/ . Embryol. exp. Morph. Vol. 38, pp. 211-216, 1977
Printed in Great Britain
211
Studies on the development of Fl embryos from
inter-strain crosses involving DDK mice
ByNOBORU WAKASUGI 1 AND MITSUYO MORITA 2
From the Laboratory of Animal Breeding, Faculty of Agriculture,
Nagoya University
SUMMARY
The development of the early mouse embryos was investigated from 36 to 84 h after the
presumed time of fertilization. The mean number of cells constituting the embryo from the
intra-strain crosses of three strains at 84 h of development was as follows: DDK, 22-7; BS,
43-1 and 1TES, 620. A significant difference was observed in the cleavage rate of the embryo
between DDK and the other two strains.
The Fx embryos from the crosses of BS females x DDK males and ITES females x DDK
males showed almost the same progress of development as BS and ITES embryos, respectively. Therefore, it is concluded that the early development of the Fx embryos is regulated
mainly by the factors from the mother.
The morulae from the semi-sterile cross, DDK females x BS males, showed conspicuously
small cell numbers as compared with their litter-mates that developed to the blastocyst stage.
It is inferred that the discrimination is already made during cleavage between the fortunate
survivors and the lethal embryos due to the incompatibility between the cytoplasmic factor of
DDK eggs and alien spermatozoa.
INTRODUCTION
Certain variations have been recognized in the preimplantation development
of the mouse embryos (Braden, 1958; Dickson, 1967) and investigations have
been performed on the development of ¥x embryos between strains with
different characteristics of embryonic development. Whitten & Dagg (1961)
showed an influence of spermatozoa on cleavage rate with embryos from reciprocal crosses between inbred strains. McLaren & Bowman (1973) found that the
development of F1 embryos is regulated mainly by factors from the mother.
The ¥x embryos between DDK females and males from other strains mostly die
during the process of trophoblast formation, the cause of which is postulated
to be genetically-determined incompatibility between the cytoplasmic factor
of DDK eggs and alien spermatozoa (Wakasugi, Tomita & Kondo, 1967;
Wakasugi, 1973, 1974).
1
Author's address: Laboratory of Animal Breeding, Faculty of Agriculture, Nagoya
University, Nagoya, Japan, 464.
2
Author's address: Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan, 606.
212
N. WAKASUGI AND M. MORITA
As a difference was found in cleavage rate of the embryo between DDK and
other strains, detailed investigation has been performed on increase in cell
number during preimplantation development. The present report is concerned
with the development of F x embryos from the fertile crosses between DDK males
and females from other strains and that of Fx embryos from the semi-sterile cross
between DDK females and alien males during the preimplantation stages.
MATERIALS AND METHODS
The inbred strains of mice used in the present study were DDK, BS and ITES.
The characteristics of DDK have been described by Staats (1972). BS is a substrain of IXBL, of which description has been given elsewhere together with
that of ITES (Kondo, 1975).
Mice aged over 60 days were used for the experiments. A female was caged
with a male and examined for the presence of a vaginal plug each morning. A
female with a plug was transferred to a new cage and 3 a.m. of the day of plug
discovery was counted as hour 0 of embryonic development. Embryos were
examined at the three stages 36,60 and 84 h of development. Embryos were taken
out in phosphate-buffered saline, either from the oviduct or from the uterine
horn according to the stage of development. The oviduct was cut into short
pieces and its contents were squeezed out with surgical needles. The uterine horn
was flushed with a pipette.
At the 8-cell stage or younger, cells were counted under the light microscope.
Beyond the 8-cell stage, the number of nuclei, counted by the method of
Tarkowski (1966), was taken as the number of cells constituting the embryo.
RESULTS
Incidence of abnormal eggs from intra-strain crosses of the three inbred
strains, DDK, BS and ITES, was 23-0% 11-6% and 9-2%, respectively,
throughout the periods examined. Almost the same incidence was observed in
the females used for inter-strain crosses. The abnormal eggs were degenerate for
the most part and they were thought to have been abnormal already at ovulation.
Table 1 shows the number of cells constituting the embryo from intra-strain
crosses of the three strains, reciprocal crosses between DDK and BS and the
cross of ITES females x DDK males at 36, 60 and 84 h of development. At 36 h,
almost all embryos were at the 2-cell stage and some eggs had started the second
cleavage. At 60 and 84 h, differences between strains in the average cell number
were statistically significant. The F± embryos from the cross of DDK females x
BS males showed slightly smaller cell number than DDK embryos both at 60
and 84 h. The difference at 60 h was significant. The F x embryos from the cross
of BS females x DDK males showed similar mean cell numbers as BS embryos on
the whole, although they showed larger mean cell number at 60 h and the difference was significant. The F x embryos from the cross of ITES females x DDK
F± embryos from inter-strain crosses involving DDK mice
213
2-0 |-
ITES/ITES99
1-8
//xDDKo'o'
1-6
1 1-4
.E
l-
2
o 1-0
•y;
«g 0-8
p
= 0-6
s5
o
i-J 0-4
0-2
36
48
60
72
84
Time after fertilization (h)
Fig. .1. Increase in cell number in embryos from intra-strain crosses of DDK, BS
and ITES and reciprocal crosses between DDK and BS and the cross of ITES females
x DDK males.
males showed similar mean cell numbers as ITES embryos throughout the period
examined.
Figure 1 shows, on a logarithmic scale, the increase in cell number in the
embryo from the six types of crosses with regression lines based on the least
square method. Statistically significant differences in cleavage rate were seen
between DDK and the other two strains. The F x embryos from the cross of DDK
females x BS males show slightly slower increase than DDK embryos, though
the difference is not significant. The F x embryos from the cross of BS females x
DDK males show almost the same increase as BS embryos and those from the
cross of ITES females x DDK males show the same cleavage rate as ITES
embryos, significantly greater than that of DDK embryos.
The embryos at 84 h were classified into five stages; 6-8 cell, morula, early
blastocyst, late blastocyst and zona-free. Early and late blastocysts were taken to
be those in which the volume of the blastocoele was respectively less than or
214
N. WAKASUGI AND M. MORITA
Table 1. Cell number in embryos from intra-s train crosses of DDK, BS and ITES
and reciprocal crosses between DDK and BS and the cross of ITES females x DDK
males at 36, 60 and 84 h of development
No. of cells constituting the embryo at
Types of crosses
Female
Male
36 h
2-0 ±0-0* (48)
20±00(ll)
2-1 ± 0 1 (72)
2 0 ± 0 0 (41)
2-3 ±0-2 (20)
2-1 ±0-1 (42)
DDKxDDK
DDK x BS
BS x DDK
BS xBS
ITES x DDK
ITES x ITES
60 h
84 h
61 ±0-3 (44)
4-2 ±0-3 (19)
9-8 ±0-5 (46)
7-4 ± 0-2 (40)
12-6±10(17)
14-4±l-2 (39)
22-7 ±1-2 (42)
19-9 ±2-6 (22)
41-7 ±2-5 (43)
431 ±2-8 (31)
600±l-9(21)
620±l-4(42)
* Mean ± standard error.
The number in parenthesis indicates the number of embryos examined.
Table 2. Classification of embryos from intra-strain crosses of DDK, BS and ITES
and reciprocal crosses between DDK and BS and the cross of ITES females x DDK
males at 84 h of development according to embryonic stage and their cell number
and mitotic index
Total
Types of
no c\\
llsj* Ul
A
No. of embryos at the stage of
(
n
mbryos
Female male examined
DDK x DDK
42
DDKxBS
22
BS
x DDK
43
BS
xBS
31
ITES x DDK
21
ITES x ITES
42
6-8 Cell
Morula
3
29
(7-0 ±0-6)* (22-5 + 1-1)
(7-4 %)t
—
18
(15-3 + 1-7)
(36%)
—
26
(32-1+2-5)
(3-2%)
17
—
(33-8 ±2-6)
(4-7%)
8
—
(600 ±2-7)
(1-3%)
—
8
(58-9 ±3-4)
(2-5 %)
Early
blastocyst
Late
blastocyst
Zona-free
5
(23-3 + 2-0)
(7-1 %)
4
(40-3 + 2-1)
(3-7%)
2
(50-5 ±2-5)
(119%)
4
(49-8 + 6-2)
(5-5%)
3
(51-3 ±7-2)
(3-3 %)
3
(57-7 + 1-4)
(4-1 %)
5
(330 + 2-4)
(30%)
—
—
15
(57-2+1-9)
(1-1 %)
9
(560 ±1-9)
(2-2%)
10
(62-5 ±2-1)
(2-6%)
20
(63-0 ±1-6)
(3-9%)
* Mean cell number ± standard error.
t Mitotic index.
—
1
(58)
d-7%)
—
11
(63-6 + 3-5)
(6-3 %)
Fx embryos from inter-strain crosses involving DDK mice
215
more than half that of the embryo. A frequency distribution of the embryos according to this classification is presented in Table 2, together with the average cell
number and the mitotic index. Three DDK embryos showing the slowest development were at 6-, 7- and 8-cell stages, respectively. Although it was difficult to
tell whether these embryos were living or dead, they were regarded as normal,
since some morulae consisting of 10-15 cells were observed among DDK embryos. There was no constant cell number at which the embryo develops to the
blastocyst stage, for the mean cell number of DDK early blastocysts was 23-3
and of ITES morulae, 58-9.
There were no late blastocysts among the F± embryos from the cross of
DDK females x BS males, and a striking difference in cell number was observed
between morulae and early blastocysts. In addition, the Fx morulae showed a
low mitotic index, whereas DDK embryos at the morula and early blastocyst
stages showed higher mitotic indices although their cell numbers were small.
Frequency distribution of the F x embryos from the cross of BS females x DDK
males and their mean cell numbers and mitotic indices were almost the same as
those shown by BS embryos. The same was observed between the F x embryos
from the cross of ITES females x DDK males and ITES embryos except that
there were no zona-free embryos among the F± embryos.
DISCUSSION
McLaren & Bowman (1973) found that the difference in progress of embryonic
development between C57BL/McL and C3H/BiMcL strains was due to the
time of the first cleavage. Finding a difference in the time of pronucleus formation, they suggested that the difference is due to the time interval between copulation and fertilization of eggs. Krzanowska (1972), with in vitro experiments,
has shown a difference in the time of dispersion of cumulus cells and removal of
zona pellucida among inbred strains of mice. Braden (1958) has shown that the
time of dissolution of the cumulus and the time of the first cleavage is related to
the genotype of the female. Nicol & McLaren (1974) have reported that the
time interval that the sperm requires to reach the site of fertilization is regulated
by the genotype of the female. On the other hand, Whitten & Dagg (1961)
showed the difference in cleavage rate of the embryo between BALB/c and 129
strains. The present study has established a difference in cleavage rate of the
embryo between DDK and the other strains. Thus, two factors may affect the
development of early mouse embryos, time at which the first cleavage occurs and
cleavage rate.
The F x embryos from the crosses of BS females x DDK males and ITES
females x DDK males showed similar progress of development to BS and
ITES embryos respectively. These observations are accordant with the results
reported by McLaren & Bowman (1973). However, the facts that the F x embryos
from the cross of BS females x DDK males showed larger cell number than BS
216
N. WAKASUGI AND M. MORITA
embryos at 60 h of development and no zona-free embryos were observed
among those from the cross of 1TES females x DDK males suggest that factors
from the male may have some effects on development of F x embryos. This view
is consistent with the observation on development of the F1 embryos between
BALB/c and 129 strains (Whitten & Dagg, 1961).
The Fj embryos from the semi-sterile cross, DDK females x BS males, showed
slightly slower progress of development than DDK embryos, although the difference was not significant as a whole. However, the morulae from this cross
showed a conspicuously small mean cell number and a low mitotic index as
compared with DDK morulae, whereas their litter-mates that developed to the
blastocyst stage showed a larger mean cell number than DDK blastocysts. These
facts may be taken as the indication that the discrimination between lethal
embryos and fortunate survivors has already been made at this stage of development. It is considered that the abnormality or delay of development due to
the incompatibility between the cytoplasmic factors of DDK eggs and alien
spermatozoa begins to appear during cleavage.
The authors wish to thank Professor K. Kondo for his encouragement and criticism and
Dr A. McLaren for reading the original manuscript. They are also grateful to Mrs Hayakawa
for taking care of animals. This work was supported by a grant-in-aid (No. 012204) for the
scientific research from the Ministry of Education, Science and Culture of Japan.
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BRADEN,
{Received 27 July 1976, revised 30 October 1976)