Lipoprotein and phospholipid distribution in human follicular fluids*

FERTILITY AND STEIULITY Copyright' 1985 The American Fertility Society

Vol. 43, No.3, March 1985 Printed in V.SA.

Lipoprotein and phospholipid distribution in human follicular fluids *

Bertrand-Pierre Perret, M.D.t:j:§ Jean Parinaud, M.D.II Huguette Ribbes, M.D.:!: Jean-Pierre Moatti, M.D.:!: Georges Pontonnier, M.D.II Hugues Chap, Ph.D.:!:§ Louis Douste-Blazy, Ph.D,§ Laboratoire de Biochimie III, Centre Hospitalier Regional La Grave; Laboratoire de Fecondation in vitro, Institut National de la Sante et de la Recherche Medicale (lNSERM) U 168, and INSERM U 101, Biochimie des Lipides, Toulouse, France

Human preovulatory follicular fluids, obtained in the course of stimulated cycles, were analyzed for their lipid and protein compositions. By combining different methods, a single class of lipoprotein, high-density lipoprotein, was detected in all cases. The phospholipid distribution revealed an accumulation of lysophosphatidylcholine. These data are discussed in view of the possible roles of the follicular fluid during fertilization and in relation to the high levels of estradiol and progesterone measured in those fluids. Fertil Steril 43:405, 1985

The chemical composition of follicular fluids (FF) from various species has been well documented. I Among other peculiarities, very high levels of steroids are encountered in the FF, although subject to wide variations. I, 2 The rate and rhythm of synthesis of these steroids all along the cycle may depend on complex regulatory mechanisms, such as the balance between follicle-stimulating hormone and luteinizing hormone, and the availability of the different Received March 26, 1984; revised and accepted October 19, 1984. *Supported by Ministere de I'Industrie et de la Recherche (MIR 83.C.0669). tReprint requests: Bertrand-Pierre Perret, M.D., Laboratoire de Biochimie III, H6pital de la Grave, Place Lange, 31052 Toulouse Cedex, France. :j:Laboratoire de Biochimie III, Centre Hospitalier Regional La Grave. §INSERM U 101, Biochimie des Lipides. IILaboratoire de Fecondation in vitro, INSERM U 168. Vol. 43, No.3, March 1985

lipoprotein substrates within the follicle. 3 The predominance of high-density lipoprotein (HDL) has already been demonstrated by cholesterol measurements in the density fractions at different stages of follicular development. 4 , 5 The present study confirms these observations on human preovulatory follicles obtained in the course of a hyperstimulated cycle, when a copious progesterone (P) secretion had already started in the follicle. This was achieved by combination of three different· analytical methods. The phospholipid composition in the whole fluids and in the different fractions was also determined and compared with that found in normal plasma. MATERIALS AND METHODS COLLECTION OF THE FLUIDS

Twenty-five FF samples were obtained from 12 patients in an in vitro fertilization program. OvuPerret et al. Lipids in follicular fluid

405

lation was hyperstimulated using a combination of clomiphene dtrate, human menopausal gonadotropin, and human chorionic gonadotropin. Preovulatory follicles were punctured during laparoscopy .. Only the cleanest fluids, with evidence of no blood cell contamination, were retained. Aliquots were withdrawn for hormone analysis, and the data were compared with cytomorphologic criteria to determine the follicle maturation level. 2 The remaining was kept for biochemical characterization.

HORMONE MEASUREMENTS

17p-Estradiol, P, and 4-androstene-3,17-dione were measured by radioimmunoassay. 3H-Iabeled hormones and specific antibodies were from Biomerieux, Charbonieres, Lyon, France and Amersham, Les Ulis, Paris, France.

BIOCHEMICAL PARAMETERS

Total and free cholesterol were measured by the cholesterol oxidase/cholesterol esterase technique. Triglycerides were also assayed enzymatically, using commercial kits (Boehringer, Mannheim, FRG). Protein was measured according to Lowry et al. 6 , using bovine serum albumin as a standard. Total phospholipids were determined by the lipid phosphorus content following lipid extraction. 7 Sodium, potassium, chloride, carbon dioxide, and calcium were measured by routine conventional methods. Protein electrophoresis was run on commercial Cellogel plates (Sebia, Issy les Moulineaux, France) with the Ponce au red dye. Lipoprotein profile was performed likewise on pre coated polyacrylamide plates (Lipofilm Sebia) using Sudan black. Apolipoproteins Al and B (apo-A I , apo-B) were measured by laser immunonephelemetry using specific antisera against purified human apoproteins (Hyland Diagnostics, Travenol, Costa Mesa, CA).

PHOSPHOLIPID COMPOSITION

Lipids were extracted by the Bligh and Dyer procedure8 after acidification with 0.01 ml formic acid/ml fluid. The different phospholipids were separated by thin-layer chromatography on Silicagel60, 0.25 mm in thickness (Merck AG, Darmstadt, FRG) with the solvent system of Skip ski et aL9 The lipid spots were analyzed for their phosphorus content. LIPOPROTEIN ISOLATION

Follicular fluids (2 to 4 ml) were raised to a density (d) of 1.063 gm/ml by addition of a KBr solution of d = 1.35 (5 M KBr, 0.15 M NaCI, pH 7.00) and were then centrifuged in a 40.3 rotor (Beckman Instruments, Palo Alto, CA) at 120,000 gav. The d < 1.063 gm/ml fraction containing very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) was separated by the tubeslicing technique. The infranatant was brought to a density of 1.21 gmlml with solid KBr and submitted to 40 hours of ultracentrifugation. The d = 1.063 to 1.21 gm/ml fraction (HDL) and the d > 1.21 gm/ml fraction (protein) were separated likewise.

RESULTS Various biochemical parameters were assayed on 15 to 25 FF, in a preliminary approach. The ionic equilibrium was essentially similar to that observed in peripheral plasma. Protein showed slightly lower values: 49 ± 12 nig/ml (mean ± standard deviation [SD)) (Table 1), although the electrophoretic patterns were identical to those found in normal plasma (not shown). In addition, calcium was found somewhat lowered in FF-1.8 ± 0.3 f.Lmollml (mean ± sm. Lipid parameters were profoundly altered, as compared with plasma. Results are summarized in Table 1. Triglycerides were always found very low, while cholesterol and phospholipid data were about 15% to 25% of their counterparts in plasma. Only 10% of

Table 1. Protein and Lipid Concentrations in Human FE'" Cholesterol

Triglycerides

Phospholipids

Protein

Apo-A 1 b

Apo-B

JJ.mollml

JJ.1nollml

f,Lmo/lm/

mg'ml

mgml

mg,m/

0.08 (0.035)

0.235 (0.05)

0.63 (0.16)

49.1 (12.0)

1.10 (0.3)

< 0.36

Total

Free

JJ.mollml

0.81 (0.16)

"Values represent mean ± SD (in parentheses) from 15 measurements. bApo-AI and Apo-B were determined in 12 samples. 406

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Fertility and Sterility

Table 2; Steroid Leuefs in FF" Steroid p 17j3-estradiol 4-Androstene3,17-dione

Level,

2.8().434.00 jLmolll 0.55-19.20 ILmol/l 0.07-3.80 ILmolll

Mean

'" 19.00 "'3.70 '" 0.50

aData represent the overall concentration range measured in the fluids and the mean value for each steroid.

the total cholesterol was in a nonesterified form versus 25% to 30% in a normal plasma. HORMONE LEVELS

High levels of steroids were measured in those FF, obtained from patients given gonadotropin and just before ovulation, P being predominant (Table 2). Yet great individual differences were noted among the follicles. However, in most cases, the values were 50 to 1000 times higher than those measured in the plasma at the same period.

heaviest fractions contained up to 28% to 30% of .the total; essentially ,as lysolecithin. PHOSPHOLIPID COMPOSITION

Choline-containing phospholipids were widely predominant in those FF, with sphingomyelin sharing up to 20% of the total, essentially as observed in a normal plasma (Table 4). However, a relative increase of lysophosphatidylcholine at the expense of phosphatidylcholine was noted in all samples: 18.5% and 53.4%, respectively, versus 6.0% and 66% in plasma. The distribution among the density fractions shows the lysocompounds mainly isolated in the d > 1.21 gmlml fraction, whereas the majority of phosphatidylcholine is recovered with HDL. Phosphatidylethanolamine and small amounts comigrating with phosphatidy lserine/phosphatidy linosi tol were also detected. Thus, the HDL isolated from FF exhibits a phospholipid composition similar to that of plasma HDL.10 DISCUSSION

LIPOPROTEIN PROFILE

A single class oflipoprotein behaving like HDL was detected in the fluids by several methods. Polyacrylamide gel electrophoresis of entire lipoproteins showed in all cases only one band migrating as an a-lipoprotein. The determination of apolipoprotein content in 12 fluids revealed the constant presence of apo-A1-1.10 ± 0.30 mg/ml (mean ± SD)', while apo-B values were always below the limits of detection (by sample concentration in two cases, an approximate value of 0.05 mg/ml was calculated for apo-B). Ultracentrifugal isolation ofthe lipoprotein classes was carried out on nine FF, leading to three major fractions: the d < 1.063 gm/ml fraction (VLDL + LDL), the d = 1.063 to 1.21 gm/ml fraction (HDL), and the d > 1.21 gm/ml fraction, containing the bulk of protein. Cholesterol, phospholipids, and protein were assayed, and the results are expressed per milliliter original fluid (Table 3). Cholesterol could be accurately determined only in the HDL density range. Most of the protein was recovered in the bottom fraction, but the HDL contained 0.9 ± 0.15 mg/ml (mean ± SD) as measured chemically. It is noteworthy that this value is rather close to that found by immunonephelemetry for apo-Ab which is expected to constitute the predominant HDL apoprotein. Phospholipids were mostly isolated in the HDL density range, but the Vol. 43, No.3, March 1985

The present study points out the almost exclusive presence of HDL as a lipoprotein class in human preovulatory FF, thus confirming previous observations by Simpson and co-workers. 4 This was evidenced by combining three different methods: the lipoprotein profile obtained on polyacrylamide gel electrophoresis, the measurement of apo-A1 versus apo-B in the fluids, and the data from ultracentrifugal isolations. No material floating in the low-density ranges was detected, and cholesterol was entirely recovered with HDL. The chemical composition of the isolated HDL looks very similar to that of plasma total HDL,10 except for a low free cholesterol/total cholesterol Table 3. Lipid and Protein Distribution Among Lipoprotein Density Classes in Human FF Cholesterol

Density class gmlml

d < 1.063 (VLDL + LDL) d = 1.063-1.21 (HDL) d> 1.21 (protein)

Total

Free

Phospho· lipids

Protein

fLmollml

fLmollml

fLmollml

mglml

< 0.05

ND b

ND

0.63 (0.08) < 0.05

< 0.05

0.56 (0.10) 0.22 (0.10)

0.04 (0.01) 0.90 (0.15) 45.0 (6.7)

ND

aValues represent mean ± SD and are expressed per milli· liter of original fluid. bND, not detectable. Perret et a1.

Lipids in follicular fluid

407

F

. Table 4. Phospholipid Distribution in Human FF and Among the Different Lipoprotein Classes a % Total phospholipids in FF

Whole fluid HDL fraction (d = 1.063-1.21) Protein fraction (d > 1.21)

100 72 (5.6) 28 (5.6)

Normal plasma

%

Sphingomyelin

19.6 (3.0) 14.6 (3.9) 9.0 (4.7) 20 (2.0)

%

Lysophospha- % Phosphatidyl- % Phosphatidyl- % Phosphatidyltidylcholine choline serine/inositol ethanolamine 18.4 (2.9) 4.6 (1.4) 50.2 (4.8) 6 (2.0)

53.4 (2.6) 69.4 (3.3) 35.8 (4.4)

3.4 (1.6) 5.0 (2.0) 1.2 (2.0)

66 (5.0)

8 (20)

5.0 (1.5) 6.4 (2.0) 2.1 (3.0)

aYalues are expressed as a percentage of total phospholipids in the different fractions. They are the means ± SD from eight experiments.

ratio, but no attempt was made to separate HDL subfractions. In addition, the d > 1.21 gmlml fractions may contain some very high-density lipoprotein; yet their major lipid constituents were found to be lysophosphatidylcholine, which binds with a high affinity to serum albuminY Various proteins present in the FF are in equilibrium with levels in plasma. 1, 5 The follicular membrane and the granulosa layer may act as a barrier and a molecular sieve, retaining large molecules. I, 5 The differences in particle size of HDL--molecular weight, 3.105 d; diameter, 90 A-and of LDL--molecular weight, 1.5 106 d; diameter, 215 A12-eould thus explain the absence ofthe latter in the fluid. A local synthesis ofHDL, or a specific utilization of this lipoprotein class in the follicle, has not yet been ruled out. The presence ofHDL may be related to the high steroidogenic activity of the follicle cells. However, their amount in the fluid does not exceed that in plasma. On the other hand, several studies indicate that LDL is a better candidate to provide cholesterol for steroidogenesis in various tissues, including corpus luteum maintained in culture. 13 , 14 The presence of HDL alone could thus impede the P secretion by granulosa cells during the follicular phase, or could even cause a cellular cholesterol efflux. 3 That such processes are actually occurring within the follicle needs to be demonstrated. Also at variance with this mechanism are our observations made at the preovulatory phase following a hormonal stimulation, since they show simultaneously the almost absence of LDL together with an already copious P secretion in the follicle. A mechanism of cholesterol efflux has also been evoked, accompanying sperm capacitation. 15 The spermatozoa would undergo a negative cholesterol gradient when in the presence of uterine cells and uterine and oviductal secretions. 15, 16 In this scheme, the decrease in sperm cholesterol would result in a membrane destabilization. 15 Induction 408

Perret et al. Lipids in follicular fluid

of sperm capacitation in the presence of FF has also been reported in various in vitro systems; yet the factors responsible for such a phenomenon are not clearly defined. 17 The particular wealth in steroids, especially estradiol may be of importance. Conversely, among the plasma lipoproteins, HDL is involved in promoting a free cholesterol efflux from cells and other lipoproteins. 18, 19 Moreover, HDL is the preferred substrate of the enzyme lecithin:cholesterol acyltransferase (LCAT), which allows the storage of cholesterol as cholesterol ester molecules, thus increasing the efficiency of the cholesterol removal. 19 We have recent evidence in the laboratory that whole FF are able to pick up labeled free cholesterol from human erythrocytes. Part of the exchanged sterol is in turn esterified during the same incubation (i.e., 25% in 6 hours). Whether such processes are effective toward sperm cells and result in a net lowering of the cellular cholesterol mass needs to be demonstrated, and the contribution of the HDL system as against that of other FF components in an in vitro system has to be estimated. The phospholipid composition of the whole FF revealed a slight accumulation of lysophosphatidylcholine, which was not associated with the HDL particle, but with the heaviest fractions, probably after binding to albumin. The origin of this accumulation is so far unexplained. It may reflect an increased lipolytic activity in FF, as compared with plasma: either a phospholipase A l JA 2-like activity or an increased LCAT activity. Whether these lysolecithins appear only at the latest stages of follicular development or earlier is not known. Whatever their origin, due to their detergent-like properties,2~ the presence of the lysolecithin may be of importance in the processes of membrane fusion, occurring during fertilization. Acknowledgment. Thanks are due to M. C. Yialette for excellent secretarial assistance. Fertility and Sterility

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12. Eisenberg S: Plasma lipoprotein conversions: the origin of low-density and high-density lipoproteins. In The Lipoprotein Structure, Edited by AM Scanu, FR Landsberger. New York, The New York Academy of Sciences, 1980 p

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13. Carr BR, Sadler RK, Rochelle DB, Stalmach MA, MacDonald PC, Simpson ER: Plasma lipoprotein regulation of progesterone biosynthesis by human corpus luteum tissue in organ culture. J Clin Endocrinol Metab 52:875, 1981 14. Carr BR, Parker CR Jr, Milewich L, Porter JC, MacDon~ld PC, Simpson ER: The role of low-density, highdenSIty and very low-density lipoproteins in steroidogenesis by the human fetal adrenal gland. Endocrinology 106:1854, 1980 15. Davis BK: Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval. Proc Natl Acad Sci USA 78:7560, 1981 16. Davis BK: Uterine fluid proteins bind sperm cholesterol during capacitation in the rabbit. Experientia 38:1063, 1982 17. Mukherjee AB, Lippes J: Effect of human follicular fluid and tubal fluid on human mouse and rat spermatozoa in vitro. Can J Genet Cytol 14:167, 1972 18. Glomset J A: The plasma lecithin cholesterol acyltransferase reaction. J Lipid Res 9:155, 1968 19. Glomset JA: Physiological role oflecithin cholesterol acyl transferase. Am J Clin Nutr 23:1129, 1970 20. Lange Y, Slayton JM: Interaction of cholesterol and lysophosphatidylcholine in determining red cell shape. J Lipid Res 23:1121, 1982

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