Morphometric analysis of the development of sexual dimorphism of the mouse pelvis.код для вставкиСкачать
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. 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