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Effects of Sex Hormone Disturbances on Craniofacial Growth in Newborn MiceDepartment of Orthodontics and Craniofacial Developmental Biology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; Correspondence: * corresponding author, seven{at}hiroshima-u.ac.jp
It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.
Key Words: orchiectomy • ovariectomy • craniofacial growth • newborn mice
It is well-known that sex hormones regulate bone metabolism. Estrogen deficiency is recognized as a cause of post-menopausal bone loss. On the other hand, estrogen treatment has a strong effect on bone accumulation (Riggs and Melton, 1986). Cortical and cancellous bone densities were reduced in men with hypogonadism before the peak of bone mass (Finkelstein et al., 1989). In animal experiments, it has been demonstrated that OVX and ORX induce bone loss, and that estrogen and androgen are effective in preventing bone loss during adolescence (Fujita et al., 2001a). However, the effects on bone growth immediately after birth have not been fully clarified. Although bone modeling has been examined in various conventional animal experiments, there are few studies in the literature that relate craniofacial growth to sex hormones. Craniofacial growth, in nature, presents large variations between individuals. The effects of sex hormones on the growth of bones or muscles can be greater than those of genetic or environmental factors (Morishima et al., 1995). We have recently reported, from an animal experiment, that the secretion levels of sex hormones changed the internal structure of the mandibular condyle, which is a mandibular growth site or center (Fujita et al., 2001a). The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial growth immediately after birth.
Experimental Animals Forty five-day-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were used in this experiment. The mice were divided equally into 2 experimental groups, ORX and OVX, and the corresponding sham-operated groups. Under general anesthesia with sodium pentobarbital, 10 male and 10 female mice underwent ORX and OVX 5 days after birth, by means of a stereoscopic microscope (SZX9, Olympus Optical Co., Tokyo, Japan). These mice were killed 4 wks after the surgery. Body weight was measured every 4 days. The animals were treated under the ethical regulations defined by the Ethics Committee, Hiroshima University Faculty of Dentistry.
Radioimmunoassay
Morphometric Analyses with Lateral Cephalograms
Statistical Analysis We performed analyses of variance (ANOVA) and pairwise comparisons (Fisher) to examine the differences in measured values between the groups with a confidence level greater than 95%.
Body Weight and Serum Concentrations of Testosterone and Estradiol Body weight significantly increased in the experimental groups as compared with the sham-operated groups during the entire experimental period (Table 1  ). Serum testosterone was undetectable in ORX mice and 4.0 ± 1.8 ng/mL in male sham-operated mice. Serum 17β-estradiol was also undetectable in OVX mice, whereas the concentration was 24.3 ± 10.1 pg/mL in female sham-operated mice.
Morphometric Findings for the Craniofacial Skeleton Means and standard deviations for linear and angular measurements are shown for the experimental and sham-operated groups in Tables 2 and 3, respectively. No significant differences in total skull size or neurocranium size were found between the experimental and sham-operated groups, except for Ba-S distance in the male groups. In the ORX group, all measurements of the maxilla, except for Iu-A, were significantly smaller than in the sham-operated group. In the OVX group, the distances of Mu-Pr, A-Pr, Iu-Bu were significantly smaller than in the sham-operated group. In the experimental groups, the amount of maxillary growth until 4 wks after birth was thus decreased as compared with that in the sham-operated groups, and was more prominent in ORX mice than in OVX mice.
In the experimental groups, similar to the maxilla, the amount of mandibular growth was reduced as compared with the sham-operated groups in terms of dimensional changes. In the ORX group, 6 measurement items (Pg-Go, Pg-Gn, Co-Co', Co-Gn, Co-Pg, Il-Id) were significantly smaller than in the sham-operated group. In the OVX group, the distances of Pg-Gn, Ml-Bl, Co-Co', and Il-Id were significantly smaller than in the sham-operated group. The nature of mandibular growth and the differences observed in those affected by sex hormone deficiency were almost the same as those previously described for the maxilla. Significant differences in all of the angular measurements of the neurocranium were found between the ORX and sham-operated groups, whereas no significant differences were found between the OVX and sham-operated groups. N-A-Pr was significantly larger in the ORX group. N-A-Bu and N-A-Iu were significantly larger in the OVX group. In the experimental group, it was thus shown that point A is located more posteriorly, as noted by the reduced maxillary dimensions mentioned in the preceding linear measurements. No significant differences were found in the shape of the mandibular or gonial angle (GnPg/CoGo, GnPg/CoGn) between the experimental and sham-operated groups.
Radioimmunoassay of terminal serum testosterone and estradiol confirmed the effectiveness of castration. Analysis of these data indicates that the surgeries for ORX and OVX were performed appropriately to simulate deficiency of the sex hormones. The results of this study suggest that sex hormones exert a substantial influence on craniofacial growth, due to the fact that suppression of craniofacial growth was significantly induced, as compared with sham-operated groups, by ORX or OVX in animals at 5 days old. Furthermore, the influence was more prominent in the ORX group; thus androgen seems more important for growth control. These results also suggest that sex hormones might be applied to the prevention and treatment of reduced craniofacial growth. ORX and OVX in mice or rats are regarded as suitable experimental models for studying the functions of sex hormones. However, reports on ORX and OVX performed in newborn mice and rats were previously unavailable in the literature because of the difficulty of the surgery. The function of sex hormones immediately after birth thus has been unclear. In this study, we succeeded in creating ORX and OVX to induce an experimental model of sex hormone deficiency in five-day-old mice by using a stereoscopic microscope that was not lethal to the mice. All mice were alive at the end of the experimental period. Although estrogen is not essential for normal differentiation and growth of female sexual organs during embryogenesis, it is required for sexual differentiation of the brain (Quadros et al., 2002). Androgens secreted from the embryonic orchis are locally transformed into estrogens in the brain, and the estrogen then stimulates differentiation of the brain in males (Swerdloff et al., 1992). The growth and morphology of neurocranial sutures and structures are known to reflect their functional environment (Rönning and Kylämarkula, 1982). In this study, environmental factors may greatly influence the growth of the neurocranium. It may be supposed that sex hormones have no influence on the growth of the neurocranium. The influence of sex hormones on bone after adolescence is comparatively well-known. OVX increases hypertrophic cartilage. The total amount of growth plate cartilage in OVX animals was decreased by estradiol, whereas testosterone exerts no effect in the females. ORX decreased the amount of growth plate cartilage, but increased the hypertrophic zone (Ornoy et al., 1994). Sex hormones are related to the maintenance of bone volume after the growth period (Riggs, 1991). Estrogen and androgen strongly influence normal bone modeling in the mandibular condyles of both males and females (Fujita et al., 2001a). The present study has clearly demonstrated the influence of sex hormones on bone growth immediately after birth. From morphometric analyses with lateral cephalograms, it was shown that sex hormones substantially influence craniofacial growth in newborn mice. In particular, remarkable changes in the height and length of the maxilla and mandible were found in the experimental groups, whereas no significant differences in the total size of the skull or neurocranium were found between the experimental and sham-operated groups, except for Ba-S in the ORX group. Craniofacial morphology is determined by membranous, sutural, and cartilaginous growth. Estrogen and androgen stimulate osteoclast differentiation indirectly (Bellido et al., 1995) or directly (Mizuno et al., 1994), osteoblast differentiation (Eriksen et al., 1988), and endochondral ossification (Fujita et al., 2001b). The sutures are composed of fibrous connective tissue between the membranous craniofacial bones, and grow most actively during the pre-natal to neonatal period (Babler et al., 1987). In this study, the growth of the maxilla, that was mainly determined by sutural growth, was inhibited in the experimental groups, as was that of the mandible, determined by membranous and cartilaginous growth. From these considerations, it could be assumed that sex hormone is a critical factor not only for membranous and cartilaginous growth but also for sutural growth. It is well-understood that male hormones influence bone growth. In adolescence, both sex hormones are secreted. Male hormones have a powerful effect on bone extension. Furthermore, in adolescence, male hormones promote bone maturity and cause an increase in body height and the closing of epiphyseal cartilage, thus determining adult height (Soliman et al., 1995). In this experiment, many measurements indicated a significant difference in the ORX group compared with the OVX group. However, it has been reported that, as an action mechanism of testosterone on bone cells, aromatase (P450arom) transforms testosterone into estradiol, which then further affects the cells (Tanaka et al., 1993). This indicates that estrogen may be important for bone metabolism in males as well as in females. Thus, we cannot directly compare the effects of bone growth between male and female hormones, and further studies are needed to understand the mechanisms. Also in the angular measurements, the results reflect inhibition of bone growth in the experimental groups, but no significant differences in gonial angle were found between experimental and sham-operated groups. In growing rats, decreased ramus height and a large gonial angle were found, increasing the occlusal vertical dimension (Sugiyama et al., 1999). Gonial angle may be influenced by mechanical and functional changes in occlusion and not by internal secretion changes. Our body weight results support past research (Turner et al., 1995). In a study using OVX rats on restricted diets to match body weights to those of control rats, Wronski et al.(1987) suggested that increased body weight provides partial protection against osteopenia. In this study, craniofacial growth in the experimental groups was inhibited regardless of this protective effect. In conclusion, it was strongly suggested that the suppression of sex hormone secretion in the growth phase might inhibit craniofacial growth and result in poor craniofacial development in the growth phase.
This investigation was supported in part by Grant-in-Aid 14771181 from the Ministry of Education, Science, Sports and Culture of Japan, and by institutional funds. Received for publication March 27, 2003. Revision received December 21, 2003. Accepted for publication January 14, 2004.
Journal of Dental Research, Vol. 83, No. 3,
250-254 (2004) This article has been cited by other articles:
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