Biochemical Characterization of Pre- 1 High-Density Lipoprotein from Human Ovarian Follicular Fluid: Evidence for the Presence of a Lipid Core ²,‡ Be ´atrice Jaspard, § Xavier Collet,* ,§ Ronald Barbaras, § Jeanine Manent, § Claude Vieu, § Jean Parinaud, | Hugues Chap, § and Bertrand Perret § Institut National de la Sante ´ et de la Recherche Me ´ dicale, Unite ´ 326, Phospholipides membranaires, Signalisation cellulaire et Lipoprote ´ ines, Ho ˆ pital Purpan, and UniVersite ´ Paul Sabatier, Toulouse Cedex 31059, France, and Laboratoire de Fe ´ condation in Vitro, Ho ˆ pital LagraVe, Toulouse, France ReceiVed April 26, 1995; ReVised Manuscript ReceiVed October 23, 1995 X ABSTRACT: In order to isolate pre- 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre- 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double-dimension gel electrophoresis. Apo A-I in pre- 1 HDL represented 17.6% of total apo A-I. Stokes’ radii corresponded to 3.42 nm in follicular fluid pre- 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre- 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre- 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidyl- cholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre- 1 HDL total weight. Thus, a lipid core is present in pre- 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism. High-density lipoproteins (HDL) 1 are involved in the reverse cholesterol transport from peripheral cells to the liver (Miller et al., 1985), and this process may involve several HDL subspecies (Castro & Fielding, 1988). Human plasma HDL are heterogeneous in terms of particle size, density, lipid content, and apolipoprotein composition (Eisenberg, 1984). On the basis of their electrophoretic mobility on agarose, HDL have been divided into two main popula- tions: the major subfractions with R mobility, and the others with pre- mobility (pre- HDL) (Kunitake et al., 1985; Castro & Fielding 1988; Huang et al., 1993). Most of HDL in plasma are R-HDL, and pre- HDL represent only 2-10% of total apoA-I, the main apo HDL (Kunitake et al., 1985; Neary et al., 1991). These pre- HDL are further resolved into pre- 1 , pre- 2 , and pre- 3 HDL particles according to increasing size, as assessed by two-dimensional gel electro- phoresis (Francone et al., 1989). Pre- 1 HDL are considered to be the first acceptors of cellular cholesterol (Castro & Fielding, 1988; Huang et al., 1993). These pre- 1 are also present in the interstitial fluid (Asztalos et al., 1993). In contrast to the plasma and the interstitial fluid, the human follicular fluid obtained from preovulatory follicles contains a single class of lipoproteins, HDL (Perret et al., 1985). In the ovarian follicle, granulosa cells are in direct contact with follicular fluid, but are separated from the blood capillaries by the basement membrane (Schreiber & Weinstein, 1986). In this work, we have evidenced the presence of pre- 1 HDL in the human follicular fluid, which surrounds the preovu- latory oocyte. Furthermore, we have isolated and character- ized these particles, and their lipid and protein compositions were analyzed. MATERIALS AND METHODS Preovulatory follicular fluids and plasma were obtained from patients engaged in an in Vitro fertilization program as previously described (Parinaud et al. 1993). Hyperstimula- tion was realized in a two-step procedure. The first step consists of a pituitary blockade using LH-RH analogs, which abolish endogenous secretion of FSH and LH. The second step consists of follicular stimulation achieved by injection of human menopausal gonadotropins. Preovulatory follicles were punctured during laparoscopy. Only the cleanest follicular fluids, with evidence of no blood cell contamination, were retained. Calibration standards for exclusion chromatography and polyacrylamide gel electro- phoresis were obtained from Bio-Rad (Ivry sur Seine, France) and Pharmacia (St. Quentin-en-Yvelines, France), respec- tively. Rabbit anti-human albumin was obtained from Sigma (St. Quentin Fallavier, France). Monoclonal antibodies against human apo A-I, human apo-E (3H1), and apo-AII ² This work was in part supported by a research grant from ARCOL and Fournier Laboratories, Dijon, France. ‡ This work is dedicated to the memory of Pr. Schlomo Eisenberg. * To whom correspondence should be addressed at INSERM Unite ´ 326, Ho ˆpital Purpan, 31059 Toulouse, France. Telephone: (33) 61 49 18 53; Fax: (33) 61 49 67 49. E-mail: collet@cict.fr. § Ho ˆpital Purpan and Universite ´ Paul Sabatier. | Ho ˆpital Lagrave. X Abstract published in AdVance ACS Abstracts, December 15, 1995. 1 Abbreviations: FF, follicular fluid; GLC, gas-liquid chromatog- raphy; apoA-I, apolipoprotein A-I; HDL, high-density lipoprotein(s); TG, triacylglycerol; CE, cholesteryl ester; VHDL, very high density lipoprotein(s). 1352 Biochemistry 1996, 35, 1352-1357 0006-2960/96/0435-1352$12.00/0 © 1996 American Chemical Society