EMBRYONIC MORTALITY IN RELATION TO
OVULATION RATE IN THE HOUSE MOUSE
BY J. C. BOWMAN
Institute of Animal Genetics, Edinburgh
AND R. C. ROBERTS
A.R.C. Animal Breeding Research Organization, Edinburgh 9
(Received 21 September 1957)
INTRODUCTION
An inverse relationship between the numbers of foetuses in the two horns of the
uterus in the house mouse during the last week of gestation has been reported
several times (Hollander & Strong, 1950; Runner, 1951). We recently found a
significant negative correlation between the two ovaries in respect of the number of
corpora lutea present at 18 days of gestation. In respect of implantation sites and
live embryos at 18 days, however, the correlations were much less strongly negative
and were non-significant. This reduction in the strength of the correlation points
to a diflFerential loss of eggs or of embryos, the horn receiving the greater number of
eggs suffering a proportionately greater loss. We have accordingly re-investigated
these correlations and looked for direct evidence of a differential loss. We have found
an increasing proportional loss of eggs related to the number shed within a horn,
and have shown that it occurs after fertilization but before implantation. The results
are reported below.
RESULTS
Two series of observations were made. In the first, pregnant female mice of
heterogeneous origin were dissected at 18 days of gestation. The numbers of
corpora lutea were counted as a measure of the number of eggs shed from each
ovary. The numbers of implantation sites and of live embryos in each uterine horn
were also counted. The correlation between sides within mice for corpora lutea was
— 0-436 (P<o-ooi). In agreement with our previous findings, the numbers of
implantation sites and live embryos showed lower negative correlations between
sides than the number of eggs shed. The correlation between sides for implantation
sites was —0-300 (P<o-O2) and for live embryos — O-III (N.S.).
Further analysis revealed that the loss of eggs is affected by the number of eggs
shed into the horn. Horns with a larger number of eggs suffer a proportionately
greater loss. Thus, mice with an extreme distribution of eggs between sides will
contribute largely to the negative covariance in corpora lutea counts, but as the two
horns are affected by differential loss the negative correlation is greatly reduced by
the time of implantation.
The distribution of loss of eggs and implanted embryos within uterine horns and
Table i. Distribution of amount and time of loss of eggs
Corpora
lutea
per
ovary
No.
of
ovaries
examined
Total
corpora
lutea
Fraction of
corpora lutea
not accounted
for by live
embryos at
18 days of
gestation
Loss of implants up to
18 days of gestation
Fraction of
corpora lutea
not accounted
for by implants As a fraction
of eggs shed
As a fraction
of implants
A. Within uterine horns
i
2
o
6
3
4
5
6
7
8
9
IO
10
Total
0
12
30
M
96
26
28
130
20
140
9
7
72
2
132
168
0
0-083
0-066
o-i35
0-085
0-137
0
0
0-066
0-063
0-092
0-083
0-029
0-042
O-222
0-071
0-072
O-IOI
20
0-171
0-278
0-254
0-150
O
73i
O-I55
OO75
63
0
0
0
0-083
O-I33
0-198
0-177
O-22O
0-200
O-3I9
0-097
0-034
0-056
0-298
0-476
0-I5O
0-23O
0
0-089
B. Within mice
Corpora
lutea
No.
of
per
mice
examined
mouse
8
9
6
48
12
108
6
II
15
60
165
12
11
10
132
130
3
42
0-125
0-074
0-133
0-158
0-106
0-169
0-167
16
2
1
3°
0-633
0-188
0-117
0-060
0-053
0-062
0-119
0-133
0-062
Total
66
73'
O-I55
0-075
IO
13
14
15
16
O-IO4
O-229
0-148
O-250
0-218
0-159
O-23I
0-286
0-767
0-250
O-23O
0-119
0080
0-135
0-072
0-059
0-074
O-I43
0-074
0-364
0-077
0-089
Table 2. x* analysis of loss of eggs
D.F.
A. Within uterine horns
(i) Eggs lost up to implantation
1
Linear trend
13-166
9-028
Deviations from linear trend
7
123
193-885
Heterogeneity of loss within
corpora lutea groups
(2) Eggs not accounted for by live embryos at 18 days
Linear trend
1
14-937
17-760
Deviations from linear trend
7
123
199430
Heterogeneity of loss within
corpora lutea groups
B. Within mice
(1) Eggs lost up to implantation
1
Linear trend
18-938
Deviations from linear trend
51 565
7
(2) Eggs not accounted for by live embryos at 18 days
1
Linear trend
13-501
Deviations from linear trend
7
51-485
M.S.
ft.
Source of variation
F
i3-i66>5:;:^
I •2OO--^\'n
,
2
i # 576-—— > °' 354
i4-937'^-p >5 .g88
1-621
18-938
7-366
^55
-—
*
,.„.
2 S71
***
0 oz
O'OI
0*05
O'OI
N.S.
N.S.
N.S.
i4°
J- C. BOWMAN AND R. C. ROBERTS
within mice is shown in Table i. The x2 analyses for linear trend of proportion lost
at two stages of gestation are given in Table 2. Only the linear trends of loss within
horns up to implantation and for total loss up to 18 days of gestation are significant.
The loss within horns between implantation and 18 days of gestation is very
irregular and does not seem to be related either to the number of eggs shed into the
horn or to the number of eggs implanted. We conclude therefore that before
implantation there is positive linear trend of loss of eggs with increasing numbers
shed per horn. This conclusion leads us to expect to find a similar increasing proportional loss when the data are analysed on a within-mouse basis. Our data does
suggest such a trend, though statistical analysis reveals this to be non-significant.
Table 3. Fraction of eggs not fertilized in relation to number shed per ovary
Eggs shed
per ovary
1
2
3
4
5
6
7
8
9
No. of
Fallopian
tubes
examined
Total no.
of eggs
2
2
7
7
14
21
12
11
48
75
66
13
91
8
64
9
IS
1
10
11
12
5
—
Total
82
1
5°
—
12
452
Fraction not
fertilized
0-500
0-429
0-048
0-125
0-173
0-030
0-132
0-125
o-ooo
o-oSo
—
0 250
0-126
The differential loss between horns up to implantation may be due either to a
lack of fertilization or to a failure to implant, and a second series of observations
was made to solve this problem. Females of heterogeneous origin, similar to those
used in the first series of observations, were put singly with a male between 5.0 and
5.30 p.m. and examined the following day between 9.0 and 10.0 a.m. for vaginal
plugs. Those which had mated were killed between 7.0 and 10.0 p.m. and the
eggs in each Fallopian tube were extracted. From the findings of Snell, Fekete,
Hummel & Law (1940), Snell, Hummel & Abelmann (1944) and Braden & Austin
(1954) we considered that the majority of fertilized eggs would at that time be in
the pronucleate stage. The eggs were examined by phase-contrast microscope
according to the method described by Austin & Smiles (1948). The numbers of
fertilized and non-fertilized eggs in each tube were counted. Judgement as to
whether eggs were fertilized or not was based on the description by Austin (1951)
of the formation of the pronuclei in the rat egg.
The correlation between ovaries in the numbers of eggs shed in a mouse was found
to be —0-528 (P o-ooi), which is in good agreement with the similar correlation
for corpora lutea counts. The number of eggs not fertilized per tube in relation
to the total eggs shed per ovary are shown in Table 3. In marked contrast to the
Embryonic mortality in relation to ovulation rate in the house mouse 141
implantation data, no regular trend of loss is apparent in this case. The data has
also been analysed on a within-mouse basis and as expected no trend is shown. These
results indicate that fertilization rate is not related to the number of eggs shed per
ovary or per mouse and that it does not normally limit litter size in the mouse.
DISCUSSION
Our observations indicate that the loss of implanted eggs up to 18 days of gestation
does not vary with the number of implantations in a horn, but that as the number
of -eggs shed into a uterine horn increases the probability of each individual egg
implanting decreases. The fertilization rate is not related to the number of eggs in
the horn, and therefore the factor or factors causing the variation in implantation
rate must be operating on fertilized eggs or on pre-implantation embryos.
Our results and conclusions do not entirely agree with those of other workers.
Danforth & de Aberle (1928) and McLaren & Michie (1956) found no correlation
between the two horns for the number of implantations. As mentioned earlier other
authors have reported significant negative correlations, and therefore we can only
attribute the inconsistency of the results reported to heterogeneity between mice
used at different laboratories.
The differential loss of eggs in our data could be explained if trans-uterine
migration of eggs had occurred in many of our mice. Such migration is known to
occur in rodents (Runner, 1951; Boyd & Hamilton, 1952; Young, 1953; McLaren
& Michie, 1954), but these reports suggest that its frequency is very low. For this
reason we have dismissed migration as an explanation of our results.
Previous work and ideas as to the causes of pre-implantational loss have been
reviewed by Hammond (1952), but it is impossible to decide from our present
experimental evidence which if any of the causes are applicable to our findings.
Some useful conclusions may be made, however, by comparing our results with
those of McLaren & Michie (1956).
From the results of an experiment, in which they transferred varying numbers of
3^-day-old blastocysts from donor mice to normally mated recipient mice i\ days
pregnant, they concluded that' although we have found no limit to the number of
eggs which can implant in a single uterine horn, we are beginning to approach a
limit to the number of implantations which a single horn can keep alive'. In the
data reported here the post-implantational loss was irregular and not proportionately
related to the number of implants in the horn. The reason for this apparent difference is fairly easily found. McLaren & Michie (1956) consider that in their
material the limit to the number of implantations which remain alive to 16 days of
gestation may possibly be due to ' insufficiency of corpora lutea to supply the progesterone requirements of the excessive number of implantations'. In all animals
included in our data, presented here, there were at least as many corpora lutea in
the ovary as implantations in the horn to which it corresponded, and consequently
the progesterone supply is much less likely to have been insufficient. It is possible,
however, that the total number of implants per mouse surviving to birth is limited
142
J. C. B O W M A N AND R. C. R O B E R T S
by the level of some substance circulating in the maternal blood supply—a hypothesis favoured by Runner (1951) and by Hammond (1952).
McLaren & Michie (1956) found in their experiment that the number of successful implantations, from donor and recipient sources combined, rises linearly by
increments of 0-2 for each additional egg injected. Again, this conclusion seems to
conflict with our results of an increasing proportional loss of eggs up to implantation.
However, the discrepancy may not be so serious as it first appears. If indeed there
is no increase in the fractional loss in McLaren & Michie's data, then it seems highly
probable that the increased proportionate mortality in our material occurred very
soon after fertilization, in fact between fertilization itself and the stage at which the
blastocysts were removed by McLaren & Michie for transplantation.
If this suggestion is correct we have a more accurate estimate of the time interval
during which the differential loss of eggs takes place, and this knowledge might
prove useful in further elucidation of causes of pre-implantational loss.
SUMMARY
1. Two series of observations were made to determine the time and amount of
loss of eggs in relation to the number shed per ovary and per mouse.
2. The correlations between sides within mice for eggs shed was —0-528, for
corpora lutea counts —0-436, for implantations —0-300, and for live embryos
— O-III.
3. A positive linear trend of loss of fertilized eggs with increasing numbers of
eggs per uterine horn has been shown to occur before implantation.
4. Possible causative mechanisms for the loss are discussed in relation to observations on embryonic mortality previously reported by other workers.
We wish to express our gratitude to Prof. C. H. Waddington for laboratory
facilities, to Dr D. S. Falconer for much helpful criticism and advice and to Dr B.
Woolf for statistical advice. J. C. Bowman gratefully acknowledges financial support from the Agricultural Research Council.
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