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Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell
M K Skinner1, P M Fetterolf, C T Anthony
1Department of Pharmacology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
Abstract:
A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.
Insights
A testicular paracrine factor, P-Mod-S, was purified from peritubular cells. This protein significantly enhances Sertoli cell function, impacting spermatogenesis and testicular health.
Area of Science:
- Reproductive Biology
- Cell Signaling
- Endocrinology
Background:
- Peritubular cells and Sertoli cells are crucial for testicular function.
- Interactions between these cells are vital for spermatogenesis.
- A specific paracrine factor mediating these interactions was yet to be fully characterized.
Purpose of the Study:
- To purify and characterize a novel testicular paracrine factor (P-Mod-S) from peritubular cells.
- To investigate the bioactivity of P-Mod-S on Sertoli cell function.
- To elucidate the role of P-Mod-S in cell-cell communication within the testis.
Main Methods:
- Conditioned medium from serum-free peritubular cell cultures was used for purification.
- High-pressure size exclusion chromatography and reverse-phase chromatography were employed.
- Bio-assay involved stimulation of testicular transferrin production by Sertoli cells.
- Radiolabeling was used to confirm synthesis and secretion by peritubular cells.
Main Results:
- A bioactive protein, P-Mod-S, with molecular weights of 56,000 (P-Mod-S (A)) and 59,000 (P-Mod-S (B)) was isolated.
- P-Mod-S exhibits heparin-binding affinity.
- Peritubular cells actively synthesize and secrete both forms of P-Mod-S.
- Purified P-Mod-S demonstrated significantly greater effects on Sertoli cell function (transferrin and androgen-binding protein production) than known agents, including FSH.
Conclusions:
- P-Mod-S is a potent paracrine factor secreted by peritubular cells that significantly influences Sertoli cell function.
- P-Mod-S plays a critical role in mediating mesenchymal-epithelial cell interactions within the testis.
- These interactions are essential for maintaining spermatogenesis and overall testicular function.