Nes W D, Lukyanenko Y O, Jia Z H, Quideau S, Howald W N, Pratum T K, West R R, Hutson J C
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79430, USA.
Endocrinology. 2000 Mar;141(3):953-8. doi: 10.1210/endo.141.3.7350.
Macrophages are known to release a lipophilic factor that stimulates testosterone 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 and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected 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 spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. 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. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.
已知巨噬细胞会释放一种亲脂性因子，该因子可刺激睾丸间质细胞产生睾酮。这种巨噬细胞衍生因子（MDF）被认为具有生理相关性，因为从睾丸中去除巨噬细胞会导致睾酮分泌改变和生育能力下降。本研究的目的是纯化该因子，阐明其化学结构，并确定其是否存在于睾丸中以及经睾丸内注射时是否起作用。用乙醚提取睾丸和腹膜巨噬细胞的培养基，有机相依次在C18、硅胶和氰基高效液相色谱柱上进行纯化。使用大鼠睾丸间质细胞生物测定法检测MDF，以睾酮分泌作为终点。通过气相色谱/质谱联用和核磁共振光谱对纯化物质和粗乙醚提取物进行分析。在所有三根高效液相色谱柱上，来自睾丸和腹膜巨噬细胞的MDF的洗脱时间相同。对从最后一根高效液相色谱柱获得的MDF进行气相色谱分析时，观察到一个单峰。来自腹膜和睾丸巨噬细胞的纯化物质的质谱碎片模式与25-羟基胆固醇参考制剂的相同。此外，MDF的核磁共振光谱与正宗的25-羟基胆固醇相似。当25-羟基胆固醇按照与MDF相同的纯化方案进行处理时，发现在所有三根柱上它与MDF同时洗脱，并如预期在睾丸间质细胞生物测定中引发活性。同样纯化的对照培养基不含25-羟基胆固醇或没有生物活性。睾丸的乙醚提取物含有25-羟基胆固醇，表明该化合物在生理条件下存在。同样，当将25-羟基胆固醇注射到成年大鼠的睾丸中时，3小时内睾酮产量增加。综上所述，这些数据表明巨噬细胞产生的刺激类固醇生成的亲脂性因子是25-羟基胆固醇。
