Further characterization of the testosterone inducible protein fraction from the mouse fetal male reproductive tract inducing masculine differentiation in vitro

Abstract

Recently, we identified a protein fraction with phospholipase A2 (PLA2) stimulatory activity (named as PLSP in previous publications) from the fetal male reproductive tract which induced masculine differentiation of the Wolffian duct in vitro. The role of the PLA2-stimulatory activity of this protein was not clear from the past investigations and the present study was designed to elucidate its role. PLSP as expected stimulated snake venom PLA2 and it induced masculinization not only in male explant but also in female explants in a dose-dependent manner in the absence of gonads. However, neither its PLA2-stimulatory activity nor its masculinizing activity was affected after passing it through a PLA2-Sepharose column (a compound expected to eliminate any PLA2 binding component) suggesting that the PLA2-stimulatory activity of PLSP was not at the level of PLA2 enzyme. To investigate whether modulation of the PLA2 substrate by PLSP played a role in producing PLA2-stimulatory activity, we determined the effect of increasing amounts of unlabeled substrate on the PLA2-stimulatory activity induced by PLSP. However, no change in the PLA2-stimulatory activity of PLSP by unlabeled substrate was noticed suggesting against this possibility. The PLA2-stimulatory activity of PLSP, however, was found completely abolished when it was subjected to extensive dialysis, while its protein composition and masculinizing activity remained unaffected. Thus, it appears that PLA2-stimulatory activity of PLSP is associated with a small compound co-purified with masculinizing protein of PLSP and this activity plays no role in inducing masculinization by PLSP.