An osteocalcin-deficient mouse strain without endocrine abnormalities

Abstract

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/2p.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, μCT 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/2pPro25fs17Ter 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.

Fig 1. Generation and validation of a Bglap and Bglap2 double-knockout allele (Bglap/2^dko) created by CRISPR/Cas9 gene editing.

(A)Top: Schematic (not to scale) showing the 25-kb interval on mouse chromosome 3 containing Bglap, Bglap2, and Bglap3. The locations of the guide RNAs used to produce founders with Bglap and Bglap2 intragenic or intergenic knockout alleles are indicated (arrows). Bottom, left: The Bglap/2 double-knockout (dko) allele (p.Pro25fs17Ter) deletes a 6.8-kb DNA fragment extending from Bglap exon 2 to Bglap2 exon 4. Bottom, right: A 5-bp insertion (5 bp ins) at the DNA ligation site creates a chimeric exon with a reading frame shift. The frame shift occurs after p.25 and terminates the protein 17 residues downstream (orange residues indicate wild-type sequence; blue indicates the first 4 frame-shifted residues). (B) Integrated Genomics Viewer screenshots of RNA sequencing data from wild-type and homozygous Bglap/Bglap2 double-knockout mice (Bglap/2^dko/dko). Note the absence of sequencing reads mapping to Bglap exons 2, 3, and 4, and Bglap2 exons 1, 2, and 3 in the double-knockout mice; the mapping algorithm fails to map reads to the Bglap2 chimeric exon 2 because the chimera has a short seed length and a 5-bp insertion. (C) Serum ELISA assays for male and female 6-month-old Bglap/2^dko/dko (green) and their control (black) littermates for the gamma-carboxyglutamic acid (Gla) and uncarboxylated (Glu) forms of OCN as well as osteopontin (OPN). In the box and whisker plots, each box extends from the 25th to 75th percentiles, the line represents the median, and the whiskers extend to the minimum and maximum values. The following sample sizes were used: male wild-type (n = 5), male Bglap/2^dko/dko (n = 5), female wild-type (n = 7), and female Bglap/2^dko/dko (n = 8).

Fig 2. OCN deficiency does not signficantly alter bone mass or strength as assessed by μCT and biomechanical testing.

In the interleaved box and whisker plots, each box extends from the 25th to 75th percentiles, the line represents the median, and the whiskers extend to the minimum and maximum values. (A) μCT analysis of trabecular bone parameters indicated no significant differences between male or female 6-month-old Bglap/2^dko/dko (green) and their control (black) littermates. Trabecular BV/TV, trabecular number, thickness, and separation are shown. Additional measurements are included in Table 1. The following sample sizes were used for both cortical and trabecular measurements: male wild-type (n = 10), male Bglap/2^dko/dko (n = 12), female wild-type (n = 11), and female Bglap/2^dko/dko (n = 11). (B) μCT analysis of cortical bone parameters indicated no significant differences between male or female 6-month-old Bglap/2^dko/dko and their control littermates. Cortical BMD, cortical area fraction, tissue area, and cortical thickness are shown as representative measurements. Additional measurements and details are included in Table 1, and sample sizes are as in panel A. (C) Biomechanical loading assessments indicated no significant differences between male or female 6-month-old Bglap/2^dko/dko and their control littermates. Ultimate force, energy to ultimate force, and stiffness are shown as representative measurements. Additional measurements and details are included in S1 Table and S2 Table. The following sample sizes were used: male wild-type (n = 12), male Bglap/2^dko/dko (n = 12), female wild-type (n = 18), and female Bglap/2^dko/dko (n = 14).

Fig 3. FTIR showed differences in carbonate/mineral ratio and collagen maturity between Bglap/2^dko/dko and wild-type mice.

FTIR images of cortical bone showing the spatial distribution of the variables in wild-type (n = 3) and Bglap/2^dko/dko (n = 4) female mice. Representative images show carbonate-to-mineral ratio and collagen maturity. Additional measurements and details are included in S3 Table.

Fig 4. No significant evidence that whole body weight, random-fed glucose levels, or fasting glucose values differ between Bglap/2^dko/dko and wild-type mice.

(A) Total weight for 6-month-old Bglap/2^dko/dko and wild-type males and females. Sample sizes were male wild-type (n = 11), male Bglap/2^dko/dko (n = 11), female wild-type (n = 11), and female Bglap/2^dko/dko (n = 8). (B) Five- to six-month-old female wild-type (n = 5) and Bglap/2^dko/dko (n = 5) mice were sampled for blood glucose concentration. Samples were taken on four consecutive days, 6 h into their light cycle while having ad libitum access to food. For each mouse, at least two glucose measurements were taken each day and averaged. Means and standard deviations for each genotype are shown. (C) The animals in panel 4B were then fasted for 16 h before blood glucose was again assessed. Samples were collected on two occasions approximately one week apart. At least two glucose measurements were taken on each day and averaged. The data display the means and standard deviations for each genotype. (D) After an overnight fast and at the time of euthanasia, blood glucose was measured in 6-month-old wild-type and Bglap/2^dko/dko males and females. Sample sizes are male wild-type (n = 11), male Bglap/2^dko/dko (n = 11), female wild-type (n = 11), and female Bglap/2^dko/dko (n = 8).

Fig 5. No significant evidence of fertility being affected in Bglap/2^dko/dko mice.

In the interleaved box and whisker plots, each box extends from the 25th to 75th percentiles, the line represents the median, and the whiskers extend to the minimum and maximum values. (A) The number of pups per litter and the percentage of litters per plug (n = 11 for wild-type and n = 12 for Bglap/2^dko/dko total matings for each genotype) resulting from crosses of Bglap/2^dko/dko males or their wild-type control littermates to control C57Bl/6J females. (B) Dry testis weight expressed as mg/g total body weight for wild-type (n = 4) and Bglap/2^dko/dko (n = 6) males. (C) Testis volume expressed as mm³/g of total body weight for wild-type (n = 4) and Bglap/2^dko/dko (n = 6) males. (D) Blood testosterone concentration from 6-month-old, virgin wild-type (n = 9) and littermate Bglap/2^dko/dko males (n = 6). (E) Blood testosterone concentration (ng/mL) from 10- to 15-week-old, virgin males. Analysis of wild-type (n = 7) and Bglap/2^dko/dko (n = 8) littermates shown at two different collection times for each animal (* represents p > 0.05). (F) Values from panel E plotted to show the change in testosterone of each individual from day 0 (●) to day 3 (■).