Differential expression of multiple cathepsin mRNAs in the rat testis during maturation and following lonidamine induced tissue restructuring.

In the seminiferous epithelium, germ cell development behind the blood-testis barrier involves continual degradation and renewal of inter-testicular cell junctions. This allows: (i) the translocation of developing germ cells from the basal lamina to the adluminal compartment during spermatogenesis, and (ii) the eventual release of mature spermatids into the tubular lumen during spermiation. Throughout spermatogenesis, cellular debris must also be removed from the epithelium Thus, it is conceivable that proteases, protease inhibitors, and cell junctional components are involved in these events. The present study sought to examine whether testicular cells can express multiple cathepsin mRNAs given that these proteases are involved in the degradation and processing of proteins as well as in tissue regeneration. By using total RNA isolated from primary cultures of Sertoli, Leydig, and germ cells for reverse-transcription and polymerase chain reaction (RT-PCR), the mRNAs of cathepsin B, C, D, H, L, and S were shown to be expressed by Sertoli and Leydig cells, whereas germ cells isolated from adult rats expressed all of the above cathepsin mRNAs except cathepsin D. Throughout postnatal development and maturation, the testicular steady-state mRNA levels of cathepsin B, C, D, L, and S remain relatively unchanged with the exception of cathepsin H whose mRNA level increased during maturation and peaked at 45-60 days of age. Using lonidamine, an anti-spermatogenic drug which is known to induce premature release of germ cells without affecting Leydig cell function by disrupting the inter-Sertoli-germ cell junctions, we have examined the differential expression of these cathepsin mRNAs in the testis at the time of extensive tissue restructuring. It was noted that the expression of cathepsin L and S in the testis increased significantly concomitant with the disappearance of elongate spermatids whereas the expression of cathepsin B, C, D, and H increased significantly when most of the round spermatids and spermatocytes were depleted. These results illustrate the intricate inter-relationship between these proteases in the testis during maturation and tissue restructuring.

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.