Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis

Macrophages are known to release a lipophilic factor that stimulates testos terone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility . The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular an d peritoneal macrophages were extracted with ether, and the organic phase w as sequentially purified on C-18, silica, and cyano-HPLC columns. MDF was d etected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectr oscopy. The time of elution of MDF from both testicular and peritoneal macr ophages was when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified ma -terial fro m both peritoneal and testicular macrophages was identical to that of a ref erence preparation of 25-hydroxycholesterol. Also, the nuclear magnetic res onance spectrum of MDF was similar to that of authentic 25-hydroxycholester ol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Cont rol medium purified identically did not contain 25-hydroxycholesterol or ha ve biological activity. Ether extracts of testis contained 25-hydroxycholes terol, indicating that this compound is present under physiological conditi ons; Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken togethe r, these data indicate that the lipophilic factor produced by macrophages t hat stimulates steroidogenesis is 25-hydroxycholesterol.