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Identification of the Lipophilic Factor Produced by Macrophages That Stimulates Steroidogenesis¹

Department of Chemistry and Biochemistry, Texas Tech University (W.D.N., Z.H.J.), and the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (Y.O.L., J.C.H.), Lubbock, Texas 79430; Laboratoire de Chimie des Substances Végétales, University of Bordeaux I (S.Q.), Talence, France; the Departments of Medicinal Chemistry (W.N.H.) and Chemistry (T.R.P.), University of Washington, Seattle, Washington 98195; and Zymogenetics (R.R.W.), Seattle, Washington 98195

Address all correspondence and requests for reprints to: Dr. James C. Hutson, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: jim.hutson{at}ttmc.ttuhsc.edu

	Abstract

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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.

	Introduction

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THE PRODUCTION of testosterone by Leydig cells is essential for spermatogenesis and thereby propagation of the species. After the early work of Brady (1), it has become well established that Leydig cell steroidogenesis is regulated by LH. However, it is also known that local factors play a role in the regulation of steroidogenesis. Macrophages are commonly found in direct contact with Leydig cells in unusually high numbers, thus strategically poised for paracrine involvement (2, 3, 4). More specifically, it has been shown that elimination of macrophages from the testis caused altered testosterone production and reduced fertility (5, 6, 7). Earlier studies from this laboratory demonstrated that macrophages secrete a factor capable of stimulating Leydig cells to produce testosterone in culture (8), which may mediate the stimulatory action of macrophages in vivo. We refer to this factor as macrophage-derived factor (MDF). MDF can be extracted from macrophage-conditioned medium with organic solvents and purified by HPLC (9). Such preparations stimulated testosterone synthesis in the same time frame and to a similar efficacy as hCG/LH without causing an increase in the expression of the acute steroidogenic regulatory protein (10). The significance of this phenomenon was further heightened by the finding that MDF also acted on granulosa cells of the rat ovary and adrenal cortical cells of both rats and humans (11). Taken together, these in vivo and in vitro studies indicate that MDF is an important paracrine regulator of steroidogenesis in both rats and humans. Although several lipophilic compounds are known to be secreted by macrophages (12), the identity of this steroidogenic lipid was unknown. Therefore, the primary purpose of the present study was to determine the chemical structure of MDF. Because most previous studies have been conducted in vitro, we were also interested in determining whether MDF is both present in the testis and/or acts within the testis under normal physiological conditions.

	Materials and Methods

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Materials
Adult rats (150–300 g) were obtained from Harlan Sprague Dawley, Inc. (Minneapolis, MN), and maintained under standard conditions. DMEM/Ham’s F-12 medium (DMEM/F12), Dulbecco’s PBS without calcium and magnesium (PBS), BSA (fraction V), newborn calf serum, penicillin, streptomycin, collagenase (type I), and other routine compounds were obtained from Sigma (St. Louis, MO). Organic solvents were purchased from Fisher Scientific (Fairlawn, NJ). 20-, 22(R)-, and 25-hydroxycholesterols were obtained from Steraloids (Newport, RI). 27-hydroxycholesterol [(25R)-cholest-5-ene-3ß, 26-diol] was obtained from the W. David Nes collection. The HPLC columns (C₁₈, silica and cyano; Microsorb MV, 100 angstrom, 4.6 mm x 25 cm) were obtained from Varian Chromatography Systems (Walnut Creek, CA). The 100-mm diameter culture dishes (no. 3001) were obtained from Becton Dickinson and Co. Labware (Franklin Lakes, NJ), and the 96-well plates (no. 25860) were purchased from Corning Glass Works (Corning, NY). The testosterone RIA kit was purchased from Diagnostics Systems Laboratories, Inc. (Webster, TX).

Purification of MDF
Testicular and peritoneal macrophage-conditioned media were obtained from adult rats as previously described (8, 9, 10, 11). Testicular macrophages were cultured in DMEM/F12 plus 0.1% BSA, and peritoneal macrophages were cultured in DMEM/F12 plus 0.1% BSA and 10% newborn calf serum. Briefly, after 2–3 days in culture, the media were extracted with ether and chromatographed on a C₁₈ reverse phase HPLC column using a methanol-water gradient (70–100% methanol developed linearly over 2 min at 1 ml/min). The fraction that stimulated testosterone production in the Leydig cell bioassay was then further purified on a normal phase silica HPLC column using 90% hexane/10% isopropyl alcohol as the mobile phase under isocratic conditions at 1 ml/min. The fraction with Leydig cell-stimulating activity was then chromatographed on a cyano-HPLC column using 90% hexane/10% isopropyl alcohol as the mobile phase at 1 ml/min. Two separate 10-liter preparations of peritoneal macrophage-conditioned medium and one 800-ml preparation of testicular macrophage-conditioned medium were analyzed. Control medium (without exposure to macrophages) was also purified as described above.

Leydig cell bioassay
Leydig cells were isolated from adult rats, plated in 100 µl DMEM/F12 plus 0.1% BSA at 20,000–40,000 cells/well, and maintained for 18–24 h in a CO₂ incubator at 33 C in 96-well plates as previously described (9). On the following day, the medium was removed, and 50 µl fresh medium containing samples from the various fractions from the HPLC or control preparations were added. The medium was then assayed for testosterone after 5 h using a commercial RIA with standards prepared in the same medium as that used for the Leydig cells.

Total cellular sterol content
Testicular macrophages and Leydig cells were isolated as described above and plated into 100-mm dishes in DMEM/F12 plus 0.1% BSA. After 24 h in culture, the cells were counted using a gridded eyepiece calibrated to a stage micrometer. The cells were then scraped from the dishes in methanol and sonicated for 1 min to completely disrupt the cells. The methanol was evaporated, and the sample was sonicated for an additional 1 min in 200 µl acetone. The sample was centrifuged at 14,000 x g, and the sterol content in the supernatant was determined by gas chromatography (GC) and GC/mass spectrometry (GC/MS).

GC/MS
GC/MS analysis of the HPLC isolates was performed on a Hewlett-Packard Co. 6890 series gas chromatograph (GC) equipped with an on-column injector and coupled to a Hewlett-Packard Co. 5973 mass spectrometer (MS) operated in the positive ion electron ionization mode (Hewlett-Packard Co., Palo Alto, CA). A DB-5 fused silica capillary gas chromatographic column (id, 30 m x 0.25 mm; 0.25-µm film thickness) was used (J & W Scientific, Rancho Cordova, CA) using a temperature program of 170–280 C developed over 7 min.

Nuclear magnetic resonance spectroscopy
Purified samples of MDF from peritoneal macrophages were dissolved in CDCl₃ and analyzed using a Bruker DMX750 (750 MHz) FT nuclear magnetic resonance (NMR) spectrometer.

Presence of MDF in the testis
Testes were removed from normal adult rats and decapsulated. The testes were then minced into small pieces and extracted with ether for 4 h. MDF was purified from this extract as described above. This procedure was repeated three times using 13, 16, or 8 animals. The total MDF recovered from each experiment was diluted into 170 µl culture medium and added to Leydig cells plated at a density of 16,000 cells/well in 96-well plates at 50 µl/well. Leydig cells were cultured for 22 h, and the medium was assayed for testosterone.

Actions of MDF in vivo
Adult rats were anesthetized with ether, and 100 µg 25-hydroxycholesterol was injected into the left testis through the scrotum in 100 µl PBS containing 10% ethanol. The right testis received only the solvent. After 3 h, the animals were killed by CO₂ asphyxiation, and the testes were decapsulated and homogenized in 6 ml ether. Water (6 ml) was then added, and the extract was transferred to 100-ml bottles. Ether (30 ml) was then added, and the mixture was allowed to stand for 15 min. The organic phase was removed and dried under nitrogen, and the amount of testosterone was determined by RIA.

Estimate of the rate of production of 25-hydroxycholesterol
Testicular macrophages from 10 adult rats were isolated as described above and plated into 10 100-mm dishes in 7 ml DMEM/F-12 plus 0.1% BSA. The cells were cultured for 45 h, and then 25-hydroxycholesterol was isolated as described above. The amount of 25-hydroxycholesterol in this sample was determined by comparing the peak area (UV absorbance obtained during HPLC) to that obtained with a standard curve of authentic 25-hydroxycholesterol.

Sensitivity of Leydig cells to 25-hydroxycholesterol
Leydig cells were obtained from adult rats as described above and plated into 96-well plates at approximately 42,000 cells/well in 100 µl DMEM/F-12 plus 0.1% BSA. After 18 h, 200 µl fresh medium were added to the cells, and then all medium was immediately replaced with 50 µl fresh medium containing various doses of 25-hydroxycholesterol (0, 0.001, 0.01, 0.1, 1, and 10 µg/ml). The cells were maintained for 8 h, and the medium was assayed for testosterone as described above.

	Results

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Purified MDF from either testicular or peritoneal macrophages eluted as a single peak by GC (Fig. 1). When this peak was analyzed by MS, a molecular ion at m/z 402 (M^+.) was observed (Fig. 2). Because this fragmentation pattern and mol wt indicated that MDF was a hydroxycholesterol-like compound, several oxysterol reference preparations were similarly analyzed by GC/MS [20-, 22(R)-, 25-, and 27-hydroxycholesterol]. Only 25-hydroxycholesterol was found to both elute at the same time as MDF by GC and exhibit an identical fragmentation pattern by MS (Figs. 1 and 2). The elution times (RRTc) of these authentic standards compared with cholesterol were 1.267 for 20-hydroxycholesterol, 1.310 for 25-hydroxycholesterol, 1.362 for 22(R)-hydroxycholesterol, and 1.582 for 27-hydroxycholesterol.

View larger version (19K): [in this window] [in a new window]   Figure 1. MDF from testicular and peritoneal macrophages was purified by HPLC and evaluated by GC. Resulting chromatograms are illustrated in comparison to a reference preparation of 25-hydroxycholesterol. Results indicate that the HPLC purification procedure resulted in near-homogeneous preparations of MDF, and that it elutes from the GC at the same time as 25-hydroxycholesterol.

View larger version (28K): [in this window] [in a new window]   Figure 2. The GC peaks eluting at approximately 18 min on the chromatograms illustrated in Fig. 1 were analyzed by MS. Identical spectra were observed for all three preparations, indicating that MDF is 25-hydroxycholesterol.

View larger version (16K): [in this window] [in a new window]   Figure 3. The 1H NMR spectra for MDF (bottom) and authentic 25-hydroxycholesterol (top) were very similar, with the exception of low level impurities in the MDF sample and a signal at 4.1 ppm in the 25-hydroxycholesterol spectrum that comes from residual isopropanol used to wash the tube containing this material. Both samples were dissolved in CDCl3 and analyzed using a Bruker DMX750 (750 MHz) FTNMR spectrometer.

View larger version (24K): [in this window] [in a new window]   Figure 4. Ether extracts of peritoneal macrophage-conditioned medium or testicular macrophage-conditioned medium, and a reference preparation of 25-hydroxycholesterol were purified on a C18 HPLC column and then on the silica HPLC column, each time collecting the fractions that had activity in the Leydig cell bioassay. The biologically active fractions from the silica column were then chromatographed on the cyano column, and the biological activities of those fractions are illustrated in this graph. These data illustrate that MDF and 25-hydroxycholesterol elute identically from all three HPLC columns, and all have biological activity. One hundred micrograms of the reference preparation of 25-hydroxycholesterol were assayed, whereas approximately 10 µg or less of the testicular and peritoneal macrophage sources of 25-hydroxycholesterol (MDF) were obtained (amount estimated by UV absorption).

View larger version (25K): [in this window] [in a new window]   Figure 5. A illustrates that bioactive MDF/25-hydroxycholesterol is present in normal adult rat testis. Testes were extracted with ether, and MDF/25-hydroxycholesterol was purified by HPLC and then added to the Leydig cell bioassay. Control represents Leydig cells in the absence of treatment. Treatment resulted in a statistically significant difference (P < 0.05), as determined by unpaired Student’s t test (n = 3). B illustrates that 25-hydroxycholesterol is capable of increasing testosterone production after injection into normal adult rat testis. Animals were anesthetized with ether; 100 µg 25-hydroxycholesterol were injected into the left testis, and saline was injected into the right testis. After 3 h, the testes were removed, and the total amount of testosterone per testis was determined by RIA. Treatment resulted in a statistically significant difference (P < 0.05), as determined by paired Student’s t test (n = 6).

	Discussion

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The rate-limiting step in biosynthesis of all steroid hormones is the rate of movement of cholesterol to the side-chain cleavage enzyme complex on the inner mitochondrial membrane (13). Once at this site, cholesterol is hydroxylated at the 20 and 22 positions, yielding 20,22(R)-hydroxycholesterol (14, 15, 16). The side-chain is then cleaved yielding pregnenolone, which is further metabolized to various steroid hormones (depending upon cell type) through higher capacity enzyme systems. The present findings indicate that macrophages have the potential to provide an alternate pathway for steroidogenesis, which bypasses the traditional rate-limiting step by offering 25-hydroxycholesterol as a direct substrate for side-chain cleavage (Fig. 6). The rate-limiting step for this paracrine pathway would therefore be the regulation of biosynthesis of 25-hydroxycholesterol in the macrophage. A 25-hydroxylase has been described and thereby may produce the 25-hydroxycholesterol needed for this LH-independent paracrine pathway (17). Although nothing is currently known of the regulation of 25-hydroxycholesterol production by testicular macrophages, it is unlikely that immune activation of macrophages plays a role, as activation has been shown to cause the release of inhibitors, rather than stimulators, of steroidogenesis (18).

View larger version (21K): [in this window] [in a new window]   Figure 6. This scheme, representing our working hypothesis, illustrates the known conversion of cholesterol to testosterone as influenced by the LH-regulated pathway using 20,22-hydroxycholesterol as the endogenous substrate and the proposed macrophage-regulated pathway using 25-hydroxycholesterol as an exogenous substrate. We further hypothesize that there may be a regulator of this paracrine pathway.

As mentioned earlier, testosterone levels have been shown to be altered and animals become less fertile when macrophages are removed from the testis by experimental or genetic approaches (5, 6, 7). In the experimental model, toxins have been injected into the testis that kill macrophages by apoptosis (5, 6). In the genetic model, macrophages are absent because the animals produce a mutant form of colony-stimulating factor-1, a growth factor responsible for the production of monocytes/macrophages (7). Although the effect of macrophage depletion in the genetic model appears to be mediated in part by decreased circulating levels of LH, the data also support a local macrophage-derived effect (19). In the experimentally induced macrophage depletion models, it appears that most of the effect is exerted locally. This local effect may be due to the absence of macrophage-derived 25-hydroxycholesterol, as this is the only stimulatory factor known to be present in macrophage-conditioned medium (Ref. 9 and the present data). The present studies add to these physiological studies, not only by identifying 25-hydroxycholesterol as the possible paracrine mediator, but also by demonstrating that it is present in the testes, and causes increased testosterone production when injected into the testis.

Although oxysterols are known to naturally occur as autoxidation products (21), it is clear that 25-hydroxycholesterol was not formed by autoxidation in the present studies, either in culture or during purification, as control medium (exposed to identical conditions) did not contain 25-hydroxycholesterol and lacked biological activity. Also, other oxy-sterols were not detected in ether extracts of macrophage-conditioned medium before purification.

Cooperation between cells for metabolism of steroids to their final active form is an important and conserved theme in reproductive biology. For example, theca internal cells produce androgens, which are subsequently aromatized by granulosa cells to estradiol (22, 23). Similarly androgens from Leydig cells are aromatized by Sertoli cells to estradiol (24), and androgens can be 5-reduced by target cells, yielding a more active compound (25). Thus, passage of 25-hydroxycholesterol from macrophages to neighboring steroidogenic cells for conversion to pregnenolone and ultimately more active terminal hormones may be an additional example of how evolution has reproduced this most interesting theme.

Although the most likely fate of testicular macrophage-derived 25-hydroxycholesterol is conversion to pregnenolone, it is worth noting that this sterol has been shown to act as a signaling molecule in a wide variety of cell types. For example, 25-hydroxycholesterol stimulates sphingomyelin synthesis in Chinese hamster ovary cells (26), inhibits macrophage and lymphocyte functions (27), stimulates the accumulation of intracellular calcium in smooth muscle cells (28), inhibits the growth of tumor cells (29), and induces apoptosis (30) and eicosanoid production (31) in endothelial cells. Although the mechanisms responsible for these actions have yet to be determined, a nonnuclear oxysterol-binding protein has been found (32) that is involved in the translocation of this sterol-bound protein to the Golgi apparatus (33). However, it should be emphasized that the significance of this translocation is unknown in any cell type, and therefore much more is needed to be done to determine whether this mechanism is present and operative in Leydig cells.

In summary, the present studies demonstrate that the lipophilic factor produced macrophages that stimulates steroidogenesis is 25-hydroxycholesterol. We have also shown that this oxsterol is both present within the testis and acts when injected into the testis of adult rats.

	Acknowledgments

 
The authors thank Ms. Selena Wampler, a Howard Hughes Scholar, for her assistance with the purification of MDF.

	Footnotes

 
¹ This work was supported by grants from the Advanced Research Program from the Texas Higher Education Coordinating Board, the South Plains Foundation, the Dean’s Cooperative Seed Research Grant Program, and the NIH (HD-34708; to J.C.H.) and from the Welch Foundation (D1276; to W.D.N.).

Received November 19, 1999.

	References

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1. Brady RO 1956 Biosynthesis of radioactive testosterone in vitro. J Biol Chem 193:145–148
2. Hardy MP, Zirkin BR, Ewing LL 1989 Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat. Endocrinology 124:762–770[Abstract]
3. Hutson JC 1990 Development of testicular macrophages. Biol Reprod 43:885–890[Abstract]
4. Hutson JC 1992 Development of digitations between Leydig cells and macrophages. Cell Tissue Res 267:385–389[CrossRef][Medline]
5. Bergh A, Damber J-E, van Rooijen N 1986 Liposome-mediated macrophage depletion: an experimental approach to study the role of testicular macrophages. J Endocrinol 136:407–413
6. Gaytan F, Bellido C, Morales C, Garcia M, van Rooijen N, Aguilar E 1996 In vitro manipulation (depletion versus activation) of testicular macrophages: central and local effects. J Endocrinol 150:57–65[Abstract]
7. Cohen PE, Chisholm O, Arceci RJ, Stanley ER, Pollard JW 1996 Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects. Biol Reprod 55:310–317[Abstract]
8. Yee JB, Hutson JC 1985 Effects of testicular macrophage-conditioned medium on Leydig cells in culture. Endocrinology 116:2682–2684[Abstract]
9. Hutson JC, Garner C, Doris PA 1996 Purification and characterization of a lipophilic factor from testicular macrophages that stimulates testosterone secretion. J Androl 17:502–508[Abstract/Free Full Text]
10. Lukyanenko YO, Carpenter A, Brigham D, Stocco D, Hutson JC 1998 Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages. J Endocrinol 158:267–275[Abstract]
11. Lukyanenko YO, Carpenter AM, Boone MM, Baker Jr CRF, McGunegle DE, Hutson JC Tissue and species specificity of a macrophage-derived factor involved in stimulating steroidogenesis: potential for therapeutic use in humans. Int J Androl, in press
12. Gordon S 1996 Overview: the myeloid system. In: Herzenberg LA, Weir DM, Herzenberg LA, Blackwell C (eds) Weir’s Handbook of Experimental Immunology. Blackwell, Cambridge, vol 4:153.1–153.9
13. Iida S, Papadopoulos V, Hall PF 1989 The influence of exogenous free cholesterol on steroid synthesis in culture of adrenal cells. Endocrinology 124:2619–2624[Abstract]
14. Shimizu K, Gut M, Dorfman RI 1962 20, 22-Dihydroxycholesterol, an intermediate in the biosynthesis of pregnenolone (3ß-hydroxypregn-5-en-20-one) from cholesterol. J Biol Chem 237:699–702[Free Full Text]
15. Constantopoulos G, Tchen TT 1961 Cleavage of cholesterol side chain by adrenal cortex. J Biol Chem 236:65–67[Free Full Text]
16. Takemoto C, Nakano H, Sato H, Tamaoki B 1968 Fate of molecular oxygen required by endocrine enzymes for the side-chain cleavage of cholesterol. Biochim Biophys Acta 152:749–757[Medline]
17. Lund EG, Kerr TA, Sakai J, Li W, Russell DW 1998 cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J Biol Chem 273:34316–34327[Abstract/Free Full Text]
18. Hales BD 1996 Leydig cell-macrophage interactions: an overview. In: Payne A, Hardy M, Russell L (eds) The Leydig Cell. Cache River Press, Vienna, pp 451–465
19. Cohen PE, Hardy MP, Pollard JW 1997 Colony-stimulating factor-1 plays a major role in the development of reproductive function in male mice. Mol Endocrinol 11:1636–1650[Abstract/Free Full Text]
20. Hardy, MP, Zirkin, BR, Ewing, LL 1989 Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat. Endocrinology 124:762–770
21. Smith L 1987 Cholesterol autoxidation 1981–1986. Chem Physics Lipids 44:87–125[CrossRef][Medline]
22. Dorrington JH, Moon YS, Armstrong DT 1975 Estradiol-17ß biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone. Endocrinology 97:1328–1331[Abstract]
23. Fortune JE, Armstrong DT 1977 Androgen production by theca and granulosa isolated from proestrous rat follicles. Endocrinology 100:1341–1347[Abstract]
24. Dorrington JH, Armstrong DT 1975 Follicle-stimulating hormone stimulates estradiol-17ß synthesis in cultured Sertoli cells. Proc Natl Acad Sci USA 72:2677–2681[Abstract/Free Full Text]
25. Resko JA, Roselli CE 1997 Prenatal hormones organize sex differences of the neuroendocrine reproductive system: observations on guinea pigs and nonhuman primates. Cell Mol Neurobiol 17:627–648[CrossRef][Medline]
26. Storey MK, Byers DM, Cook HW, Ridgway ND 1998 Cholesterol regulates oxysterol binding protein (OSBP) phosphorylation and Golgi localization in Chinese hamster ovary cells: correlation with stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. Biochem J 336:247–256
27. Dushkin M, Schwartz Y, Volsky N, Mustov M, Vereschagin E, Perminova O, Kozlov V 1998 Effects of oxysterols upon macrophage and lymphocyte function in vitro. Prostaglandins Other Lipid Mediators 55:219–236[Medline]
28. Zhou Q, Jimi S, Smith TL, Kummerow FA 1991 The effect of 25-hydroxycholesterol on accumulation of intracellular calcium. Cell Calcium 12:467–476[CrossRef][Medline]
29. Kato H, Horino A, Taneichi M, Fukuchi N, Eto Y, Ushijima H, Komuro K, Uchida T 1998 Macrophage inhibition of lymphocyte and tumor cell growth is mediated by 25-hydroxycholesterol in the cell membrane. Int Arch Allergy Immunol 117:78–84[CrossRef][Medline]
30. Lizard G, Deckert V, Dubrez L, Moisant M, Gambert P, Lagrost L 1996 Induction of apoptosis in endothelial cells treated with cholesterol oxides. Am J Pathol 148:1625–1638[Abstract]
31. Wohlfeil ER, Campbell WB 1997 25-Hydroxycholesterol enhances eicosanoid production in cultured bovine coronary artery endothelial cells by increasing prostaglandin G/H synthase-2. Biochim Biophys Acta 1345:109–120[Medline]
32. Dawson PA, Ridgway ND, Slaughter CA, Brown MS, Goldstein JL 1989 cDNA cloning and expression of oxysterol-binding protein, an oligomer with a potential leucine zipper. J Biol Chem 264:16798–16803[Abstract/Free Full Text]
33. Ridgway ND, Dawson PA, Ho YK, Brown MS, Goldstein JL 1992 Translocation of oxysterol-binding protein to Golgi apparatus triggered by ligand binding. J Cell Biol 116:307–319[Abstract/Free Full Text]

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