/. Embryol. exp. Morph. Vol. 36, 1, pp. 67-72, 1976
Printed in Great Britain
67
Role of karyoplasm in the
emergence of capacity of egg cytoplasm to induce
DNA synthesis in transplanted sperm nuclei
ByM. N. SKOBLINA 1
From the Institute of Developmental Biology,
USSR Academy of Sciences, Moscow
SUMMARY
The behaviour of sperm nuclei was studied both in the cytoplasm of intact toad oocytes
undergoing maturation and the cytoplasm of oocytes matured without germinal vesicles. The
behaviour of the nuclei of pronase-treated sperm injected in the mature egg cytoplasm was
shown to be exactly similar to that of the sperm nucleus after fertilization, i.e. they swelled,
synthesized DNA, and divided. No changes in such sperm nuclei could be detected in the
cytoplasm of the oocytes matured without germinal vesicles.
INTRODUCTION
In recent years Gurdon has extensively studied the mechanism of the cytoplasmic control of nuclear activity (Gurdon, 1967, 1968, 1969). One aspect of
this problem is the cytoplasmic control of nuclear division and DNA synthesis.
According to Gurdon 'DNA synthesis factor' appears during oocyte maturation, after passage of karyoplasm into the cytoplasm (Gurdon, 1967, 1968). The
question of involvement of the karyoplasm in the origin of this factor remains
open. We have attempted to answer it by studying the behaviour of the sperm
nuclei in the cytoplasm of oocytes matured in the absence of karyoplasm, and
in the cytoplasm of intact oocytes at successive stages of maturation.
MATERIALS AND METHODS
A piece of toad (Bufo viridis) ovary was excised and transferred into amphibian
Ringer solution. Separate oocytes were detached from it with the aid of forceps.
Germinal vesicles were removed from some oocytes by the method of Dettlaff,
Nikitina & Stroeva (1964). The enucleated and the intact oocytes were cultivated in Ringer solution with progesterone (5 /*g/ml) at 18-20 °C. Sperm of the
common frog {Rana temporarid) treated with pronase (Koch Light Lab.) were
used as a source of nuclei (Skoblina, 1974). Common frog sperm were used
1
Author's address: Institute of Developmental Biology, U.S.S.R. Academy of Sciences,
Moscow, U.S.S.R.
5-2
68
M. N. SKOBLINA
owing to the absence of toad males; frog oocytes are not suitable for such
experiments as they do not tolerate microsurgery well enough. Some intact
oocytes were transferred every 3 h to Ringer solution without progesterone and
simultaneously injected with a sperm suspension treated with pronase, and with
[H3]thymidine (0-04 [i Ci/oocyte, specific activity 22-4 Ci/mmole, Radiochemical
Centre, Amersham). In 24-27 h the sperm suspension and thymidine were also
injected into the oocytes which were maturing without germinal vesicles. To
know exactly the stage of maturation at which the injections were made, 10-30
intact oocytes maturing under the same conditions were fixed in San-Felice
fluid before each injection. Injected oocytes were fixed 2, 3, and 4 h after the
injection of [H3]thymidine. Serial sections 6/«n thick were prepared. Some
sections were treated with 1 mg/ml RNA-ase solution in phosphate buffer,
pH 7-5 at 37 °C. Radioautographs were obtained as usual. The Feulgen reaction
was carried out prior to the coating of sections with emulsion and after development they were counterstained with light green. From the oocytes fixed for stage
determination, 243 were sectioned; the sperm suspension and [H3]thymidine
were injected into 340 oocytes of three toad females, and in 300 oocytes the
sperm nuclei were found.
RESULTS
Examination of sections showed that the number of sperm injected into an
oocyte varied from 2 to 20. All the data refer to the behaviour of sperm nuclei
in the animal cytoplasm. The transplanted nuclei in the same egg are sometimes
at different stages of transformation; the tables include data on the most
'advanced' nuclei.
Behaviour of sperm nuclei in the cytoplasm of intact mature eggs and
oocytes matured after the germinal vesicle removal
Most sperm nuclei were markedly swollen in the cytoplasm of intact mature
eggs 2 h after injection (Fig. 1 A). At the same time the first labelled mitoses,
sometimes multipolar, occurred. Four hours after injection, swelling and dividing sperm nuclei were observed in practically all intact mature eggs (Table 1).
All of them proved to be labelled (Figs. 2, 3). The label was retained when the
sections were pretreated by RNA-ase prior to coating with emulsion. In none
of the oocytes matured after germinal vesicle removal was there either swelling
or labelling of the transplanted nuclei (Fig. 4, Table 1). Cytasters, occasionally
numerous, were visible in the cytoplasm of these oocytes.
Behaviour of sperm nuclei injected into the cytoplasm of intact
oocytes at successive stages of maturation
When the sperm suspension and [H3]thymidine were injected into the oocyte
cytoplasm within the first 6 h of progesterone-induced maturation (from the
stage of intact germinal vesicle to prometaphase of thefirstmaturation division (1))
Karyoplasm in transplanted sperm nuclei
Fig. 1
69
Fig. 2
Fig. 4
Fig. 3
Figs. 1-4. Sperm nuclei in the toad egg cytoplasm (Figs. 1,2, 3) and in the cytoplasm
of oocytes matured without germinal vesicle (Fig. 4), 2h after injection: 1, slide
without emulsion, 2-4, radioautographs.
Table 1. Behaviour of sperm nuclei in the cytoplasm of eggs and
oocytes matured in vitro without germinal vesicle
Oocytes in which sperm nuclei:
Recipients
Eggs
Enucleated
oocytes
State of oocyte
nucleus at the
moment of
injection
Total number
of oocytes
containing
sperm nuclei
No.
%
No.
%
No.
%
Metaphase II (15)*
66
52
2
52
3
100
31
0
47
0
33
0
50
0
Remained
intact
Were
swollen
Began
dividing
* In this table and the next the number of oocytes examined for stage determination is
indicated in parentheses.
70
M. N. SKOBLINA
Table 2. Behaviour of sperm nuclei in the cytoplasm of toad oocytes at
successive stages of maturation
Duration of
cultivation
in Ringer
with progesterone
Uvl Ul. w
State of
oocyte
nucleus at
uiv
111V-/11 twin
Oocytes in which sperm nuclei:
Total
rState of number of
Remained
Were
Began
oocyte
oocytes
nucleus at containing
intact
swollen
dividing
thp
mnmpnt
Hlv* 111UI11V-1.IL
cnprm
oJJCU 111
j
y
No.
%
0
0
0
0
0
0
0
100
0
0
0
0
17
85
3
15
0
0
19
8
43
6
31
5
26
41
4
11
22
53
15
36
33
11
33
11
33
11
33
25
9
36
8
31
8
31
nuclei
No.
21
21
100
0
28
28
100
21
21
Polar body I
(4), prometaphase
II (29)
PrometaphaseII (10)
20
Metaphase
II (27)
Metaphase
II (10)
Metaphase
II (7)
injection (h) of injection
of fixation
Intact
0
Intact
GV (21)
GV (25)*
GV at the
GV at the
3
surface (20) surface (3),
prometaphaselt(15)
PrometaPrometa6
phase I (10) phase I (7),
polar body I
(3), prometaphase
No.
/o
/o
n : (i)
9
12
15
18
21
Prometaphase I (3),
prometaphase II (1)
Polar body
(2), prometaphase
H(6)
Metaphase
11(9)
Metaphase
11(8)
Metaphase
H(7)
* GV, germinal vesicle. t I, first maturation division. % II, second maturation division.
no marked changes occurred in the nuclei, and they did not incorporate
[H3]thymidine (Table 2). With injection of sperm suspension and [H3]thymidine
9 h after transfer of oocytes to the progesterone solution, the first swelling and
labelled nuclei appeared only 4 h after injection. Only prometaphase and metaphase II (second division) were found in the oocytes at this time. The behaviour
of the sperm nuclei injected into oocytes 12 h after the beginning of hormonal
treatment and later, was similar to that in the intact mature eggs (Table 2). With
a single exception all swelling and dividing nuclei were labelled (Table 2). With
injection of the sperm suspension 12 h, 15 h, 18 h or 21 h after the beginning of
Karyoplasm in transplanted sperm nuclei
71
hormonal treatment (metaphase II), certain differences in the percentage of
oocytes containing swelling and dividing nuclei (Table 2) were observed,
although these were not statistically significant.
DISCUSSION
The transplantation of pronase-treated sperm from the common frog into
intact mature eggs of the toad showed that sperm nuclei swelled, synthesized
DNA, and began dividing. DNA synthesis was first detected at the metaphase
II stage. DNA synthesis in brain nuclei transplanted into maturing Xenopus
laevis oocytes was observed for the first time by Gurdon (1967). The stage of
oocyte maturation at which DNA synthesis began was not mentioned, but it
appeared to be the second maturation division (nuclei were transplanted at
metaphase I stage, their transformation was observed 90 min later). Ziegler &
Masui (1973) observed the swelling of brain nuclei (which, according to Gurdon,
is a prerequisite of the beginning of DNA synthesis) only in mature Xenopus
eggs after their activation. Hence, DNA synthesis in sperm and somatic nuclei
transplanted into maturing oocytes appears to have begun at a similar stage of
maturation.
The absence of changes in the sperm nuclei in the oocytes matured without
the germinal vesicle, showed that the karyoplasm is indispensable for the cytoplasm to acquire the capacity to induce both the transformation of sperm
nuclei into pronuclei and DNA synthesis in them. But since there exists a considerable time gap between the germinal vesicle breakdown and the beginning
of nuclear transformation, it is highly probable that certain processes occurring
in the cytoplasm after its mixing with the karyoplasm during maturation are
essential for the appearance of 'DNA synthesis factor'.
One of the candidates for the role of 'DNA synthesis factor' was DNA
polymerase (Gurdon & Speight, 1969; Gurdon & Woodland, 1969; Grippo &
LoScavo, 1972). Grippo and his co-authors have determined the activity of DNA
polymerase in extracts from intact primary and mature oocytes and oocytes
matured without the germinal vesicle, and have shown that a new DNA
polymerase appears in the oocytes during maturation and that it is absent both
in primary oocytes and those matured without germinal vesicle (Grippo et al.
1974). But Ford and Woodland have recently calculated that the rise in DNA
polymerase activity per cell occurring during oocyte maturation is insufficient
to account for the difference in DNA synthesis between these two cell types.
They have suggested therefore that factors other than DNA polymerases are
involved in the control of DNA synthesis during oocyte maturation (Ford &
Woodland, 1975).
72
M. N. SKOBLINA
REFERENCES
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oocyte maturation in anurans as revealed by the removal and transplantation of nuclei.
/. Embryol. exp. Morph. 12, 851-873.
FORD, C. C. & WOODLAND, H. R. (1975). DNA synthesis in oocytes and eggs of Xenopus
laevis injected with DNA. Devi Biol. 43, 189-199.
GRIPPO, P. & LOSCAVO, A. (1972). DNA polymerase activity during maturation in Xenopus
laevis oocytes. Biochem. biophys. Res. Commun. 48, 280-285.
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DETTLAFF,
(Received 13 October 1975; revised 12 February 1976)