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Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Mizuhashi K, Chaya T, Kanamoto T, Omori Y, Furukawa T - PLoS ONE (2015)

Bottom Line: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated.These findings suggest that Obif plays essential roles in the development of multiple tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, Japan; Department of Developmental Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, Japan.

ABSTRACT

Background: Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.

Results: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.

Conclusions: Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

No MeSH data available.


Related in: MedlinePlus

Histological analysis of distal femoral epiphysis of Obif−/− mice.(A-B) Villanueva bone staining of distal femur sections from wild-type and Obif−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and Obif−/− mice (black box) (A). In wild-type and Obif−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm (A) and 20 μm (B). Error bars show the SEM (n = 3).
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pone.0133704.g003: Histological analysis of distal femoral epiphysis of Obif−/− mice.(A-B) Villanueva bone staining of distal femur sections from wild-type and Obif−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and Obif−/− mice (black box) (A). In wild-type and Obif−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm (A) and 20 μm (B). Error bars show the SEM (n = 3).

Mentions: To further investigate the cellular mechanism for how Obif affects bone formation, we carried out bone histomorphometry on male femurs isolated from wild-type and Obif−/− mice at 8 wks (Figs 3A, 3B, and 4A–4R). First, we examined wild-type and Obif−/− epiphyseal cartilage and bone by Villanueva bone staining (Fig 3A and 3B). We detected no significant difference in the thickness of distal femoral growth plates between wild-type and Obif−/− mice (Fig 3A). In addition, osteoblasts and osteoclasts were almost unchanged in number and size between wild-type and Obif−/− mice (Fig 3B, arrowheads for osteoblasts and arrows for osteoclasts). Next, to determine several bone parameters in Obif−/− mice, we performed fluorescence microscopic imaging of isolated bone tissues following the injection of calcein and tetracycline into mice (Fig 4A–4R). Fluorescence imaging of calcein and tetracycline, which bind to calcium incorporated into the new bone, enables us to assess various bone parameters, including bone formation, bone resorption, bone mineralization, and bone volume. The bone formation parameters such as osteoid volume/bone volume (OV/BV) showed no significant difference between wild-type and Obif−/− mice, while osteoblast surface/bone surface (Ob.S/BS) and osteoblast number/bone surface (N.Ob/BS) were slightly reduced in Obif−/− mice compared with wild-type mice, but these were not statistically significant (Fig 4B, 4D, and 4E). Moreover, osteoid volume/osteoid surface (OV/OS) significantly increased in Obif−/− mice (Fig 4C). Bone resorption parameters such as eroded surface/bone surface (ES/BS) and bone resorption rate (BRs.R) showed no significant differences between wild-type and Obif−/− mice. While osteoclast surface/bone surface (Oc.S/BS) and osteoclast number/bone surface (N.Oc/BS) were slightly reduced in Obif−/− mice compared with wild-type mice, these were not statistically significant (Fig 4F–4I). In addition, the bone volume parameters such as bone volume per tissue volume (BV/TV) and trabecular number (Tb.N) were decreased and trabecular separation (Tb.Sp) were increased in Obif−/− mice (Fig 4J–4L). Although trabecular thickness (Tb.Th) was slightly reduced in Obif−/− mice compared with wild-type mice, this was not statistically significant (Fig 4M). This observation agrees with our finding obtained by μCT analysis in a previous study [7].


Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Mizuhashi K, Chaya T, Kanamoto T, Omori Y, Furukawa T - PLoS ONE (2015)

Histological analysis of distal femoral epiphysis of Obif−/− mice.(A-B) Villanueva bone staining of distal femur sections from wild-type and Obif−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and Obif−/− mice (black box) (A). In wild-type and Obif−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm (A) and 20 μm (B). Error bars show the SEM (n = 3).
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pone.0133704.g003: Histological analysis of distal femoral epiphysis of Obif−/− mice.(A-B) Villanueva bone staining of distal femur sections from wild-type and Obif−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and Obif−/− mice (black box) (A). In wild-type and Obif−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm (A) and 20 μm (B). Error bars show the SEM (n = 3).
Mentions: To further investigate the cellular mechanism for how Obif affects bone formation, we carried out bone histomorphometry on male femurs isolated from wild-type and Obif−/− mice at 8 wks (Figs 3A, 3B, and 4A–4R). First, we examined wild-type and Obif−/− epiphyseal cartilage and bone by Villanueva bone staining (Fig 3A and 3B). We detected no significant difference in the thickness of distal femoral growth plates between wild-type and Obif−/− mice (Fig 3A). In addition, osteoblasts and osteoclasts were almost unchanged in number and size between wild-type and Obif−/− mice (Fig 3B, arrowheads for osteoblasts and arrows for osteoclasts). Next, to determine several bone parameters in Obif−/− mice, we performed fluorescence microscopic imaging of isolated bone tissues following the injection of calcein and tetracycline into mice (Fig 4A–4R). Fluorescence imaging of calcein and tetracycline, which bind to calcium incorporated into the new bone, enables us to assess various bone parameters, including bone formation, bone resorption, bone mineralization, and bone volume. The bone formation parameters such as osteoid volume/bone volume (OV/BV) showed no significant difference between wild-type and Obif−/− mice, while osteoblast surface/bone surface (Ob.S/BS) and osteoblast number/bone surface (N.Ob/BS) were slightly reduced in Obif−/− mice compared with wild-type mice, but these were not statistically significant (Fig 4B, 4D, and 4E). Moreover, osteoid volume/osteoid surface (OV/OS) significantly increased in Obif−/− mice (Fig 4C). Bone resorption parameters such as eroded surface/bone surface (ES/BS) and bone resorption rate (BRs.R) showed no significant differences between wild-type and Obif−/− mice. While osteoclast surface/bone surface (Oc.S/BS) and osteoclast number/bone surface (N.Oc/BS) were slightly reduced in Obif−/− mice compared with wild-type mice, these were not statistically significant (Fig 4F–4I). In addition, the bone volume parameters such as bone volume per tissue volume (BV/TV) and trabecular number (Tb.N) were decreased and trabecular separation (Tb.Sp) were increased in Obif−/− mice (Fig 4J–4L). Although trabecular thickness (Tb.Th) was slightly reduced in Obif−/− mice compared with wild-type mice, this was not statistically significant (Fig 4M). This observation agrees with our finding obtained by μCT analysis in a previous study [7].

Bottom Line: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated.These findings suggest that Obif plays essential roles in the development of multiple tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, Japan; Department of Developmental Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, Japan.

ABSTRACT

Background: Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.

Results: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.

Conclusions: Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

No MeSH data available.


Related in: MedlinePlus