Grima J, Calcagno K, Cheng C Y
Population Council, Center for Biomedical Research, New York, New York 10021, USA.
J Androl. 1996 May-Jun;17(3):263-75.
Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [125I]collagen film assay to identify inhibitors of metalloproteases, we have purified a 22-kDa polypeptide to apparent homogeneity from primary Sertoli cell-enriched culture medium. Partial N-terminal amino acid sequence analysis revealed that this protein is similar to the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determine the similarity of rat testicular TIMP-2 to the human homolog, a full-length cDNA coding for rat testicular TIMP-2 was isolated from a rat Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat testicular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIMP-2 nucleotide and amino acid sequences, respectively. A survey of its mRNA transcripts in different tissues by northern blots revealed the presence of two mRNA species of 3.7 and 1.3 kb in the testis and brain but not in the kidney, spleen, epididymis, and liver in adult male rats. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sertoli cells, and Leydig cells have shown that only Sertoli and Leydig cells expressed TIMP-2 mRNA. These results indicate that Sertoli cells are the major source of TIMP-2 in the testis behind the blood-testis barrier (seminiferous tubule barrier). During testicular development from 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level increased steadily with an advancement of age. Such an increase in the steady-state testicular TIMP-2 mRNA level apparently is not the result of an up-regulation by germ cells because germ cells cocultured with Sertoli cells failed to elicit an increase in the Sertoli cell steady-state TIMP-2 mRNA level. The results of this study suggest that TIMP-2 secreted by Sertoli cells may play a role in tissue restructuring and germ cell migration during spermatogenesis.
我们运用多个高效液相色谱（HPLC）步骤以及高效电泳色谱（HPEC），结合[125I]胶原蛋白膜分析法来鉴定金属蛋白酶抑制剂，从富含原代支持细胞的培养基中纯化出一种22 kDa的多肽，使其达到表观均一性。部分N端氨基酸序列分析表明，该蛋白与人类金属蛋白酶组织抑制剂-2（TIMP-2）相似。为确定大鼠睾丸TIMP-2与人同源物的相似性，从大鼠支持细胞cDNA表达文库中分离出编码大鼠睾丸TIMP-2的全长cDNA并进行测序。对大鼠睾丸TIMP-2 cDNA的核苷酸序列和推导的氨基酸序列分析显示，其与人类TIMP-2核苷酸序列和氨基酸序列的同源性分别为84%和98%。通过Northern印迹法对成年雄性大鼠不同组织中其mRNA转录本的调查显示，在睾丸和脑中存在3.7 kb和1.3 kb的两种mRNA，但在肾脏、脾脏、附睾和肝脏中未检测到。使用聚合酶链反应（PCR）和Southern印迹法，利用从生殖细胞、支持细胞和间质细胞中分离的总RNA检测TIMP-2 mRNA的研究表明，只有支持细胞和间质细胞表达TIMP-2 mRNA。这些结果表明，在血睾屏障（生精小管屏障）之后，支持细胞是睾丸中TIMP-2的主要来源。在3至60日龄睾丸发育过程中，睾丸TIMP-2 mRNA的稳态水平随着年龄的增长而稳步增加。这种睾丸TIMP-2 mRNA稳态水平的增加显然不是生殖细胞上调的结果，因为与支持细胞共培养的生殖细胞未能引起支持细胞TIMP-2 mRNA稳态水平的增加。本研究结果表明，支持细胞分泌的TIMP-2可能在精子发生过程中的组织重构和生殖细胞迁移中发挥作用。
