Gender- and compartment-specific bone loss in C57BL/6J mice: correlation to season?

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.

Gender-specific changes in bone turnover and skeletal architecture in igfbp-2-null mice.

IGF-binding protein-2 (IGFBP-2) is a 36-kDa protein that binds to the IGFs with high affinity. To determine its role in bone turnover, we compared Igfbp2(-/-) mice with Igfbp2(+/+) colony controls. Igfbp2(-/-) males had shorter femurs and were heavier than controls but were not insulin resistant. Serum IGF-I levels in Igfbp2(-/-) mice were 10% higher than Igfbp2(+/+) controls at 8 wk of age; in males, this was accompanied by a 3-fold increase in hepatic Igfbp3 and Igfbp5 mRNA transcripts compared with Igfbp2(+/+) controls. The skeletal phenotype of the Igfbp2(-/-) mice was gender and compartment specific; Igfbp2(-/-) females had increased cortical thickness with a greater periosteal circumference compared with controls, whereas male Igfbp2(-/-) males had reduced cortical bone area and a 20% reduction in the trabecular bone volume fraction due to thinner trabeculae than Igfbp2(+/+) controls. Serum osteocalcin levels were reduced by nearly 40% in Igfbp2(-/-) males, and in vitro, both CFU-ALP(+) preosteoblasts, and tartrate-resistant acid phosphatase-positive osteoclasts were significantly less abundant than in Igfbp2(+/+) male mice. Histomorphometry confirmed fewer osteoblasts and osteoclasts per bone perimeter and reduced bone formation in the Igfbp2(-/-) males. Lysates from both osteoblasts and osteoclasts in the Igfbp2(-/-) males had phosphatase and tensin homolog (PTEN) levels that were significantly higher than Igfbp2(+/+) controls and were suppressed by addition of exogenous IGFBP-2. In summary, there are gender- and compartment-specific changes in Igfbp2(-/-) mice. IGFBP-2 may regulate bone turnover in both an IGF-I-dependent and -independent manner.

Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice.

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. Furthermore, 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 168-172 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.

Bone quality and bone strength in BXH recombinant inbred mice.

The relationship between bone quality and strength was studied in 11 BXH recombinant inbred (RI) strains of mice. The bone quality parameters studied were bone mineralization, microhardness, architecture, and connectivity. Previous studies have demonstrated considerable variability in bone density, biomechanical properties, and microstructure among inbred strains of mice. In particular, C3H/HeJ (C3H) mice exhibit thicker femoral and vertebral cortices and fewer trabeculae in the vertebral body compared with C57BL/6J (B6) mice, despite having similar vertebral bone strength. A set of RI mouse strains has been generated from B6 and C3H (denoted BXH) in an attempt to isolate genetic regulation of numerous traits, including bone. The objective of this study was to investigate relationships among bone quality and bone strength in femurs and vertebrae among BXH RI mice. The study involved 11 BXH RI strains of female mice (n = 5-7) as well as the B6 and C3H progenitor strains. Parameters contributing to bone quality were evaluated, including BMD, bone mineralization, microhardness, architecture, and connectivity. There was a strong correlation between femoral and vertebral BMD in all strains (P < 0.001) except in BXH-9 and -10 (P < 0.001). Within the vertebrae, cortical bone was more mineralized than trabecular bone, and a strong correlation existed between the two (P < 0.001). However, cortical microhardness did not differ from trabecular microhardness. Cortical bone was more mineralized in the femur than in the vertebrae and significantly harder, by 30%. There was a wide range in trabecular connectivity, architecture, and femur geometry among BXH RI strains. BMD explained 43% of vertebral bone strength but only 11% of femoral bone strength. Trabecular connectivity explained an additional 8% of vertebral strength, while mineralization and femur geometry explained 7% and 50% of femoral strength, respectively. Different bone quality parameters had varying influences on bone mechanical properties, depending on bone site. BMD may play a larger role in explaining bone strength in the vertebrae than in the femur. Moreover, cortical bone in the femur is harder than in vertebrae. The control of cortical bone material properties may be site-dependent.

Detecting novel bone density and bone size quantitative trait loci using a cross of MRL/MpJ and CAST/EiJ inbred mice.

Most previous studies to identify loci involved in bone mineral density (BMD) regulation have used inbred strains with high and low BMD in generating F(2) mice. However, differences in BMD may not be a requirement in selecting parental strains for BMD quantitative trait loci (QTL) studies. In this study, we intended to identify novel QTL using a cross of two strains, MRL/MpJ (MRL) and CAST/EiJ (CAST), both of which exhibit relatively high BMD when compared to previously used strains. In addition, CAST was genetically distinct. We generated 328 MRL x CAST F(2) mice of both sexes and measured femur BMD and periosteal circumference (PC) using peripheral quantitative computed tomography. Whole-genome genotyping was performed with 86 microsatellite markers. A new BMD QTL on chromosome 10 and another suggestive one on chromosome 15 were identified. A significant femur PC QTL identified on chromosome 9 and a suggestive one on chromosome 2 were similar to those detected in MRL x SJL. QTL were also identified for other femur and forearm bone density and bone size phenotypes, some of which were colocalized within the same chromosomal positions as those for femur BMD and femur PC. This study demonstrates the utility of crosses involving inbred strains of mice which exhibit a similar phenotype in QTL identification.

Congenic mice provide in vivo evidence for a genetic locus that modulates serum insulin-like growth factor-I and bone acquisition.

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.

Early neoplastic and metastatic mammary tumours of transgenic mice detected by 5-aminolevulinic acid-stimulated protoporphyrin IX accumulation.

A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg(-1)) was administered to mouse strains that develop mammary tumours of various histological subtypes upon expression of the transgenic oncogenes HRAS, Polyoma Virus middle T antigen, or Simian Virus 40 large T antigen in the mammary gland. Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380-440 nm). Detection of mouse mammary tumours at the stage of ductal carcinoma in situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients.

Congenic mice reveal sex-specific genetic regulation of femoral structure and strength.

Genetic linkage studies in C3H/HeJ (C3H) and C57BL/6J (B6) mice identified several chromosomal locations or quantitative trait loci (QTL) linked to femoral volumetric bone mineral density (vBMD). From QTL identified on chromosomes (chr) 1, 4, 6, 13, and 18, five congenic mouse strains were developed. In each of these mice, genomic DNA from the QTL region of the donor C3H strain was transferred into the recipient B6 strain. Here we report the effects of donated C3H QTL on femoral structure, cortical vBMD and bending strength. Femoral structure was quantified by the polar moment of inertia (Ip) at the mid-diaphysis, which reflects the bending or torsional rigidity of the femur. Although the C3H progenitor mice have a smaller Ip than B6 progenitor mice, the congenic mice carrying the C3H segment at Chr 4 had significantly increased Ip in both males and females, giving these mice stronger femora. In female mice from the congenic Chr 1 strain, Ip was increased whereas male mice from the Chr 1 strain had smaller femoral cross-sections and significantly reduced Ip. This sex-specific effect on femoral structure was seen to a lesser extent in Chr 18 congenic mice. In addition, cortical vBMD was measured using peripheral quantitative computed tomography. Cortical vBMD was similar among most congenic strains except in Chr 6 congenic mice, where cortical vBMD was significantly less in females, but not in males. We conclude that (1) chromosomal QTL from C3H mice, which are genetically linked to total femoral vBMD, also regulate femoral structure; (2) the QTL on Chr 4 improves femoral structure and strength; (3) QTL on Chr 1 and 18 impart sex-specific effects on femoral structure; and (4) the QTL on Chr 6 imparts a sex-specific effect on cortical vBMD and femoral strength.

IGF-I regulates osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand in vitro and OPG in vivo.

IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 micro g/LIGF-I (13 nM) decreased OPG expression by 37.0 +/- 1.8% (P < 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 micro g/liter ( approximately 6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1-7 ng/ml at 48 h (P < 0.05). Similarly, IGF-I at 100 micro g/liter (13 nM) increased RANKL mRNA expression to 353 +/- 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18-26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 micro g/kg.d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39-45 nM) and a 20% reduction in serum OPG (P < 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone.

Construction of a BAC contig for a 3 cM biologically significant region of mouse chromosome 1.

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.

Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice.

The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice.

Genetics and bone. Using the mouse to understand man.

The rationale for use of inbred strains of mice in bone research is well recognized and includes: a) practical factors (economics of scale, rapid development of adult status, pre-existing knowledge, down-sized technologies) and b) proven methodologies for genetic studies (polygenic trait analyses, mapping tools, genomic sequencing, methods for gene manipulation). Initial investigations of inbred strains of mice showed that femoral and lumbar vertebral volumetric bone mineral density (BMD, mg/mm(3)) by pQCT varied in excess of 50% for femurs and 9% in vertebral BMD. Two strains - low BMD C57BL/6J (B6) mice and high BMD C3H/HeJ (C3H) - were investigated for insights to their BMD diversity. B6C3F2 females derived from intercrossing B6C3F1s were raised to adult skeletal status at 4 months, then necropsied for phenotyping of bone and genotyping of genomic DNA. 1000 F2 females were genotyped for PCR product polymorphisms on all 19 autosomes at approximately 15 cM. Genome wide analyses for genotype-phenotype correlations showed 10 chromosomes (Chrs) carried genes for femoral and 7 Chrs for vertebral BMD. LOD scores ranged from 2.90 to 24.4, and percent of F2 variance accounted for ranged from 1 to 10%. Analyses of main effects revealed both dominant-recessive and additive inheritance patterns. Both progenitor strains carried alleles with positive and negative effects on BMD of each bone sites. A remarkable array of additonal skeletal phenotypes (femur and vertebral geometry, strength measures, serum markers) also proved polygenic in nature, with complex segregation patterns. Verification of BMD quantitative trait loci (QTLs) was undertaken by creating congenic B6 strains carrying individual QTL regions from C3H. Following 6 cycles of backcrossing a QTL-containing region from C3H to the B6 strain, N6F2 congenic strain mice were aged to 4 months, then genotyped for the QTL region and phenotyped for skeletal traits. Comparison of mice homozygous for C3H alleles versus homozygous for B6 alleles in the QTL regions showed that femoral BMD increased or decreased significantly in congenic strains, as was predicted from F2 data. Gender differences specific to BMD QTLs have been revealed, as have more than 30 additional phenotypes associated with cortical and trabecular structural parameters and biomechanical properties.

Is skeletal mechanotransduction under genetic control?

Studies of twins have established that peak bone mass is about 70% heritable. The skeletal response to exercise contributes to peak bone mass, as mechanical loading increases skeletal mass during growth and development. It is possible that the skeletal responsiveness to mechanical loading is under genetic control, so that some individuals will build stronger bones with exercise. This appears to be the case in mice. Long bones in mice of the C3H/He strain are largely unresponsive to mechanical loading. Ironically, this strain of mice has very high bone density. Perhaps the genes that regulate BMD are not the same as those that regulate mechanical loading response. Studies of recombinant inbred and congenic strains derived from C3H mice will help to identify genes influencing bone size, density and responsiveness to mechanical loading.

Mouse genetic model for bone strength and size phenotypes: NZB/B1NJ and RF/J inbred strains.

The relationships of bone size, bone strength, and bone formation were investigated in two strains of mice, NZB/B1NJ and RF/J. Measurement of the femur midshaft size by peripheral quantitative computed tomography (pQCT) showed that the RF/J mice had a 32% greater cross-sectional area than NZB/B1NJ mice at 10 weeks of age, and a 38% greater cross-sectional area at 22 weeks of age. Body weight in the RF/J mice was 10% higher at 10 weeks but 9% lower at 22 weeks. Bone strength was determined by a three-point bending method. In agreement with the difference in bone cross-sectional area, the femurs of the RF/J mice were stronger (80% greater) and stiffer (80% greater) than the bones of the NZB/B1NJ mice. To determine whether periosteal bone formation played a role in the greater size of the RF/J mice, the mice were injected with tetracycline to label areas of new bone formation. Histomorphometrical analysis of the femur diaphysis demonstrated higher rates of periosteal bone formation (131% greater) and of periosteal forming surface (81% greater) in RF/J than in NZB/B1NJ mice. We conclude that a high rate of periosteal bone formation increases bone size and strength in RF/J mice when compared with NZB/B1NJ mice. The NZB/B1NJ and RF/J mice should be an excellent model to investigate the genes that regulate femur size and strength.

Melanocyte and gonad activity as potential severity modifying factors in C3H/HeJ mouse alopecia areata.

Circumstantial evidence has previously suggested gonad derived steroid hormones and melanogenesis related antigens may modify human alopecia areata (AA). AA-like hair loss can be induced in C3H/HeJ mice after skin allografts from spontaneous AA-affected mice. This inducible model was used to evaluate hormones and hair follicle melanocyte presence as disease-severity modifiers. Ten females and 9 males were gonadectomized and received AA-affected allografts. All gonadectomized mice had 2-4 weeks delay in AA onset relative to non-gonadectomized controls. Two females and 4 males failed to develop any AA by 25 weeks after grafting. The experiment was repeated with gonadectomized female and male mice plus non-gonadectomized mice subcutaneously implanted with silastic capsules containing 80 microg 17beta estradiol or 10 mg 5alpha dihydrotestosterone, respectively. Five of 11 ovariectomized and 9 of 11 non-ovariectomized, estradiol supplemented females developed AA with extremely rapid progression. Three of 8 castrated, but none of 11 non-castrated, dihydrotestosterone-supplemented males expressed AA. In a separate study, 14 mice were freeze-branded, producing white hair on the dorsal lumbar region, and later received full-thickness allografts. Thirteen mice developed patchy pigmented and non-pigmented hair loss. One mouse developed diffuse, pigmented hair loss, but with white hair survival persisting 25 weeks after grafting. The results suggest that gonadal steroid hormones can modulate C3H/HeJ mouse AA where estradiol promoted rapid progression of AA while dihydrotestosterone increased resistance to AA onset. In general, both pigmented and non-pigmented C3H/HeJ mouse hair is susceptible to AA. Murine AA susceptibility and severity clearly involves an interplay between genetic and epigenetic factors.

Defining the genetics of osteoporosis: using the mouse to understand man.

Very low bone mineral density (BMD) is now considered as diagnostic of osteoporosis. Moreover, many women who are osteopenic eventually develop osteoporotic fractures. Hence, bone density testing has occupied center stage in the diagnosis and treatment of this disorder. In addition, over the last several years, BMD has been utilized as the phenotype of choice for defining heritable markers for osteoporotic fractures. However, genetic studies in humans have been limited to some degree by the tremendous heterogeneity among populations, as well as multiple genetic, heritable and environmental determinants of the BMD phenotype. Recent advances in technology have afforded investigators the opportunity to study acquisition and maintenance of BMD in small animals. Along with newer knockout and transgenic strategies, quantification of mouse bone mass has advanced our understanding of both the biologic and genetic determinants of bone density. In this review, we will examine the use of the mouse to map the heritable factors that regulate bone acquisition. We will also examine the role of newer technology to decompose the bone density phenotype into components that are amenable to genetic studies. This review will focus on three models: (1) healthy inbred (2) recombinant inbred, and (3) congenic strains of mice. Progress in this area with these strains has been rapid, and a summary of several quantitative trait loci (QTLs) is provided. The future of the mouse as a tool to map the genes that define the osteoporosis syndrome is extremely promising.

Quantitative trait loci for femoral and lumbar vertebral bone mineral density in C57BL/6J and C3H/HeJ inbred strains of mice.

Significant differences in vertebral (9%) and femoral (50%) adult bone mineral density (BMD) between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice have been subjected to genetic analyses for quantitative trait loci (QTL). Nine hundred eighty-six B6C3F2 females were analyzed to gain insight into the number of genes that regulate peak BMD and their locations. Femurs and lumbar vertebrae were isolated from 4-month-old B6C3F2 females at skeletal maturity and then BMD was determined by peripheral quantitative computed tomography (pQCT). Estimates of BMD heritability were 83% for femurs and 72% for vertebrae. Genomic DNA from F2 progeny was screened for 107 polymerase chain reaction (PCR)-based markers discriminating B6 and C3H alleles on all 19 autosomes. The regression analyses of markers on BMD revealed ten chromosomes (1, 2, 4, 6, 11, 12, 13, 14, 16, and 18) carrying QTLs for femurs and seven chromosomes (1, 4, 7, 9, 11, 14, and 18) carrying QTLs for vertebrae, each with log10 of the odds ratio (LOD) scores of 2.8 or better. The QTLs on chromosomes (Chrs) 2, 6, 12, 13, and 16 were unique to femurs, whereas the QTLs on Chrs 7 and 9 were unique to vertebrae. When the two bone sites had a QTL on the same chromosome, the same marker had the highest, although different, LOD score. A pairwise comparison by analysis of variance (ANOVA) did not reveal significant gene x gene interactions between QTLs for either bone site. BMD variance accounted for by individual QTLs ranged from 1% to 10%. Collectively, the BMD QTLs for femurs accounted for 35.1% and for vertebrae accounted for 23.7 % of the F2 population variances in these bones. When mice were homozygous c3/c3 in the QTL region, 8 of the 10 QTLs increased, while the remaining two QTLs on Chrs 6 and 12 decreased, femoral BMD. Similarly, when mice were homozygous c3/c3 in the QTL region for the vertebrae, five of the seven QTLs increased, while two QTLs on Chrs 7 and 9 decreased, BMD. These findings show the genetic complexity of BMD with multiple genes participating in its regulation. Although 5 of the 12 QTLs are considered to be skeleton-wide loci and commonly affect both femurs and vertebrae, each of the bone sites also exhibited unique QTLs. Thus, the BMD phenotype can be partitioned into its genetic components and the effects of these loci on normal bone biology can be determined. Importantly, the BMD QTLs that we have identified are in regions of the mouse genome that have known human homology, and the QTLs will become useful experimental tools for mechanistic and therapeutic analyses of bone regulatory genes.

Effects of dietary restriction on appendicular bone in the SENCAR mouse.

Peptide hormones, cytokines, and growth factors regulate cellular metabolism by stimulating second messenger signal transduction cascades in target tissues. A mutation in the regulatory domain of protein kinase C (PKC) in SENCAR (sensitive to carcinogenesis) mice renders them extremely sensitive to diacylglycerol and phorbol esters, resulting in rapid growth, high free radical generation, carcinogenesis, and metabolic bone disease. Dietary restriction (DR) normalizes PKC and ameliorates adverse downstream effects, including carcinogenesis, in SENCAR mice. We hypothesized that DR sufficient to ameliorate carcinogenesis would prevent or delay the early onset of metabolic bone disease in SENCAR mice. Male mice were assigned to 1 of 4 feeding groups from 10 to 16 weeks of age (the critical period when metabolic bone disease develops): ad libitum (AL)-fed; AL antioxidant (0.07% thioproline)-fed; 40% DR; or 40% DR antioxidant-fed. Femoral bone mass was determined gravimetrically. Tibial total, cortical, and trabecular bone mineral density (BMD) were determined by quantitative computed tomography. Body weight, femoral bone mass, and tibial cortical BMD were lower in DR than in AL mice. However, tibial total and trabecular BMD were higher in DR than in AL mice. Serum calcitonin, the hormone that inhibits the osteoclastic bone resorption that is most notable in trabecular bone, was 2-fold higher in DR than in AL-fed mice. Dietary thioproline had no major effects. Thus, DR sufficient to ameliorate carcinogenesis in SENCAR mice did not prevent early-onset metabolic bone disease, but it had a beneficial effect on tibial trabecular BMD that occurred at the apparent expense of cortical BMD. DR in SENCAR mice was also associated with elevated serum calcitonin, which may inhibit osteoclastic resorption and account for trabecular bone conservation in this model. In conclusion, PKC or the downstream metabolic processes regulated by it appear to play previously unrecognized roles in the regulation of tibial trabecular BMD and serum calcitonin in SENCAR mice.

The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages.

Our purpose in this investigation was to determine if we could reduce cage changing frequency without adversely affecting the health of mice. We housed mice at three different cage changing frequencies: 7, 14, and 21 days, each at three different cage ventilation rates: 30, 60 and 100 air changes per hour (ACH), for a total of nine experimental conditions. For each condition, we evaluated the health of 12 breeding pairs and 12 breeding trios of C57BL/6J mice for 7 months. Health was assessed by breeding performance, weanling weight and growth, plasma corticosterone levels, immune function, and histological examination of selected organs. Over a period of 4 months, we monitored the cage microenvironment for ammonia and carbon dioxide concentrations, relative humidity, and temperature one day prior to changing the cage. The relative humidity, carbon dioxide concentrations, and temperature of the cages at all conditions were within acceptable levels. Ammonia concentrations remained below 25 ppm (parts per million) in most cages, but, even at higher concentrations, did not adversely affect the health of mice. Frequency of cage changing had only one significant effect; pup mortality with pair matings was greater at the cage changing frequency of 7 days compared with 14 or 21 days. In addition, pup mortality with pair matings was higher at 30 ACH compared with other ventilation rates. In conclusion, under the conditions of this study, cage changes once every 14 days and ventilation rates of 60 ACH provide optimum conditions for animal health and practical husbandry.

Variation in bone biomechanical properties, microstructure, and density in BXH recombinant inbred mice.

To test the hypothesis that factors associated with bone strength (i.e., volumetric bone mineral density [vBMD], geometry, and microstructure) have heritable components, we exploited the 12 BXH recombinant inbred (RI) strains of mice derived from C57BL/6J (B6; low bone mass) and C3H/HeJ (C3H; high bone mass) progenitor strains. The femurs and lumbar vertebrae from each BXH RI strain were characterized for phenotypes of vBMD, microstructural, biomechanical, and geometrical properties. Methods included bending (femur) and compression (vertebra) testing, peripheral quantitative computed tomography (pQCT), and microcomputed tomography (microCT). Segregation patterns of femoral and vertebral biomechanical properties among the BXH RI strains suggested polygenic regulation. Femoral biomechanical properties were strongly associated with femoral width in the anteroposterior (AP) direction and cortical thickness--geometric properties with complex genetic regulation. Vertebral vBMD and biomechanical properties measured in BXH RI strains showed a greater variability than either B6 or C3H progenitors, suggesting both progenitor strains have independent subsets of genes that yield similar vBMD and strength. The microCT and pQCT data suggested that the distribution of vertebral mineral into cortical and trabecular compartments is regulated genetically. Although the B6 and C3H progenitors had similar vertebral strength, their vertebral structures were markedly different: B6 had good trabecular bone structure and modest cortical bone mineral content (BMC), whereas C3H had high cortical BMC combined with a deficiency in trabecular structure. These structural traits segregated independently in the BXH RI strains. Finally, vertebral strength was not correlated consistently with femoral strength among the BXH RI strains, suggesting genetic regulation of bone strength is site specific.