PURIFICATION AND CHARACTERIZATION OF A LIPOPHILIC FACTOR FROM TESTICULAR MACROPHAGES THAT STIMULATES TESTOSTERONE PRODUCTION BY LEYDIG-CELLS

Testicular macrophages have been shown to secrete a factor that stimul ates testosterone production by Leydig cells. The purpose of this inve stigation was to purify and characterize this factor. Medium was colle cted from 24- to 48-hour cultures of testicular macrophages isolated f rom adult rats. This medium induced a sevenfold increase in testostero ne production by cultured Leydig cells. When the medium was extracted with ether, all biological activity was found in the organic phase, in dicating that the factor was lipophilic. The ether extract was then fr actionated on a C-18 reversed-phase high-performance liquid chromatogr aphy (HPLC) column, using a gradient of acidified methanol as the mobi le phase. Leydig cell-stimulating activity eluted at approximately 11 minutes. Standards of testosterone, dihydroepiandrosterone (DHEA), pre gnenolone, progesterone, dihydrotestosterone (DHT), and prostaglandin E(2) (PGE(2)) all had elution times of between 5 and 6 minutes, under identical column conditions, The biological activity of the HPLC-purif ied fraction was partly resistant to boiling but was completely abolis hed by Dextran-coated charcoal treatment. Biological activity of testi cular macrophage-conditioned medium was not abolished following chymot rypsin treatment, indicating that this molecule was not a hydrophilic peptide. It was found that the factor obtained by reversed-phase HPLC could be further purified by normal-phase HPLC. The results of this in vestigation demonstrate that the testicular macrophage-derived factor that stimulates testosterone production by Leydig cells can be purifie d by organic extraction and HPLC, and that it is a highly potent chymo trypsin-resistant heat-stable lipophilic factor.