/. Embryol. exp. Morph. Vol. 31, 3, pp. 635-642, 1974
635
Printed in Great Britain
Diploid and haploid mouse
parthenogenetic development following in vitro
activation and embryo transfer
By M. H. KAUFMAN 1 AND R. L. GARDNER 2
From the Physiological Laboratory, Cambridge, U.K.
SUMMARY
Parthenogenetic mouse embryos were selected following //; vitro activation, and transferred
to the oviducts of pseudopregnant recipients. Decidua was evoked by 50-56 % of diploid
parthenogenones compared to 35-1 % of haploid embryos with a single pronucleus, 37-5 % of
immediate cleavage eggs and 77 % of fertilized eggs (controls). On day 4, 587 % of diploid
parthenogenones were morphologically normal morulae or blastocysts; over 90 % of these
' normal' embryos evoked decidua when retransferred to recipients compared to 89 %
of abnormal embryos flushed from the 'transfer' sides, suggesting that only 'normal'
embryos could evoke decidua. Potentially diploid parthenogenones remained diploid on
chromosomal examination on day 4.
INTRODUCTION
This paper describes a technique which permits a high rate of development
to the Jate preimplantation and early postimplantation stages of selected
types of mouse parthenogenones induced by in vitro activation (Graham,
1970, 1971, 1972; Kaufman, 1973a, b; Kaufman & Surani, 1974). The
advantage of the in vitro approach is that it combines a very high rate of
activation (Kaufman, 1973a) with the ability to regulate the proportion of
the various types of parthenogenones induced. Thus by altering two major
variables, the postovulatory age of oocytes at the time of activation (Kaufman,
1973 a), and the osmolarity of the culture medium during the period immediately
after activation (Graham, 1972; Kaufman & Surani, 1974), it is possible to
obtain largely haploid or diploid parthenogenones. Eggs activated in vitro
and incubated throughout the induction period in medium of normal osmolarity
will give rise to mainly haploid parthenogenetic embryos. Approximately 98 %
of the parthenogenones induced by the activation of eggs from females killed
19-20 h after the HCG injection of superovulation possess a single haploid
pronucleus and second polar body (2 P.B.). Activation under similar conditions
of oocytes isolated from females killed approximately 25 h after HCG results
1
Author's address: Zoology Department, Tel-Aviv University, 155 Herzl Street, Tel-Aviv,
Israel.
2
Author's address: Zoology Department, Oxford University, Oxford, 0X1 3PS, U.K.
636
M. H. KAUFMAN AND R. L. GARDNER
in almost 60 % of activated eggs undergoing immediate cleavage. Under both
circumstances between 70 and 80 % of oocytes are usually activated. Very few
diploid parthenogenones are obtained.
Suppression of second polar body extrusion occurs in 56 % of the activated
population when eggs are incubated in low osmolarity medium (3 volumes
medium, 2 volumes distilled water) during the 2 h period directly after
hyaluronidase treatment when they would normally be completing their second
meiotic division. The frequency of activation is unaffected by this treatment.
In a recent series of experiments (Kaufman & Surani, 1974) almost 99% of
eggs which retained their second polar body developed two pronuclei. A
common first cleavage metaphase spindle develops, which gives rise to a
potentially heterozygous diploid embryo (Kaufman, 19736).
Pronuclear development is usually apparent within 6 h of hyaluronidase
treatment, so that selection of embryos for oviduct transfer (Tarkowski, 1959)
can be made at this stage.
MATERIALS AND METHODS
Recipients
Recipients for oviduct and uterine transfer were selected by vaginal inspection
(Champlin, Dorr & Gates, 1973) at about 10 p.m., and those in oestrus were
placed with vasectomized males. At 8 to 10 a.m. the following morning females
were checked for evidence of mating, and oviduct transfers were carried out
either in the morning or on the afternoon of finding the vaginal plug, that is,
on day 1 of pseudopregnancy. The uterine transfers were carried out in the
early afternoon of day 3.
Control series of transfers
Separate control series were carried out by each author when early pronucleate fertilized eggs from (C57B1 x A 2 G)F 1 females mated to Fx males
were transferred to recipients at about midday on day 1 of pseudopregnancy.
Diploid parthenogenetic transfers
Eggs from (C57B1 x A2G)FX females killed approximately 20 h after HCG
were incubated for 2\ h in low osmolarity medium directly after hyaluronidase
treatment (Kaufman & Surani, 1974). Eggs were either activated at 2 a.m.
and transferred to recipients between 9 a.m. and 12 noon (bilateral series), or
activated at 9-9.30 a.m. and transferred between 3 and 6 p.m. on day 1 of
pseudopregnancy (unilateral series). In these two series potentially diploid
parthenogenones with two pronuclei were transferred to recipients within 6-9 h
of activation.
Mouse parthenogenetic development
637
Haploid parthenogenetic transfers
In addition to diploid parthenogenones, haploid eggs with a single pronucleus and second polar body and immediate cleavage eggs were also obtained
following low osmolarity treatment of eggs, as described in the previous section.
These two classes of eggs were transferred separately to recipients within
6-9 h of activation.
Format of experiment
Three series of experiments were performed. In series I, fertilized eggs from
(C57B1 x A a G ^ females mated to F x males were transferred unilaterally or
bilaterally to the oviducts of female mice on day 1 of pseudopregnancy. The
recipients were examined for uterine implantation sites on day 6 or day 7. In
series II, diploid parthenogenones from Fx females were similarly transferred
to the oviducts of another group of recipients. Some of the recipients were
examined for uterine implantation sites on day 6 or day 7, while the uterine
horns of the remainder were flushed at midday on day 4. The embryos recovered
from these females were examined and divided into the following three
categories: morphologically normal blastocysts, morphologically normal
morulae, and abnormal or degenerate eggs. Some of the blastocysts and morulae
were retransferred to the uteri of recipients on day 3 of pseudopregnancy and
the remainder were made into air-dried preparations for chromosome analysis.
Some of the abnormal and degenerate eggs were also retransferred to the
uteri of day 3 recipients, and the uteri examined for implantation sites on
day 6 or day 7. In series III, two classes of haploid parthenogenetic eggs were
transferred to the oviduct on day 1 of pseudopregnancy and the uteri examined
for implantation sites on day 6 or day 7.
RESULTS
Series I. Control transfers
In both the unilateral and bilateral series of transfers 77 % of transferred
eggs had evoked decidua when the uteri of recipients were examined on day 6
or 7 of pseudopregnancy. These results are presented in Table 1.
Series If. Diploid parthenogenones
In the bilateral transfer series 50 %, and in the unilateral series 56 % of
diploid parthenogenones evoked a decidual response when recipients were
killed and their uteri inspected on day 6 or 7 of pseudopregnancy (see Table 1).
In series 'a' of the control and diploid parthenogenetic transfers, 5 or 6 eggs
were usually transferred bilaterally to each oviduct, while in series '/>' a similar
number of eggs were always transferred unilaterally (Table 1). No decidua
were found in the uterine horns on the non-transferred sides in series '/>'
when these were inspected on day 6 or 7.
Diploid
parthenogenones
II.
Unilateral
Bilateral
(a) Bilateral
(b) Unilateral
{a) Bilateral
(b) Unilateral
71
167
55
89
35
67
Total number
of eggs
transferred
56
94
40
89
35
57
Total eggs
transferred
to females
with implants
10
18
11
16
7
12
Total no.
of
recipients
(oviducts)
8
13
9
16
7
10
Recipients
with
implants
(uteri)
21
33
20
50
771
77-2
27
44
transfers
Unilateral
Diploid
parthenogenones
308
transferred
pocy*5
Group
tr\ fpm^iipc?
Total no.
hi 1 JI tprj* 1
288
withL embryos
1t l l l d l t O
Total eggs
transferred
Unilateral
or
\JX
31
recipients
of
Total no.
29
recovered
ViJ 11 KJl J \J&
Recipients
from which
f
100
Morulae
^
11 \J111
I v l 1 IOIWJ
from fptnjilp^
% of embryos
recovered
69
58-7
Blastocysts with embryos
Total no. of embryos
recovered
37-5
351
500
56-2
% of embryos
implanting, in
females with
implants
Total no.
of
decidua
Table 2. Diploid parthenogenetic embryos transferred on day 1 of pseudopregnancy, analysed on day 4
(ii) Immediate
cleavage class5
III. Haploid
parthenogenones
(i) Single
pronucleus
+ 2 P . B . class
Fertilized eggs
(controls)
I.
Group
Unilateral
or
bilateral
Table 1. Transfers to the oviduct of female recipients on day 1 of pseudopregnancy, analysed on day 6 or day 7
D
-
X
as
oo
fn
50
, L. G A R D N
Mouse parthenogenetic development
639
Fig. 1. Group of parthenogenetic blastocysts, phase
contrast. x250.
The number of morulae and blastocysts recovered when the uterine horns
of other recipients were flushed with culture medium at approximately midday
on day 4 are presented in Table 2. Twenty-nine out of 31 uterine horns yielded
morulae and/or blastocysts. In addition to morphologically normal morulae
and blastocysts (Fig. 1), grossly abnormal or degenerate eggs were often
isolated. It was not possible to determine the source of these abnormal eggs in
the 'transfer' sides. Degenerate eggs could either result from transferred
parthenogenones which failed to develop, or eggs ovulated by the recipient.
As activated eggs were all transferred unilaterally in this series (9 or 10 ova/
horn), contralateral horns were also flushed to determine the morphology of
their contents. Without exception only eggs undergoing degeneration were
found to be present. The 29 recipients from which late preimplantation embryos
were recovered yielded 100 morulae and 69 blastocysts out of the 288 activated
eggs transferred at the one-cell stage. If all of the eggs transferred were recovered
}()
E M B 31
Diploid parthenogenones
(i) Morulae
(ii) Blastocysts
(iii) Abnormal and
fragmented eggs
Groups
Unilateral
Unilateral
Unilateral
Unilateral
or
bilateral
transfers
12
30
30
3
6
6
Total no.
of
recipients
Overall value for six recipients, 8-9 %.
18
30
45
Total no.
of eggs
transferred
Total eggs
transferred in
females with
decidua
2
6
4
Recipients
with
decidua
10
29
4
Total no.
of
decidua
83-3
96-7
13-3*
% of embryos
implanting, in
females with
decidua
Table 3. Implantation rate on day 6 or 7 of embryos flushed from recipients on day 4 and retransferred
to the uteri of other recipients on day 3
ffl
z
D
o
>
>
Z
d
O
OS
Mouse parthenogenetic development
641
from the uterine horns of the recipients, this represents a minimum of 58-7 %
normal development. As most uteri were flushed at about midday, the majority
of morulae recovered would probably have reached the blastocyst stage by
the evening of day 4. Some of these parthenogenetic morulae and blastocysts
were retransferred to the uterine horns of recipients on the 3rd day of pseudopregnancy. Recipients were killed on day 6 or 7 of pseudopregnancy and the
number of decidual sites determined. These results are presented in Table 3.
Twenty-nine out of 30 blastocysts evoked a decidual response and all six
recipients had decidua, whereas 10/12 morulae evoked a decidual response
in two recipients, the third having no decidua. Blastocysts generally evoked
a larger decidual reaction than morulae. Histological examination is being
carried out to assess the morphology of these post-implantation conceptuses.
In a parallel series, abnormal and fragmented eggs isolated from 'transfer'
sides on the 4th day of pseudopregnancy were retransferred unilaterally to
the uteri of recipients on the 3rd day of pseudopregnancy (seven or eight ova/
horn). Recipients were killed on day 6 or 7, and the number of decidual sites
determined. These results are presented in Table 3. Two out of six recipients
had no decidua, while the remaining 4 females each had a single decidual site.
These decidua were all very small compared to those obtained when diploid
parthenogenetic blastocysts were retransferred under similar experimental
conditions. It is possible that a few morphologically abnormal morulae may
have been transferred in this series and evoked a response similar to that
previously observed when diploid parthenogenetic morulae were retransferred
to recipients. The four decidual swellings were not at the sites of transfer,
which suggests that they were not traumatic in origin.
Sixty-three of the diploid parthenogenetic morulae and blastocysts flushed
from recipients on day 4 were examined by the air-drying technique (Tarkowski,
1966) and at least one suitably spread metaphase was examined from each
embryo. A total of 75 diploid metaphase spreads were scored. One early morula
with 12 blastomere nuclei had 2 haploid metaphases present. This may have
resulted from the transfer of an egg which underwent 'delayed immediate
cleavage' (Graham, 1971, 1972). This abnormal behaviour might also explain
the low cell number.
Series III. Haploid parthenogenones
A low implantation rate was observed when haploid eggs with a single
pronucleus and second polar body and immediate cleavage eggs were transferred to recipients (see Table 1), possibly because of the greater degree of
asynchrony between embryos and recipients at the time of implantation
(Kaufman, 19736)- A decidual response was evoked on day 6 of pseudopregnancy by 35-1 % of haploid eggs with a single pronucleus and 37-5 % of
immediate cleavage eggs.
40-2
642
M. H. KAUFMAN AND R. L. GARDNER
DISCUSSION
The similarity between the proportion of morphologically normal embryos
recovered from the 'transfer' sides (58-7%, 169/288), and the proportion of
such embryos which were able to evoke a decidual reaction when recipients
were examined on day 6 or 7 of pseudopregnancy (54-3 %, 70/129), suggests
that only 'normal' embryos were responsible for the observed decidual sites.
The high decidual response obtained by retransferring parthenogenetic morulae
and blastocysts to the uteri of recipients (92-9 %, 39/42), compared to the low
response obtained when abnormal embryos and fragmented eggs were transferred (8-9 %, 4/45), would seem to confirm this hypothesis. However, it
remains possible that some of the decidua observed on days 6 and 7 of pseudopregnancy were caused by degenerating eggs of donor origin.
The main advantage of the technique described here over in vivo activation
(Tarkowski, Witkowska & Nowicka, 1970) is that it allows the investigator
to select the type or types of parthenogenones he wishes to examine during
the pre- or early post-implantation period. The advantages and disadvantages
of the various methods of activation have been discussed in a recent review
by Tarkowski (1971). However, the method outlined in this paper allows the
incidence of cytokinetic failure leading to mosaicism in individual classes of
parthenogenones, and their respective rates of cleavage, to be examined in
detail. This should also greatly increase the possibility of developing selected
parthenogenetic cell lines suitable for genetical research.
We wish to thank Professor C. R. Austin and Miss Janet Rossant. The work was
supported by the Medical Research Council and the Ford Foundation.
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(Received 18 September 1973, revised 30 October 1973)