Purification, cDNA cloning, and developmental changes in the steady-state mRNA 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 [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.
Grima
J
J
Population Council, Center for Biomedical Research, New York, New York 10021, USA.
Calcagno
K
K
Cheng
C Y
CY
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D001921:Brain / Q000201:enzymology; D002478:Cells, Cultured / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D015723:Gene Library; D005854:Germ Cells / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012708:Sertoli Cells / Q000201:enzymology; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D013739:Testosterone / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2
D018076:DNA, Complementary; D011480:Protease Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D013739:Testosterone; D005640:Follicle Stimulating Hormone