/. Embryo!, exp. Morph. Vol. 35, 1, pp. 191-196, 1976
Printed in Great Britain
Influence of conceptus number and weight on the
amount of foetal fluid in the mouse
By ANNE McLAREN1, MARILYN B. R E N F R E E 2 A N D
HUGH C. HENSLEIGH3
From the Department of Animal Genetics, University of Edinburgh, U.K.
SUMMARY
The relation of extra-embryonic fluid weight to litter number and foetal and placental
weight was studied in mice on the 18th day of pregnancy, in litters both of experimentally
reduced and of normal number. Partial regression analysis showed that litter number and
foetal weight both exerted a negative effect on fluid weight; placental weight had no significant
effect. Increased foetal weight reduced weight locally; on the other hand the effect of litter
number was exerted systemically, throughout both horns of the uterus.
INTRODUCTION
The quantity as well as the quality of amniotic fluid seems to be important
for normal foetal development in mammals, yet little is known of the factors
that regulate it. A study of foetal fluids from the 12th to the 20th day of gestation
in the mouse (Renfree, Hensleigh & McLaren, 1975) showed that the amount
of fluid was related both to the day of gestation, reaching a peak on the 17th
day, and to the weight of the foetus. The present study seeks to throw further
light on the determinants of foetal fluid volume, by relating it to litter size, and
to both placental and foetal weight, on a single day of gestation.
Since control litters vary little in size, operative procedures were applied to
reduce litter size, either by reducing the number of implantations in the uterus
as a whole, or by preventing implantation in one of the two uterine horns. This
design should allow local (i.e. within-horn) effects of uterine crowding to be
distinguished from systemic effects.
1
Author's address: M.R.C. Mammalian Development Unit, University College, Wolfson
House, 4 Stephenson Way, London NW1 2HE, U.K.
2
Author's address: School of Environmental & Life Sciences, Murdoch University,
Murdoch, Western Australia 6153.
3
Author's address: Department of Anatomy, University of Oregon Health Sciences
Center, Portland, Oregon 97201, U.S.A.
192
A. McLAREN, M. B. RENFREE AND H. C. HENSLEIGH
MATERIAL AND METHODS
The 32 litters studied were all from the randomly bred Q strain of mice. They
belonged to the following groups :
1. Untreated pregnancies from the earlier study (eight litters) (Renfree et al.
1975).
2. Additional untreated pregnancies (six litters).
3. Pregnancies in which a hypodermic needle was inserted into the ovarian
end of each uterine horn 2\ days post coitum (p.c.) (four litters).
4. Pregnancies in which a small volume of 10% calf serum in phosphatebuffered saline, as used in egg transfer, was injected into the ovarian end of
each uterine horn 2\ days p.c. (four litters).
5. Pregnancies from transfers of embryos 3 | days p.c. (derived from spontaneous ovulation) to the uteri of pseudopregnant recipients (mated to vasectomized males) 2\ days/?.c, in the same serum-saline mixture (four litters).
6. Pregnancies in which one oviduct was removed \\ days p.c. (six litters).
Treatments 3-5 would all be expected to result in a reduced total number of
implantations (McLaren & Michie, 1956), distributed between both uterine
horns; treatment 6, on the other hand, would result in half the normal number
of implantations, all confined to a single horn.
Mice were killed between 11.00 and 15.00 h ofthe 18th day of gestation (i.e.
17^ days p.c). The numbers of live and dead embryos in each uterine horn were
recorded. Each live conceptus was removed and weighed, then the foetus,
placenta and membranes were dissected out, blotted, and weighed individually.
The amount of fluid was obtained by subtraction of these weights from the total.
The average weights of foetus, fluid, and placenta plus membranes for each
uterine horn and each litter were calculated.
RESULTS
Litter size varied from 3 in the experimentally reduced litters, up to 15 in the
controls (Fig. 1). Analysis of variance revealed no significant difference among
the different groups in slope or position of the regression lines of fluid weight
on number in the litter. The groups were therefore combined for further analysis.
Using simple linear regressions (Fig. 2), mean fluid weight was directly related
to placental weight, and inversely related to litter size (Fig. 1). No significant
effect of foetal weight was seen.
Interpretation ofthe simple regressions is complicated by the strong negative
effect of litter size on both foetal and placental weight in the mouse (McLaren,
1965; McCarthy, 1965), confirmed on the present data (Fig. 2). In order to assess
the effects on fluid weight ofthe other three variables independently, we subjected
the data to partial regression analysis (Fig. 3). The inverse relation of fluid
193
Foetal fluid in the mouse
12
14
Number
in litter
niter
jNumoer in
Fig. 1. The dependence of fluid weight (w) on litter size (/) for six groups of pregnant
mice, with the calculated regression line for the groups combined (r=— 7-20 ±
1-45). x , Group .1; + , group 2; • , group 3; O, group 4; • , group 5; • , group 6.
Litter size
-13-85
+ 5-45*
—4 91
+ 0-84***
Foetal weight
Placental weight
-7-20 + 1-45*
-0001
±0-60
+ 1-042
±0-221***
Fluid weight
Fig. 2. Simple linear regression coefficients ( r ± s . E . ) of fluid weight (w) (mg) o n foetal
weight ( / ) , placental weight (p) a n d litter size (/), a n d of foetal a n d placental weight
on litter size. n = 32, *P<005,
***P<0-001.
Regression e q u a t i o n s : w= 2 0 8 0 7 - 0 0 0 1 / , w= 37-80+ 1 0 4 2 p, w- 2 6 2 - 4 9 - 7-20 /,
/ = 1 0 6 0 - 2 6 - 1 3 - 8 5 /, p = 2 0 0 - 2 6 - 4 - 9 1 /.
Foetal weight
-0100
+ 0045*
Litter size
-6-51 + 213"
Placental weight
+0-423
+ 0-292
Fluid weight
Fig. 3. Partial linear regression coefficients ( r ± s . E . ) of fluid weight (mg) o n foetal
weight, placental weight a n d litter size. // = 32, * P < 0 0 5 , * * P < 0 0 1 . Multiple
regression equation: w = 283-82-0-100/+0-423jp-6-51 /.
13
EMB 35
194
A. MCLAREN, M. B. RENFREE AND H. C. HENSLEIGH
weight with litter size was still significant; the effect of placental weight decreased
and was no longer significant, suggesting that litters with large placentas had
large quantities of fluid only because they tended to contain small numbers of
foetuses; and a slight but significant inverse relation with foetal weight was now
seen, previously obscured by the marked dependence of foetal weight on litter
size.
Table 1. Local and systemic effects offoetal number, foetal weight and
placental weight on fluid weight (mg), in 24 2-horn pregnancies
Regression coefficients
(±S.E.)
A
Fluid weight on:
Local effect
Systemic effect
Foetal number
Foetal weight
Placental weight
1-976 ±2-440
-0162 ±0064*
0-317±0-367
*P<0-05; ***P< 0-001.
-10-32 ±3-56***
00086 ±0045
-0092 ±0-276
In species with a bicornuate uterus, such as the mouse, the accumulation of
foetal fluid could be affected by the number or weight of the foetuses in the same
uterine horn, through some local effect of, for example, crowding; alternatively,
a systemic effect could operate, according to which fluid accumulation may be
affected by foetuses in either horn equally. Foetal growth, for example, is known
to be more affected by the presence of other foetuses in the same, rather than
in the opposite, uterine horn (Healy, McLaren & Michie, 1960; McCarthy,
1965); on the other hand placental growth is subject to systemic rather than
to local effects of foetal number (McLaren, 1965).
In order to separate local effects (bt) from systemic effects (bs), we applied
the strategy derived by Healy, McLaren & Michie (1960), using only those
females with foetuses in both horns (« = 24). The regression coefficients for this
subset of the data did not differ significantly from those for the data as a whole.
Using in turn mean fluid weight, mean foetal weight, mean placental weight and
number of foetuses for the two horns of each uterus, we first formed the differences between the two horns (D) and sums of the two horns (S). We then applied
the following relations, bt = bD, bs = ^ (bs-bD), where bD is the appropriate
partial regression of differences on differences, and bs the corresponding
partial regression of sums on sums.
The results, shown in Table 1, are clear-cut. Placental weight again proves
irrelevant to fluid weight. Foetal weight bears a significant inverse relation to
fluid weight locally, so that if foetal weight is reduced by crowding or any other
local factor, the amount of fluid increases (Fig. 4). This effect operates within the
individual conceptus, so that within horns there exists in general a highly
significant negative correlation between foetal weight and amount of fluid: of
43 horns analysed from the 24 2-horn pregnancies, the correlation was negative
Foetal fluid in the mouse
195
in 33 and positive in only 10. Foetal number, on the other hand, has no local
effect other than that operating via foetal weight, but does exert a highly
significant systemic effect, such that larger litters are associated with smaller
amounts of fluid.
-60
-200
-160
-120
-80
-40
0
+40
+80
.Foetal weight difference, left-right (mg)
+ 120
Fig. 4. The local effect of foetal weight on fluid weight, for two-horn pregnancies
only, with the calculated regression line for the groups combined. Symbols as in
Fig. 1.
DISCUSSION
The finding that foetal weight exerts a local influence on the amount of foetal
fluid suggests that pressure may be involved. With a relatively large foetus, the
increased pressure within the amniotic cavity might favour the passage of water
from this embryonic compartment into the maternal circulation (via the vitelline
circulation and placenta). The increased pressure would not be transmitted to
the opposite horn, probably not even to neighbouring foetuses in the same
horn. In contrast, foetal number has a systemic effect of foetal fluid, so that an
extra foetus reduces the average amount of foetal fluid throughout both horns
of the uterus. The systemic effect of foetal number on foetal and placental weight
has been attributed by Eckstein, McKeown & Record (1955) and by Healy et ah
(1960) to haemodynamic factors.
No effect of litter size on foetal fluid was detected in our earlier study, in
13-2
196
A. M C L A R E N , M. B. R E N F R E E A N D H . C. H E N S L E I G H
which we examined the day-to-day changes in amount of foetal fluid from the
12th to the 20th day of gestation. Since the present study was confined to the 18th
day, this difference may suggest that the effect of litter size is not apparent
during the earlier stages of gestation. Alternatively, it may be that the effect
does not show up unless litter size is experimentally reduced, since the data of
Fig. 1 show little trend for litters of normal size (8-12 young).
We are grateful to the Ford Foundation for financial support.
REFERENCES
& RECORD, R. G. (1955). Variation in placental weight according
to litter size in the guinea-pig. /. Endocr. 12, 108-114.
HEALY, M. J. R., MCLAREN, A. & MICHIE, D. (1960). Foetal growth in the mouse. Proc. R.
Soc. B 153, 367-379.
MCCARTHY, J. C. (1965). Genetic and environmental control of foetal and placental growth
in the mouse. Anim. Prod. 7, 347-361.
MCLAREN, A. (1965). Genetic and environmental effects on foetal and placental growth in
mice. /. Reprod. Fert. 9, 79-98.
MCLAREN, A. & MICHIE, D. (1956). Studies on the transfer of fertilized mouse eggs to uterine
foster-mothers. I. Factors affecting the implantation and survival of native and transferred
eggs. /. exp. Biol. 33, 394-416.
RENFREE, M. B., HENSLEIGH, H. C. & MCLAREN, A. (1975). Developmental changes in the
composition and amount of mouse fetal fluids. /. Embryol. exp. Morph. 33, 435-446.
ECKSTEIN, P., MCKEOWN, T.
{Received 17 September 1975)