/. Embryol. exp. Morpli. Vol. 41, pp. 233-243, 1977
Printed in Great Britain © Company of Biologists Limited 1977
233
Development of interspecific hybrids of Mus
By JOHN D. WEST,1 WILLIAM I. FRELS, 1
VIRGINIA E. PAPAIOANNOU, 2 JAMES P. KARR 3
AND VERNE M. CHAPMAN 1
From the Department of Molecular Biology,
Roswell Park Memorial Institute, Buffalo
SUMMARY
Artificial insemination has been used to produce interspecific mouse hybrids. Mus musculus x Mus cervicolor cervicolor hybrids failed to complete more than a few cleavage divisions
but both M. mmculus x M. dunni and M. musculus x M. caroli hybrids completed preimplantation development. These hybrid embryos are heterozygous for various X-linked enzymes and
may provide a useful genetic system for studying X-chromosome inactivation during early
development. Further development of M. musculusx M. caroli hybrids was studied: several
completed foetal development; a few survived to maturity but none has yet reproduced.
INTRODUCTION
The genetics of the mouse is the best documented of any laboratory mammal.
Numerous mutations have been catalogued and are maintained in laboratory
strains and stocks. In addition, considerable genetic variation for cell surface
antigens, enzymes and other proteins is present among existing inbred strains.
Despite this extensive array of genetic variants and mutations, surveys of wild
mice have established that laboratory stocks contain only a portion of the
total genetic variation present in the species gene pool (Hunt & Selander, 1973;
Chapman, 1973). It is particularly unfortunate that there are few variants of
X-linked enzyme loci (Lyon, 1972) which would be suitable to study X-chromosome inactivation in early mouse embryos.
Sampling of wild or feral mice and especially distantly related, but still interfertile subspecies of Mus musculus has provided a means of increasing the
number of polymorphisms in laboratory mice. Prominent examples include the
subspecies M. musculus castaneus from Thailand (Chapman, 1973), M. musculus
molossinus from Japan (Dev et al., 1975) and M. musculus musculus from
Denmark (Chapman, 1975). The polymorphisms found among feral-derived
1
Author's address: Department of Molecular Biology, Roswell Park Memorial Institute,
666 Elm Street, Buffalo, New York 14263, U.S.A.
2
Author's address: Department of Zoology, University of Oxford, South Parks Road,
Oxford, 0X1 3PS, England.
3
Author's address: Department of Medicine, Roswell Park Memorial Institute, 666
Elm Street, Buffalo, New York 14263, U.S.A.
234
J. D. WEST AND OTHERS
mice include the X-linked variants of a-galactosidase from M. m. molossinus
(Lusis & West, 1976) and phosphoglycerate kinase (PGK) in M. musculus
musculus (Nielsen & Chapman, 1977).
In recent years samples of Mus species have been trapped in Southeast Asia
by Dr J. T. Marshall and have been made available to many laboratories. We
have established colonies of the species M. caroli, M. dunni and M. cervicolor
cervicolor in our laboratory. These species have the same chromosome number
as M. musculus but differ from M. musculus in a number of biochemical characters, and are reproductively isolated from each other and from M. musculus.
Two of these species, M. caroli and M. dunni, are particularly interesting because
they differ from M. musculus in the electrophoretic mobility of several X-linked
enzymes. In addition, the X chromosome of M. dunni is metacentric and is
readily distinguishable from any other chromosome of either M. dunni or M.
musculus (Makvong, Marshall, Pathak & Hsu, 1975).
We have used artificial insemination to achieve interspecific hybridization in
an attempt to incorporate the genetic variation that exists between Mus species
into the laboratory mouse gene pool. Previously, M. musculus x M. caroli
hybrids have occasionally developed to mid-gestation (Chapman, 1971, unpublished; Chapman & Karr, 1974, unpublished); these foetuses have been
used in studies of satellite DNA (Sutton & McCallum, 1972) and for establishing the X-chromosome linkage of glucose-6-phosphate dehydrogenase (G6PD),
hypoxanthine-phosphoribosyl transferase (HPRT) and phosphoglycerate kinase
(PGK) (Chapman & Shows, 1976).
In this paper we report the production of adult M. musculus x M. caroli
hybrids and the embryonic development achieved by M. musculus x M. dunni
and M. musculus x M. c. cervicolor hybrids.
MATERIALS AND METHODS
(a) Mice
Randomly bred HA(ICR) Swiss mice, from the Roswell Park Production
Colony at West Seneca, New York, represented the species M. musculus. This
strain was developed by selecting for fecundity (Hauschka & Mirand, 1973;
McWhirter & McWhirter, 1976). The stocks of M. caroli, M. c. cervicolor and
M. dunni are maintained in our laboratory and are descended from mice kindly
supplied by Dr J. T. Marshall, in Bangkok. M. caroli and M. c. cervicolor are
both Thailand species and M. dunni is an Indian species. Some comparisons
between the four species are shown in Fig. 1 and Table 1. More extensive
characterization has been reported by Marshall (1972a, b) and Rice (1974).
Interspecific mouse hybrids
235
Table 1. Comparison of four Mus species
Mean weight at 42 days (g)
A
Species
M. musculus
strain HA(ICR)
M. caroli
M. c. cervicolor
M. dunni
vJColdUUIL
Period
(days)*
females
mean±s.E.|
males
mean±s.E.t
40
19-21
22-5 ±0-4
280 ±0-5
40t
40J
17-18
17-18
—
10-6 ±0-3
10-9 ±0-3
5-5 ±0-2
12-6 + 0-3
13-4±0-3
6-6 ±0-2
Chromosome
number (2n)
40t
* The gestation period was determined by timed matings for M. caroli and M. c. cervicolor
but was not determined for M. dunni.
f 20 mice were weighed in each group.
% Marshall 1972o, b; Makvong et ai, 1975.
(b) Artificial Insemination
The method of insemination was similar to those described by Leckie, Watson and Chaykin (1973) and by Maddock & Dawson (1974). Female HA(ICR)
mice were induced to ovulate with intraperitoneal injections of 4 i.u. pregnant
mares serum gonadotrophin (Sigma), followed 48 h later by 4 i.u. human chorionic gonadotrophin (Ayerst). Ovulation occurred 12-13 h after the second
injection and the mice were inseminated 12-14 h after this injection. Two
injection timetables were used, resulting in insemination at either 8 a.m. or
at 11 p.m. For studies of post-implantation stages, 5 mg medroxyprogesterone
acetate (Depoprovera, Upjohn) was injected intraperitoneally 4 days after insemination.
Sperm were allowed to swim clear of finely chopped cauda epididymides in a
small volume of pH 7-2 phosphate-buffered saline (PBS) containing 0-3 %
bovine serum albumin. (For HA(ICR) males approximately 0-3 ml PBS was
used per male and for M. caroli and M. dunni the volume of PBS was reduced
to about 0-2 ml and 0-1 ml per male respectively.) A small sample of sperm suspension was diluted 50-fold in 10 % formol saline and the sperm concentration
was determined using a haemocytometer counting chamber. The number of
sperm inseminated usually ranged from 2 x 106 to 5 x 106 per mouse although in
early experiments a minimum of 106 was tolerated. The sperm suspension was
drawn into a 1 ml disposable tuberculin syringe fitted with a 4 cm 22-gauge
blunted needle, bent 2 cm from its tip to an angle of about 110°.
Each female was prepared for insemination by inserting a vibrating brass
rod into the vagina for about 20 seconds. The mouse was then etherised and
taped by the base of the tail to the stage of a Wild M5 dissecting microscope.
The microscope head was positioned to view the cervix while the vagina was
dilated with curved forceps. The cervix was then penetrated with the blunted
needle. After insemination with 0-05 ml sperm suspension the vagina was
236
J. D. WEST AND OTHERS
Fig. 1. Adult female mice of the four species used in the experiments. From left to
right: Mus dunni, Mus cervicolor cervicolor, Mus caroli, Mus musculus (random
bred albino strain HA(ICR)).
plugged by one or two cotton wool balls soaked in isotonic saline. The vibrating
brass rod and cotton wool plugs serve to stretch the vagina. This stimulation
induces the formation of functional corpora lutea necessary for the maintenance of pregnancy (McGill, 1970). Using this method of artificial insemination,
two people can inseminate forty female mice in about 90 min (including preparation time), with the help of an anaesthetist.
Preimplantation embryos were recovered from the fallopian tubes or uteri
by flushing either with PBS or with PBI medium plus 10 % foetal calf serum
(Whittingham & Wales 1969). Embryos were cultured in Whitten's medium
(Whitten, 1971) under paraffin oil at 37 °C in a gas phase of 5 % O2, 5 % CO 2
and 90 % N 2 . Blastocysts were surgically transferred to pseudopregnant
HA(ICR) females.
RESULTS AND DISCUSSION
Our artificial insemination technique normally results in fertilization in both
fallopian tubes. Two-cell embryos were recovered from 17 of 20 HA(ICR)
females 30-34 h after inseminating with HA(ICR) sperm (Table 2, Experiments
1 and 3). Thirteen of these females had two-cell embryos in both fallopian tubes,
Interspecific mouse hybrids
237
Fig. 2. Preimplantation embryos recovered between 84 and 86 hours after artificial
insemination. (A) M. musculus x M. musculus embryos, (B) M. musculus x M. caroli
hybrid embryos. Both groups include normal blastocysts but none of the hybrid
blastocysts has yet completely expanded.
three had embryos only in the right tube and one had embryos only in the left
tube.
Table 2 shows the preimplantation development of the three types of interspecific hybrids and typical preimplantation embryos are illustrated in Fig. 2.
The sham insemination with PBS instead of sperm suspension indicates that
gynogenesis did not occur. M. musculus x M. c. cervicolor hybrids rarely underwent even the first cleavage division and none completed preimplantation
development.
A significant percentage of M. musculus x M. caroli hybrids developed to the
blastocyst stage, though perhaps more slowly than the M. musculus x M.
musculus control embryos. At 78-82 h after insemination (Table 2, Experiments
1 and 2), about one third of the control embryos but very few of the M. musculus x
M. caroli hybrid embryos had reached the blastocyst stage. By 96-100 h most
of the cleaving hybrid embryos were blastocysts. The efficiency of control inseminations in Experiments 1 and 2 was high and for the first 82 h of pregnancy
89 % (62/70) of the females inseminated with M. musculus sperm contained
cleaving embryos. At 96-100 h the percentage of control females from which
embryos could be recovered by flushing the uteri dropped to 40% (6/15),
probably because implantation had already begun. The percentage of females
with hybrid embryos at 96-100 h (53 %; 8/15) was not much lower than the
16
EMB 41
3
2
1
sperm type
30-34 (sham)
M. musculus
M. caroli
M.c. cervicolor
54-58 M. musculus
M. caroli
M.c. cervicolor
78-82 M. musculus
M. caroli
M.c. cervicolor
78-82 M. musculus
M. caroli
96-103 M. musculus
M. caroli
30-34 M. musculus
M. dunni
54-58 M. musculus
M. dunni
78-32 M. musculus
M. dunni
after
Expt. inseminno.
ation)
Age
(hr
15
15
15
15
15
15
15
25
25
25
15
15
15
15
5
5
5
5
5
5
No. of
females
inseminated
0
12
11
4
15
10
3
21
12
5
14
8
6
8
5
5
4
5
5
4
335
277
239
271
198
138
94
439
253
255
297
193
119
155
197
109
79
61
190
144
42
23
36
36
17
39
65
14
68
78
18
42
43
61
17
46
8
44
11
39
No. of Abnormal
or
ova and
embryos degenerate
58
21
41
62
12
20
31
6
17
19
4
14
8
4
23
22
3
25
0
15
1-cell
0
56
23
1
2
19
1*
2
10
1
1
4
0
1
59
32
0
3
1*
1*
2-cell
* Only one embryo recovered at this stage.
No. of
females
pregnant
< 1*
4
1
3
0
0
80
20
3
3
4
0
3
0
0
1*
8
0
0
0
0
0
0
49
4
1*
4
2
0
5-8
< 1*
18
17
2
1
4
1
< 1*
0
0
0
3-4
0
0
0
0
4
1
0
36
1
0
48
31
10
9
0
0
8
0
48
30
Morula
% of embryos and ova at each developmental stage
0
38
13
0
0
0
0
28
1
39
19
0
0
38
0
0
0
0
0
0
Blastocyst
Table 2. Preimplantation development of embryos following artificial insemination of Mus musculus HA{ICR) females
O
H
on
H
w
to
oo
239
Interspecific mouse hybrids
Table 3. Postimplantation development following artificial
insemination o/Mus musculus HA{ICK) females
Age (days
No. of
•fV*mci 1*^c
JCIIldlCo
clllCl
insemination)
8
14
19
19i
20i
Sperm type
M. musculus
M. caroli
M. musculus
M. caroli
M. musculus
M. caroli
M. musculus
M. caroli
M. musculus
M. caroli
M. dunni
No. of
vr
f
rNU.n nUl
No. of implantations
f p m a 1f*c
1CI llctlCo
insemi- females with live
nated pregnant foetuses Mole
10
10
30
30
60
62
60
60
31
286
28
5
2
7
3
20
10
26
21
15
89
5
—
7
2
20
1
25
4
13
8
0
—
20
8
8
36
41
62
1
339
35
>
•
Dead
foetus
Live
foetus
Total
—
0
3
5
17
23
14
28
92
2
—
79
4
224
1
280
4
235
13
0
100
12
99
15
237
54
344
80
264
444
37
overall pregnancy rate of 59 % (41/70) of these mice at earlier times, suggesting
that implantation of M. musculus x M. caroli hybrid embryos occurs later than
M. musculus x M. musculus control embryos. Some M. musculus x M. dunni
hybrids also developed to the blastocyst stage (Table 2, Experiment 3), in the
same time (78-82 h) as the M. musculus x M. musculus controls.
Table 3 shows the postimplantation development of M. musculus x M. musculus controls and M. musculus x M. caroli and M. musculus x M. dunni hybrids.
The overall percentage of control females with postimplantation embryos is
rather low (38 %; 73/191) and lower still for those bearing M. musculus xM.
caroli hybrids (28 %; 125/448). The postimplantation mortality of the M. musculus x M. caroli hybrids is extremely high (97 % by 20^ days after insemination).
Most of these deaths were detected as 'moles', indicating that development
failed before 11-12 days after insemination (Edwards & Fowler, 1959). Thus
most of the hybrid embryos died before implantation and most of those that
did implant died soon afterwards. However, eighteen M. musculus x M. caroli
hybrids were delivered live by Caesarian section at 19, 19^ or 20^ days post
insemination. The hybrids were usually small and few survived after fostering
to nursing mothers. Figure 3 illustrates three of these hybrid foetuses delivered
at 19^ days. One survived 21 h but received no milk from its foster mother and
another initially received some milk but was later neglected by its foster mother
and survived for 49 h.
Four hybrids (all females) delivered at 20^ days have survived to maturity
but have not yet reproduced. The surviving hybrids were all fostered to M. caroli
mothers and superficially resemble this species in gross morphology (Fig. 4).
The body weights of the four female hybrids at six weeks were respectively 1 l-0g,
13-2 g, 15-3 g and 16-3 g, which are all between the mean of 10-6 g for 6-week-old
240
J. D. WEST AND OTHERS
Fig. 3. Newborn mice. Top row: three M. musculus x M. caroli hybrids delivered by
Caesarian section at 19£ days after insemination. These hybrids were fostered and
survived 2-5 h, 21 h, and 49 h, respectively (from left to right). Bottom row:
left, M. musculusxM. musculus control delivered 19^ days after insemination;
centre, newborn M. musculus from natural mating; right, newborn M. caroli
from natural mating.
M. caroli females and the mean of 22-5 g for M. musculus females (Table 1). The
activity of both M. musculus and M. caroli genes in the hybrids was detected in
haemolysates using starch gel electrophoresis of the X-linked enzyme PGK
(Beutler, 1969; Chapman & Shows, 1976). The heterozygous expression of an
X-linked gene shows that the hybrids were chromosomally as well as phenotypically female. Postimplantation development of M. musculus x M . dunni
hybrids has yet to be investigated thoroughly, but preliminary results show that
implantation of these hybrids sometimes occurred (Table 3).
The development achieved by the three groups of interspecific hybrids together with the control group is summarized in Table 4. Despite the low success
rate, the adult M. musculus x M. caroli hybrids which have been produced show
that the M. musculus and M. caroli genotypes can be successfully combined to
form a viable mouse. It is tempting to conclude that the failure of M. musculus x
M. c. cervicolor hybrids to develop reflects a greater evolutionary distance
between M. musculus and M. c. cervicolor than between M. musculus and
241
Interspecific mouse hybrids
Fig. 4. M. caroli female (pregnant) with two M. caroli offspring (19 days after birth)
and a M. musculus x M. caroli hybrid (19 days after delivery). The young hybrid
is the third from the left and is already larger than its two adopted 'sibs'. At
six weeks this hybrid weighed 153 g.
Table 4. Summary of development achieved by four groups of embryos,
expressed as number of embryos per female inseminated
Stage of development
Preimplantation
(3 or more cleavages
Total
at 78-100 h after AI)* postimplantation
M. musculus x
M. musculus
M. musculus x
M. caroli
M. musculus x
M. dunni
M. musculus x
M. c. cervicolor
Live at 19-20^
days after AI
13-23f
5-46
4-89
4-47
1 35
004
13-20
1-32
0
—
—
0
* AI = artificial insemination.
t Probably understimated as some embryos will have implanted at this time.
242
J. D. WEST AND OTHERS
either M. caroli or M. dunni. Although this conclusion is compatible with the
evolutionary tree derived for M. musculus, M. caroli and M. c. cervicolor by
Rice (1974), on the basis of single copy DNA's, it is not clear that embryonic
development of interspecific hybrids is a reliable guide to the taxonomic relationships between the species (see Maddock & Dawson, 1974 for discussion). We
do not yet know why so many of the hybrids die.
In an unsuccessful attempt to increase the yield of M. musculus x M. caroli
hybrids at term, preimplantation hybrid embryos were recovered from 98
females on the fourth day after artificial insemination. The abnormal or retarded embryos were discarded and the remaining normal-appearing blastocysts
and morulae were either transferred in groups of 5 to 12 to females in their
third day of pseudo-pregnancy or were cultured overnight and then transferred
to females in their third day of pseudopregnancy. This procedure was designed
both to allow more time for the more slowly developing hybrid embryos to
complete preimplantation development and also to increase the number of
normal hybrid embryos in each female. The females were killed and dissected
at mid-gestation. The rate of implantation was fairly high (26 implantation
sites, from 54 embryos transferred, in six females that became pregnant); however, these were mostly moles or empty decidual swellings. Only three normal
foetuses were recovered.
The successful development of significant numbers of both M. musculus x M.
caroli and M. musculus x M. dunni hybrids at least to the blastocyst stage provides a useful system for some genetic studies of early development. Chapman
& Shows (1976) have already used cultured cells from a female M. musculus x M.
caroli foetus to demonstrate the location of genes for three enzymes (G6PD,
HPRT and PGK) on the X-chromosome in both M. caroli and M. musculus.
The availability of female hybrid embryos, heterozygous for these three Xlinked enzymes, provides a useful system for studying X-chromosome inactivation in early mammalian embryos.
If the M. musculus x M. caroli hybrids are interfertile with M. musculus,
M. caroli genes could be transferred to the genomes of laboratory mice, thereby
greatly enlarging the available genetic variation for both X-linked and autosomal loci. The fertility of these hybrids is now being tested.
We wish to thank Dr J. T. Marshall for providing our foundation stocks of Mus caroli, M.
c. cervicolor and M. dunni, and Mary Caye Deisler for help in maintaining these stocks. We
are grateful to the National Institutes of Health for financial support. J. W. was a recipient of
a Lalor Foundation Fellowship and V.P. was a recipient of a Cancer Research Campaign
(London) Grant and a Yamagiwa-Yoshida Memorial International Cancer Study Grant.
Interspecific mouse hybrids
243
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