PURIFICATION, CDNA CLONING, AND DEVELOPMENTAL-CHANGES IN THE STEADY-STATE MESSENGER-RNA LEVEL OF RAT TESTICULAR TISSUE INHIBITOR OF METALLOPROTEASES-2 (TIMP-2)

Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [I-125]collagen film assay to identify inhibitors of metallop roteases, we have purified a 22-kDa polypeptide to apparent homogeneit y from primary Sertoli cell-enriched culture medium. Partial N-termina l amino acid sequence analysis revealed that this protein is similar t o the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determ ine the similarity of rat testicular TIMP-2 to the human homolog, a fu ll-length cDNA coding for rat testicular TIMP-2 was isolated from a ra t Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat tes ticular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIM P-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 ra ts. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sert oli cells, and Leydig cells have shown that only Sertoli and Leydig ce lls expressed TIMP-2 mRNA. These results indicate that Sertoli cells a re the major source of TIMP-2 in the testis behind the blood-testis ba rrier (seminiferous tubule barrier). During testicular development fro m 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level i ncreased 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.