Language：Japanese   Publisher：日本繁殖生物学会  

【目的】卵巣内の卵母細胞は減数分裂第一分裂前期の状態を維持し，LHサージにより再開する。C型ナトリウム利尿ペプチド(CNP)は，受容体(NPR2)により合成されたcGMPを介して減数分裂の停止維持に関わることが後期胞状卵胞でのみ報告されている。本研究では，CNPシグナルによる減数分裂抑制効果がより早期卵胞で機能しているか調べ，さらに，LHによる減数分裂再開への関与についても解析をおこなった。 【方法】 1．Npr2遺伝子に機能欠失型の突然変異をもつマウスについて，25日齢の卵巣切片を作成し，卵母細胞における核の状態を観察した。2．25日齢の正常マウスとPMSG，hCGを投与した正常マウス卵巣におけるCNP (Nppc) 遺伝子および受容体(Npr2)遺伝子の発現パターンを解析した。3．正常マウス卵巣より単離した顆粒膜細胞を培養し，アンフィレギュリンおよびEGFレプター阻害剤によるNppc遺伝子の発現量への影響を解析した。【結果】 Npr2遺伝子に変異をもつマウスの卵巣では，前胞状卵胞までの卵母細胞は正常マウスと同様にGV期であったが，初期胞状卵胞以降の半数以上の卵母細胞では染色体が観察され，減数分裂が異常に再開していた。初期胞状卵胞では卵胞外側および卵母細胞周囲の顆粒膜細胞にNppcおよびNpr2遺伝子はそれぞれ発現し，PMSG投与48時間後の胞状卵胞では，顆粒膜細胞と卵丘細胞において発現していた。また，Nppc遺伝子の発現はhCG投与後に顕著に減少した。培養顆粒膜細胞では，Nppc遺伝子の発現量はアンフィレギュリン添加により減少し，EGFレセプター阻害剤の前処理により発現量の低下は認められなかった。以上の結果より，CNP/NPR2シグナルは，胞状卵胞期以降の卵母細胞の減数分裂の停止維持に関わる体細胞由来の必須因子であることが示された。また，LHサージによる減数分裂再開には，CNPによる減数分裂停止維持効果の低下が関与している可能性が示された。

DOI： 10.14882/jrds.106.0.P-75.0

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DOI： 10.1111/j.1740-0929.2009.00678.x

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repro23 is an autosomal recessive mutation of the mouse generated by the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis program at The Jackson Laboratory. The repro23/repro23 homozygous mouse shows male-specific infertility caused by defective spermatogenesis. In the present study, we investigated the testicular pathology of the affected mouse and performed linkage analysis to determine the chromosomal localization of the repro23 locus. Histological examination of the affected testis showed that the seminiferous epithelium of the repro23/repro23 mice contained spermatogonia and early stage spermatocytes, but no spermatids or spermatozoa. Immunohistochemical staining for Hsc70t, a spermatid specific protein, confirmed the absence of elongating spermatids. These findings indicated interruption of the spermatogenesis during meiosis in the repro23/repro23 mouse. By linkage analysis using 137 affected mice of F₂ progeny obtained from crosses between repro23/repro23 female and JF1/Ms (+/+) male mice, the repro23 locus was mapped to 2.2-Mb region of mouse chromosome 7. Although this region contains several potential candidate genes for the repro23 mutation, no gene already identified as a cause of defective speramatogenesis was in this region. Therefore, the gene responsible for the repro23 mutation is suggested to be a novel gene which plays an essential role in mammalian spermatogenesis.<br>

DOI： 10.1538/expanim.58.525

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Other Link： https://jlc.jst.go.jp/DN/JALC/00340059676?from=CiNii

DOI： 10.1186/1471-2156-10-60

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Hemophilia A is a severe congenital bleeding disorder characterized by subcutaneous hematoma and hemorrhage into muscles resulting from a deficiency of blood coagulation factor VIII. The authors have recently reported two cases of hemophilia A in Japanese Brown cattle and identified a nucleotide substitution in the factor VIII gene, resulting in an amino acid substitution of Leu to His, as a possible cause of the deficiency. In the present study, a simple and effective polymerase chain reaction (PCR)-based diagnostic method was developed to identify carriers of this disorder, using a mismatch primer in combination with restriction enzyme digestion. The PCR reaction amplified a 118 bp fragment, which was not digested by the BspT104I restriction enzyme in affected animals but was digested into two fragments in normal animals. Both digested and undigested fragments were observed in carrier animals. This method was applied to identify the carriers of hemophilia A in a population of Japanese Brown cattle. By screening 155 DNA samples from Japanese Brown cattle, except for the dam of the two probands, no carriers were identified. It was therefore concluded that the probands represent isolated cases of hemophilia A, and that the frequency of the mutant allele in the Japanese Brown cattle population is very low. © 2006 Japanese Society of Animal Science.

DOI： 10.1111/j.1740-0929.2006.00329.x

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Ellis-van Creveld (EvC) syndrome is a genetic disorder with mutations in either EVC or EVC2 gene. Previous case studies reported that EvC patients underwent orthodontic treatment, suggesting the presence of craniofacial bone phenotypes. To investigate whether a mutation in EVC2 gene causes a craniofacial bone phenotype, Evc2 knockout (KO) mice were generated and cephalometric analysis was performed. The heads of wild type (WT), heterozygous (Het) and homozygous Evc2 KO mice (1-, 3-, and 6-week-old) were prepared and cephalometric analysis based on the selected reference points on lateral X-ray radiographs was performed. The linear and angular bone measurements were then calculated, compared between WT, Het and KO and statistically analyzed at each time point. Our data showed that length of craniofacial bones in KO was significantly lowered by similar to 20% to that of WT and Het, the growth of certain bones, including nasal bone, palatal length, and premaxilla was more affected in KO, and the reduction in these bone length was more significantly enhanced at later postnatal time points (3 and 6 weeks) than early time point (1 week). Furthermore, bone-to-bone relationship to cranial base and cranial vault in KO was remarkably changed, i.e. cranial vault and nasal bone were depressed and premaxilla and mandible were developed in a more ventral direction. Our study was the first to show the cause-effect relationship between Evc2 deficiency and craniofacial defects in EvC syndrome, demonstrating that Evc2 is required for craniofacial bone development and its deficiency leads to specific facial bone growth defect. Anat Rec, 299:1110-1120, 2016. (C) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/ar.23353

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Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) (c) 2016 Japanese Teratology Society

DOI： 10.1111/cga.12153

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Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) (c) 2016 Japanese Teratology Society

DOI： 10.1111/cga.12153

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The effective population size of Japanese Black cattle has been significantly reduced and maintaining the genetic diversity of the population is important for breeding of Japanese Black cattle. For the conservation of genetic diversity, use of different lines of the breed with unique genetic characteristics for breeding, instead of intensive use of sires of few particular lines as common in current breeding of Japanese Black cattle, will be effective to prevent the genetic homogenization of the population. In the present study, we performed genetic characterization a population of a rare line of Japanese Black cattle, which has been originated from ancestral &quot;Tsuru-ushi&quot; in Okayama prefecture. By using microsatellite markers, allelic richness, and average observed and expected heterozygosity are 3.48, 0.514, and 0.511, respectively, and these values were lower than those of most of the Japanese Black cattle local subpopulations compared. The result of the clustering analyses indicated that the animals of the rare line formed a single group with a cluster that was clearly distinguished from the other populations. Sequence analysis of mitochondrial D-loop region revealed that only two haplotypes were observed in the population and 80% of the animals in the population possess a single haplotype. However, the other haplotype was a novel unique haplotype that has not been reported in cattle. Genotyping of six genes associated with important traits revealed that &lt;i&gt;SREBP1&lt;/i&gt; and &lt;i&gt;NCAPG&lt;/i&gt; loci were fixed for a single allele in the population and more than 90% of animals possess an allele of &lt;i&gt;MC1R&lt;/i&gt; locus. These findings indicated that while genetic diversity of the population of the rare line is lower than those of the Japanese Black cattle local subpopulations, this population has unique genetic characteristics that were distinguished from the other populations and, therefore, the rare line is important for maintaining genetic diversity of Japanese Black cattle.

DOI： 10.2508/chikusan.87.1

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Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. (C) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/dvg.22879

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Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.

DOI： 10.1371/journal.pgen.1005433

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本繁殖生物学会  

DOI： 10.14882/jrds.107.0_OR1-2

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

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The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs.

DOI： 10.1538/expanim.63.11

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DOI： 10.5924/abgri.42.11

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Ggt1(dwg/dwg) mice are spontaneous mutant mice with a nucleotide deletion in the Ggt1 gene. They are characterized by dwarfism, cataract, and coat color abnormality. These abnormalities in the external appearance of Ggt1(dwg/dwg) mice closely resemble those of previously reported GGT1-deficient mice, Ggt1(tm1Zuk/tm1Zuk) (Ggt1(-/-)) and Ggt1(enu1/enu1), generated by gene targeting or ENU mutagenesis. However, whether the pathological features of Ggt1(dwg/dwg) mice are also similar to those of the Ggt1(-/-) and Ggt1(enu1/enu1) mice remains unclear. To clarify the pathogenesis of Ggt1(dwg/dwg) mice, we physiologically and histologically investigated the abnormalities of Ggt1(dwg/dwg) mice in this study. First, we analyzed the activity of GGT1 and GSH levels in Ggt1(dwg/dwg) mice. GGT1 activity in the Ggt1(dwg/dwg) mice was reduced to approximately 4.0% of that in the wild-type mice. Plasma and kidney GSH levels were markedly increased, while eye and liver GSH levels were markedly decreased, in the Ggt1(dwg/dwg) mice. Notably, no significant difference in survival rate was observed between the Ggt1(dwg/dwg) and wild-type mice, whereas high mortality was reported in the Ggt1(-/-) and Ggt1(enu1/enu1) mice. Growth retardation, degeneration of lens fibers, and an increased number of osteoclasts in the Ggt1(dwg/dwg) mice were reversed by administration of N-acetyl-L-cysteine, a precursor of GSH synthesis. Thus, we conclude that the abnormalities of Ggt1(dwg/dwg) mice are caused by alteration of the GSH levels due to the depression of GGT1 activity and that Ggt1(dwg/dwg) mice will be a useful model for GGT deficiency with peculiar features.

DOI： 10.1538/expanim.62.151

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Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.

DOI： 10.1155/2013/760236

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Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C-type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression. Mol. Reprod. Dev. 79: 795802, 2012. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/mrd.22114

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Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity. Reproduction (2012) 144 187-193

DOI： 10.1530/REP-12-0050

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Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity. Reproduction (2012) 144 187-193

DOI： 10.1530/REP-12-0050

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Long bone abnormality (lbab/lbab) is a strain of dwarf mice. Recent studies revealed that the phenotype is caused by a spontaneous mutation in the Nppc gene, which encodes mouse C-type natriuretic peptide (CNP). In this study, we analyzed the chondrodysplastic skeletal phenotype of lbab/lbab mice. At birth, lbab/lbab mice are only slightly shorter than their wild-type littermates. Nevertheless, lbab/lbab mice do not undergo a growth spurt, and their final body and bone lengths are only similar to 60% of those of wild-type mice. Histological analysis revealed that the growth plate in lbab/lbab mice, especially the hypertrophic chondrocyte layer, was significantly thinner than in wild-type mice. Overexpression of CNP in the cartilage of lbab/lbab mice restored their thinned growth plate, followed by the complete rescue of their impaired endochondral bone growth. Furthermore, the bone volume in lbab/lbab mouse was severely decreased and was recovered by CNP overexpression. On the other hand, the thickness of the growth plate of lbab/+ mice was not different from that of wild-type mice; accordingly, impaired endochondral bone growth was not observed in lbab/+ mice. In organ culture experiments, tibial explants from fetal lbab/lbab mice were significantly shorter than those from lbab/+ mice and elongated by addition of 10(-7) M CNP to the same extent as lbab/+ tibiae treated with the same dose of CNP. These results demonstrate that lbab/lbab is a novel mouse model of chondrodysplasia caused by insufficient CNP action on endochondral ossification.

DOI： 10.1007/s00223-011-9567-0

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Development of the male gonads is a complex process with interaction of various cells in the gonads including germ, Sertoli, Leydig, and myoid cells. TF is a mutant rat strain showing male pseudohermaphroditism, with agenesis of Leydig cells and androgen deficiency controlled by an autosomal single recessive gene (mp). The mp locus was mapped on the distal region of rat chromosome 7 by linkage analysis, but the gene responsible for the mp mutation has not been identified. In this study, we performed fine linkage mapping and sequence analysis to determine the causative gene of the mp mutation, and performed an immunohistochemical study using a Leydig cell-specific marker to investigate detailed phenotypes of the mutant rats during the testicular development. As a result, we found a missense mutation of the gene encoding Desert hedgehog (Dhh) in the mutant rat, which could result in loss of function of the DHH signaling pathway. Histochemical examination revealed remarkably reduced number of fetal Leydig cells and lack of typical spindle-shaped adult Leydig cell in the mp/mp rats. These phenotypes resembled those of the Dhh-null mice. Additionally, testosterone levels were significantly lower in the mp/mp fetus, indicating androgen deficiency during embryonic development. These results indicate that the mutation of the Dhh gene may be responsible for the pseudohermaphrodite phenotypes of the mutant rat, and that the Dhh gene is probably essential for the development of Leydig cells. Reproduction (2011) 141 217-225

DOI： 10.1530/REP-10-0006

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Language：English   Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

Natriuretic peptide receptor B (NPR-B), which has high affinity for C-type natriuretic peptide (CNP) and synthesizes intracellular cGMP, may be involved in gastrointestinal tract (GIT) regulation. A mutant allele of the NPR-B-encoding gene (Npr2) is responsible for the phenotype of the short-limb dwarfism (SLW) mouse. Homozygosity for this autosomal-recessive gene (slw/slw) leads to dwarfism and death before weaning because of milk retention in the stomach and intestinal distention. To elucidate the relationship between CNP/NPR-B signaling and GIT function, we investigated the association between Npr2 mutation and the GIT phenotype in slw/slw mice. The pylorus and large intestine of the mutants did not respond to CNP stimulation; further, they showed pyloric lumen narrowing with randomly aligned circular muscle cells. Comparison of the cGMP and neuronal marker distribution in GIT tissues confirmed cGMP expression in neuronal tissues. An Auerbach&apos;s plexus and submucosal tissues of the mutants didn&apos;t express cGMP and expressed Ca(2+). In contrast, those of normal mice (controls) expressed both cGMP and Ca(2+). Sequencing revealed that the causative Npr2 mutation was a 7-base deletion in exon 8, resulting in a frameshift and premature termination codon appearance. Therefore, the GIT phenotype of slw/slw mice is because of a CNP/NPR-B-signaling defect caused by an Npr2 mutation. These results facilitate better understanding of the role of CNP/NPR-B signaling in GIT motility. (Am J Pathol 2010, 177:822-828; DOI: 10.2353/ajpath.2010.091278)

DOI： 10.2353/ajpath.2010.091278

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publisher：SPRINGER  

The dwg and dwg (Bayer) are allelic mutations of the mouse that are characterized by dwarfism, cataracts, and coat color change in homozygotes. The Ggt1 gene encodes gamma-glutamyltransferase 1 (GGT1), an extracellular membrane-bound enzyme that is critical for glutathione homeostasis. Both the dwg locus and Ggt1 gene are localized on mouse chromosome 10, and the phenotypes of GGT1-deficient mice with targeted disruption of the Ggt1 gene show remarkable similarities with those of dwg/dwg and dwg (Bayer) /dwg (Bayer) mice. This evidence led us to hypothesize that the Ggt1 gene is responsible for dwg and dwg (Bayer) mutations. In this study we characterized dwg mutations by investigating their association with the Ggt1 gene. Histological analysis revealed reduced numbers of proliferative and hypertrophic chondrocytes in the growth plate of dwg/dwg mice, which are characteristic abnormalities observed in GGT1-deficient mice. To identify the causative mutations of dwg mutations, we analyzed the Ggt1 gene in dwg/dwg and dwg (Bayer) /dwg (Bayer) mice. In dwg/dwg mice, 13 nucleotides on exon 7 of the Ggt1 gene were deleted, resulting in the generation of aberrant transcripts due to disrupted pre-mRNA splicing. Furthermore, dwg (Bayer) /dwg (Bayer) mice had a 46.7-kb deletion containing complete coding sequences of Ggt1 and AI646023 genes and the first exon of the Ggt5 gene, which is closely related to the Ggt1 gene as a member of the GGT gene family. These results indicate that both dwg and dwg (Bayer) have defective mutations of the Ggt1 gene. Thus, we concluded that mutations in the Ggt1 gene are responsible for the phenotypes of dwg/dwg and dwg (Bayer) /dwg (Bayer) mice.

DOI： 10.1007/s00335-009-9221-6

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Multiple ocular defects (MOD) in cattle is an autosomal recessive hereditary disorder characterized by dysplasia of the lens, retinal detachment, persistence of the hyaloid artery, and microphthahmia, The locus responsible for MOD has been mapped to the proximal region of bovine chromosome 18. In the present Study, we refined the localization of the MOD locus to a 1.0-Mb interval by haplotype analysis using a pedigree of affected animals. Comparison of nucleotide sequence of genes in this region revealed a one-nucleotide insertion in the WFDC1 gene, which resulted in a frame shift mutation and premature termination codon at the middle of the protein. WFDC1 is a small secretory protein containing a WAP-type four disulfide core domain. Specific expression of Wfdc1 was observed in the lens, retina, and optic nerves of embryonic and adult mouse eyes by immunohitochemical staining and in situ hybridization. The present finding demonstrated the essential role of WFDC1 in mammalian eye development. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygeno.2009.04.001

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.08.139

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Language：English   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and. female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3 beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.

DOI： 10.1262/jrd.19191

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Language：English   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.

DOI： 10.1262/jrd.19186

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Language：English   Publisher：BLACKWELL PUBLISHING  

Forelimb-girdle muscular anomaly is an autosomal recessive disorder of Japanese black cattle characterized by tremor, astasia and abnormal shape of the shoulders. Pathological examination of affected animals reveals hypoplasia of forelimb-girdle muscles with reduced diameter of muscle fibres. To identify the gene responsible for this disorder, we performed linkage mapping of the disorder locus using an inbred pedigree including a great-grand sire, a grand sire, a sire and 26 affected calves obtained from a herd of Japanese black cattle. Two hundred and fifty-eight microsatellite markers distributed across the genome were genotyped across the pedigree. Four markers on the middle region of bovine chromosome 26 showed significant linkage with the disorder locus. Haplotype analysis using additional markers in this region refined the critical region of the disorder locus to a 3.5-Mb interval on BTA26 between BM4505 and MOK2602. Comparative mapping data revealed several potential candidate genes for the disorder, including NRAP, PDZD8 and HSPA12A, which are associated with muscular function.

DOI： 10.1111/j.1365-2052.2007.01679.x

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Language：English   Publisher：INT PRESS EDITING CENTRE INC  

Koala (Koa) and hairy ears (Eh) mutations of mice are associated with chromosomal inversions in the distal half of chromosome 15. Since these two mutant mice show some common phenotypic features including extra hair on pinna and craniofacial dysmorphogenesis and have similar inverted regions, we determined the inverted regions of these two chromosomal inversions to examine whether a common gene is responsible for the phenotypes of these two mutants. The inverted regions were identified as the recombination-suppressed regions by linkage analysis. The length of the recombination-suppressed regions of Koa and Eh were approximately 52 and 47 Mb, respectively, and these inverted regions were not the same. These results indicate that the phenotypes of Koa and Eh mutant mice are likely to be caused by different genes.

DOI： 10.1538/expanim.57.73

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Language：English   Publisher：OXFORD UNIV PRESS INC  

Short-limbed dwarfism (SLW) is a new mutant mouse characterized by a dwarf phenotype with markedly short body, limbs, and tail. In the present study, we investigated the skeletal phenotypes of the SLW mouse and determined the chromosomal localization to identify the gene responsible for the phenotypes (s/w). Skeletal preparations stained with alcian blue and alizarin red revealed that longitudinal growth of the extremities of the affected (s/w/s/w) mice was significantly reduced in comparison with that of normal mice, whereas the positions and numbers of skeletal elements were normal. Histological examination of tibial growth plates of the affected mice showed that the numbers of proliferating and hypertrophic chondrocytes were obviously diminished. These phenotypes resembled to those of human chondrodysplasias caused by defective chondrocyte proliferation and differentiation. We mapped the s/w locus on an 11.7-cM interval of the proximal region of mouse chromosome 4 by linkage analysis. Furthermore, allelism test using Npr2(cn) locus, a mutant allele of Npr2 gene encoding a natriuretic peptide receptor B, revealed that s/w locus is an allele of the Npr2 gene. These results suggest that the dwarf phenotype of the SLW mouse is caused by the disturbed endochondral ossification, and a mutation in the Npr2 gene is expected to be responsible for the phenotypes of the SLW mouse.

DOI： 10.1093/jhered/esm065

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

J-GLOBAL

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The hairy ears (Eh) mutation in the mouse originated from neutron irradiation experiments and is associated with chromosomal inversion on chromosome 15. Eh/+ mice have small pinna and extra hairs on the pinna but the phenotypic features of Eh/Eh mice are unclear. In this study we found that Eh/Eh mice died shortly after birth and had a cleft palate caused by impaired growth of palate shelves. Because genes located on the breakpoints of inversion are likely to be responsible for the defects associated with chromosomal inversions, we determined the breakpoints of the Eh inversion. We used a new genetic method that uses recombinant chromosomes resulting from crossing over between two overlapping inversions to determine the breakpoints. Koa is a mouse mutation associated with inversion of chromosome 15, which partially overlaps with the Eh inversion. We made Eh +/+ Koa double heterozygotes and obtained the recombinant chromosomes possessing deletion and duplication of the regions flanked by the breakpoints of both inversions, which were generated by crossing over within the overlapped region of these inversions. By defining the deleted regions we identified the breakpoints of the Eh inversion. We then examined the expression of genes in the vicinities of the breakpoints and found ectopic expression of the Hoxc5 gene and a transcript with unknown function in the developing palate of Eh/Eh mice, which is likely to be responsible for the cleft palate.

DOI： 10.1007/s00335-007-9015-7

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publisher：INT PRESS EDITING CENTRE INC  

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomi. c sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.

DOI： 10.1538/expanim.55.491

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Mice carrying the piebald mutation exhibit white coat spotting due to the regional absence of neural crest-derived melanocytes. We reported previously that the piebald locus encodes the Ednrb gene and that piebald mice express low levels of structurally intact Ednrb mRNA and EDNRB protein (Hosoda, K., Hammer, R. E., Richardson, J. A., Baynash, A. G., Cheung, J. C., Giaid, A., and Yanagisawa, M. (1994) Cell 79, 1267-1276). Here, we report that both the life span of the Ednrb mRNA and the promoter activity of the Ednrb gene are indistinguishable between wild-type and piebald mice. Introns 2-6 of the Ednrb gene in piebald mice were correctly excised with an efficiency indistinguishable from those in wild-type mice in exon trapping experiments. We found that the piebald allele of the Ednrb gene has a 5.5-kb retroposon-like element in intron 1 possessing canonical sequences of a polyadenylation signal and a splice acceptor site. Abnormal hybrid transcripts carrying exon 1 of the Ednrb gene and a portion of the 5.5-kb element are expressed in piebald mice. The insertion of the 5.5-kb element into a heterologous intron in a mammalian expression vector markedly reduced the expression of the reporter gene. Premature termination and aberrant splicing of the Ednrb transcript caused by the retroposon-like element in intron 1 lead to a reduced level of the normal Ednrb transcript, which is responsible for the partial loss-of-function phenotype of piebald mice.

DOI： 10.1074/jbc.M512618200

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DOI： 10.1111/j.1740-0929.2006.00329.x

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Congenital multiple ocular defects (MOD) in Japanese black cattle is a hereditary ocular disorder with an autosomal recessive manner of inheritance, showing developmental defects of the lens, retina, and iris, persistent embryonic eye vascularization, and microphthalmia. In the present study, we mapped the locus responsible for the disorder by linkage analysis using 240 microsatellite markers covering the entire bovine genome and an inbred pedigree obtained from commercial herds. The linkage analysis demonstrated a significant linkage between the disorder locus and markers on the proximal region of bovine Chromosome (BTA) 18 with the maximum LOD score of 5.1. Homozygosity mapping using the haplotype of the linked markers further refined the critical region. The results revealed the localization of the locus responsible for MOD in an approximately 6.6-cM region of BTA18. Comparison of published linkage and radiation hybrid (RH) maps of BTA18 with its evolutionary ortholog, human Chromosome (HSA) 16, revealed several potential candidate genes for the disorder including the MAF and FOXC2 genes.

DOI： 10.1007/s00335-005-0043-x

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Factor XI deficiency in Japanese black cattle is an hereditary mild bleeding disorder with an autosomal recessive mode of inheritance. To characterize the molecular lesion causing factor XI deficiency in cattle, we isolated an entire coding region of the bovine F11 gene, which comprises 15 exons and 14 introns, and determined its nucleotide sequences. Comparison of the nucleotide sequences of the F11 gene between affected and unaffected animals revealed an insertion of 15 nucleotides in exon 9 of the affected animals. The insertion results in a substitution of one amino acid with six amino acids in a highly conserved amino acid sequence in the fourth apple domain of factor XI protein. Genotyping of the F11 gene in 109 Japanese black cattle revealed that the insertion clearly corresponded to the factor XI activities of the animals. We therefore concluded that the insertion of 15 nucleotides in the F11 gene is the causative mutation for factor XI deficiency in Japanese black cattle. Genotyping of the F11gene by detecting the insertion will be an effective DNA-based diagnostic system to prevent incidence of the disease.

DOI： 10.1007/s00335-004-2462-5

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F-2 progeny mapped the cn locus on an similar to 0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in +/+ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-of-function mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal.

DOI： 10.1074/jbc.C500024200

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Publisher：Japanese Society of Animal Breeding and Genetics  

DOI： 10.5924/abgri2000.32.2_133

CiNii Article

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Language：English   Publisher：Blackwell Publishing  

The aim of the present study was to demonstrate estrogen receptor (ER) α (ER-α) and ER-β expression in the bursa of Fabricius and cultured bursal epithelial cells (CBEC) by reverse transcription (RT)-polymerase chain reaction (PCR) using a primer set design based on the sequences of chicken ER-α complementary DNA (cDNA) and chicken ER-β-cDNA. In the case of ER-α, RT-PCR products of the expected size (698 base pairs (bp)) were amplified from cDNA derived from the bursa and CBEC, and from the oviduct, which was used as a positive control. All of the products were cleaved into two fragments of 476 bp and 222 bp by treating the products with restriction nuclease Hind III, indicating that these RT-PCR products from the bursa, CBEC and genital organs were the same. In contrast, in the case of ER-β, PCR products of the expected size (303 bp) were not detected in the bursa of Fabricius or CBEC, or in the oviduct and ovary. These results indicate that ER-α is expressed in the bursa of Fabricius and in CBEC, but ER-β is not expressed in the bursa or CBEC, or in the female genital organs of chickens.

DOI： 10.1111/j.1740-0929.2005.00264.x

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Language：English   Publisher：UNIVERSAL ACADEMY PRESS INC  

YPC is a mutant mouse strain with defective hair growth characterized by thin, short hairs and poorly developed hair bulbs and dermal papillae. To identify the gene associated with the phenotype, we performed genome-wide linkage analysis using 1010 backcross progeny and 123 microsatellite markers covering all chromosomes. The mutant locus (ypc) was mapped to a 0.2-cM region in the proximal part of mouse chromosome 1. This 0.2-cM region corresponds to a 450-kb region of genome sequence that contains two genes with known functions and five ESTs or predicted genes with unknown functions. Sequence analysis revealed a single C-to-A nucleotide substitution at nucleotide 1382 in the Sgkl gene, causing a nonsense mutation at codon 461. Sgkl encodes serum and glucocorticoid-inducible kinase-like kinase (SGKL), which belongs to a subfamily of serine/threonine protein kinases and has been suggested to have a role downstream of lipid signals produced by activation of phosphoinositide 3-kinase (PI3K). In the mutant SGKL, a serine residue in the C-terminal end of the protein (Ser486), which is indispensable for activation of SGKL upon phosphorylation, is abolished by premature termination. Specific expression of the Sgkl gene in the inner root sheath of growing hair follicles was also identified by in situ hybridization. Therefore, we concluded that the nucleotide substitution in the Sgkl gene is the causative mutation for defective hair growth in the ypc mutant mouse and that the signaling pathway involving SGKL plays an essential role in mammalian hair development.

DOI： 10.1093/dnares/11.6.371

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Language：English   Publisher：WILEY-LISS  

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplasia characterized by short limbs, postaxial polydactyly, natal teeth, and dysplastic nails. The Ellis-van Creveld (EVC) gene, which is mutated in patients with EvC syndrome, has been identified by positional cloning. However, the physiological roles of the EVC gene have not been elucidated. Histopathological analyses of EvC syndrome have shown disturbed chondrocytic phenotypes during cartilage development. We therefore postulated that the EVC gene is a critical factor for chondrocytes during endochondral ossification. The present study focuses on the relationship between the Evc gene and chondrocytes, and examines Evc gene expression in the rat tibial growth plate at the mRNA and protein levels. Evc mRNA in tibial epiphyseal cartilage was expressed at postnatal day (P) 1, P28, and P56 by RT-PCR. Immunohistochemical analyses localized the Evc protein mainly in prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate in the tibia during the embryonic and postnatal periods. Evc mRNA was also detected in prehypertrophic and hypertrophic chondrocytes by in situ hybridization. These results indicate that the Evc gene functions mainly in the prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate. The data presented here are important for future studies of the underlying mechanism of chondrodysplasia in EvC syndrome. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20059

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Language：English   Publisher：INT PRESS EDITING CENTRE INC  

We have established a new mouse strain with vertebral deformities caused by an autosomal single recessive mutation (oma). The mutant mice showed short trunk and short and kinky tail. The skeletal preparations of newborn and prenatal mice showed disorganized vertebrae and numerous vertebral and rib fusions which are thought to be caused by patterning defects at the stage of somitegenesis. Linkage analysis localized the oma locus on the proximal region of mouse chromosome 7 close to DII3 gene. DII3 is the gene involved in the Notch signaling pathway and null-mutation of the gene has been reported to cause vertebral deformities. The phenotypic similarity between oma and DII3 null-mutant mice suggests that the causative gene for the oma mutant is the DII3 gene. We, therefore, investigated the nucleotide sequence of the DII3 gene of the oma mouse and found a single nucleotide substitution of G to T which causes missense mutation of glycine to cysteine at codon 409. Since the amino acid substitution is a nonconservative amino acid substitution at the conserved portion of the DII3 protein, and the substitution is specific to the mutant mice, we concluded that the nucleotide substitution of the DII3 gene is responsible for the skeletal deformities of the oma mouse.

DOI： 10.1538/expanim.53.129

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Language：English   Publisher：AMER SOC BONE & MINERAL RES  

Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059,were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts.
Introduction: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis.
Materials and Methods: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy.
Results: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells.
Conclusion: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.

DOI： 10.1359/jbmr.2003.18.7.1198

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Language：English   Publisher：NATL ACAD SCIENCES  

Chondrodysplastic dwarfism in Japanese brown cattle is an autosomal recessive disorder characterized by short limbs. Previously, we mapped the locus responsible for the disease on the distal end of bovine chromosome 6. Here, we narrowed the critical region to approximate to2 cM by using linkage analysis, constructed a BAC and YAC contig covering this region, and identified a gene, LIMBIN (LBN), that possessed disease-specific mutations in the affected calves. One mutation was a single nucleotide substitution leading to an activation of a cryptic splicing donor site and the other was a one-base deletion resulting in a frameshift mutation. Strong expression of the Lbn gene was observed in limb buds of developing mouse embryos and in proliferating chondrocytes and bone-forming osteoblasts in long bones. These findings indicate that LBN is responsible for bovine chondrodysplastic dwarfism and has a critical role in a skeletal development.

DOI： 10.1073/pnas.152337899

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Language：English   Publisher：HISTOCHEMICAL SOC INC  

Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor family, is an osteoclastogenesis inhibitory factor. We investigated the localization of OPG in rat tibia using a specific peptide antibody to clarify the role of OPG in bone remodeling. OPG reactivity was mainly seen on bone surfaces. In bone matrices, OPG was also localized on cartilage/bone interfaces and cement lines. However, labeling was scarcely detected in the region of contact between osteoclasts and stromal cells. Some osteoblasts and osteocytes showed weak labeling. Immunoreactivity was not seen in chondrocytes or osteoclasts. Immunoelectron microscopic observation revealed that OPG is localized on the bone surfaces under osteoclasts. These findings suggest that OPG derived from osteoblast lineage cells and/or serum may be concentrated on resorbed bone surfaces and subsequently on cement lines. OPG may play an important role in the prevention of excess bone resorption by inhibiting differentiation and activity of osteoclasts in bone remodeling.

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Language：English   Publisher：WILEY-LISS  

Fine structural and cytochemical studies were performed to clarify the pattern of medullary bone calcification, specifically in relation to sulfated glycosaminoglycans, by using estrogen-induced medullary bone of male Japanese quails. Tibiae were collected at 4 and 7 days after estrogen treatment. Medullary bone had developed inward toward the marrow cavity, and calcification had begun near the cortical bone and deeper parts of the trabeculae, accompanied by wide osteoid at extending tips and surface areas of the trabeculae. Sulfated glycosaminoglycans, detected by high iron diamine (HID), were distributed in the matrix in a pattern similar to that of calcified matrix of the trabeculae. Cortical bone was negatively stained by HID. In undecalcified specimens, calcified nodules were seen in areas undergoing calcification. Globular structures composed of fine filamentous materials, a marginal dense layer, and central core, were also observed in the matrix of decalcified specimens. Both the calcified nodules and globular structures showed the same distribution pattern, i.e., they were dispersed at surface areas and coalesced in the deeper areas of the matrix. The globular structures were exclusively positive for HID-thiocarbohydrazide-silver protein (HID-TCH-SP) stain, indicating the localization of sulfated glycosaminoglycans. These results strongly suggest that medullary bone calcification progresses by the coalescence of calcified nodules and that sulfated glycosaminoglycans play an important role for the regulation of globular calcification. Anat Rec 264:25-31, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/ar.1101

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

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Language：English   Publisher：INT PRESS EDITING CENTRE INC  

DOI： 10.1538/expanim.50.359

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Language：English   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

We investigate, by the immunogold method, the localization of keratan sulfate (KS) proteoglycan in rat calvaria in order to clarify the detailed process of intramembranous ossification. KS was localized in bone nodules corresponding to calcified nodules, close to the saggital suture of calvaria, The immunoreactivity decreased in fully calcified regions distant from the suture. Electron microscopic observation revealed that KS was distributed in and around matrix vesicles, among collagen fibrils at the initial crystal deposition stage, and then concentrated in bone nodules. According to the progress of mineralization, KS tended to be localized in the peripheral region of the nodules. In addition, these nodules came in contact with collagen fibrils which also showed KS-positive reactivity. In cell organelles of osteoblasts, KS was detected in the Golgi apparatus. These findings suggest that osteoblasts in intramembranous ossification sites actively synthesize KS. IIS in the calcified nodules, as well as other glycosaminoglycans in osteoid, may play an important role in additional and/or collagenous calcification by trapping calcium ions through its negative charge.

DOI： 10.1679/aohc.64.109

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Language：English   Publisher：Japanese Society of Animal Science  

In the present study, we isolated promoter region of mouse insulin receptor-related receptor (Irr) gene and analyzed its expression in various mouse tissues and embryos in different stages. The nucleotide sequence of the promoter region of the Irr gene showed the presence of putative promoter elements and binding sites for trans-acting factors. The expression analysis revealed the strong expression in kidney, stomach, and embryo at the stage of 14.5 days. These findings were basically consistent with the previously reported findings of rat and human, but we found several differences in the sequence and expression pattern of the gene between mouse and rat or human.

DOI： 10.2508/chikusan.72.169

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Language：Japanese   Publisher：歯科基礎医学会  

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CiNii Books

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Language：English   Publisher：INT PRESS EDITING CENTRE INC  

DOI： 10.1538/expanim.49.67

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Language：English   Publisher：Maruzen Co. Ltd.  

Gastrointestinal glutathione peroxidase (GSHPx-GI) is an enzyme expressed in intestinal epithelial cells and may reduce hydroperoxides generated from the ingested diet. We isolated a genomic clone containing the mouse Gpx2 gene encoding 190 amino acids of GSHPx-GI. This gene is composed of two exons and an intron. The nucleotide sequence of the coding region was 89.9% identical with that of the human GPX2 gene. A TGA opal codon predicted to encode a selenocysteine was identified at codon 40. A genomic clone containing a pseudogene for the Gpx2 gene was also isolated. The nucleotide sequence of the pseudogene was 98.3% identical with that of the mouse Gpx2 gene and showed characteristics of a processed pseudogene. Linkage analysis using backcross mouse progeny indicated the mouse Gpx2 gene and its pseudogene to be located on mouse chromosomes 12 and 7, respectively.

DOI： 10.1292/jvms.60.651

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PubMed

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Language：English   Publisher：Maruzen Co. Ltd.  

Gastrointestinal glutathione peroxidase (GSHPx-GI) is an enzyme expressed in intestinal epithelial cells and may reduce hydroperoxides generated from the ingested diet. We isolated a genomic clone containing the mouse Gpx2 gene encoding 190 amino acids of GSHPx-GI. This gene is composed of two exons and an intron. The nucleotide sequence of the coding region was 89.9% identical with that of the human GPX2 gene. A TGA opal codon predicted to encode a selenocysteine was identified at codon 40. A genomic clone containing a pseudogene for the Gpx2 gene was also isolated. The nucleotide sequence of the pseudogene was 98.3% identical with that of the mouse Gpx2 gene and showed characteristics of a processed pseudogene. Linkage analysis using backcross mouse progeny indicated the mouse Gpx2 gene and its pseudogene to be located on mouse chromosomes 12 and 7, respectively.

DOI： 10.1292/jvms.60.651

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PubMed

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Language：English   Publisher：Universal Academy Press Inc.  

Congenital aganglionosis rat (AR) is a mutant with an autosomal recessive gene (sl), which shows megacolon caused by the absence of myenteric ganglion cells and white coat-color with a small pigmented spot on the head. Recently, targeted disruption of the endothelin-B (ETB) receptor gene (EDNRB) in the mouse has been reported to cause aganglionic megacolon and coat color spotting resembling the phenotypes of the sl/sl rats. To identify the mutation responsible for the phenotypes of the sl/sl rats, we determined the nucleotide sequences of the EDNRB genes of the sl/sl rats and found that a 301-bp region intervening between direct repeat sequences was deleted in the EDNRB gene, and the deletion produces various transcripts due to aberrant splicing.

DOI： 10.1093/dnares/3.2.101

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PubMed

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Language：English   Publisher：Universal Academy Press Inc.  

Congenital aganglionosis rat (AR) is a mutant with an autosomal recessive gene (sl), which shows megacolon caused by the absence of myenteric ganglion cells and white coat-color with a small pigmented spot on the head. Recently, targeted disruption of the endothelin-B (ETB) receptor gene (EDNRB) in the mouse has been reported to cause aganglionic megacolon and coat color spotting resembling the phenotypes of the sl/sl rats. To identify the mutation responsible for the phenotypes of the sl/sl rats, we determined the nucleotide sequences of the EDNRB genes of the sl/sl rats and found that a 301-bp region intervening between direct repeat sequences was deleted in the EDNRB gene, and the deletion produces various transcripts due to aberrant splicing.

DOI： 10.1093/dnares/3.2.101

Scopus

PubMed

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