Ability of trypsin in mimicking germ cell factors that affect Sertoli cell secretory function

• Published in: Journal of cellular physiology Vol. 168; no. 1; pp. 123 - 133
• Main Authors: Aravindan, G. Rolands, Pineau, Charles P., Bardin, C. Wayne, Cheng, C. Yan
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.07.1996
• Subjects: Amino Acid Sequence; Animals; Cell Separation - methods; Cells, Cultured; Germ Cells - physiology; Male; Molecular Sequence Data; Proteins - metabolism; Rats; Rats, Sprague-Dawley; Sertoli Cells - secretion; Spermatogenesis; Trypsin - pharmacology
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/(SICI)1097-4652(199607)168:1<123::AID-JCP15>3.0.CO;2-8

A biological factor that inhibits the in vitro secretion of testin by Sertoli cells was purified to apparent homogeneity from conditioned medium of germ cells isolated using trypsin. Partial N‐terminal amino acid sequence analysis of the purified germ cell factor revealed a sequence of NH2‐IVGGYTXAAN. Comparison of the sequence with the existing protein database revealed that it is homologous to trypsin. Immunoprecipitation experiments using either [15S]‐labeled germ or Sertoli cell proteins and a monospecific anti‐trypsin antibody failed to demonstrate the synthesis and secretion of trypsin by these testicular cells, suggesting the isolated factor is the residuary trypsin that was used for isolating germ cells from seminiferous tubules. Subsequent experiments revealed that trypsin per se can inhibit the secretion of Sertoli cell testin and clusterin dose‐dependently, whose effect can be prohibited by soybean trypsin inhibitor (STI). In view of these findings, a nonenzymatic procedure was deemed necessary to prepare germ cell conditioned medium (GCCM) to assess whether an authentic biological factor(s) is indeed present. Four batches of conditioned medium of germ cells isolated by a mechanical procedure without the use of trypsin were fractionated by sequential Mono Q anion exchange and C8 reversed‐phase HPLC. 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 specialized junctions between Sertoli and germ cells, the identification of a germ cell factor(s) that affects its secretion by Sertoli cells suggests a dynamic biochemical relationship between these cell types in the seminiferous epithelium. © 1996 Wiley‐Liss, Inc.