ABILITY OF TRYPSIN IN MIMICKING GERM-CELL FACTORS THAT AFFECT SERTOLI-CELL SECRETORY FUNCTION

A biological factor that inhibits the in vitro secretion of testin by Sertoli cells was purified to apparent homogeneity from conditioned me dium of germ cells isolated using trypsin. Partial N-terminal amino ac id sequence analysis of the purified germ cell factor revealed a seque nce of NH2-IVGGYTXAAN. Comparison of the sequence with the existing pr otein database revealed that it is homologous to trypsin. Immunoprecip itation experiments using either [S-35]-labeled germ or Sertoli cell p roteins and a monospecific anti-trypsin antibody failed to demonstrate the synthesis and secretion of trypsin by these testicular cells, sug gesting the isolated factor is the residuary trypsin that was used for isolating germ cells from seminiferous tubules. Subsequent experiment s revealed that trypsin per se can inhibit the secretion of Sertoli ce ll testin and clusterin dose-dependently, whose effect can be prohibit ed by soybean trypsin inhibitor (STI). In view of these findings, a no nenzymatic procedure was deemed necessary to prepare germ cell conditi oned medium (GCCM) to assess whether an authentic biological factor(s) is indeed present. Four batches of conditioned medium of germ cells i solated by a mechanical procedure without the use of trypsin were frac tionated by sequential Mono Q anion exchange and C8 reversed-phase HPL C. When these fractions were monitored for testin modulatory activity using an in vitro bioassay with primary cultures of Sertoli cells, it was shown that GCCM prepared by this procedure indeed contained testin modulatory bioactivity. Since testin is a novel component of speciali zed junctions between Sertoli and germ cells, the identification of a germ cell factor(s) that affects its secretion by Sertoli cells sugges ts a dynamic biochemical relationship between these cell types in the seminiferous epithelium. (C) 1996 Wiley-Liss, Inc.