Identification and Partial Purification of Embryonic Mouse Genital Protein(s) Stimulating Phospholipase-A₂and Inducing Masculinization in Vitro*

Abstract

The present study was designed to test whether phospholipase-A2 stimulatory protein (PLSP) has any role during androgen-induced masculine differentiation. Thus, an investigation was made to identify such a protein in the fetal genital tract and to test whether this protein can produce masculinization in vitro. Fetal tracts (15/batch) containing genital ducts and urogenital sinus from male, female, and testosterone-exposed (40 mg/kg.day, from days 13-17 of gestation) female embryonic mice on day 18 of gestation were fractionated using Bio-Rad P-100 gel filtration, DEAE-cellulose, and carboxymethyl-Sephadex chromatography. Phospholipase-A2 (PLA2) stimulatory activity was identified at every step of purification. The final preparation stimulated both bee venom and mouse genital PLA2; however, it had no effect on PLC. The preparation lost its PLA2 stimulatory activity after pronase treatment. The partially purified PLSP fraction produced two bands (63K and 55K), as determined by sodium dodecyl sulfate-gel electrophoresis, and its PLA2 stimulatory activity appeared at the region of 55K on a P-100 gel filtration column. PLSP was also identified in female and testosterone-exposed female genital tracts. However, the specific activity of the female PLSP was much lower than that of the male or testosterone-exposed females. Sodium dodecyl sulfate-gel analysis of 2-3 micrograms partially purified PLSP revealed the presence of a faint 55K band in the females compared to the presence of a darker 55K band in the male and testosterone-exposed female. The intensity of the 63K band was similar in both sexes. PLSP from the male and testosterone-exposed females maintained and stimulated the Wolffian duct, whereas PLSP from the female tract had no masculinizing effect. Thus, the masculinizing activity of the PLSP preparation appears to correlate with its PLA2 stimulatory activity and 55K band intensity, suggesting the role of PLA2 stimulatory protein in masculine differentiation.

Short-Chained Alcohols Make Membrane Surfaces Conducive for Melittin Action: Implication for the Physiological Role of Alcohols in Cells

“…This endogenous cationic peptide is structurally similar to bee venom melittin and is recognized by anti-melittin antibodies [ 56 ]. Another membrane-active peptide which regulates PLA 2 activity via modulation of the packing of the lipid phase of membranes was isolated from murine embryonic genital tracts [ 35 , 57 , 58 ]. Two decades ago, a mitochondria-derived peptide, humanin (HN), was identified in the brain of Alzheimer patients [ 59 ].…”

The 72-Kilodalton Protein of the Male Reproductive Tract is Differentially Expressed and Developmentally Regulated during Wolffian Duct Differentiation of the Fetal Mouse1

“…Our initial studies were focused primarily on the first event of the Wolffian duct differentiation process and were designed to determine whether the 72-kDa protein isolated from the fetal male reproductive tract [15] can stabilize the Wolffian duct structure. The protein was indeed able to induce this process as determined by its ability to maintain the Wolffian duct structure [15,16,22] as well as by the ability of its antibody to regress this structure [17]. In the present paper, we provide further evidence in support of the masculinizing role of this protein by demonstrating its specific expression in Wolffian duct structures in response to Wolffian duct differentiation.…”

“…This, in turn, is believed to stimulate specific mRNA transcripts and to result in synthesis of a protein with masculinizing activity. Recently, we identified a 72-kDa protein from the developing male reproductive tract of mouse fetuses that was able to stabilize the Wolffian duct in the absence of fetal testis or testosterone [15]. Testosterone appeared to stimulate the level of this protein, and cyproterone acetate, an anti-androgen receptor, was shown to reduce the level of the protein in the fetal reproductive tract [16].…”