Histological analysis of distal femoral epiphysis of <i>Obif</i>^−/− mice.

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

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

<div><p>Background</p><p>Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that <i>Obif</i> (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that <i>Obif^−/−</i> 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 <i>Obif^−/−</i> mice.</p><p>Results</p><p>First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that <i>Obif^−/−</i> 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 <i>Obif^−/−</i> mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that <i>Obif</i> is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the <i>Obif^−/−</i> testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in <i>Obif^−/−</i> mice compared with wild-type mice, although this was not statistically significant.</p><p>Conclusions</p><p>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 <i>Obif</i> is required for normal bone mineralization and late testicular differentiation <i>in vivo</i>. These findings suggest that <i>Obif</i> plays essential roles in the development of multiple tissues.</p></div

<i>Obif</i> is required for normal spermatogenesis.

<p><b>(A)</b> Gross appearance of testes in male wild-type and <i>Obif</i>^−/− mice at 12 wks. <b>(B)</b> Comparison of testicular weight in wild-type (white box), <i>Obif</i>^+/− (grey box), and <i>Obif</i>^−/− (black box) mice at 5 wks and 12 wks (n = 6). <b>(C)</b> Comparison of epididymis weight between wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(D)</b> Comparison of sperm number from cauda epididymis between wild-type, <i>Obif</i>^+/−, and <i>Obif</i>^−/− at 16 wks and 24 wks (n = 4). <b>(E)</b> The level of serum testosterone in wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(F)</b> H&E staining of testis sections from wild-type and <i>Obif</i>^−/− mice at 12 wks. Scale bars represent 5 mm (<b>A</b>), and 50 μm <b>(F)</b>. Error bars show the SEM. *P < 0.05.</p

Obif protein is O-glycosylated at serine residue 36.

<p><b>(A)</b> Potential O-glycosylation sites of human and mouse Obif proteins. The predicted amino acid sequences of human OBIF (NP_859075.2) and mouse Obif (NP_666274.1) were aligned by the ClustalW program (<a href="http://clustalw.ddbj.nig.ac.jp/" target="_blank">http://clustalw.ddbj.nig.ac.jp/</a>). Asterisks indicate identical amino acids. Colons and periods indicate similar amino acids. Yellow boxes indicate potential O-glycosylation sites. Red bracket indicates extracellular domain. Blue underline indicates N-terminal signal peptides. Green box indicates single transmembrane domain. (<b>B-C</b>) Analysis of O-glycosylation sites in the mouse Obif protein. Constructs of pCAGGS expression vector (CAG), FLAG-tagged GFP (GFP), or FLAG-tagged mObif with or without mutation(s) (wild-type (WT), S36A, S43A, T54A, T60A, T67A, or S36A/S43A/T54A/T60A/T67A (ALL)) were transfected into HEK293T cells. The HEK293T cells were cultured for 24 h. The cell lysates were analyzed by Western blotting using an anti-FLAG M2 antibody <b>(B)</b>. FLAG-tagged constructs expressing GFP (GFP), human CD55 (hCD55), or wild-type mouse Obif (mObif-WT) were transfected into HEK293T cells cultured in standard medium. The HEK293T cells were cultured for 24 h, and subsequently cultured for 3 days in medium with or without benzyl-GalNAc. The cell lysates were analyzed by Western blot analysis using the anti-FLAG M2 antibody <b>(C)</b>. Arrowheads indicate the 60 kDa band of O-glycosylated mObif. Arrows indicate the 37 kDa band is a nascent form of mObif-WT. Benzyl-GalNAc, benzyl 2-acetamido-2-deoxy- α-D-galactopyranoside, an O-glycosylation inhibitor.</p

Fertility parameters.

<p>Male mice at 12 wks were placed with ICR fertile females at 10 wks for 28 days. Results are shown as mean ± SEM. All data were analyzed with the <i>F</i>-test to determine normality, and the appropriate <i>t</i>-test was applied at the level of 5%.</p><p>Fertility parameters.</p

Loss of <i>Obif</i> impaired bone growth.

<p><b>(A)</b> CR lengths from wild-type (white box) and <i>Obif</i>^−/− mice (black box) at 8 wks. <b>(B)</b> Longitudinal bone lengths of radius, humerus, tibia, and femur in wild-type (white box) and <i>Obif</i>^−/− mice (black box) at 8 wks. CR length, crown-rump length. Error bars show the SEM (n = 5). *P < 0.05.</p

<i>Obif</i>^−/− mice showed abnormal bone formation and bone mineralization.

<p><b>(A)</b> Fluorescence microscopic images of calcein (indicated by arrowhead) and tetracycline (indicated by arrow) staining of distal femur sections from wild-type and <i>Obif</i>^−/− mice. Scale bar represents 100 μm. <b>(B-R)</b> Bone histomorphometric analyses of the distal femur sections from wild-type (white box) and <i>Obif</i>^−/− mice (black box) at 8 wks. Parameters for bone formation <b>(B-E)</b>, bone resorption <b>(F-I)</b>, bone volume <b>(J-M)</b>, and mineralization <b>(N-R)</b> were analyzed. OV/BV, osteoid volume per bone volume; OV/OS, osteoid volume per osteoid surface; Ob.S/BS, osteoblast surface per bone surface; N.Ob/BS, osteoblast number per bone surface; ES/BS, eroded surface per bone surface; Oc.S/BS, osteoclast surface per bone surface; N.Oc/BS, osteoclast number per bone surface; BRs.R, bone resorption rate; BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; MAR, mineral apposition rate; BFR/BS, bone formation rate per bone surface; Aj.Ar, adjusted MAR; Omt, osteoid maturation time; Mlt, mineralization lag time. Error bars show the SEM (n = 5). *P < 0.05.</p