Effects of 2500, 2900 and 3400 με on proliferation of primary osteoblast-like cells derived from female and male WT (A), <i>Lrp5</i>^−/− (B) and <i>LRP5</i>^G171V (C) mice.

<p>Changes in absolute number of cells between static and strain of both genotypes and genders are shown. Results are mean ± SEM of three independent experiments. Experiments were repeated 3 times. No significant differences at 2500 and 2900 με were observed. ***p<0.001 and *p<0.05 compared with the static control within the gender. (D) The effects of 3400 με on proliferation of primary osteoblast-like cells derived from female and male <i>LRP5</i>^G171V and <i>Lrp5</i>^−/− mice and their WT littermates. Percentage differences between static and strain of both genotypes and genders are shown. Results are the mean ± SEM of three independent experiments. Experiments were repeated 3 times. There were no significant differences between groups.</p

Wnt16 mRNA expression is not affected by unloading in young female mice.

<p>Wnt16 expression was unaffected by unloading for (A) 3, 6, 12 and 24 hours, and after (B) two weeks of unloading in young female mice. However, (C) Sost gene expression was significantly increased after two weeks of unloading. Gene expression was determined by quantitative RT-PCR and normalized relative to β2-microglobulin. Bars represent the mean ± SEM, ** = p<0.01 vs. sham operated control, N = 6. SN = sciatic neurectomy.</p

Percentage of apoptosis in osteoblast-like cells 48 hours after treatment with 0.1% (A), 2.5% (B) and 10% (C) serum.

<p>The TUNEL staining was used to determine the percentage of apoptotic cells in primary osteoblast-like cells derived from male and female WT_HBM, <i>Lrp5</i>^−/− and <i>LRP5</i>^G171V mice. Results are mean ± SEM of three independent experiments. Groups with the same letter are not significantly different.</p

Cell population doubling time of primary osteoblast-like cells derived from female and male <i>Lrp5</i><b>^−/−</b>, <i>LRP5</i><b>^G171V</b> and their WT littermates.

<p>Doubling time in days between 2 and 8 days of culture of primary osteoblast-like cells isolated from female and male <i>Lrp5</i>^−/−, <i>LRP5</i>^G171V and their WT littermates. Cell doubling time were calculated using GraphPad Prism v5.0 software for Windows (GraphPad Software Inc., San Diego, CA) by nonlinear regression (exponential growth equation) analysis.</p

Wnt16 and ERβ expression are increased in cortical bone of ERα depleted female mice.

<p>(A) Wnt16, (B) ERβ, (C) Axin2, (D) OPG, (E) Rankl and (F) the OPG:Rankl ratio were quantified in young ERα depleted female mice and compared to WT mice. Gene expression was determined by quantitative RT-PCR and normalized relative to β2-microglobulin. Bars represent the mean ± SEM. * = p<0.05, ** = p<0.01,*** = p<0.001 vs. WT mice, N = 6.</p

Wnt16 expression is not acutely affected by axial mechanical loading.

<p>(A) Wnt16 expression was unaffected 1, 6, 12 and 24 hours after a single episode of axial mechanical loading. However, (B) Egr2 expression was significantly up-regulated in young female mouse tibias after 1 hour. Gene expression were determined by quantitative RT-PCR and normalized relative to β2-microglobulin. Statistical analysis was by paired t-test. Bars represent the mean ± SEM. *** = p<0.001 vs. non-loaded control, N = 6–10.</p

Wnt16 expression is decreased in femoral cortex of aged mice.

<p>Wnt16 expression was significantly lower in aged compared to young female (A) cortical bone and (B) bone marrow but unaffected by age in (C) kidney. Wnt16 expression was significantly lower in aged compared to young male (D) cortical bone and (E) bone marrow but unaffected by age in (F) kidney. Wnt16 expression was determined by quantitative RT-PCR and normalized relative to β2-microglobulin. Bars represent the mean ± SEM, * = p<0.05, ** = p<0.01 *** = p<0.001 vs. young females, N = 6–8.</p

Ageing does not significantly alter expression of Axin2, OPG, or Rankl, in female mice but cortical bone Rankl expression is lower in aged than young male mice.

<p>(A,E) Axin2, (B,F) OPG, (C,G) Rankl and (D,H) the OPG:Rankl ratio were quantified in cortical bone from young and aged (A-D) female and (F-H) male mice by quantitative RT-PCR and normalized relative to β2-microglobulin. Bars represent the mean ± SEM, * = p<0.05, N = 8.</p

Tamoxifen treatment enhances Wnt16, Opg and Rankl expression in cortical bone of young female mice.

<p>(A) Wnt16, (B) Axin2, (C) OPG and (D) RANKL expression was quantified by qRT-PCR in ovariectomized young female mice treated with vehicle or tamoxifen. Bars represent the mean ± SEM. * = p<0.05, ** = p<0.01 vs. vehicle, N = 8.</p

Estrogen treatment enhances Wnt16 expression in cortical bone of young female mice.

<p>(A) Both a low (gray bars) and a 20 times higher (black bars) dose of 17β-estradiol (E2) restored the uterus weight. (B) Wnt16, (C) Axin2, (D) OPG, (E) Rankl and (F) the OPG:Rankl ratio were determined by qRT-PCR in cortical bone from young female mice subjected to sham surgery or ovariectomized and then treated with vehicle, low dose or high dose of E2. Following qRT-PCR analysis, the sham-operated group was normalized to 100%, as indicated by the horizontal dashed line. Bars represent the mean ± SEM. ** = p<0.01,*** = p<0.001 vs. vehicle, N = 10.</p