Estradiol esterification in the human preovulatory follicle

Steroids 66 (2001) 889 – 896

Estradiol esterification in the human preovulatory follicle Luisa Ciglianoa, Maria Stefania Spagnuoloa, Brian Daleb, Marco Balestrieria, Paolo Abresciaa,c,* a

Dipartimento di Fisiologia Generale ed Ambientale, Universita` di Napoli Federico II, Via Mezzocannone 8 – 80134 Napoli, Italy b Center for Reproductive Biology, Villa del Sole, Via Manzoni 15– 80123 Napoli, Italy c International Institute of Genetics and Biophysics, C.N.R., Via Marconi 10 – 80121 Napoli, Italy Received 11 December 2000; received in revised form 12 March 2001; accepted 16 March 2001

Abstract In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme Km and Vmax values were lower with estradiol than with cholesterol. Higher estradiol linoleate/ estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Steroid; Estradiol; Follicle; Granulosa cells; Ovulatory cycle

1. Introduction In the preovulatory follicle, progesterone (P) is mostly produced by the theca cells and partly used for the synthesis of androgens, which are aromatized to a great extent in the granulosa cells. Part of progesterone and aromatase-produced estradiol (E) diffuse into the follicular fluid and, then, into the circulation. After the LH surge, the granulosa cells, which have all the biochemical machinery for steroidogenesis [1– 4], increase their production of P [5–7], which inhibits the conversion of cholesterol (C) to C esters [8], and reduces the aromatase activity [5– 6,9 –12]. Thus the follicular levels of P and E (or their ratio) change during the late preovulatory period as the luteinization process progresses. These levels have been proposed as markers of human * Corresponding author. Tel.: ⫹39-081-2535095; fax: ⫹39-0812535090. E-mail address: [email protected] (P. Abrescia).

follicular function, in order to evaluate oocyte quality or predict the success of programs of assisted conception. The LH surge modifies not only the cell function but also the permeability of the blood-follicle barrier [13–15]. The sieving properties of the barrier, during follicle development, permit free diffusion of negatively charged proteins with the same Mr of albumin or lower, while reducing or blocking the transport of heavier proteins [13–17]. This explains why the HDL population, spanning Mrs from 62 000 to 180 000 [18], is the only class of lipoproteins present in the mature follicle [16 –21]. In the same context Haptoglobin (Hpt), penetrating to a limited extent in human healthy follicles until an ovulatory stimulus is given, was suggested to be used as a marker of the permeability of the follicle wall [22]. HDL provides C esters to steroidogenic cells via the selective uptake pathway, in which only C esters are transferred while the lipoprotein is not internalized [23,24]. In human granulosa cells, C esters cytoplasmic storage [25,26] or conversion to C for steroidogenesis [27] was recently

0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 0 1 ) 0 0 1 2 4 - 6

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described. Homeostasis of cell C involves a balance between the influx and the efflux processes, both requiring lipoproteins for transport in the extracellular fluids. The excess of C must be removed from all peripheral cell types, since it can destabilize the plasma membrane structure and lead to dysfunction or death [28 –30]. In particular, C accumulation in granulosa cells can enhance the biosynthesis of P, which might reduce the aromatase activity [5– 6,9 –12] and induce atresia [31]. The C removal from the cell, known as ‘reverse cholesterol transport,’ is promoted by the enzyme lecithin-cholesterol acyltransferase (LCAT, E.C. 2.3.1.43). This enzyme transfers an acyl chain from HDL lecithin to C, thus producing C esters which are taken up by the lipoprotein for target liver cells [28]. The efflux of C esters from granulosa cells to HDL was previously described [17,18] or suggested [32,33]. Thus the bi-directional traffic of C esters between granulosa cells and the extracellular environment, required for cell C homeostasis, involves HDL. LCAT and HDL participate in another phenomenon, that is the esterification of estrogens and the delivery of the resulting estrogen esters to circulation [34]. The E esters display prolonged hormonal action [35] and powerful antioxidant activity [36,37]. These esters are sequestered in fatty tissues, in which they represent a protected store of preformed hormone and where they are freed of the acyl chain by properly stimulated esterases for their action [38]. Thus C and E are both substrates for LCAT in follicular fluid. As the preovulatory luteinization of granulosa cells progresses, C is increasingly used for steroidogenesis and therefore should be less available for the enzyme activity. Accordingly it can be hypothesized that, after the LH surge, the C esterification in the fluid decreases while the production of E esters increases. To support this hypothesis, we studied whether changes in the follicular level of C esters or E esters occur during the preovulatory period. In this paper, we report data on the level of Hpt penetration into the follicle and on the follicular concentrations of P, E, C, E esters and C esters from women undergoing an assisted reproduction program. In particular, we analyzed C linoleate (lin-C) and E linoleate (lin-E), which are the major ester forms for C [39] and E [40], respectively. The results are discussed in terms of LCAT activity in healthy follicles with different P production, aromatase activity and barrier permeability.

2. Experimental 2.1. Materials Antibodies, Hpt (mixed phenotypes), bovine serum albumin (BSA) fraction V (BSA), human serum albumin (HSA), chemicals of the highest purity, protein markers and HPLC standards were purchased from Sigma-Aldrich [Milan, Italy]. HDL was purchased from Calbiochem [Inalco,

Milan, Italy]. [1␣,2␣-3H]Cholesterol (45 Ci/mmole) and [C-4, 14C]Estradiol (53 mCi/mmole) were obtained from NEN Life Science [Boston, MA, USA]. Columns for highperformance liquid chromatography (HPLC) and Sil-G plates for thin layer chromatography (TLC; thickness 0.25 mm) of Macherey-Nagel [Du¨ ren, Germany] were used. Organic solvents were purchased from Romil [Cambridge, UK]. Superdex S-200 was purchased from Pharmacia-Biotech [Cologno Monzese, Italy] and polystyrene 96-wells plates from Corning-Costar [Concorezzo, Italy]. 2.2. Follicular fluid preparation Multiple follicular stimulation, cycle monitoring and oocyte retrieval were performed using standard protocols [22]. Follicular fluids (0.8 –1.0 ml) from each patient were individually collected into separate disposable plastic tubes, freed of suspended cells by centrifugation (400 g, 20 min, 4°C) and, if free of blood contamination, analyzed within 2 h. Mature oocytes were identified as having expanded cumulus, corona radiata greater than one half the oocyte diameter, distinct zona pellucida and clear ooplasma, according to criteria reported elsewhere [41]. In this study, only patients producing at least one embryo were considered and only follicular fluids associated with mature oocytes were analyzed. The program of experiments with follicular fluids was approved by the Institutions which the authors are affiliated to (Universita` di Napoli Federico II, Villa del Sole). All the patients were informed about the use of their fluids for the here described analysis and gave their consent. 2.3. Titration of hormones and lipids The concentration of P, lin-E and E, in 27 separate follicular fluids from 19 women, was determined. Each fluid (200 ␮l) was vigorously shaken with one volume of hexane and, then, the upper phase was carefully removed. The mixture was likewise extracted two more times. The three organic phases were combined and dried under nitrogen stream. The residue was dissolved in 50 ␮l of acetonitrile and 20 ␮l were aliquoted for injection onto the HPLC column (4.6 mm ⫻ 25 cm). Octadecyl groups bound to 5 ␮m particles of spherical silica (100 Å pore diameter, 20% carbon loading, endcapped silanol) constituted the stationary phase. Synthetic E 17␤-linoleate (C-18:2), prepared by the method of Mellon-Nussbaum et al. [42], and commercial P and E were treated as the samples and used as standards in HPLC. In particular, the standard amounts were assessed by spectrophotometry, using 15 849 and 1995 as molar extinction coefficient for P and E (or lin-E), at ␭ of 241 and 280 nm respectively. HPLC was performed, at 1.5 ml/min, by Shimadzu [Kyoto, Japan] model LC-10AD pumps equipped with the diode array UV detector model SPD-M10A and the fluorescence detector model RF-551 (setting: ␭ex 61 275 nm, ␭em 61 308 nm). The chromatog-

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raphy was carried out by 100% of acetonitrile/water mixture (50:50, v:v) from 0 to 7 min, followed by a linear gradient of methanol (0 –100%) from 7 to 12 min and, finally, 100% of methanol from 12 to 25 min. The first isocratic phase fractionated E (detected by UV absorbance or fluorescence), the gradient eluted P (detected by UV absorbance) and the second isocratic phase isolated lin-E (mainly detected by fluorescence) from other E esters. To avoid contamination the column was washed, after each run, with 100% of methanol for 10 min and, then, 100% of the acetonitrile/ water mixture for 10 min. Separation and quantitation of follicular fluid lin-C and free C was carried out essentially according to a published procedure [43]. In details, to 10 ␮l of sample 90 ␮l of water and, then, 200 ␮l of ethanol were added. The mixture was shaken for 2 min with 2 ml of ice-cold hexane. After careful removal of the hexane extract, the lower phase was likewise extracted two more times. The three extracts were combined and dried under nitrogen stream. The residue was dissolved in 200 ␮l of eluent solution (acetonitrile/isopropanol 57:43, v:v) and 20 ␮l were processed by HPLC, using a column (3.9 mm ⫻ 15 cm) with the same stationary phase as above. The chromatography was performed at 40°C in isocratic conditions with 1 ml/min flow rate, and the UV detector was set at 205 nm. Each analyte was titrated only when its elution was monitored as isolated and homogeneous peak on the chromatogram. Therefore concentration values were accurately determined in all the fluids for P and C, but only in 24, 22 and 21 fluids for E lin-E and lin-C respectively. 2.4. Determination of Hpt penetration in the follicle Follicular fluids (N ⫽ 27) and homologous plasma were analyzed for their Hpt concentration by ELISA [22]. Fluids or plasma exhibiting traces of blood were not used. A calibration curve was obtained by assaying the immunoreactivity of 0.1– 0.25– 0.50 – 0.75–1.0 –2.0 ng of purified protein. These amounts were prepared from a mother solution, titrated by a colorimetric assay [44] using BSA as standard. The Hpt phenotype was determined by electrophoresis as previously reported [22]. The Hpt penetration in the follicle was calculated as P ⫽ (Concentration in follicular fluid/ Concentration in plasma) ⫻ 100 [22]. Only penetration data from samples with the phenotype 1–2 were considered in this study, because hemoglobin-free samples with the other phenotypes (Hpt 1–1: 2/5; Hpt 2–2: 4/9) were too few for statistical analysis. 2.5. LCAT assay Follicular fluid or plasma samples were treated by 0.1% dextran sulfate (DS, Mr 50 000) according to Albers et al. [45] and, then, 3 ␮l aliquots from the soluble protein fraction were used in the standard LCAT assay. The enzyme activity was determined by measuring the conversion of 3 H-labeled C to 3H-labeled C esters, or 14C-labeled E to

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C-labeled E esters, in the presence of HDL. The reaction mixture (40 ␮l), containing 1.32 nmoles of labeled substrate (2.84 or 0.11 ␮Ci/nmole specific activity for C or E respectively) in 80 mM tri-sodium citrate, was incubated at 37°C for 3 h. The effective substrate concentration was measured by HPLC using aliquots of radioactive standard with known specific activity. The cholesterol concentration used in the assay was lower than its critical micellization concentration (CMC), as determined by light scattering [46]. The reaction was stopped by ethanol, the lipid extracted by hexane and, finally, fractionation of C and C esters was carried out by TLC essentially as previously reported [45], but the mobile phase was the mixture petroleum ether/diethyl ether/acetic acid (90:30:1, v:v:v). Fractionation of E and E esters was carried out by TLC using petroleum ether/diethyl ether/ acetic acid (60:60:1, v:v:v) as mobile phase. C, E and C esters were visualized under iodine vapor, while E esters were detected just for their radioactivity. The layers containing the labeled analytes were cut and analyzed by scintillation. The enzyme activity was expressed as units (nmoles of C incorporated per hour) per ml of sample. LCAT activity was assayed in whole follicular fluid, following treatment with DS, in the presence or absence of cholesterol. In detail, a pool of follicular fluids (N ⫽ 3) from Hpt 1–2 women was treated with 0.1% DS at room temperature for 20 min. The mixture was centrifuged (6000 g, 10 min, 18°C) and the supernatant was divided in 200 ␮l aliquots. The aliquots, in triplicates, were supplemented with different amounts of cholesterol (6.16, 15.40, 30.80 ng/200 ␮l). Samples incubated with no C added served as control. After 45 min of incubation at 37°C, the samples were processed as described above, but HPLC (equipped with fluorescence detector) instead of TLC was used to separate E from esters of E. One sample, without C added, was not incubated in order to evaluate the amount of preexisting lin-E. The ratio of the lin-E amount with the E amount was calculated for each sample. The procedure of analysis by HPLC is reported above. 2.6. Determination of LCAT Km and Vmax Measurements of LCAT Km and Vmax for E or C were carried out using enzyme solutions free of Hpt. Follicular fluids from three healthy women with phenotype Hpt 2–2 were pooled and processed by DS as reported above. The soluble proteins (500 ␮l) were fractionated on a column (2.5 cm ⫻ 33 cm) of Superdex S-200 in 1 mM EDTA containing TBS at 1 ml/min flow rate. After 48 ml of draining, fractions of 1.5 ml were collected and analyzed for their content of Hpt by ELISA. The elution of LCAT was monitored by incubating 6 ␮l of each fraction with 0.6 ␮g of purified HDL in the standard reaction mixture. LCAT containing fractions, free of detectable Hpt, were pooled and the resulting protein solution (0.91 mg/ml) used to study the enzyme kinetics. Aliquots of 5 ␮l of the partially purified LCAT solution were used in the standard assay mixture.

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The enzyme was incubated with different concentrations (4.97, 8.61, 12.28, 15.92, 23.22, 30.53, 37.83 ␮M) of C or E. Five replicates for each concentration were analyzed. The assay was carried out as described above. Samples containing substrate concentration below 23.22 ␮M (N ⫽ 4 ⫻ 5) were used to obtain trend curves on the Lineweaver-Burk plot: intercepts of 1/V ⫽ f (1/[S]) with abscissa and ordinate were calculated, for each substrate, to obtain the values of ⫺1/Km and 1/Vmax. 2.7. Statistical analysis In protein assays, Hpt titration and LCAT detection, the samples were processed at least in triplicate while, in the LCAT quantitative assays (titration in plasma or follicular fluid, determination of Km and Vmax) four replicates were used. Averages ⫾ standard deviations (SD) were calculated from the experimental data. Titration of P, C, lin-C, E and lin-E by HPLC was carried out on duplicates. The program ‘Graph Pad Prism 3’ (Graph Pad Software, San Diego, CA, U.S.A) was used to obtain trend curves, perform regression analysis and calculate significance.

3. Results 3.1. Titration of P and E in follicular fluid. Determination of the Hpt penetration In the large antral follicle, the LH surge stimulates progesterone production and inhibits aromatase activity. LHdependent structure modification of the blood-follicle barrier is associated with enhanced transport of heavy blood proteins into the follicle. Follicular fluids (N ⫽ 24), collected from women undergoing an assisted conception program, were analyzed by HPLC for their level of P and E, which were found ranging from 0.36 to 13.60 ␮g/ml (1.15– 43.24 ␮M) and 0.09 to 0.66 ␮g/ml (0.33–2.42 ␮M) respectively. The value of the ratio of P with E (P/E, w/w) was calculated. The statistical analysis of the data indicated that the P level was negatively correlated with the E level (r ⫽ ⫺0.513, P ⬍ 0.01). The P/E ratio was positively correlated with the P level (r ⫽ 0.857, P ⬍ 0.01) while negatively correlated with the E level (r ⫽ ⫺0.689, P ⬍ 0.01). These data are in agreement with the present information on the levels of P, E and P/E (or E/P) in the developing follicle. Samples of fluids (N ⫽ 12) and homologous plasma from women with the phenotype Hpt 1–2 were analyzed for their Hpt concentration, and the level of Hpt penetration in the follicle was calculated. This level was found positively correlated with the P level (r ⫽ 0.686, P ⬍ 0.01) or the P/E ratio (r ⫽ 0.860, P ⬍ 0.05), while negatively correlated with the E level (r ⫽ ⫺0.708, P ⬍ 0.05). This experiment suggests that an inverse relationship might exist between the follicular fluid level of E and the permeability level of the

blood-follicle barrier, as represented by the amount of Hpt penetration into the follicle. 3.2. Analysis of lin-E/E ratio and LCAT activity in follicular fluids The levels of E and lin-E were analyzed in a subset of follicular fluids (19/27). Values ranging from 0.33 to 2.42 ␮M (E) or 0.08 to 0.63 ␮M (lin-E) were found. The lin-E/E molar ratio, which reflects the efficiency of E esterification in the follicle, was negatively correlated with the E level (r ⫽ ⫺0.75, P ⫽ 0.0002) (Fig. 1A) and not correlated with the in vitro assayed LCAT activity (Fig. 1B). The data suggest that the enzyme activity is not stimulated in vivo by increased E concentration and, moreover, similar (or equal) amounts of enzyme activity are associated with different levels of product. 3.3. In vitro esterification of E and C The absence of a relationship between the physiological lin-E production and the enzyme availability, as evaluated by the in vitro assay, suggests that the catalytic properties of LCAT (Km, Vmax) might be different for the two different substrates (i.e. E and C) and that the amount of lin-E produced might be influenced by the concentration of free C. The enzyme activity, as a function of the substrate concentration in the assay mixture, was analyzed. The C esterification rate was found directly related to the C concentration, in all the ranges of values used (from 5.0 to 37.8 ␮M), while the E esterification rate displayed a similar relationship only with E concentrations lower than 16 ␮M (Fig. 2A). Over this value (23.2, 30.5 or 37.8 ␮M), the higher concentration was present, the lower esterification rate was found. In particular it can be predicted that, when both the substrates are present in the same LCAT assay mixture at the concentration of 30 ␮M, the production of C esters should be about twice as much as the production of E esters. As a matter of fact, in these experimental conditions, 2.73 ⫾ 0.15 SD and 1.33 ⫾ 0.11 SD nmoles/h of C esters and E esters were produced respectively. The values of substrate concentrations lower than 16 ␮M and the related values of enzyme activity were used to draw the points of the Lineweaver-Burk function 1/V ⫽ f (1/[S]). The trend curves were determined, as 1/V ⫽ 0.1394/[C] ⫹ 0.1235 (r 2 ⫽ 0.9672) and 1/V ⫽ 0.1199/[E] ⫹ 0.235 (r 2 ⫽ 0.9302), and the values of their intercepts with abscissa and ordinate were calculated. The enzyme apparent Km was 14.2 and 31.3 ␮M for E and C respectively. The apparent Vmax was 4.3 and 8.1 units/ml for E and C respectively. The above kinetic studies suggest that C should effectively compete with E for LCAT. Such a C competition might be responsible of the large differences in lin-E synthesis found at similar values of enzyme activity (see Fig. 1B). In other words, different C concentrations should inhibit differently the E esterification. This hypothesis was supported by the finding of

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Fig. 1. Correlations of the lin-E/E ratio with E or in vitro LCAT activity. E and lin-E were titrated in follicular fluid by HPLC with fluorescence detector. The titers, expressed as ␮moles/l, were used to determine molar lin-E/E ratios. These values were plotted against the E concentration (Panel A) or the LCAT activity (Panel B) for linear regression analysis. The enzyme assay was carried out in vitro by using aliquots of fluids and radioactive E as precursor. Each sample was analyzed in duplicate and the average was used: differences of the experimental values from the average were lower than 5%. The statistic program Graph Pad Prism 3 performed the regression analysis and the calculation of P ⫽ 0.0002.

decreased lin-E production in follicular fluids supplemented with increasing amounts of C (Fig. 2B). 3.4. Correlations among lin-E/E, lin-C/C, Hpt penetration, and E Linoleoyl chains are the major fatty acid residues of C [39] or E esters [40]. We assumed the levels of lin-E and lin-C as reflecting the levels of the total populations of E esters and C esters respectively. As mentioned above, the molar concentrations of E and lin-E were measured in follicular fluids by

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Fig. 2. Kinetics of lin-E or lin-C synthesis. A pool of follicular fluids (N ⫽ 3) was treated by 0.1% dextran sulfate and the resulting protein solution was divided in aliquots. Panel A: An aliquot of protein solution was fractionated by chromatography with Superdex S-200. The fraction containing partially purified LCAT was analyzed for the enzyme activity, using different amounts of radioactive C (squares) or E (circles) in the standard reaction mixture. The activity is expressed as units/ml. Each point represents the average from four replicates: standard deviations (never found higher than 4% of the mean) are not represented, in order to avoid a dull reading. Panel B: Aliquots of protein solution were supplemented with different amounts of free C and, then, incubated as such for 45 min. The lin-E/E ratio was calculated from the levels of lin-E and E, as measured by HPLC with fluorescence detector. The filled circles represent the levels of lin-E/E ratio in the incubated samples. The level of lin-E/E ratio in control samples, neither incubated nor supplemented with cholesterol, is shown (open circle). Each point represents the mean from three replicates: error bars indicate standard deviations.

HPLC, and the lin-E/E ratio was calculated. The lin-C/C ratio was determined in the same way and, like the lin-E/E ratio, was not correlated with the in vitro LCAT activity. These ratios were plotted one versus the other (Fig. 3). This plotting was done with data from 15 fluids, because all the required analytes were measured only in this subset of fluids. The two parame-

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Fig. 3. Correlation between lin-C/C and lin-E/E levels in follicular fluid. The follicular levels of lin-C and C were determined by HPLC with UV detector, and used to calculate the molar lin-C/C ratio. The molar lin-E/E ratio was calculated from the follicular levels of lin-E and E, as measured by HPLC with fluorescence detector. Each point represents the average from two determinations: differences of the experimental values from the average were lower than 5%. The program Graph Pad Prism 3 performed the regression analysis and the calculation of P ⫽ 0.022.

ters were found to be negatively correlated (r ⫽ ⫺0.584, P ⫽ 0.022). The data suggest that higher levels of E esters might be associated with lower amounts of free C in the follicular fluid, and vice versa. The levels of the lin-C/C and the lin-E/E ratio were correlated (P ⫽ 0.0005 and P ⫽ 0.002 respectively) with the Hpt penetration (r ⫽ ⫺0.942 and r ⫽ ⫹0.907 respectively), as shown in Fig. 4A. Moreover, the E concentration was significantly correlated not only with the lin-E/E level (r ⫽ ⫺0.687, P ⫽ 0.001; see also Fig. 1A) but also with the lin-C/C level (r ⫽ 0.789, P ⫽ 0.0005) (Fig. 4B). This experiment suggests that in the preovulatory follicle high levels of lin-C are associated with lower amounts of penetrated Hpt, that is with working blood-follicle barrier and aromatase activity.

4. Discussion The LCAT dependent production of lin-E in follicular fluids was found higher at lower E levels and, conversely, lower when higher amounts of E were present. This finding, if the physiological amount of the enzyme in the fluid does not essentially change, appears to contrast basic principles of enzymology. High levels of E esters might be expected if the follicular amount of LCAT would increase as the permeability of the blood-follicle barrier increases. However, this enzyme displays Mr ⫽ 65 000 [47] and pI ⫽ 4.4 – 4.7 [48] which are compatible with free diffusion through the barrier at any time during all the follicular periods. As a matter of fact, the LCAT activity was not found reduced in follicular fluid compared with plasma [39]. In order to explain why different amounts of E-esters are synthesized with similar LCAT levels, one could suppose that the more

Fig. 4. Correlations of lin-C/C and lin-E/E levels with Hpt penetration or E levels. The molar ratios lin-C/C (squares, left scale) and lin-E/E (triangles, right scale) were calculated from the follicular levels of the single analytes, measured by HPLC as detailed in the text. The data were plotted versus the Hpt penetration (Panel A; P ⬍ 0.002) or the E ␮molar level (Panel B; P ⫽ 0.0005). The Hpt penetration was calculated as P ⫽ [titer in fluid/titer in plasma] ⫻ 100. Each point represents the average from two determinations: differences of the experimental values from the average were lower than 5%. The program Graph Pad Prism 3 performed the regression analysis.

ovulation approaches the more an unknown follicular factor might stimulate enzyme activity. Actually the penetration of HDL, which provides a phospholipid substrate for the LCAT activity, does not increase before ovulation [17,19]. Similarly the follicular level of the apolipoprotein A-1, which is exposed on the HDL surface and required for the LCAT activity [28,49], does not change in the preovulatory period [39]. Therefore, the possibility that higher levels of E esters are produced by higher amounts of enzyme or higher enzyme stimulation should be ruled out.

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The lin-E/E ratio was not correlated with the in vitro LCAT activity and, as mentioned above, higher lin-E/E ratio was associated with lower E levels. These findings, taken together, triggered the hypothesis that only part of this activity esterifies E and that such a fraction increases as the aromatase production decreases. This hypothesis was supported by the fact that a major role for LCAT is the C esterification, which is a crucial step in the reverse C transport. This esterification is required to remove the excess of free C from any cell that does not succeed in storing (as C esters, by the enzyme ACAT) or converting it to other molecules for excretion (e.g. steroid hormones). When exceeding C is not eliminated, it accumulates into the cell plasma membrane or might even crystallize, thus causing cell dysfunction or death [28 –30]. The granulosa cells, in the preovulatory period, increase their P production by using the internal stores [25,26,50] and increasing the uptake of C esters [51,52]. Thus the more the cells luteinize the less C should be released (in ester form), and the C esters/C ratio should decrease. In this case the LCAT activity might be more available for the E esterification. In other words, we propose to evaluate the LCAT activity as differently engaged in the effluxes of E and C, i.e. in producing lin-E and lin-C, as ovulation approaches. As a matter of fact, the lin-E/E ratio was found positively correlated with the Hpt penetration while negatively correlated with the E level. This finding suggests that increasing enzyme activity be addressed to the HDL-mediated transport of E (in ester forms) as the granulosa cells increase their C utilization for P production. The negative correlation of lin-C/C with lin-E/E seems to give further support to the above hypothesis. It is worth noting that differences between lin-C/C and lin-E/E do not seem to be accounted for by differences of LCAT affinities or catalytic rates with the two distinct substrates, since these parameters closely lie in the same order of magnitude. On the basis of the kinetic parameters, low rates of E esterification are expected when high amounts of C are available for the enzyme activity. Actually we found that follicular fluid supplementation with free C resulted in decreased production of lin-E. In conclusion, our data suggest that the activity of LCAT is not distributed in a fixed way between E esterification and C esterification during the preovulatory period, and support the hypothesis that higher levels of E esters might be produced when granulosa cells use C for steroidogenesis. Further experiments are required to ascertain whether follicular LCAT actually prevents cell structure and function damages derived from C accumulation, and what is the physiological significance of estrogen esterification in different phases of the preovulatory period. However, we show here that different lin-E/E ratios can be detected in follicles with different E levels, and report for the first time the use of follicular Hpt levels to evaluate the permeability of the blood-follicle barrier. We suggest that the lin-E/E ratio and the Hpt level

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might be considered as markers of the follicle physiology for clinical or research purposes.

Acknowledgments The authors are deeply indebted to Marialaura De Simone for collecting plasma and follicular fluids. Special thanks to Dr. P. Bergamo for assistance in the analysis of cholesterol and cholesteryl esters. This research was supported partly by a grant POP-96 (5.4.2) from Regione Campania, and partly by funds from the University of Naples Federico II (Ric. Dipart. 98-99)

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