/. Embryol. exp. Morph. Vol. 37 pp. 203-209, 1977
Printed in Great Britain
203
A method for enucleating oocytes of
Xenopus laevis
By C. C. FORD 1 AND J. B. GURDON 2
From the School of Biological Sciences, University of Sussex and
MRC Laboratory of Molecular Biology, University
Postgraduate Medical School, Cambridge
SUMMARY
A method is described for the enucleation and complete healing of Xenopus oocytes so that
the enucleated oocytes withstand multiple injections and culture for several days. The oocytes
are defolliculated and enucleated manually and allowed to heal in a potassium phosphate
buffer. Oocytes enucleated in this way support RNA synthesis by injected HeLa-cell nuclei 3
days later. This method is valuable in providing a low-background system in which transcription of injected nuclei can be studied.
INTRODUCTION
The oocyte of Xenopus laevis has provided a valuable assay system for the
study of transcription and translation following micro-injection of nuclei or
messenger RNA (Gurdon, 1968; Gurdon, Lane, Woodland & Marbaix, 1971;
Lane & Knowland, 1975; Gurdon, Partington & de Robertis, 1976). In studying
the synthetic activities that occur following injection of nuclei into oocytes it is
important to eliminate possible transcription by the host nucleus. In addition,
the time period of the experiments should be sufficiently long for the synthetic
activities of the inserted nuclei to be detected. For these purposes a method has
been developed by which oocytes can be enucleated and fully healed so as to
withstand further injection and remain synthetically active for at least 3 days.
Previous methods of enucleating oocytes have been used with Rana species
to study the role of nucleus and cytoplasm in hormone-induced maturation
(Dettlaff, Nikitina & Stroeva, 1964; Smith & Ecker, 1969). The healing solution
(Steinberg's saline) used by Smith & Ecker with Ranapipiens does not give high
levels of healing with Xenopus oocytes. For this reason the following enucleation
method has been developed.
1
Author's address: School of Biological Sciences, University of Sussex, Falmer, Brighton.
BN1 9QG, U.K.
2
Author's address: MRC Laboratory of Molecular Biology, University Postgraduate
Medical School, Hills Road, Cambridge. CB2 3EJ, U.K.
204
C. C. FORD AND J. B. GURDON
MATERIALS
Ovaries removed from mature females were cut up to give small clusters of
oocytes, and maintained in modified Barth saline-Hepes buffered (MBS-H).
This solution contains 88 mM-NaCl, 1-0 mM-KCl, 0-33 mM-Ca(NO3)2, 0-41 mMCaCl2, 0-82 mM-MgSO4, 2-4 mM-NaHCO3, 10 IHM Hepes pH 7.4. Benzyl penicillin (Sodium salt, 1-6 x 103 i.u./mg) and streptomycin sulphate (Glaxo Laboratories, Greenford, Essex) were added to give a concentration of 10 mg/litre.
Other reagents were 'Analar' grade and were obtained from BDH Chemicals,
Poole, Dorset.
The healing solution contained 90 HIM potassium phosphate pH 7-2, 10 mMNaCl, 1 mM-MgSO4. A stock potassium phosphate solution was prepared by
adding 450 mM-KH2PO4 to 450 mM-K2HPO4 to give a final pH of 7-2 (usually
37:100 by volume respectively).
BD Yale Microlance syringe needles were obtained from Becton Dickinson
U.K. Ltd, York House, Empire Way, Wembley, Middlesex.
All manipulations were performed at room temperature (19-22 °C) using a
low power binocular microscope (approx. x 35). Oocytes were placed in clean
glass Petri dishes for all manipulations, unless otherwise stated.
Enucleation procedure
Step 1. Separated oocytes with attached follicular material are placed in
MBS-H containing an additional 60 mM-NaCl (made by adding 0-175 g NaCl
to 50 ml MBS-H) and left for 10 min.
Step 2. Using sharpened watchmaker's forceps the follicular layers are
removed from around the oocyte (Fig. 1A). A few follicle cells may not be
removed by this procedure. Some 10-15 follicle cells are seen round an equatorial 10 /an section of a defolliculated oocyte (ca. 5 % of the number round an
unmanipulated oocyte). However, for enucleation, sufficient material has been
removed when the oocyte appears flaccid and sticks readily to clean glass surfaces. Defolliculated oocytes must be completely undamaged, since even the
smallest tear results in excessive cytoplasmic leakage during subsequent
treatment.
Step 3. Defolliculated oocytes are transferred immediately to MBS-H.
Oocytes can be accumulated at this step.
Step 4. Small numbers of defolliculated oocytes are transferred to halfstrength MBS-H (MBS-H diluted 1:1 with H2O). Each oocyte is rotated so that
it sticks to the glass dish with the animal pole uppermost, and left for 5 min.
Step 5. A small incision is made obliquely at the animal pole of each oocyte
with a 26-gauge (10/45) syringe needle (Fig. IB, C, D). The needle should be
inserted to about one quarter of the diameter of the oocyte so that when removed
the animal pole has a slit about one fifth the oocyte's diameter.
Step 6. After a few minutes any exuding yolk is carefully dislodged and the
Oocyte enucleation
205
0-5 mm
Fig. 1. Photographs of theenucleation procedure. (A) Oocyte with follicular sheath
partially removed. (B) DefoUiculated oocyte and syringe needle. (C) Syringe needle
inserted in the animal pole of the oocyte. (D) Oocyte with an incision at the
animal pole. (E) Germinal vesicle emerging through the incision. (F) Enucleated
oocyte with the germinal vesicle beside it. (G) Enucleated oocyte with slit partially
closed. (H) Fully healed enucleated oocyte.
14
EMB 37
206
C. C. FORD AND J. B. GURDON
Table 1. Healing of enucleated oocytes
Time in healing
solution (min)
Healed at
5h*
Healed at
24 h*
Survival at
4 days
0
15
50
60
105
135
300
330
2/4
7/7
6/6
6/6
3/4
3/4
1/4
4/7
0/4
5/7
6/6
6/6
3/4
3/4
0/4
2/7
0
1
5
2
1
0
0
0
Oocytes were defolliculated and enucleated as described in the text and then left in the
healing solution for various times. Subsequently the cells were cultured in MBS-H at 19 °C
and the number of healthy healed oocytes scored as a fraction of the number of oocytes
originally present.
* 2/4 means 2 out of 4 operated oocytes had a healed-over incision when examined 5
(or 24) h later. The wounds of some of those oocytes which appeared healed at 5 h had
reopened by 24 h.
oocyte squeezed with forceps until the translucent germinal vesicle (GV) appears
in the slit. Over the next 10 min the GV slowly emerges from this slit (Fig. 1E).
Sometimes the oocyte needs an additional squeeze to help the GV emerge. If
necessary the GV is dislodged (Fig. 1F) merely by moving the dish from side to
side. Occasionally the GV bursts and these oocytes are discarded.
Step 7. The enucleated oocyte is immediately transferred to the phosphate
healing solution. Oocytes are left in this solution for 60-90 min. The incision
should now be largely covered over by the black pigmented oocyte surface
(Fig. 1G, H). The most successfully enucleated oocytes will heal in less than 1 h.
Those that have not healed within 2 h do not do so thereafter.
Step 8. The oocytes are transferred to MBS-H. In successfully enucleated
oocytes the enucleation slit will have been completely eliminated (Fig. 1H) and
will not reopen for as long as the oocyte survives.
Factors affecting the success oj the method
Variation of each major step in the procedure indicates that the time for which
oocytes are left at steps 3, 4 and 8 is not critical. In steps 1 and 2 the total time
of exposure to MBS-H containing additional NaCl should not be more than
30 min. In a test of physiological normality of the oocytes, normal maturation
(Schorderet-Slatkine, 1972) in response to progesterone was obtained in 59 % of
defolliculated oocytes exposed for less than 30 min to MBS-H containing
additional NaCl, and in 65 % of control oocytes not exposed to this buffer.
However, only 25 % of oocytes exposed to the high NaCl solution for 40 min
showed normal maturation.
The precise composition of the healing solution is not critical in that moderate
Oocyte enucleation
207
variations in concentration of components and slight variation in pH (6-7-7-4)
gave similar results. However, the step that is extremely important is the length
of time for which enucleated oocytes are left in the healing solution (Table 1).
Incubation for more than 2 h in healing solution reduces their survival thereafter
and does not increase the proportion of successfully healed oocytes.
Re-nucleation of enucleated oocytes
To demonstrate the usefulness of this enucleation method, we have injected
nuclei into enucleated oocytes. This is technically more difficult than injecting
oocytes as usual, because the lack of follicle cells makes the oocytes fragile and
sticky. A procedure which has proved successful is to place each enucleated
oocyte in a small drop of MBS-H on a siliconized slide. The injection pipette
containing a suspension of 100-200 nuclei in about 35 nl (Gurdon, 1976) is
inserted into the oocyte by pushing the oocyte against the inside wall of the drop
(Fig. 2 A). The injected oocytes are then transferred to an agar-coated Petri dish
where they are cultured for a few days.
In an experiment of this kind, oocytes were defolliculated, enucleated, healed
and maintained in MBS-H. The next day the oocytes were injected as described
above with HeLa nuclei and incubated for a further 3 days. They were then
injected, for a second time, with about 30 nl of [3H]GTP, and fixed 8 h later.
The oocytes appeared to be in good condition after all these manipulations.
When sectioned and examined histologically, numerous apparently healthy
HeLa nuclei could be seen (Fig. 2B, C). The criteria of healthy nuclei and their
frequency of occurrence are described in detail by Gurdon (1976). Subsequent
autoradiography showed that all nuclei had incorporated label (Fig. 2D).
Nuclear labelling of this kind represents RNA synthesis as judged by ribonuclease sensitivity (for details see Gurdon et al. 1976). The normal appearance
and synthetic activity of the oocytes and injected nuclei demonstrates that the
enucleation technique and healing procedure are reasonably harmless. Such
oocytes may certainly be used for experiments for several days after enucleation,
and can withstand at least two subsequent injections.
Survival of enucleated oocytes
In two series of experiments, enucleated oocytes were prepared as described
above, and half of them (i.e. 12) were re-nucleated with HeLa nuclei within 24 h
of enucleation. Both groups were cultured in MBS-H. After 3-4 days at 19 °C
all 12 enucleated oocytes had come to appear abnormal, and looked dead by the
fifth day. In contrast, six of the re-nucleated oocytes looked healthy on the fifth
day. The number of oocytes in these experiments was small. Nevertheless, the
results suggest that enucleated oocytes do not normally survive for more than
3-4 days, whereas re-nucleated oocytes are rescued, at least temporarily, from
death.
14-2
208
C. C. FORD AND J. B. G U R D O N
A
B
Drop of medium
Injection needle
\
Siliconized
slide
200 urn'_ •--'.
> ; ' v . • V** V-$
Fig. 2. The injection of enucleated oocytes. (A) Diagram of an enucleated oocyte
being injected. (B) Low power view of a section of an enucleated oocyte injected
with HeLa nuclei. (C) High power view of injected HeLa nuclei. (D) Autoradiograph
of injected HeLa nuclei showing preferential labelling over the nuclei.
DISCUSSION
The enucleation procedure we have described has enabled us to prepare
enucleated oocytes capable of withstanding multiple injections and survival for
several days. The defolliculated oocytes respond to a hormonal test (progesterone induced maturation) to the same extent as unmanipulated oocytes. These
features make oocytes manipulated by this method eminently suitable for
studying the long-term synthesis of RNA by injected nuclei in the absence of a
host nucleus. In these circumstances the labelled RNA detected cannot have
been synthesized by, and transferred from, the host nucleus. We consider the
GV to be fully removed by this procedure because it emerges with its membrane
round it and because histological examination of sectioned oocytes fails to
reveal any fragments. The long-term culture of oocytes increases the chances of
accumulating sufficient newly synthesized RNA to detect their protein products
(Gurdon, De Robertis & Partington, 1976).
The present procedure differs from that developed by Smith & Ecker (1969) in
Oocyte enucleation
209
two major respects. Firstly MBS-H containing additional NaCl is used to aid
defolliculation. In this high NaCl solution the oocyte becomes flaccid and
partially separated from the follicular sheath. This greatly facilitates removal of
the follicular sheath without damaging the oocyte.
The second major difference is the healing solution. Steinberg's solution (used
by Smith & Ecker) in our hands did not give complete healing as frequently as
the phosphate solution described here. This phosphate solution almost completely prevents exudation of cytoplasm from the freshly enucleated oocyte.
Perhaps for this reason the oocyte membrane has sufficient time to heal over
before it is prevented from doing so by a plug of exuding yolk. Irrespective of
the precise role of the phosphate solution, this method has the advantages that
enucleated oocytes heal with a high frequency and subsequently survive after
renucleation, for up to 5 days.
We thank Dr B. C. Goodwin and Dr R. A. Laskey for their comments on this manuscript.
We are grateful to the Medical Research Council and Cancer Research Campaign for
financial support.
REFERENCES
T. A., NIKITINA, L. A. & STROEVA, O. G. (1964). The role of the germinal vesicle
in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei.
/. Embryo!, exp. Morph. 12, 851-873.
GURDON, J. B. (1968). Changes in somatic cell nuclei inserted into growing and maturing
amphibian oocytes. /. Embryol. exp. Morph. 20, 401-414.
GURDON, J. B. (1976). Injected nuclei in frog oocytes: fate, enlargement and chromatin
dispersal. /. Embryol. exp. Morph. (In the press.)
GURDON, J. B., DE ROBERTIS, E. M. & PARTINGTON, G. (1976). Injected nuclei in frog oocytes
provide a living cell system for the study of transcriptional control. Nature, Loud. 260,
116-120.
GURDON, J. B., LANE, C. D., WOODLAND, H. R. & MARBAIX, G. (1971). Use of frog eggs and
oocytes for the study of messenger RNA and its translation in living cells. Nature, Lond.
233, 177-182.
GURDON, J. B., PARTINGTON, G. A. & DE ROBERTIS, E. M. (1976). Injected nuclei in frog
oocytes: RNA synthesis and protein exchange. /. Embryol. exp. Morph. (In the press.)
LANE, C. D. & KNOWLAND, J. S. (1975). The injection of RNA into living cells: the use of
frog oocytes for the assay of mRNA and the study of the control of gene expression. In
The Biochemistry of Animal Development, vol. 3 (ed. R. Weber), pp. 145-182. New York:
Academic Press.
SCHORDERET-SLATKINE, S. (1972). Action of progesterone and related steroids on oocyte
maturation in Xenopus laevis. An in vitro study. Cell Differen. 1, 179-189.
SMITH, L. D. & ECKER, R. E. (1969). Role of the oocyte nucleus in physiological maturation
in Ranapipiens. Devi Biol. 19, 281-309.
DETTLAFF,
{Received 13 August 1976)