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Effects of Sex Hormone Disturbances on Craniofacial Growth in Newborn Mice

Department of Orthodontics and Craniofacial Developmental Biology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;

^* corresponding author, seven{at}hiroshima-u.ac.jp

	ABSTRACT

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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

	INTRODUCTION

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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.

	MATERIALS & METHODS

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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
The concentrations of total testosterone and 17ß-estradiol in the serum were measured by radioimmunoassay with a commercially available kit (Diagnostic Products Corp., Los Angeles, CA, USA), in accordance with the manufacturer’s guidelines.

Morphometric Analyses with Lateral Cephalograms
Lateral cephalograms were taken of all of the mice, by means of a rat and mouse cephalometer (RM-50, Asahi Roentgen Industry Co., Kyoto, Japan). The head of each animal was fixed firmly with a pair of ear rods oriented perpendicularly to the median sagittal plane. The cephalogram was taken with dental occlusal film (DF-50, Eastman Kodak, Rochester, NY, USA) under an electric condition of 6 mA and 20~25 Kvp with an exposure time of 3.0 sec. Cephalograms were scanned by means of an imaging scanner (GT-9000, Epson, Tokyo, Japan) and enlarged 5X. Cephalometric analysis was performed with the use of a personal computer (Power Book G4, Apple Japan Inc., Tokyo, Japan) according to the modified method of Kiliaridis et al.(1985). Landmarks and measurement items were established for the present study, as depicted in the Fig.

View larger version (19K): [in this window] [in a new window]   Figure. Landmarks and measurement items used for the cephalometric analyses. Po Most posterior point on cranial valut N Point the nasofrontal suture A Most anterior point on nasal bone E Intersection between frontal bone and most superior-anterior point of the posterior limit to the ethmoid bone S Intersection between posterior border of basisphenoid and the tympanic bulla Ba Most posterior and inferior point of occipital condyle Pr Most inferior and anterior point on alveolar process of premaxilla Bu Point on premaxilla between jawbone and lingual surface of upper incisors Mu Point on intersection between maxillary bone and mesial surface of upper first molar Iu Most prominent point between incisal edges of upper incisors U1 Point on mesial occulusal fossa of upper first molar U1' Crossing point on A-N perpendicular to A-N from U1 U2 Point on distal occulusal fossa of upper second molar U2' Crossing point on A-N perpendicular to A-N from U2 Pg Point on most inferior contour of lower border of mandible, adjacent to incisors Mn Point in deepest part of antegonial notch curvature Gn Point on most inferior contour of angular process of mandible Go Most posterior point of angular process of mandible Co Most posterosuperior point of condylar process Co' Crossing point on Pg-Gn perpendicular to Pg-Gn from Co Id Most inferior and anterior point on alveolar process of mandible Bl Point on intersection between lingual surface of lower incisor and anteriormost part of lingual alveolar bone Ml Point on intersection between the mandibular alveolar bone and mesial surface of first molar Il Most prominent point between incisal edges of lower incisor L1 Point on mesial occulusal fossa of lower second molar L1' Crossing point on Pg-Gn perpendicular to Pg-Gn from L1 L2 Point on distal occulusal fossa of lower second molar L2' Crossing point on Pg-Gn perpendicular to Pg-Gn from L2

	RESULTS

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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 17ß-estradiol was also undetectable in OVX mice, whereas the concentration was 24.3 ± 10.1 pg/mL in female sham-operated mice.

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.

	DISCUSSION

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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.

	ACKNOWLEDGMENTS

 
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 March 27, 2003; Last revision December 21, 2003; Accepted January 14, 2004

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