of 7-10 frozen nuclei or an equivalent volume of frozen cytoplasm is collected
in an envelope, the envelope is pressure-sealed. Sealed envelopes are brought out of the dry box
and weighed on a Cahn Electrobalance along with the corresponding control envelopes (see
section on Controls below). The mouths of the envelopes are opened, and the samples are dried
for 18-24 h at 50 °C over PjO 6 , reweighed, and the % dry weight and total water content of
each sample determined (see Table 1).
Determination of Na and K concentrations
Following dry weight determination, the sides of the Parafilm envelope are carefully opened
to a flat sheet which, with the adhering sample, is placed in a 5-ml polypropylene container
(Bel Art No. F-17890) with either 2 ml (nuclear samples and controls) or 4 ml (cytoplasmic
samples and controls) of distilled water containing less than 6 parts per io 9 Na. The containers
are tightly sealed and placed in boiling water for 30 min. This extraction procedure was adopted
when it was found to give the same results as were obtained with oocytes which were competely
dissolved in hot nitric acid.
The Na and K concentrations of the samples are determined by emission flame photometry
using a Zeiss PMQ II spectrophotometer with an FM-I flame attachment with hydrogenoxygen flame. (The galvanometer resistance of the spectrophotometer was altered to give a
fourfold increase in sensitivity.) In order to prevent interference from recirculating vaporized
sample and from dust particles, air from the flame was directly vented from the room by an
exhaust system; a filter enclosure was built around the flame housing so that air drawn into the
Na and K in the nucleus and cytoplasm of oocytes
9
area of the flame was filtered. Concentrations of Na and K in the experimental samples generally
ranged from 001 to 0-30 mg Na/1. and from o-i to 0 8 mg K/l. No interference of Na with K
readings, or vice versa, was found at these concentrations.
Controls
Appropriate volumes of solutions containing Na at experimental concentrations were exposed
to the materials and tools used in the micro-dissection procedure, and changes in the Na content of these solutions were monitored. They were found to be negligible.
Each Beem capsule holds 2 Parafilm envelopes, one to contain the cell fragment and the other
to serve as control. A control envelope goes through every step of the procedure with its
respective experimental envelope and is similarly analysed for dry weight, Na, and K. Na and K
contents of control envelopes range from less than 1-15% of that of experimental samples
(experimental envelope plus nuclei or pieces of cytoplasm). The dry weights, Na, and K contents of each experimental sample are corrected for the corresponding control values.
RESULTS AND OBSERVATIONS
Diameters of frozen nuclei range from 200 to 400 fim among the species studied; the
size of nuclei from the oocytes of a given species is quite constant. Results of the
Table 1. Values for dry weight, Na and K concentrations of nuclei and pieces of
cytoplasm of oocytes of 4 species of Amphibia
(Na and K concentrations expressed as /Jequiv./ml of nuclear or cytoplasmic water. Each
nuclear measurement was done on a separate group of 7-10 nuclei.)
/iequiv Na/ml HSO /iequiv. K/ml H,O
% Dry weight
,
>*
Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm
f
Organism
Eurycea b. bislineata
(two-lined
salamander)
Mean
S.E.
Desmognathus 0.
ochrophaeus
(Allegheny
salamander)
Mean
S.E.
Rana pipiens
(Leopard frog)
Mean
S.E.
Rana catesbtana
(Bullfrog)
Mean
S.E.
21-8
291
2V3
261
±22
21-5
215
21'5
±o-o
15-5
155
15-5
155
±o-o
203
18-4
207
19-8
±0-7
61-5
632
636
62-8
±16
67-4
62-3
648
64-5
64-7
± i-o
52-8
55-3
S7'2
5S'i
±i-3
5i-9
48-8
49-5
50-1
±1-2
I4-3
162
15-5
I5-3
±o-6
159
12-5
14-2
839
75-i
91-8
83-6
±4-8
65-8
73-9
79-1
80-2
74-8
II9-9
130-1
I2O-I
123-4
±3'4
II2-0
145-8
128-9
±16-9
78-3
992
97-2
91 6
±67
76-2
79'3
86-2
826
8I-I
±2-2
±i-7
±3-3
7-2
7'5
7'3
7'3
5°'9
5I-S
75-o
±o-o
±8-o
168
84-2
71-8
8o-i
128-4
81-7
93-8
82-4
168
78-7
128-4
860
59-i
±37
127-5
122-5
129 3
1264
± 2'O
99-7
94-2
87-5
93-8
±3-5
±3-9
io
T.J. Century, I. R. FenicM and S. B. Horowitz
analysis of oocytes of the 2 salamanders and the 2 frogs for water content and Na and K
concentrations are given in Table 1 with the means and standard errors of the means
(S.E.) of the measurements for each species. The nuclei of the 4 species studied
contain 74-85% water. Frog oocyte cytoplasm is 45-50% water, while salamander
cytoplasm is 35-40 % water. This difference in cytoplasmic water content is presumably due to a denser concentration of yolk platelets in the salamander oocytes.
In Rana oocytes, which are pigmented, the nucleus appears as a small, white, hard
piece of ice embedded in a dark, only slightly softer matrix of cytoplasm. In the unpigmented salamander oocytes, on the other hand, the light-yellow cytoplasm has a
much softer, almost buttery consistency, a property which facilitates isolation and
cleaning of the nucleus which, as in the frog oocyte, is hard and white. The difference
in the mechanical properties of the cytoplasm of frog and salamander oocytes appears
to reflect the difference in water content.
The K/Na ratio in the nucleus of these oocytes is about 8, while the cytoplasmic
K/Na ratio is close to unity. On a water basis, the nucleo-cytoplasmic asymmetry of
Na is much greater than, and in the opposite direction from, that of K; nuclear Na
concentrations are only 12-21% of cytoplasmic while nuclear K concentrations are
135-159% of cytoplasmic. On a water basis, the cytoplasm of these cells contains a
small excess of monovalent cations compared with the nucleus; the ratio of cytoplasmic to nuclear (Na + K) ranges from I-I to 1-25.
DISCUSSION
The Na concentration in the nucleus is only 0-12-0-20 that in the cytoplasm, while
K is 1-4-1-6 times more concentrated in the nucleus than in the cytoplasm. These
results differ from previous determinations reported in the literature.
Naora et al. (1962) found cytoplasmic Na and K concentrations in R. pipiens
oocytes similar to those reported here (compare Tables 1 and 2). However, nuclear
concentrations of Na and K were, respectively, 17 and 2 times those given here. The
difference between the present results and those of Naora et al. may be attributable to
Table 2. Means ± S.E. and ranges {where given) of nuclear and cytoplasmic
Na and K concentrations in amphibian oocytes from previous reports in the
literature expressed as fiequiv.\ml of nuclear or cytoplasmic water
/tequiv.
Na/ml HaO
K/ml H2O
Organism
Nucleus Cytoplasm Nucleus Cytoplasm
Reference
Triturus cnstatus
(Newt)
67
123
148
118
±11
±3
±i°
±4
(44-95) (118-132) (121-164H114-134)
Riemann et al.
(1969)
Rana pipiens
(Leopard frog)
281
±28
88
±7
258
±26
106
Naora et al.
±8
(1962)
Notes
Assuming density of
1-i and 60% dry wt.
for cytoplasm, density
1-05, 20% dry wt.
for nuclei
Na and K in the nucleus and cytoplasm of oocytes
11
technique. Naora et al. found that when oocytes were frozen on glass slides in contact
with dry ice, some oocytes cracked upon freezing. This crack extended from the surface of the oocyte to the nucleus, and could be exploited to readily isolate the nucleus
from the cytoplasm. The Na and K determinations of Naora et al. were done on
nuclei isolated in this manner. We have attempted to reproduce this technique in our
laboratory, and while we have found that some cells crack upon freezing, these cracks
do not facilitate separating nucleus from cytoplasm.
The results of Naora et al. may reflect the slower rate of freezing which was used.
These authors used dry ice (— 78 °C), and the rate of freezing may have been sufficiently slow to permit marked displacements of cellular water as Trump (1969)
found in other cells. This displaced water shows up as large extracellular ice crystals
around a highly distorted cell. Such water movements are likely to be accompanied by
displacements of cellular electrolytes as the freezing front advances through the cell.
Cells which have been rapidly quenched at —155 °C, on the other hand, show little
displacement of the cellular water, which shows up as minute ice crystals uniformly
distributed throughout the cell (Trump, 1969). Other than this, we cannot account for
the results of Naora et al. except to suggest that since their technique involved analysis
of only that 10% of the cells which showed a surface crack upon freezing, their
results may describe the distribution of Na and K in an abnormal population of
cells.
Recently, Riemann, Muir & Macgregor (1969) dissected various sizes of immature
and mature Triturus oocytes under oil at room temperature and analysed known
volumes of nuclei and cytoplasm for Na and K (water contents were not determined).
When expressed in terms of the nuclear and cytoplasmic water contents of our salamander oocytes (see Table 1), their data show that Na in the nucleus of the mature
Triturus oocyte (> 1-5 mm diameter) is only one-half as concentrated as in the
cytoplasm, while the nuclear K concentration is 1-2 times that of the cytoplasm.
Cytoplasmic Na and K concentrations were found to be of similar magnitude (Table 2).
Our results and those of Riemann et al. differ principally in that K/Na ratios in nuclei
isolated under oil are less than one-fourth of the K/Na ratios of nuclei isolated by
the frozen microdissection technique. Since the ratio of total nuclear Na + K to total
cytoplasmic Na + K obtained with each of the two techniques is the same, 0-9, it may
be that when the oocyte is broken open under oil at room temperature, equimolar
amounts of Na and K move down their respective gradients; Na moves from the
cytoplasm into the nucleus while K moves out of the nucleus into the cytoplasm.
Abelson & Duryee (1949) have shown radioautographs of frozen sections of R.
pipiens oocytes which had been exposed to MNa for 30 min to 1 h. Grain density over
the nucleus was much greater than over the surrounding cytoplasm, and it was concluded that the nucleus had at least twice the 24Na concentration of the cytoplasm on a
volume basis. Naora et al. (1962) have repeated these experiments with similar results.
32
Na influx into Eurycea oocytes has been studied in this laboratory using radioautographic and extractive analysis (Horowitz & Fenichel, 1970). For short influx
times, the radioautographs show a nuclear to cytoplasmic ratio of 2-6:1 on a volume
basis, confirming the earlier radioautographic results.
12
T.J. Century, I. R. Fenichel and S. B. Horowitz
The radioautographic picture (high nuclear 23Na) appears to contradict the results
of the analysis of frozen microdissected nuclei (low nuclear fflNa); however, two further
observations resolve this apparent contradiction. First, 22Na exchange in amphibian
oocytes shows 2 distinct kinetic fractions: a rapidly exchanging fraction, comprising
about 15 % of t n e total c e u Na, which exchanges completely with a2Na in about 2 h,
and a large slowly exchanging fraction which exchanges with a half-time of the order
of days. Second, the radioautographic results for short influx times, when expressed
on a water basis, give a nuclear : cytoplasmic 22Na ratio of near unity, showing that the
rapidly exchanging fraction is uniformly distributed between the nucleus and cytoplasm. Since the microdissected frozen nuclei are only about 15% as concentrated
in B Na as the cytoplasm, these results are interpreted to mean that nuclear Na consists entirely of rapidly exchanging Na which is freely diffusible and nearly uniformly
distributed in the nuclear and cytoplasmic water while the great excess of cytoplasmic
Na is attributable to slowly exchanging Na sequestered in the cytoplasm.
The actual nucleo-cytoplasmic ratio of rapidly exchanging Na in the Eurycea
oocyte is 1-3 on a water basis (Horowitz & Fenichel, 1970). The distribution of
total K in the Eurycea oocyte shows a similar nucleo-cytoplasmic ratio, 1-35 (see
Table 1), as does the rapidly exchanging fraction of glycerol in the R. pipiens oocyte
(Horowitz & Fenichel, 1968). Ling, Ochsenfeld & Karreman (1967) have reported a
slowly exchanging water fraction in R. pipiens oocytes comprising up to 0-25 of the
oocyte water. Water in this fraction would presumably not be acting as solvent for the
rapidly exchanging solutes mentioned above. A sequestered, non-solvent cytoplasmic
water fraction of 0-23 would reduce the nucleo-cytoplasmic ratios of rapidly exchanging Na and glycerol to unity.
The similarity in the distribution of total K to that of rapidly exchanging Na and
glycerol suggests that, unlike Na, all of the cellular K is freely diffusible. The recent
demonstration by Dick & McLaughlin (1969) that the activity coefficient of Na in
immature Bufo oocytes is less than half that in free solution, while the activity of K is
unchanged from that in free solution provides direct support for this speculation.
This work 13 based in part on a thesis to be submitted by T. J. C. to the Department of
Biology, University of Pennsylvania, in partial fulfillment of the requirements for the degree of
Doctor of Philosophy. It was supported in part by a U.S. Public Health Service Training
Grant (G-T1-GM-517-10) and grants from the National Science Foundation (GB 8032) and
the National Institutes of Health (GM 14886-01).
REFERENCES
ABELSON, P. H. & DURYEE, W. R. (1949). Radioactive sodium permeability and exchange in
frog eggs. Biol. Bull. mar. biol. Lab., Woods Hole 96, 205-217.
DICK, D. A. T. & MCLAUGHLIN, S. G. A. (1969). The activities and concentrations of sodium
and potassium in toad oocytes. J. Phystol., Lond. 205, 61-78.
HOROWITZ, S. B. & FENICHEL, J. R. (1968). Analysis of glycerol- 3 H transport in the frog
oocyte by extractive and radioautographic techniques. J. gen. Phystol. 51, 703-730.
HOROWITZ, S. B. & FENICHEL, I. R. (1970). Analysis of sodium transport in the amphibian
oocyte by extractive and radioautographic techniques. J. Cell Biol. (in the press.)
Na and K in the nucleus and cytoplasm of oocytes
13
LING, G. N., OCHSENFELD, M. M. & KARHEMAN, G. (1967). Is the cell membrane a universal
rate-limiting barrier to the movement of water between the living cell and its surrounding
medium? J. gen. Phystol. 50, 1807-1820.
NAORA, H., NAORA, H., IZAWA, M., ALLFREY, V. G. & MLRSKY, A. E. (1962). Some observations
on differences in composition between the nucleus and cytoplasm of the frog oocyte. Proc.
natn. Acad. Sci. U.S.A. 48, 853-859.
RIEMANN, W., MUIR, C. & MACGREGOR, H. C. (1969). Sodium and potassium in oocytes of
Tnturiis cristatiis. J. Cell Sci. 4, 299-304.
TRUMP, B. F. (1969). Effects of freezing and thawing on cells and tissues. In Autoradiography of
Diffusible Substances (ed. L. J. Roth & W. E. Stumpf), pp. 211-240. New York and London:
Academic Press.
{Received 3 December 1969)