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Morphometric analysis of the development of sexual dimorphism of the mouse pelvis.

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THE ANATOMICAL RECORD 224:490-494 (1989)
Morphometric Analysis of the Development of
Sexual Dimorphism of 1:he Mouse Pelvis
TAISEN IGUCHI, SATOKO IRISAWA, YUGO IWKAZAWA, YASUO UESUGI, AND
NOBORU TAKASUGI
Department of Biology, Yokohama City University, Set0 22-2, Kanazawa-ku, Yokohama
236 (T.I., Y.F., Y.U., N.T.) and Laboratory of Bildogy, Tokyo Kaseigakuin University,
Aihara-cho 2600, Machida, Tokyo 194-02 (S.I.), Japan
Sex differences in the innominate bone of C57BLlTw mice were
ABSTRACT
studied morphometrically from the day of birth to 120 days of age. In neonatal
male and female mice, a small cartilaginous spine was found on the basal part of
ischium. This process disappeared in males within 24 hours after birth, whereas in
females it remained until at least 30 days. Other sexual differences in the pubis
and the ischium appeared at 30 and 120 days, respectively. The pubis in female
mice was longer and thinner than that in the males, and the ischium in male mice
was shorter and thicker than that in the females. Thirty-day-old female mice
treated neonatally with testosterone or 5a-dihydrotestosterone possessed pubic
bones shorter and thicker than those of the age-matched untreated females. Pubes
in male mice castrated a t the day of birth were thinner than those in intact males.
These findings suggest that the shape of the innominate bone is transformed to the
male type under the influence of early postnatal androgen.
Sexual dimorphism of the pelvis has been described
in pocket gophers, guinea pigs, and mice (Chapman,
1919; Todd, 1925; Gardner, 1936). A pair of innominate
bones, ossa coxae, in mice is composed of four separate units: ilium, ischium, pubis, and acetabulum,
which are joined at the ventral midline by the pubic
symphysis to form the pelvis. The innominate bone is
connected dorsomedially with the sacrum by the iliosacral joint. Gardner (1936)has reported that there is
no difference in the shape of the innominate bone in
young male and female mice; however, after sexual
maturity, the pubic bone in females is thinner than in
males. Long-term administration of estrogenic hormones to male mice induces the female-type pelvis
with thin pubic bones, indicating that sex hormones
have a role in pelvic morphogenesis. Stein (1957)
pointed out the difficulty in expressing this skeletal
difference quantitatively. Festing (1972) emphasized,
however, that morphometric analysis is useful in demonstrating mandibular variations. Bailey (1986) also
reported a precise morphometric analysis of genetic differences in the shape of the mouse mandible. Computer
analysis has revealed that genetic differences are
present in the shape of the innominate bones of different strains of mice (Love11et al., 1986);however, sexual
dimorphism of the pelvis has not yet been demonstrated by such analysis. The present study was aimed,
therefore, at examining morphometrically normal development of the mouse pelvis of both sexes, as well as
the effects of neonatally administered androgens
thereon.
mercial diet (CA-1, CLEA, Tokyo) and tap water ad
libitum. Mice of both sexes were killed by ether anesthesia at ages of 0 (day of birth), 1, 5 , 10, 20, 30, and
120 days. Male mice castrated on day 0 were killed at
30 days. In addition, female mice given daily injections
of 20 p,g testosterone (TI and 20 p,g 5a-dihydrotestosterone (DHT) (Sigma, St. Louis, MO) dissolved in
0.02 ml sesame oil for 5 days starting on day 0 were
killed at 30 days. Skeletons of these animals were
stained by a modified differential method for cartilage
and calcified bone (Inoue, 1976; McLeod, 1980). The
skin, viscera, eyes, and adipose tissue were removed,
and the carcass was fixed in 95% ethanol for 4 days and
then placed in acetone for 1 day. These skeletal specimens were stained with 0.015% alcian blue 8GS
(Merck, Darmstadt) and 0.005% alizarin red S (Merck)
dissolved in 70% ethanol containing 5% acetic acid at
room temperature for 2 days. After washing in tap water For 2-3 minutes, the specimens were placed in 1%
aquleous KOH for 2-3 days for mice at 0-10 days of age
and for a week or longer for mice at 20-120 days of age,
until the skeletons became clearly visible. The specimens were transferred to 20% glycerin containing 1%
aquieous KOH, 50% and 80% glycerin in succession,
and then stored in 100% glycerin.
Seven parameters of the innominate bone were chosen for analysis: longitudinal length of the innominate
bone (IL), distances from the upper edge of the pubic
symphysis to its lower edge (SP), from the center of the
acetabulum to the lower edge of the ischium (AI), from
the lower ischium edge to the lower pubis edge (IP),
MATERIALS AND METHODS
Mice of the C57BL/Tw strain were kept under 12
hours light112 hours dark at 23-25°C and given a com0 1989 ALAN
R. LISS, INC.
Received August 9, 1988; accepted December 15, 1988.
491
SEXUAL DIMORPHISM IN MOUSE PELVIS
!I
SP
Fig. 1. Parameters of the left innominate bone. Longitudinal
length of the innominate bone (IL). SP, distance from the upper edge
of the pubis to the lower edge; AI, distance from the center of acetabulum (AC) to the lower edge of ischium; IP, distance from the lower
ischium edge to the lower pubis edge, AP, distance from AC to the
upper pubis edge. IW and PW, widths of ischium and pubis.
greater than in the age-matched females, although until 30 days, values in the males were not different from
those in the females. AP, IP, and SP showed no sexual
difference during the observation period.
Percent ratios of IP, AI, AP, and PW to IL showed
similar values until 30 days of age in both male and
female mice. However, ratios of SP and IW to IL rose
with age in both sexes (Table 2). The ratio of each parameter to IL did not differ between male and female
mice a t ages of 1-20 days. By contrast, a t 30 days, the
ratio of AP to IL (AP/IL) in female mice was significantly greater than that in male mice. PW/IL in female
mice was significantly smaller than that in male mice.
At 120 days, ratios of AI, IW, and PW to IL were significantly different in males and females. No significant differences in IP/IL and SP/IL were found between
the male and female mice even a t 120 days.
There was no significant difference in length of the
innominate bone between 30-day-old, intact male mice
and castrated male or neonatally T- or DHT-injected
female mice (Table 3). In castrated male mice, PW/IL
was significantly lower than in intact male mice (Table
4). AP/IL and IP/IL in female mice treated neonatally
with T or DHT were significantly lower than in untreated females. However, PW/IL in neonatally androgenized female mice was significantly higher than in
untreated females.
DISCUSSION
Sexual dimorphism in the shape of the innominate
bone was reported in adult mice but not in young mice
(Gardner, 1936). Stein (1957) reported that sexual and
genetic differences are present in the pelvic girdle of
adult mice, although the sexual difference in the pelvis
was illustrated but not discussed. On the other hand,
Festing (1972) pointed out that morphometric techniques are useful in demonstrating mandibular variations. Bailey (1986) reported a precise analysis of the
genetic relationships between interlandmark distances
of the mouse mandible. In the present study, sexual
RESULTS
dimorphism of the innominate bone was found in
In the innominate bone of male and female mice a t C57BL/Tw mice as early as 1day of age: in females, the
day 0 , the acetabulum, the pubic symphysis, the basal ischium had a small spinous process of cartilage in the
part of the ischium, and the upper part of the ilium basal part until a t least 30 days of age; in males, such
were still cartilaginous, whereas remaining parts process disappeared the day after birth. In male rats
were already ossified. A small spinous process of carti- and mice, higher levels of plasma testosterone were
lage was found in the basal part of the ischium in male detected in a perinatal period than those detected in a
(n = 30) and female (n = 30) mice at day 0. There was no later immature period, but not in the females (Pointis
sexual difference in morphology of the innominate et al., 1980; Slob et al., 1980; Pang and Tang, 19841,
bone at day 0. However, the spinous process of the is- suggesting that the early postnatal disappearance of
chium disappeared in 1-day-oldmale mice (n = 30) (Fig. the small spinous process in male mice results from the
2). By contrast, this process remained in the females at higher androgen levels during the perinatal period.
The present study demonstrated that at ages 30 and
least until 30 days of age. The ossified region of the
innominate bone expanded with age (Fig. 2). The upper 120 days, ratios of two and four parameters to IL in
end of the ilium, the symphysial part of the pubis, and male mice were different from those in the agethe basal part of the ischium were cartilaginous a t 20 matched females, respectively, although no such sexdays; by 30 days, all pelvic bones were ossified except ual differences were found until 30 days, indicating
for the cartilaginous junctions between the pair of pu- that some sexual differences in the pelvic bones appear
bes and between the pubis and the ischium on the an- during the prepubertal period. Serum androgen levels
tiacetabular side. The innominate bone was completely in male mice increase a t between 30 and 50 days postpartum (Selmanoff et al., 19771, suggesting that in
ossified a t 120 days of age.
There was no significant difference in the longitudi- some male pelvic bones, the shape is determined by
nal length of the innominate bone between male and prepubertally secreted androgen. However, Gardner
female mice a t all ages examined (Table 1). In male (1936) showed that a long-term administration of esmice at 120 days, however, IW, PW and A1 became trogen caused a reduction of the pubis width in male
and from the center of the acetabulum to the upper
edge of the pubis (AP), and the widths of the ischium
(IW) and of the pubis (PW) (Fig. 1). The lengths and
widths were measured by a Color Image Analyzer
CIA-102 (Olympus, Tokyo). Ratios of these lengths and
widths to IL were calculated as percentages. IL increased with age, but no sexual difference in IL was
found between intact male and female mice regardless
of age. Data were analysed by Student’s t-test.
492
T.IGUCHI ET AL.
Fig. 2. The left innominate bone. A Female mouse on day 0.
x 10.2. B 1-day-old female mouse. x 10.2. Note small spinous process
of cartilage in the basal part of the ischium (arrow head). C: 20-
dayold female mouse. x 4.3 D 120-day-old female mouse. x 4. E:
Male mouse at day 0. x 10.2. F: 1-day-old male mouse. x 10.2. G
20-day-old male mouse. x 4.3 H: 120-day-old male mouse. x 4.
mice. Because in the present study, the ratio of the
ischium width to IL in 120-day-old female mice was
significantly smaller than in 30-day-old females, ovarian estrogen secreted postpubertally may participate
in the formation of the female pelvic bones.
McLusty and Naftolin (1981) reported that in rats
and mice neural sexual differentiation takes place during the critical period within 10 days after birth, the
first 5 postnatal days being most effective. The present
study showed that the ratio of the pubis width to IL in
neonatally androgen-treated females at 30 days was
greater than in the age-matched untreated females,
whereas this ratio was smaller in neonatally castrated
30-day-old males than in age-matched intact males.
This finding may indicate that pubis width is increased
by early postnatal androgen. By contrast, the ratio of
the ischium width to IL in 120-day-oldfemale mice was
smaller than in the age-matched males, suggesting
that the ischium width is relatively decreased by prepubertal andlor postpubertal estrogen.
As there are genetic differences in the shape of the
pelvic bones (Love11et al., 1986) and age differences in
the sensitivity of the bones to androgen, as shown in
the present study, further morphometric studies on the
pelvic bones of different strains of mice and their responsiveness to androgens given at different ages are
needed for further elucidation of the development of
sexual dimorphism of the pelvis.
ACKNOWLEDGMENTS
We thank Professor Howard A. Bern of the Zoology
Department a t the University of California at Berkeley and Guest Professor of Yokohama City University
for his valuable advice and critical reading of this
manuscript. This work was supported by Grants-in-Aid
for Fundamental Scientific Research and for Encouragement of the Young Scientist from the Ministry of
Education, Science and Culture, Japan.
493
SEXUAL DIMORPHISM IN MOUSE PELVIS
TABLE 1. Sequential changes in the body weight and parameters of the innominate bone'
Age
Number Body weight
(days) Sex ofmice
(g)
1
M
30
1.2 f 0.01'
F
30
1.2 f 0.03
5
M
10
2.3 f 0.12
F
10
2.4 f 0.13
10
M
10
4.8 f 0.25
F
10
4.3 f 0.17
20
M
10
5.5 0.32
F
10
5.7 f 0.35
30
M
10
11.2 f 0.44
10
10.1 f 0.53
F
120
M
10
22.1 f 0.84
F
10
20.9 0.51
*
*
IL
5.0 f 0.12
4.7 f 0.08
5.9 ? 0.13
6.3 f 0.10
7.2 f 0.23
7.2 f 0.20
10.9 f 0.35
11.0 f 0.12
11.3 f 0.24
11.3 f 0.32
17.2 f 0.35
16.3 0.42
*
A1
2.3 t 0.08
2.2 f 0.07
2.9 f 0.07
3.0 ? 0.05
3.4 f 0.11
3.4 f 0.07
4.9 f 0.16
4.8 f 0.14
5.1 f 0.09
5.1 f 0.16
7.4 t 0.16
6.5 f 0.15*
AP
2.7 f 0.09
2.6 f 0.06
3.2 f 0.06
3.4 f 0.08
3.8 f 0.09
3.9 f 0.09
5.4 f 0.17
5.5 f 0.05
5.8 f 0.07
6.1 f 0.13
7.6 f 0.10
7.9 f 0.13
Lengths (mm)
SP
IP
0.4 f 0.02 1.8 f 0.06
0.4 f 0.03 1.8 ? 0.06
0.5 f 0.03 2.1 2 0.10
0.5 f 0.02 2.2 f 0.09
0.5 f 0.06 2.7 f 0.14
0.5 f 0.05 2.7 f 0.12
1.7 f 0.07 4.0 f 0.17
1.7 f 0.07 4.1 f 0.04
2.0 f 0.05 4.7 f 0.10
2.0 f 0.04 4.7 f 0.16
3.2 f 0.13 5.5 f 0.15
3.1 f 0.11 5.2 f 0.14
IW
0.6 f 0.04
0.6 f 0.02
0.8 f 0.07
0.8 f 0.05
1.1f 0.06
1.1 f 0.02
2.1 f 0.09
2.0 f 0.05
2.1 f 0.06
2.1 f 0.06
3.2 f 0.06
2.7 f 0.07**
PW
0.3 t 0.02
0.3 f 0.01
0.4 f 0.02
0.4 f 0.01
0.5 f 0.02
0.5 f 0.02
0.8 f 0.02
0.8 f 0.02
0.9 2 0.04
0.8 2 0.03
1.4f 0.03
1.1f 0.04**
'For abbreviations, see Figure 1.
2Mean 2 S.E.
*P < 0.002.
**P < 0.001vs. M (Student's t-test).
TABLE 2. Sequential changes in percent ratios of parameters of the innominate bone to the
longitudinal length of the bone (1L)l
Age
(days)
1
5
10
20
30
120
Sex
M
F
M
F
M
F
M
F
M
F
M
F
Number
ofmice
30
30
10
10
10
10
10
10
10
10
10
10
A1
46 f 0.8'
47 k 1.3
49 f 0.5
48 f 0.2
47 f 1.0
47 f 0.7
45 f 0.3
44 f 0.6
45 f 0.8
45 f 0.2
43 f 0.4
40 f 0.5**
AP
54 f 0.8
55 f 0.6
54 f 0.4
54 t 0.7
53 f 0.6
54 f 0.7
50 f 0.6
50 f 0.4
51 t 0.4
54 f 0.6**
44 f 0.5
48 ? 0.8**
Percent ratios to IL
SP
IP
36 f 0.6
8 f 0.4
9 f 0.6
38 f 0.8
36 f 1.1
8 f 0.4
35 f 1.5
8 t 0.2
38 f 1.8
7 f 0.7
38 f 1.1
7 f 0.5
37 f 0.5
16 f 0.3
37 f 0.4
15 t 0.5
42 f 0.8
18 0.6
42 f 0.7
18 0.6
32 f 0.7
19 f 0.5
32 f 0.7
19 f 0.5
*
*
IW
12 f 0.6
13 f 0.5
14 f 0.9
13 f 0.6
15 f 0.6
15 f 0.5
19 f 0.5
18 f 0.4
19 f 0.2
19 f 0.2
19 f 0.3
17 f 0.3**.***
PW
6 t 0.3
6 10.2
6 f 0.2
6 f 0.1
7 t 0.1
7 t 0.1
7 t 0.2
7 f 0.2
8 t 0.3
7 f0.3*
8 f 0.1
7 f 0.2**
'For abbreviations, see Figure 1 and Table 1.
2Mean f SE.
*P < 0.05.
**P < 0.001 vs. M.
***Female mice aged 30 days vs. females aged 120 days; IW ratio P < 0.001 (Student's t-test).
TABLE 3. Lengths of IL, AI, AP, IP, SP, IW, and P W to IL in 30-day-old,untreated male (M) and female (F),
neonatally castrated male (CM), neonatally androgenized female (F +T, F + DHT)'
Number
of mice
Treatment examined
IL
8
11.3 0.31'
F
8
12.1 f 0.10
F+T
8
11.8 f 0.13
F + DHT
8
11.4 f 0.03
M
CM
8
11.2 t 0.38
~
*
A1
5.0 f 0.15
5.3 f 0.06
5.2 t 0.03
5.1 f 0.08
5.0 f 0.17
AP
6.1 f 0.08
6.1 f 0.03
5.9 f 0.06
5.8 f 0.08
5.6 f 0.22
Lengths (mm)
SP
IP
4.7
f
0.07
2.0 f 0.07
2.1 f 0.05 4.4 t 0.08**
2.0 f 0.09 4.5 f 0.09
2.1 f 0.06 4.6 t 0.09
2.0 f 0.13 4.4 t 0.11*
IW
2.1 f 0.02
2.3 rt 0.03****
2.2 f 0.05
2.2 5 0.03*
2.1 f 0.10
PW
0.8 f 0.02
0.9 f 0.02***
0.9 f 0.03*****
0.9 f 0.02***
0.7 f 0.02***,*****
'For other abbreviations, see Figure 1.T, testosterone; DHT, 5a-dihydrotestosterone. F and M groups in Tables 3 and 4 are different from those
in Tables 1 and 2.
ZMeanf SE.
*P < 0.05.
**P < 0.02.
***P< 0.01.
****P < 0.001 vs. F.
*****P < 0.001 vs. M (Student's t-test).
T. IGUCHI ET AL.
494
TABLE 4. Percent ratios of AI, AP, IP, SP, IW and P W to 11, in 30-day-old, untreated male (M) and female (F),
neonatally castrated male (CM), neonatally androgenized female mice (F + T, F + DHT)'
Treatment
F
F+T
F + DHT
M
CM
Number
of mice
examined
8
8
8
8
8
A1
*
44 0.62
44 & 0.6
44 t 0.7
45 -+ 0.4
45 t 0.7
AP
54 * 0.8
51 k 0.5**
50 t 0.7****
51 ? 0.7**
50 t 0.8***
:Percent ratios to IL
SF'
IP
18 t 0.6
42 -+ 0.7
17 t 0.7
17 t 0.8
18 t 0.6
17 _t 1.0
37 t 0.8******
38 t 0.6*****
40 t 0.8
40 t 0.8
IW
19 t 0.2
19 t 0.3
19 t 0.3
19 t 0.3
19 ? 0.5
PW
7
8
8
8
7
0.3
t 0.3*
t 0.2*
t 0.2*
t 0.2*******
k
'For abbreviations, see Figure 1.
2Mean t SE.
*P < 0.05
**P < 0.02
***P < 0.01
****P < 0.005
*****P < 0.002
******P < 0.001 vs. F.
******* P < 0.01 vs. M (Student's t-test).
LITERATURE CITED
Bailey, D.W. 1986 Genes that affect morphogenesis of the murine
mandible. Recombinant-inbred strain analysis. J . Hered.,
77: 17-25.
Chapman, R.N. 1919 A study of correlation of the pelvic structure and
the habits of certain burrowing mammals. Am. J. Anat., 25:
185-219.
Festing, M. 1972 Mouse strain identification. Nature, 238:351-352.
Gardner, W.U. 1936 Sexual dimorphism of the pelvis of the mouse, the
effect of estrogenic hormones upon the pelvis and upon the development of scrota1 hernias. Am. J . Anat., 59:459-483.
Inoue, M. 1976 Differential staining of cartilage and bone in fetal
mouse skeleton by alcian blue and alizarin red S. Cong. Anom.,
16:171-173.
Lovell, D.P., F.M. Johnson, and D.B. Willis 1986 Quantitative genetic
variation in the skeleton of the mouse: XI. Description of variation
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McLeod, M.J. 1980 Differential staining of cartilage and bone in
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McLusty, N.J., and F. Naftolin 1981 Sexual differentiation of the
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Pan,:, S.F., and F. Tang 1984 Sex differences in the serum concentrations of testosterone in mice and hamsters during their critical
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Pointis, G., M.-T. Latreille, and L. Cedard 1980 Gonado-pituitary relationships in the fetal mouse a t various times during sexual
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postnatal sex differences in plasma and gonadal testosterone and
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