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Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of “osteocyte mechanosomes” - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca(2+) channels clustered around α(v)β(3) integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca(2+) indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca(2+) signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca(2+) signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca(2+) signaling osteocytes and loading was unchanged at 2 days post-OVX. However, it was altered markedly at 28 days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca(2+) signaling) osteocytes; however, at higher strains the numbers of Ca(2+) signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of α(v)β(3) foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for α(v)β(3) and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.