精子鞭毛蛋白質の遺伝子発現、機能解析と新規免疫グロブリンスーパーファミリーの探索

金沢大学医薬保健研究域医学系精巣に発現する免疫グロブリンスーパーファミリー分子は、昨年解析したMC31/CE9の他、neural cell adhesion molecule(NCAM)が報告されているのみである。そこで、NCAMの免疫グロブリン様構造のアミノ酸配列を利用して、この部分に対応する核酸の塩基配列でマウスのESTデータベースをスクリーニングして相同なアミノ酸配列をもつ新規遺伝子を探索した。この方法では蛋白質をコードする核酸の全塩基配列を決定できなかったので、マウス精巣よりmRNAを抽出して作製したcDNAライブラリーをスクリーニングして全塩基配列を決定した。データベースを参照したところ得られた遺伝子は新規の免疫グロブリンスーパーファミリー遺伝子であることがわかった。また、そのアミノ酸構造の解析から、細胞膜に局在することが推測され、細胞外領域に3個の免疫グロブリン様構造をもち細胞膜を1回貫通し細胞内領域をもつことが分かった。ノーザンハイブリダイゼーション法により精巣に発現するmRNAの大きさと発現量を解析したところ、2.1kと4.5kベースの2種類のmRNAが存在することが分かった。また、in situハイブリダイゼーション法によりこのmRNAを発現する細胞をマウス精巣において同定したところ、精祖細胞から早期の精母細胞にmRNAが強く発現することが分かった。そこで、この新規免疫グロブリンスーパーファミリー遺伝子の名称をspermatogenic immunoglobulin superfamily(SgIGSF)と命名した。さらに、精巣以外の器官・組織におけるSgIGSFのmRNAの発現を解析したところ、大脳・肝臓・腎臓・精巣上体においてもmRNAが発現していることが分かったが、大脳と精巣上体では4.5kベースのmRNAのみが発現していた。また、心臓ではSgIGSFのmRNAは発現していなかった。研究課題/領域番号:11770010, 研究期間 (年度):1999 – 2000出典：「精子鞭毛蛋白質の遺伝子発現、機能解析と新規免疫グロブリンスーパーファミリーの探索」研究成果報告書　課題番号11770010（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （ https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-11770010/ ）を加工して作

新規接着分子の精子発生における機能解析およびそれと相互作用する分子の探索

金沢大学医薬保健研究域医学系精子発生は、内分泌など様々な因子によって調節される複雑な過程であるが、生殖細胞とセルトリ細胞の細胞膜を介する相互作用も重要な調節機構であると考えられる。しかしながら、この相互作用に関与する分子についてはまだ十分にわかっていない。そこで申請者は生殖細胞とセルトリ細胞に発現する接着分子に着目し、マウス精巣のcDNAライブラリーより免疫グロブリンスーパーファミリーに属する新規遺伝子をクローニングした。この分子は細胞膜を一回貫通する膜蛋白質で、細胞外に3つの免疫グロブリン様ドメインをもつ。精巣において、mRNAは生殖細胞のみに発現し、主として精祖細胞から合糸期の精母細胞に局在したことからSpermatogenic Immunoglobulin Superfamily (SgIGSF)と命名した。次に、SgIGSFに対する特異的抗体を作製し、光学および電子顕微鏡レベルの免疫組織化学を行ったところSgIGSFは中間型精祖細胞から厚糸期早期の精母細胞とステップ7以降の精子細胞の細胞膜に局在することがわかった。生殖細胞の細胞膜に発現するSgIGSFの機能を解析するため、精巣の蛋白抽出物から抗SgIGSF抗体と反応する分子を免疫沈降法により分離し、初代培養セルトリ細胞と反応させたところ、免疫沈降産物は培養セルトリ細胞の細胞膜と結合することがわかった。また、この時SgIGSFは生殖細胞同士の接着部位に強く発現することが観察されたので、生殖細胞同士のホモフィリックな結合に関与することが示唆された。さらに、COS-7細胞にSgIGSFを遺伝子導入してSgIGSFとタグ蛋白質との融合蛋白質を産生させた。この融合蛋白質を含む蛋白質抽出物を培養セルトリ細胞と反応させ、タグ蛋白質に対する抗体で検出すると免疫沈降産物と同様にセルトリ細胞の細胞膜と結合した。以上の結果から、生殖細胞に発現するSgIGSFはセルトリ細胞の細胞膜上に存在する分子とヘテロフィリックな結合をする接着分子としても機能することが示唆された。研究課題/領域番号:13770005, 研究期間(年度):2001-2002出典：「新規接着分子の精子発生における機能解析およびそれと相互作用する分子の探索」研究成果報告書　課題番号13770005（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（ https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-13770005/）を加工して作

Identification and functional characterization of uric acid transporter Urat1 (Slc22a12) in rats

AbstractUric acid transporter URAT1 contributes significantly to reabsorption of uric acid in humans to maintain a constant serum uric acid (SUA) level. Since alteration of SUA level is associated with various diseases, it is important to clarify the mechanism of change in SUA. However, although expression of mRNA of an ortholog of URAT1 (rUrat1) in rats has been reported, functional analysis and localization have not been done. Therefore, rat rUrat1 was functionally analyzed using gene expression systems and isolated brush-border membrane vesicles (BBMVs) prepared from rat kidney, and its localization in kidney was examined immunohistochemically. Uric acid transport by rUrat1 was chloride (Cl−) susceptible with a Km of 1773μM. It was inhibited by benzbromarone and trans-stimulated by lactate and pyrazinecarboxylic acid (PZA). Cl− gradient-susceptible uric acid transport by BBMVs showed similar characteristics to those of uric acid transport by rUrat1. Moreover, rUrat1 was localized at the apical membrane in proximal tubular epithelial cells in rat kidney. Accordingly, rUrat1 is considered to be involved in uric acid reabsorption in rats in the same manner as URAT1 in humans. Therefore, rUrat1 may be a useful model to study issues related to the role of human URAT1

Quantitative Analysis of the Cellular Composition in Seminiferous Tubules in Normal and Genetically Modified Infertile Mice

The aim of this study was to establish a quantitative standard for the cellular composition in seminiferous tubules at each stage of spermatogenesis in the mouse testis, and thereby evaluate abnormalities in the infertile mouse testis. We applied a combination of lectin histochemistry for acrosomes and immunohistochemistry for various specific cell markers, both of which were visualized with fluorescence, on paraffin sections of the testis. We first examined seminiferous tubules from normal mice and counted the number of each cell type at each stage of spermatogenesis. We then examined seminiferous tubules from genetically modified mice deficient (-/-) for one of the cell adhesion molecules, nectin-2 or nectin-3, and compared the number of each cell type at each stage of spermatogenesis with the corresponding value in normal mice. In both nectin-2-/- and nectin-3-/- mice, which are infertile despite the apparently normal morphology of the seminiferous epithelia, we measured a progressive loss in the later-step spermatids, with significantly lower numbers of step 11–16 spermatids in nectin-3-/- mice and step 15–16 spermatids in nectin-2-/- mice as compared with that in normal control mice. The present study demonstrated that a quantitative analysis of cellular compositions at different stages in seminiferous tubules was useful for evaluating abnormalities in spermatogenesis

Compensatory Upregulation of Myelin Protein Zero-Like 2 Expression in Spermatogenic Cells in Cell Adhesion Molecule-1-Deficient Mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells

Expression and Localization of the Cell Adhesion Molecule SgIGSF during Regeneration of the Olfactory Epithelium in Mice

Spermatogenic immunoglobulin superfamily (SgIGSF) is a cell adhesion molecule originally discovered in mouse testis. SgIGSF is expressed not only in spermatogenic cells but also in lung and liver epithelial cells and in neurons and glia of the central and peripheral nervous systems. In the present study, we examined the expression and localization of SgIGSF in mouse olfactory epithelium before and after transection of the olfactory nerves, by RT-PCR, Western blotting and immunohistochemistry. In normal olfactory mucosa, SgIGSF showed 100 kDa in molecular weight, which was identical with that in the lung but different from that in the brain. SgIGSF was expressed on the membrane of all olfactory, sustentacular and basal cells, but more abundantly in the apical portions of the olfactory epithelium where the dendrites of olfactory cells are in contact with sustentacular cells. After olfactory nerve transection, mature olfactory cells disappeared in 4 days but were regenerated around 7–15 days by proliferation and differentiation of basal cells into mature olfactory cells through the step of immature olfactory cells. During this period, both the mRNA and protein for SgIGSF showed a transient increase, with peak levels at 7 days and 11 days, respectively, after the transection. Immunohistochemistry showed that the enriched immunoreactivity for SgIGSF at 7–11 days was localized primarily to the membrane of immature olfactory cells. These results suggested that, during regeneration of the olfactory epithelium, the adhesion molecule SgIGSF plays physiological roles in differentiation, migration, and maturation of immature olfactory cells

A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

Producing antibodies usually takes more than three months. In the present study, we introduce a faster way of producing polyclonal antibodies based on preparation of the recombinant oligopeptide as antigen followed by immunization of rats. Using this method, we produced antisera against two mouse proteins: ERGIC-53 and c-Kit. An expression vector ligated with a pair of complementary synthetic oligodeoxyribonucleotides encoding the protein was introduced into bacteria, and the recombinant oligopeptide fused with the carrier protein glutathione-S-transferase was purified. Wistar rats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads, followed by a booster injection ­after 2 weeks. One week after the booster, the sera were collected and examined for the antibody titer by immunohistochemistry. Antisera with 1600-fold titer at the maximum were obtained for both antigens and confirmed for their specificity by Western blotting. Anti-­ERGIC-53 antisera recognized acinar cells in the sublingual gland, and anti-c-Kit antisera recognized spermatogenic and Leydig cells in the testis. These antisera were applicable to fluorescent double immunostaining with mouse monoclonal or rabbit polyclonal antibodies. Consequently, this method enabled us to produce specific rat polyclonal antisera available for immunohistochemistry in less than one month at a relatively low cost

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

金沢大学医薬保健研究域薬学系Purpose: Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. Methods: MTX was intraperitonealy administered to mrp1 gene knockout (mrp1(-/-)) and wild-type (mrp1(+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. Results: mrp1(-/-) mice more severely decreased body weight, food and water intake than mrp1(+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1(-/-) mice was observed, whereas the damage was only partial in mrp1(+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1(-/-) and mrp1(+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1(-/-) mice was much higher compared to mrp1(+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1(+/+) mice, but not in mrp1(-/-) mice. Conclusion: Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity. © 2009 Springer Science+Business Media, LLC