/ . Embryol. exp. Morph. Vol. 40, pp. 259-263, 1977
Printed in Great Britain © Company of Biologists Limited 1977
259
Weight differences in rat embryos prior
to sexual differentiation
By WILLIAM J. SCOTT 1 AND JOSEPH F. HOLSON 2
From the Children's Hospital Research Foundation and Department
of Pediatrics, College of Medicine, University of Cincinnati
SUMMARY
Sex of day-12 rat embryos was determined using Barr body counts made on spreads of
amniotic membranes examined histologically. Embryonic weight, protein content and rate of
thymidine incorporation were compared in male and female embryos. Male embryos were
found to be heavier and accordingly to contain more protein on absolute but not on per unit
weight basis. The rate of thymidine incorporation did not differ in the two sexes. Since
gonadogenesis in day-12 rat embryos is rudimentary, with gonadal differentiation of sex not
yet apparent, the increased weight suggests that sex-linked genes exist which influence body
growth prior to gonadal endocrine activity.
INTRODUCTION
Two recent reports (Barr, 1973; Scott, Butcher, Kindt & Wilson, 1972) have
documented that teratogenic agents such as acetazolamide and cadmium can
selectively malform the limbs of female embryos more seriously and more often
than of male embryos. An interesting feature of both of these experiments was
that the teratogen was administered (day 10 and/or 11) before any gonadal
differentiation was morphologically apparent. This implies that some differences
in early embryonic development exists between the sexes that is unrelated to
the humoral functions associated with the sex organs at later stages.
A recent study (Holson, Scott, Gaylor & Wilson, 1976) measured the effect
of length of mating period on interlitter variability. Examination of three variables according to sex was also undertaken to determine whether differences
existed in day-12 rat embryos and constitutes the results presented here. The
variables examined were embryo weight, protein content and rate of thymidine
incorporation. In a separate group of animals fetal weight on day 20 of gestation
was determined in the two sexes.
1
Author's address: Children's Hospital Research Foundation, Elland and Bethesda
Avenues, Cincinnati, Ohio 45229, U.S.A.
2
Author's address: National Center for Toxicological Research, Jefferson, Arkansas
72079, U.S.A.
17-3
260
W. J. SCOTT AND J. F. HOLSON
Table 1. Mean weight of male and female rat embryos on day 12
No.
Litter
males
Mean
wt (mg)
No.
females
Mean
wt (mg)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
6*
7
7
7
7
5
8
9
5
3
4
6
9
7
7
9
7
7
5
5
22-45
20-49
15-34
18-72
15-75
23-51
16-75
15-69
20-43
21-71
22-54
16-50
13-76
24-89
19-37
24-78
20-67
21-39
18-77
1600
8
5
8
3
6
9
7*
7
10
11
9
6
0
5
2
3
5
4
7
4
18-17
18-51
15-92
15-55
14-29
18-72
15-63
1901
17-61
21-64
20-82
15-56
—
21-26
1813
21-64
19-12
22-59
1602
14-20
mean±std. dev.
mean±std. dev.
19-27±4-19
18-29±3-27
t == 2 0 8 ; P < 0 0 5
* One embryo three S.D.'S below mean omitted.
MATERIALS AND METHODS
The mating procedures and biochemical techniques have been described in
detail previously (Holson et al. 1976). To determine the sex of each embryo the
technique described by Bianchi & de Bianchi (1966) was followed. Essentially
this consists of spreading the acetic acid-fixed amnion on a glass slide and
staining it with carbol fuchsin. In these studies an embryo was designated
female if 25-55 % of its amniotic cells contained a chromatin body adjacent to
the nuclear membrane. Males had less than 15% of such cells. These figures
are somewhat higher than those reported by Bianchi & de Bianchi (1966) and
can most likely be attributed to our counting of all chromatin bodies adjacent
to the nuclear membrane, some of which probably were not Barr bodies. In
spite of this slight discrepancy it should be mentioned that a striking dichotomy
consistently exists between male and female cells, rarely leaving doubt as to
proper classification. Sex of the 20-day fetus was determined by two independent
observers using ano-genital distance as the criterion.
Comparison of mean values was made by conventional t test and paired t
test whereby male and female values could be compared within each litter. In
Sex-related weight differences in embryos
261
Table 2. Mean protein content of male and female rat embryos on day 12
No.
Litter
1
2
3
4
5
6
7
10
11
12
13
14
15
16
17
18
19
20
males
Mean
protein
content O g )
No.
females
960
8
899
5
8
728
7
2
7
816
7
724
6
959
9
5
637
7*
8
812
11
3
826
9
4
6
646
6
0
9
698
987
7
5
2
7
770
1021
3
9
7
5
774
823
7
4
5
894
7
775
5
4
mean ± std. dev.
mean ± std. dev.
821 ±142
749 ±113
t = 417; P < 001
6*
7
Mean
protein
content (/Jg)
780
836
716
672
693
759
601
771
794
621
—
847
763
912
711
855
768
689
* One embyro three S.D.'S below mean omitted
advance it was decided to exclude values lying outside three standard deviations
of the mean. Such values were considered to represent atypical embryos.
RESULTS
Table 1 compares day-12 mean male embryo weight to mean female embryo
weight from 20 litters. In 17 of these litters mean male weight exceeded mean
female weight and there was also a significantly larger mean weight of male
embryos when all of one sex were combined to form an overall mean for each
sex (t = 2-08; P < 0-05). This type of comparison, however, could be distorted
by a preponderance of one sex in a particular litter, as was illustrated in litter 13
which was the lightest litter examined and consisted wholly of males. This,
however, resulted in an artifactual lessening of the difference between the
overall means. To achieve a more meaningful comparison, a paired t test was
performed comparing mean male to mean female weight within each litter.
Those litters in which the number of either sex did not represent 25 % of the
total litter were arbitrarily excluded from the comparison. On this basis 17
litters were compared resulting in further confirmation that males weighed
more than females (t = 3-66, P < 0-01) on day 12 of gestation.
262
W. J. SCOTT AND J. F. HOLSON
Table 3. Mean rate of thymidine incorporation of male and
female rat embryos on day 12
No. males
CPM/mg
No. females
7
8
5
8
3
4
6
7
7
9
7
5
5
307
292
274
299
331
288
321
272
297
285
206
334
334
8
3
9
7
11
9
6
5
2
3
5
7
4
CPM/mg
294
278
284
318*
318
314
332
286
318
287
213
321
332
mean±std. dev.
mean + std. dev.
292 ±38
302 ±35
t = 1-71; 01 > P > 005
* One embryo three
S.D.'S
below mean omitted.
Table 2 presents values for mean protein content in male and female embryos
from 18 litters on day 12. As would be expected from the weight data, males
had a higher absolute protein content, both by comparing means (t — 4-17,
P < 0-01) and by paired t test of males and females within a litter (t = 4-67,
P < 0-01), but on a per unit of weight basis there was no difference.
Table 3 illustrates the rate of DNA synthesis as measured by thymidine
incorporation per unit of weight in male versus female embryos. Comparison
of means by t test (t = 1-71, P < 005) or intralitter means by paired t test
(t = 1-07, P < 0-2) indicates no significant difference between the sexes in this
regard. Interpretation of this result should be cautious, however, since the
data reflect proliferative rate for only the 2 h of exposure to tritiated thymidine,
while weight and protein content were cumulative growth characteristics.
When fetal weights on day 20 were compared it was evident from casual
inspection that males (mean = 3-482 g± 0-289 S.D.) were heavier than females
(mean = 3-246 g± 0-250 S.D.) which was statistically verified by comparing
the means (t = 5-91, P < 0-01) and by paired t of intralitter means {t = 6-28,
P < 0-01).
DISCUSSION
That males are significantly heavier than females at birth has been repeatedly
documented in a number of mammalian species. Recently, for example, Bruce &
Norman (1975) found male rat fetuses one day prior to expected parturition
to weigh 5-4% more than female rat fetuses. Since this difference increases
Sex-related weight differences in embryos
263
with advancing age of the animal it has long been assumed that sex hormones
influence such growth differences. Results of gonadectomy experiments have
tended to confirm this (Grunt, 1964).
The results of the present study once again demonstrate that the near term
male fetus is larger than the female. Of much greater interest are the results of
studies on day-12 embryos. All embryos of this age could be regarded as
physiologically neuter since sexual differentiation of gonads has not occurred,
yet male embryos already weighed more than female embryos and possessed
proportionately more protein. These data suggest that sex-linked genes exist
which influence embryonic growth, prior to and independently of the products
of the definitive sex organs. Adding to this conviction are the data of Honnebier
& Swaab (1973) who concluded that fetal hypothalamic function did not
contribute to heavier human male infants since the birth weight of anencephalic
male children was about 5 % more than that of anencephalic girls.
In regard to teratogenesis, these results emphasize the need to analyze malformation patterns and rates according to sex. They also point out the need for
caution in indiscriminate pooling of litter mates for biochemical studies during
early organogenesis, a frequent practice due to the small size of the embryo at
this time. Particular note should be taken of the ease and reliability of this
technique for diagnosing the sex of the embryo. It is not only simple and quick
to perform, but also it uses an extra-embryonic tissue, the amnion, thus leaving
the intact embryo for other purposes.
Completion of this work was greatly facilitated by the excellent technical assistance of
Mrs Claire Schreiner and Mrs Christine Grant. Supported in part by NIH grant HD06526.
REFERENCES
BARR, M. (1973). The teratogenicity of cadmium chloride in two stocks of Wistar rats.
Teratology 7, 237-242.
BIANCHI, N. O. & DE BrANCHi, M. S. A. (1966). Technique for investigation of sex chromatin
in amniotic membrane of rat foetuses. Nature, Lond. 212, 1593.
BRUCE, N. W. & NORMAN, N. (1975). Influence of sexual dimorphism on foetal and placental
weights in the rat. Nature, Lond. 257, 62-63.
GRUNT, J. A. (1964). Effects of adrenalectomy and gonadectomy on growth and development
in the rat. Endocrinology 75, 446-451.
HOLSON, J. F., SCOTT, W. J., GAYLOR, D. W. & WILSON, J. G. (1976). Reduced interlitter
variability in rats resulting from a restricted mating period and reassessment of the 'litter
effect.' Teratology 14, 135-142.
HONNEBIER, W. J. & SWAAB, D. F. (1973). The influence of anencephaly upon intrauterine
growth of the fetus and placenta and upon gestation length. /. Obstet. Gynaec. Br. Common.
80, 577-588.
SCOTT, W. J., BUTCHER, R. E., KINDT, C. W. & WILSON, J. G. (1972). Greater sensitivity of
female than male rat embryos to acetazolamide teratogenicity. Teratology 6, 239-240.
{Received 8 December 1976, revised 24 January 1977)