/ . Embryo!, exp. Morph. Vol. 60, pp. 93-97, 1980
Printed in Great Britain © Company ofBiologists Limited 1980
93
A method for visualizing the
chromosomes of the second polar body of
the mouse egg
J. MODLINSKI 1 AND ANNE McLAREN 2
From the MRC Mammalian Development Unit, London
SUMMARY
A second-polar-body nucleus inserted by microinjection into a different mouse egg
entered, into mitosis synchronously with the pronuclei of the host egg. The extent to which
the karyotype of the polar body can yield information on the karyotype of the donor egg
is discussed.
INTRODUCTION
The second polar body, extruded from the egg at the time of fertilization, is
in principle capable of reactivation, as shown by its successful reincorporation
into the egg, for example as a result of hypotonic treatment (Opas, 1977).
Efforts to produce chromosome preparations from the second polar body have
so far, however, been unsuccessful: during normal development, if it divides
at all it does so unpredictably, out of synchrony with the embryo. C. E. Ford
suggested to us that it might be possible to induce the second polar body to
enter mitosis by injecting it into a fertilized egg, and this we have now
confirmed in mice.
MATERIALS AND METHODS
Donor and recipient females from several different strains (Q, MF1, C57BL)
were used. Donor females that had ovulated spontaneously or after treatment
with pregnant mare's serum followed by human chorionic gonadotrophin were
killed between 10 a.m. and 12 noon on the morning that a vaginal plug
was found. The fertilized eggs were recovered, and the zona pellucida was
removed by brief pronase treatment and subsequent pipetting, to release the
second polar body. It is also possible to extract the second polar body without
removing the zona pellucida, by sucking it through a slit made in the zona into
a mouth-controlled holding pipette (Figs 1-3). The slit is made with a glass
needle near the second polar body. As recipients, fertilized eggs from
1
Author's address: Department of Embryology, Institute of Zoology, University of
Warsaw, 00-927/1 Warsaw, Poland.
2
Author's address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London NW1 2HE, U.K.
7
EMB 60
94
J. MODLINSKI AND A. McLAREN
Ma)
4(c)
Chromosomes of the mouse second polar body
95
spontaneously ovulating females were recovered between 11 a.m. and 1 p.m. After
removal of the surrounding cumulus cells by hyaluronidase treatment, the eggs,
which at this stage contained two medium-sized pronuclei, were cultured for
half an hour in 10/tg/ml cytochalasin B in medium 16 (Whittingham, 1971),
to facilitate injection (Modlinski, 1980). An egg was then held by a suction
pipette attached to a Leitz micromanipulator; a second polar body was sucked
into a fine micropipette (internal diameter 30-50 % of the diameter of the
second polar body) attached to a Beaudouin injection syringe, rupturing its
cell membrane. The injection technique was basically similar to that used to
inject embryonic nuclei into eggs (Modlinski, 1978). The micropipette was
inserted through the zona pellucida and into the vitellus of the recipient egg, as
quickly as possible so as to minimize the time for which the nucleus was
exposed to the medium. Injection, however, was carried out very slowly, to
minimize damage to the recipient egg, and after 30-60 sec the micropipette
was very slowly withdrawn. The injected eggs were transferred to cytochalasinfree medium 16 with five washes, and maintained at 36-5 °C in an atmosphere
of 5 % CO 2 in air. Between 8 p.m. and 10 p.m. that evening the surviving eggs
were placed in a drop of medium 16 containing Colcemid (0-2/tg/ml), and
chromosome preparations were made by an air-drying technique (Tarkowski,
1966) the following morning.
RESULTS
Of 65 fertilized eggs into which second polar bodies were injected, 50 disintegrated within minutes of injection, and 15 (23-1 %) survived. Air-dried
preparations were made from 11, 2 were lost, and 2 were cultured for 60 h,
reaching the 6- to 8-cell stage. All 11 air-dried preparations contained three
nuclei (male and female pronuclei of the fertilized egg, plus the injected polar
body nucleus): in six all three nuclei were arrested in metaphase, and 20
chromosomes could be counted in each (Fig. 4); in a further two eggs, only
two of the three nuclei were in metaphase, probably the two pronuclei.
FIGURES
1-4
Fig. 1. Donor egg sucked into holding pipette, so as to express second polar body
(arrowed) through slit in zona.
Fig. 2. Second polar body expressed through zona, but still connected to egg by thin
cytoplasmic bridge. Note the 'mid-body' (arrowed).
Fig. 3. Second polar body and attached 'mid-body', now separated from egg. Note
slit in zona (arrowed) through which polar body was expressed.
Fig. 4. Fertilized mouse egg blocked in first cleavage metaphase, after injection of
a second polar body nucleus, (a) Three haploid metaphase plates are seen,
corresponding to the male and female pronucleus and the injected nucleus.
(b-d) The three metaphase plates at higher magnification. 20 chromosomes could
be counted in each.
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J. MODLINSKI AND A. McLAREN
DISCUSSION
Our study has shown that the second polar body can give rise to a metaphase
plate if inserted into the cytoplasm of a fertilized egg, but at present we cannot
detect which of the three metaphase plates comes from which source. This
problem could be overcome, either by the use of chromosome markers such
as T6, or by using as recipient an egg of a different species. The latter strategy
was used by Rudak, Jacobs & Yanagimachi (1978), in their successful attempts
to karyotype human spermatozoa by allowing them to penetrate into zona-free
hamster eggs.
Could the present technique be used in a similar way, to give information
as to whether an egg undergoing in vitro fertilization with a view to subsequent
transfer has a normal karyotype ? Chromosomal assessment of embryos prior
to transfer is feasible in species with large blastocysts, such as rabbits (Gardner
& Edwards, 1968) and cows (Hare et al. 1976), since a fragment of trophectoderm tissue can be removed for karyotyping without damage to the embryo.
Small blastocysts, such as those of mouse and human, contain less than
a hundred cells during the preimplantation period, and removal of any of
these might prejudice survival. If the second polar body of a fertilized egg
intended for subsequent transfer were to be used for karyotypic analysis, it
would need to be extracted without removing the zona pellucida, so as to
facilitate subsequent culture of the donor egg. The success rate, at present
only about 10 %, would also need to be improved.
The polar body can of course provide no information on the normality of
the fertilizing spermatozoon: but to what extent might its karyotype reflect
that of the egg? By far the most common chromosome abnormality arising
during gametogenesis is aneuploidy due to non-disjunction, with autosomal
trisomies and X chromosome monosomy (XO) accounting respectively for about
50 and 15 % of human chromosome anomalies (de Grouchy, 1976). Autosomal
monosomies are only very rarely observed, presumably because they are lost
before the pregnancy is recognized. Aneuploidy is thought to arise more
frequently during oogenesis than spermatogenesis, e.g. more than twice as
frequently for human trisomy 21 (Hansson & Mikkelsen, 1978; Langenbeck,
Hansmann, Hinney & Honig, 1976). All such aneuploidy should be detected
in the second polar body, since if non-disjunction occurred at the first meiotic
division, egg and second polar body would have the same number of chromosomes, while if it occurred at the second meiotic division (only 10-20 % of
trisomy 21 cases) trisomy of the egg would be detected as monosomy of the
second polar body and vice versa.
Triploidy due to failure of second-polar-body formation would also of course
be detected, by absence of the second polar body.
Structural chromosome anomalies transmitted from the mother, less common
than aneuploidy, would present greater difficulties. If the mother is heterozygous
Chromosomes of the mouse second polar body
97
for a reciprocal translocation, for example, it would be difficult to predict the
extent of chromosome imbalance of the egg nucleus., from the karyotype of
the second polar body. The probability that any duplication or deficiency would
be present in both egg and second polar body would depend partly on the
distance of the breakpoint from the centromere, since chromosome regions
not separated from their centromeres by chiasmata would segregate at the
first meiotic division rather than the second. It would also, however, as C. E.
Ford has pointed out to us, depend on the segregation at second meiotic
anaphase of the unequal dyads (composed of one normal and one structurally
rearranged chromatid) that could arise as a result of crossing-over between
displaced chromosome segments (see Ford, 1969). The impossibility in such
an event of inferring the karyotype of the egg nucleus from that of the second
polar body nucleus would apply also to inversions and to insertions. With
Robertsonian translocations, on the other hand, unequal dyads are not
generated, and egg and second polar body will share a common chromosome
constitution.
J. M. is grateful to EMBO for a Fellowship, and to MRC for laboratory facilities.
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(Received 18 February 1980, revised 7 March 1980)