Seasonal variation in bone mineral density (BMD) has been documented in humans, and has been attributed to changes in 25-hydroxyvitamin D [25(OH)D] synthesis. To test the hypothesis that seasonal changes in bone mass occur in laboratory mice, we measured body composition, femoral bone phenotypes, and serum bone markers in 16-wk-old male and female C57BL/6 (B6) mice during the summer (June–August) and winter (December–February) months at The Jackson Laboratory in Bar Harbor, Maine. Both male and female B6 mice had higher volumetric BMD in the summer than winter. Females showed reduced trabecular bone, whereas males showed changes in bone volume. Males, but not females, had higher insulin-like growth factor 1 in summer than in winter, and only males showed an increase in body weight during the winter. No seasonal differences in serum TRAP5b, osteocalcin, or 25(OH)D were noted for either sex. We conclude that seasonal variation in skeletal and body composition parameters in B6 mice is significant and must be considered when performing longitudinal phenotyping of the skeleton. Further studies are needed to determine the environmental factors that cue seasonal changes in body composition and the mechanisms that produce these changes.

The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Further-more, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F= 16.49, P=0.0002), while no significant effect was found on total femur BMD (F= 2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice. (C) 2007 Elsevier Inc. All rights reserved.

We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6. C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i. e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.

Mice of the inbred mouse strain C3H/HeJ have been shown to be homozygous for a chromosomal inversion on Chromosome (Chr) 6. The inversion encompasses about 20% of the chromosome from ∼73 Mb to ∼116 Mb. The importance of this finding is that linkage crosses using C3H/HeJ will show no recombination in this region of Chr 6. The inversion has no apparent effect on the phenotype of C3H/HeJ mice and its presence should not affect biological studies; however, use of C3H/HeJ mice for genetic analysis of Chr 6 should be avoided or the results interpreted with the inversion in mind. The inversion has been named In(6)1J (inversion Chr 6, Jackson 1).

UNLABELLED:Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively. INTRODUCTION:BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1). MATERIALS AND METHODS:After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines. RESULTS:Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (-6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = -0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb. CONCLUSIONS:Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.

There is a subset of women who experience particularly rapid bone loss during and after the menopause. However, the factors that lead to this enhanced bone loss remain obscure. We show that patterns of bone loss after ovariectomy vary among inbred strains of mice, providing evidence that there may be genetic regulation of bone loss induced by estrogen deficiency.Introduction: Both low BMD and increased rate of bone loss are risk factors for fracture. Bone loss during and after the menopause is influenced by multiple hormonal factors. However, specific determinants of the rate of bone loss are poorly understood, although it has been suggested that genetic factors may play a role. We tested whether genetic factors may modulate bone loss subsequent to estrogen deficiency by comparing the skeletal response to ovariectomy in inbred strains of mice.Materials and Methods: Four-month-old mice from five inbred mouse strains (C3H/HeJ, BALB/cByJ, CAST/EiJ, DBA2/J, and C57BL/6J) underwent ovariectomy (OVX) or sham-OVX surgery (n = 6-9/group). After I month, mice were killed, and mu CT was used to compare cortical and trabecular bone response to OVXResults: The effect of OVX on trabecular bone varied with mouse strain and skeletal site. Vertebral trabecular bone volume (BV/TV) declined after OVX in all strains (-15 to -24%), except for C3H/HeJ. In contrast, at the proximal tibia, C3H/HeJ mice had a greater decline in trabecular BV/TV (-39%) than C57BL/6J (-18%), DBA2/J (-23%), and CAST/EiJ mice (-21%). OVX induced declines in cortical bone properties, but in contrast to trabecular bone, the effect of OVX did not vary by mouse strain. The extent of trabecular bone loss was greatest in those mice with highest trabecular BV/TV at baseline, whereas cortical bone loss was lowest among those with high cortical bone parameters at baseline.Conclusions: We found that the skeletal response to OVX varies in a site- and compartment-specific fashion among inbred mouse strains, providing support for the hypothesis that bone loss during and after the menopause is partly genetically regulated.

The spontaneous development of juvenile-onset ovarian granulosa cell tumors in mice of the SWXJ-9 recombinant inbred strain is a model for juvenile-type granulosa cell tumors that appear in very young girls. To expedite gene discovery in this mouse model of childhood cancer, we did a gene mapping study with the SWXJ-9 recombinant inbred strain and the evolutionarily divergent Mus musculus castaneus (CAST/Ei) strain as a mapping partner. Our mapping strategy focused on autosomal determinants of susceptibility with a backcross scheme that exploited a paternal, parent-of-origin effect for a X-linked gene (Gct4) that strongly supports granulosa cell tumor development. Of 1,968 backcross females examined, we detected 81 granulosa cell tumor-bearing animals and compared their allelic inheritance patterns to non-tumor-bearing siblings in a case-control analysis. The results of our study have confirmed an important locus on mouse chromosome (Chr) 4 (Gct1) and have revealed new loci for granulosa cell tumor susceptibility (Gct7-Gct9) on Chrs 1, 2, and 13 with susceptibility alleles contributed by the SWXJ-9 progenitor. Two novel gene-gene interactions supportive for granulosa cell tumor development were also observed between loci on Chrs 17 and 18 and loci on Chrs 2 and 10. Our data substantiate the evidence that Gct1 on Chr 4 is a fundamental oncogene for granulosa cell tumorigenesis in mice and has identified additional interacting autosomal loci that support tumor development.

Targeted gene studies have demonstrated the importance of insulin-like growth factor-I (IGF-I) for osteoblast (OB) differentiation and the acquisition of peak bone mineral density (BMD). The skeletal response to allelic differences in IGF-I expression can also be measured in vivo, using congenic mice. We created a congenic strain with reduced (approximately 20%) circulating IGF-I (C3H.B6-6T [6T]) by backcrossing a small genomic region (30 cM) of Chromosome 6 (Chr6) from C3H/HeJ (C3H) onto a C57Bl/6J (B6) background. 6T female mice have lower serum IGF-I (P < 0.001 vs. B6) but similar growth hormone (GH) and serum IGF binding protein (IGFBP) concentrations as B6. At 16 weeks of age, congenics have greater body fat (P < 0.02 vs. B6) despite less total body weight, and exhibit smaller femoral cross-sectional size (P = 0.001), reduced cortical thickness (P < 0.001) and lower trabecular BV/TV (P < 0.05) than B6. 6T mice also have suppressed serum leptin (P < 0.01), but compared to B6 have similar markers of bone resorption (i.e., urine CTx and serum TRAP 5B). At 8 weeks of age, skeletal IGF-I mRNA from long bones was reduced by 40% (P < 0.05) as were liver mRNA transcripts (i.e., 50%, P < 0.01). Osteoblast progenitors from the bone marrow of 6T mice formed less colony forming unit fibroblasts by crystal violet staining than B6 (P < 0.007) and had significantly reduced alkaline phosphatase-positive colonies than B6(P < 0.0001). In addition, staining of bone marrow with oil red O revealed greater numbers of adipocytes in 6T than B6. Several candidate genes in the Chr6 QTL were excluded by lack of strain-related expression differences in bone, but genes positively regulating adipocyte differentiation including Alox 5 and PPAR-γ require further study as either "pathway" or candidate genes. In summary, allelic differences in a QTL on Chr6 result in altered IGF-I gene expression, changes in OB lineage allocation, and reduced peak bone mass. Congenic mice are useful models not only for mapping genes related to bone mass but also for elucidating the biology underlying various skeletal phenotypes associated with more subtle manipulation of the mouse genome.