An osteocalcin-deficient mouse strain without endocrine abnormalities

Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc(-)) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2(p.Pro25fs17Ter)) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, mu CT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc(-)allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2(pPro25fs17Ter) allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution. Author summary Cells that make and maintain bone express proteins that function either locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton, while the latter, such as fibroblast growth factor 23, enable the skeleton to communicate with other organ systems. Mutations that affect the functions of most bone-cell-expressed proteins cause diseases that have similar features in humans and other mammals such as mice, for example, brittle bone diseases for type 1 collagen mutations and hypophosphatemic rickets for mutations in fibroblast growth factor 23. Our study focuses on another bone-cell-expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as a hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose, male fertility, muscle mass, brain development, behavior, and cognition. We therefore decided to generate a new strain of osteocalcin knockout mice that could also be used to investigate these nonskeletal effects. To our surprise, the osteocalcin knockout mice we created did not significantly differ from wild-type mice for the three phenotypes we examined: bone strength, blood glucose, and male fertility. Our data are consistent with findings from osteocalcin knockout rats but are inconsistent with data from the original osteocalcin knockout mice. Because we do not know why our new strain fails to recapitulate the phenotypes previously reported for another knockout mouse stain, we have donated our mice to a public repository so that they can be easily obtained and studied in other academic laboratories.