Mol. Hum. Reprod. Advance Access originally published online on April 20, 2007
Molecular Human Reproduction 2007 13(6):373-379; doi:10.1093/molehr/gam019
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Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists
1 Laboratoire de Biologie de la Reproduction, CECOS Franche-Comté/Bourgogne EA Génétique et Reproduction 3185, CHU Dijon, Dijon, France 2 Service de Gynécologie Obstétrique, Hôpital du Bocage, Dijon, France 3 INSERM UMR 866, Faculté de Médecine, 7 Bd Jeanne d'arc, BP87900, Dijon 21079, France 4 IFR 100, Faculté de Médecine, Dijon, France 5 Institut Pasteur de Lille, Département d'Athérosclérose, Lille F-59019, France; Inserm, U545, Lille F-59019 France; Université de Lille 2, Faculté de Pharmacie et Faculté de Médecine, Lille F-59019, France
6 Correspondence address. INSERM UMR 866, Faculté de Médecine, 7 Bd Jeanne d'arc, BP87900, Dijon 21079, France. Tel: +33 3 80 39 32 63; Fax: +33 3 80 39 34 47; E-mail: david.masson{at}chu-dijon.fr
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Abstract |
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Progesterone production by luteal cells is dependent on the supply of cholesterol by lipoproteins. The aim of this study was to determine whether the liver X receptors (LXRs) contribute to cholesterol homeostasis and progesterone secretion in human luteinized granulosa cells. Cells were isolated from follicular aspirates of patients undergoing in vitro fertilization. Luteinization was induced by a 7-day treatment with human chorionic gonadotrophin. LXR beta was expressed at higher levels than LXR alpha in granulosa cells and its expression was increased during luteinization. Treatment of luteinized granulosa cells by LXR agonists induced a significant time- and dose-dependent reduction in progesterone secretion (50% reductions after a 7-day treatment with 1-µM of either GW3965 or T0901317). mRNA levels of steroidogenic genes including steroidogenic acute regulatory protein and P450 side-chain cleavage were only moderately affected by LXR activation, with a significant reduction that was observed at 10 µM agonist concentration. Cellular cholesterol was markedly reduced after treatment with LXR agonists as a result of an increased cholesterol efflux that was related to the induction of LXR target genes (ABCA1, ABCG1, apo E, PLTP). Our study identifies LXRs as new, key actors contributing to regulation of cholesterol metabolism and steroidogenesis in luteinized granulosa cells.
Key words: cholesterol/Liver X receptor/progesterone
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Introduction |
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Liver X receptors (LXRs alpha and beta) belong to the nuclear receptor superfamily. They are activated by certain oxysterols and are involved in the control of cholesterol homeostasis in the body (Repa and Mangelsdorf, 2000). As LXRs regulate transcription of many genes involved in the reverse cholesterol transport pathway, synthetic LXR agonists have been mostly considered in initial studies as promising drugs in the prevention and treatment of atherosclerosis. It is noteworthy that beyond regulation of genes maintaining cholesterol balance, LXR agonists might also affect the function of other specific tissues that rely tightly on cholesterol biosynthesis for normal function. For instance, LXR alpha was recently found to act as a sterol sensor in the adrenal gland, where it maintains intracellular cholesterol below potentially toxic levels (Cummins et al., 2006), and LXR activation in cultured adrenal cells inhibited expression of steroidogenic genes and decreased steroid hormone production (Nilsson et al., 2006). In addition, LXRs were also shown to be involved in reproductive biology in mice, and in particular LXR beta was found to be essential for gonad function in male mice through regulation of lipid homeostasis in the Sertoli cells (Robertson et al., 2005). Finally, female mice lacking LXR alpha and/or beta are hypofertile and show oocyte dysfunction (Steffensen et al., 2006).
A critical feature of ovarian function is the differentiation of the ovulatory follicle into the corpus luteum, which produces steroid hormones (mainly progesterone) that are required for the initiation and the maintenance of pregnancy (Christenson and Devoto, 2003). The human corpus luteum has the remarkable capacity to produce large quantities of progesterone (up to 40 mg daily), and insufficient availability of the precursor cholesterol is thought to be a major limiting factor for progesterone biosynthesis (Christenson and Devoto, 2003). Although luteal cells can synthesize cholesterol on a de novo basis, this endogenous synthetic pathway is thought to play only a minor role under normal conditions (Gwynne and Strauss, 1982). In fact, it has been suggested that the major sources of cholesterol for the corpus luteum are either the endocytosis of cholesterol-rich, low-density lipoproteins (LDL) through the LDL-receptor pathway (Brannian and Stouffer, 1993) or the selective uptake of cholesterol esters from high-density lipoproteins (HDL) through the scavenger receptor BI (SR-BI) pathway (Azhar et al., 1998; Trigatti et al., 1999; Azhar and Reaven, 2002). A subsequent steroidogenic process in the corpus luteum requires the conversion of cholesterol first into pregnenolone and then into progesterone. These two steps are catalyzed by P450 side-chain cleavage (P450scc) and by 3ß-hydroxysteroid deshydrogenase (3ßHSD), respectively (Christenson and Devoto 2003). Interestingly, the critical step, which is governed by steroidogenic acute regulatory protein (StAR), is the movement of cholesterol from the outer to the inner mitochondrial membrane (Christenson and Strauss, 2000; Stocco, 2001). The whole steroidogenic process is under the control of multiple factors, including several nuclear receptors. The orphan nuclear receptor steroidogenic factor-1 (SF-1) is a master regulator of steroidogenesis, in particular through the control of the expression of StAR (Parker et al., 2002; Val et al., 2003). In addition, the orphan nuclear receptor liver receptor homolog (LRH-1) is also highly expressed in the corpus luteum where it stimulates the expression of StAR, P450scc and 3ßHSD (Hinshelwood et al., 2003; Peng et al., 2003; Kim et al., 2004, 2005). Recently, it has been suggested that peroxisome proliferator activated receptor (PPAR) gamma plays a role in reproductive biology. Moreover, specific PPAR gamma agonists were found to be able to modulate granulosa cell proliferation and steroid hormone synthesis (Lohrke et al., 1998; Froment et al., 2003). Although LXRs are other well-known nuclear receptors involved in cellular cholesterol homeostasis, to date their action on steroidogenesis has been explored only in adrenal cells, and their presence and functionality in the corpus luteum have never been addressed. In addition, the impact of synthetic LXR agonists on cholesterol homeostasis and progesterone production in granulosa cells that occurs mainly during luteinization is unknown. These points were explored in the present study in human luteinized granulosa cells which were treated with increasing doses of synthetic LXR agonists.
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Materials and Methods |
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Isolation and culture of Granulosa cells
Human luteinized granulosa cells were isolated from follicular aspirates of patients undergoing in vitro fertilization and embryo transfer at the Center of Sterility (CHU Dijon). All the patients had given informed consent, and the protocol was approved by the local ethics committee. The cells were taken from patients who had received a follicle-stimulation regime, including injection of 10 000 IU of human chorionic gonadotrophin (hCG) (Gonadotrophine chorionique endo®, Organon) 36 h before oocyte retrieval. After oocyte isolation, follicular fluids obtained from three to seven patients were pooled and granulosa cells were isolated as previously described (Stewart and Vandevoort, 1997). The cells were plated at a density of 2 x 105 cells/well on 35 mm 6-well dishes and cultured in 1:1 Dulbecco's modified Eagles's medium (DMEM)-Ham's F-12 medium supplemented with 10% fetal calf serum (Invitrogen, Cergy Pontoise, France) and antibiotics (100 UI/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml fungizone) at 37°C in a 95% air–5% CO2 humidified environment. The medium was changed daily in all experiments. hCG was added to the culture medium at a baseline concentration of 0.02 IU/ml from day 1 through day 7 of culture. This constant baseline dose of hCG simulates a normal non-conceptive luteal phase and was selected from the dose–response study on the basis of its ability to maintain physiological profiles of steroid and relaxin secretion (Stewart and Vandevoort, 1997). The cells obtained from the same pool of donors were used throughout one experiment (from day 0 to day7). The results presented in the study are representative of three different experiments performed with distinct pools of donors.
Luteinized granulosa cells were treated daily by the potent and specific LXR agonists T0901317 or GW3965 (Schultz et al., 2000; Collins et al., 2002) (kindly provided by Genfit, Lille, France) dissolved in dimethylsulphoxide (DMSO) (0.1, 1 and 10 µM, final concentrations) or by DMSO only (control treatment). DMSO dilutions (1/1000) were identical in all cases.
Human peripheral blood monocytes were obtained from healthy donors at the ‘Etablissement Français du Sang’ and purified as previously described (Sordet et al., 2002). They were differentiated into macrophages by culturing them for 7 days in the presence of macrophage colony-stimulating factor as described previously (Sordet et al., 2002). Macrophages were used as control because these cells expressed both human LXR alpha and beta at high levels (Repa and Mangelsdorf, 2000).
Progesterone measurement
At day 1, 3 and 7, media were collected and stored frozen until hormone concentrations were determined. Before hormone determination, the media were diluted 200 x in human male plasma containing no significant amount of progesterone. Progesterone measurements were performed by electrochemiluminescence using commercial kits (Elecsys, Roche, Switzerland). No significant levels of progesterone were found in the culture medium containing 10% calf serum prior to cell culture using the same experimental conditions.
Cholesterol efflux
The cholesterol efflux was assessed as described with minor modifications (Liao et al., 2002). Briefly, the luteinized granulosa cells cultured in 6-well plates were treated with GW3965 LXR agonist (1 µM) in DMEM-Ham's F-12 medium supplemented with 10% FCS for 6 days and then labelled with [3H]cholesterol (0.2 µCi/ml) for 5 h. The cells were gently washed three times with fresh serum-free medium, and then serum-free medium containing ApoA-I (10 µg/ml) (Sigma-Aldrich, Lyon, France) was added. After a 1-h or 2-h incubation, the medium was recovered and centrifuged to remove any detached cells. After the 2-h incubation time, the cells were also washed and lysed in 1 M NaOH. Aliquots of medium and cell lysates were assayed by liquid scintillation. The results represent the radioactivity recovered in the medium as a percentage of the total radioactivity (medium + cell lysate). All of the experiments were conducted in triplicate.
Determination of cellular cholesterol and lathosterol
The cells were homogenized with a handheld ultrasonic homogenizer for 15 s in a mixture of potassium hydroxide 10 mol/l (60 µl) and ethanol containing epicoprostanol (Steraloids, Wilton NH) as the internal standard (concentration 200 ng/ml), and the tubes were incubated for 45 min at 56°C. After incubation, 5 mL of hexane and 1 mL of water were added; the tubes were shaken, centrifuged and the organic phase was evaporated. One hundred microlitre of a mixture of bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (Acros Organics, Geel, Belgium) 4/l v/v (Gambert et al., 1979) were added; the tubes were incubated for 1 h 45 min at 80°C, evaporated, and 100 µl of hexane was added. Analysis of sterol trimethylsilylethers was performed by GCMS in a 6890 gas chromatograph coupled with a 7673 Mass Detector (Agilent Technologies). The column was an HP-5MS 30 m x 250 µm (Agilent technologies), helium was used as the carrier gas. The GLC operating conditions were as follows: injector temperature was 250°C and oven temperature was programmed, after injection, at a rate of 15°C/min to 280°C then at a rate of 2°C/min to 300°C. The MSD operating conditions for EI-MS were: source temperature 230°C, ionising voltage 70 eV. SIM mode was used for analysis, and the selected ions (m/z) were 368 for cholesterol, 458 for lathosterol and 370 for epicoprostanol, respectively. The concentration of cholesterol was determined from the ratio of the peak area corresponding to the internal standard. Levels were determined by comparison of this ratio with a standard curve of known amounts of cholesterol.
Filipin staining
Granulosa cells were rinsed twice with PBS and then fixed with 3% paraformaldehyde for 30 min at room temperature. They were then incubated with filipin in PBS (100 µg/ml) for 30 min at room temperature. Finally the cells were incubated a further 5 min with glycine (1.5 mg/ml in PBS) and rinsed twice for 5 min with PBS. The staining was visualized with a fluorescent microscope. Cellular staining was calculated as the ratio between the stained area and the total area ratio for each cell (number of pixels above threshold per cell/total pixels per cell)
RNA isolation and real-time RT–PCR
RNA levels were quantified by reverse transcription followed by real-time PCR using an ABI PRISM Sequence Detection System (Applied Biosystems, Courtaboeuf, France) (Masson et al., 2004). Total RNA was isolated from granulosa cells using RNeasy mini kit RNA (Qiagen, Courtaboeuf, France). Briefly, 1 µg of RNA was reverse-transcribed into cDNA using MuMLV reverse transcriptase and oligo dT (Invitrogen, Cergy Pontoise, France); 50 ng of the cDNA mixture were used for each PCR. Reactions were performed by using the Quantitect SYBR Green amplification kit (Qiagen, Courtaboeuf, France) following the instructions provided by the manufacturer. Relative mRNA levels were calculated using the 
Ct method. Values were normalized to cyclophilin levels, which remained constant as compared with 28 s RNA levels. The specificity of the PCR was monitored by both melting curve analysis of the amplification products and agarose gel electrophoresis. Primer sequences and typical Ct values (observed in untreated cells) are given in Table 1.
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Statistical analysis
Mann–Whitney test was used to determine the differences between data means.
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Results |
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Expression of LXR isoforms in human luteinized granulosa cells
In order to assess whether LXR isoforms are expressed at significant levels in human granulosa cells, quantitative and qualitative analysis of LXR alpha and beta mRNAs was performed by RT–PCR. As shown in Fig. 1, specific amplification products of 176 and 207 base pairs could be detected in human granulosa cells, corresponding to LXR alpha and beta isoforms, respectively. The
C(t) values with the cyclophilin housekeeping gene were 7.2 and 4.2 for LXR alpha and LXR beta, respectively. These data indicate that LXR beta is the prominent isoform in granulosa cells. LXR beta mRNA levels were similar to those observed in human primary macrophages, whereas LXR alpha mRNA levels were about four times lower in granulosa cells than in macrophages (P < 0.05 Mann–Whitney) (Fig. 1).
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Differential regulation of LXR alpha and beta during luteinization of granulosa cells
Expression of LXR alpha and beta genes was also determined during in vitro luteinization of granulosa cells as obtained by a 7-day treatment with 0.02 IU/ml hCG (Fig. 2). As previously observed, stimulation of granulosa cells with hCG induced a transcriptional burst of genes involved in progesterone synthesis. After 3 days of stimulation, P450scc, 3ßHSD, StAR and SR-BI mRNA levels were, respectively, 22, 12, 8 and 5 times higher than those retrieved in granulosa cells before hCG treatment (Fig. 2). A similar, though weaker, induction was observed at day 7. Accordingly, induction of progesterone secretion was markedly increased during hCG treatment, by a factor of 4 and 5 after 3 and 7 days of treatment, respectively. Interestingly, under the same conditions, induction of LXR beta mRNA levels was markedly increased by a factor of six at day 3 and day 7. In contrast, LXR alpha mRNAs were unchanged after 3 or 7 days of hCG treatment, respectively. These data indicate that LXR alpha and beta are differentially regulated during luteinization of granulosa cells. In order to address whether LXR beta expression was positively regulated by hCG, cells were cultured for three days in the presence or in the absence of hCG. Whether hCG was added or not, an increase in LXR beta mRNA was retrieved after 3 days of culture with no differences between treated and untreated cells (data not shown).
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Effects of LXR agonists on the expression of genes involved in progesterone synthesis in luteinized granulosa cells
To determine whether LXR activation had a direct effect on the expression of steroidogenic target genes, the mRNA levels of P450scc, 3ßHSD and StAR were determined in luteinized granulosa cells, which were treated with or without T0901317 (1 µM, 3 days) and with or without GW3965 (0.1, 1 and 10 µM, 7 days) LXR agonists. As shown in Fig. 3, 3ßHSD mRNA level was not significantly affected by LXR agonist treatment. mRNA levels of P450scc and StAR tended to be only slightly reduced by LXR agonist treatment, and the statistical significance of differences was reached only for the highest GW3965 concentration studied (Fig. 3).
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Effect of LXR agonists on cholesterol content of luteinized granulosa cells
Besides the expression level of steroidogenic genes, cholesterol availability is another rate-limiting factor of progesterone biosynthesis in luteinized granulosa cells. As shown in Fig. 4, when luteinized granulosa cells were treated with 1 µM T0901317 or GW3965 for 7 days, a significant decrease in cellular cholesterol content was observed as compared with luteinized granulosa cells treated with DMSO alone (
8 µg/million cells versus 5 µg/million cells, P < 0.05 Mann–Whitney) (Fig. 4). A significant, 4-fold decrease in cellular lathosterol content [reflecting the activation level of the cholesterol biosynthesis pathway (Bjorkhem et al., 1987)] was also observed. Cholesterol content was found to fall in similar proportions; there was thus no significant change in the lathosterol to cholesterol ratio after GW3965 treatment (Fig. 4). Accordingly, analysis of the filipin fluorescent signals demonstrated that GW3965 significantly reduced the cellular area positively stained by filipin, in particular with a reduction in the fluorescence signal in the perinuclear region (Fig. 5).
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As shown in Fig. 6, mRNA levels of several LXR target genes involved in cholesterol efflux were markedly increased in a dose-dependent manner by LXR agonist treatment as assessed by real-time RT–PCR. A significant induction of PLTP, ABCA1, apoE and ABCG1 genes was observed with an agonist concentration as low as 0.1 µM. Moreover, ABCA1, apoE and ABCG1 mRNA levels were increased by a factor of up to 3, 13, 5 and 10, respectively, with the 10 µM GW3965 agonist concentration (Fig. 6A). LXR alpha, but not LXR beta, mRNA levels were also significantly increased by the LXR agonist treatment. In contrast, neither SR-BI (the major HDL receptor) nor LDL-R mRNA levels were affected by LXR activation. Concordant observations were made with T0901317 LXR agonist after 3 days of treatment (Fig. 6B). GW3965 treatment increased isotopic cholesterol efflux towards free apoA-I by upto 40% after 1 and 2 h of incubation (P < 0.05 in both cases) (Fig. 6C). This provides clear evidence that LXR agonist treatment does affect cholesterol exchanges.
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LXR activation by synthetic agonists decreases progesterone secretion in luteinized granulosa cells
To investigate whether LXR activation and associated changes in the expression level of target genes could affect cell metabolism, progesterone secretion was assessed in luteinized granulosa cells, which had been treated for 3 or 7 days with either of the T0901317 and GW3965 LXR agonists. As shown in Fig. 7, and as compared with the untreated DMSO control, a 7-day treatment with T0901317 induced a significant dose-dependent reduction in progesterone secretion by luteinized granulosa cells, with a mean percentage decrease ranging from 50% with the 1 µM dose to 75% with the 10 µM dose. Concordant results were obtained with the GW3965 agonist, with a 30% reduction in progesterone concentrations in the medium after 3 days of treatment with the lowest 0.1 µM dose of the agonist. Again, the effects were accentuated after 7 days of treatment, with a dose-dependent reduction in progesterone concentration ranging from 30% to 50% in the 0.1–10 µM GW3965 concentration range studied (Fig. 7).
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Discussion |
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Results of the present study demonstrate that LXRs, mainly LXR beta, are expressed at significant levels in granulosa cells, in which they are able to modify the expression of a number of genes involved in cholesterol homeostasis. Alterations in luteinized granulosa cells treated with increasing concentrations of non-steroidal, synthetic LXR agonists are characterized by an increase in cholesterol efflux, a reduction in cellular cholesterol content and a decrease in progesterone secretion.
Previous studies reported that LXR alpha expression is restricted to the intestine, liver, adipose tissue and macrophages, whereas LXR beta expression appears to be ubiquitous, in particular with high levels being found in mouse oocytes.(Repa and Mangelsdorf, 2000) (Steffensen et al., 2006). Although not directly assessed at the protein level in the present study, both LXR alpha and LXR beta mRNA were found to be expressed at substantial levels in granulosa cells. LXR beta is the most prominent isoform in granulosa cells, and most interestingly, only LXR beta not LXR alpha expression was found to be up-regulated during hCG-induced luteinization. No evidence for a direct effect of hCG on LXR beta expression could be obtain. This might be due to the fact that all patients have already received an ovulatory dose of hCG prior to oocyte retrieval. Expression of steroidogenic genes was found to be only marginally affected by LXR agonists, suggesting that none of these genes was tightly dependent on LXRs for normal expression levels in luteinized granulosa cells. The observations described above suggest that the LXR beta isoform might play a major role in triggering specific events that are known to occur mostly during the luteinization phase, particularly progesterone synthesis.
Insufficient cholesterol is known to limit the rate of progesterone biosynthesis in luteinized granulosa cells, and the present study provides additional support by showing that the decreased bioavailability of cellular cholesterol, induced by treating luteinized granulosa cells with LXR agonists, reduces progesterone production. First, LXR agonist treatment produced a significant drop in the cellular cholesterol content assessed by gas chromatography/mass spectrometry analysis. Second, the parallel decrease in lathosterol, a cholesterol precursor, indicated that cholesterol biosynthesis is unable to compensate for the cellular cholesterol depletion induced by LXR agonists. Third, the efflux of cellular cholesterol was substantially accelerated after treatment of luteinized granulosa cells with LXR agonists as shown by both a significant rise in cellular cholesterol efflux and the significant induction of relevant genes, including PLTP, ABCA1, apoE and ABCG1. Indeed, PLTP, ABCA1, ABCG1 and apoE are recognized as major genes involved in the regulation of cholesterol efflux, and all of them are well-characterized LXR targets (Costet et al., 2000; Laffitte et al., 2001). Our data demonstrate that these genes are present and responsive to LXR in granulosa cells and are able to stimulate the apo A-I-mediated cholesterol efflux in this context. Interestingly, this pathway is likely to be of physiological relevance since large amounts of pre-beta HDL (the primary acceptors of cellular cholesterol) are present at high levels in human follicular fluid (Jaspard et al., 1996). In contrast, although SR-BI, the major HDL receptor, was described as an LXR target in the liver (Malerod et al., 2002), it is worthy to note that in granulosa cells (present study) and other steroidogenic organs (testes and adrenal glands), SR-BI expression is not induced by LXR activation (Robertson et al., 2005; Cummins et al., 2006).
Besides cholesterol homeostasis, we observed that expression of steroidogenic genes, in particular P450scc and StAR, was slightly decreased by treatment with synthetic LXR agonists at the highest concentration. Recent studies by Cummins et al. indicated that StAR expression is significantly induced by LXR alpha but not LXR beta in mouse adrenal cells, and a concordant, though weaker tendency was observed in human adrenal cells. In addition, they characterized a functional LXRE on the mouse StAR gene promoter (Cummins et al., 2006). In contrast, Nilsson et al. (2006) reported that the treatment of cultured adrenal cells by LXR agonists inhibited expression of StAR and P450 scc and consequently decreased adrenal steroid hormone production. The difference between our study and that of Cummins et al. was we used a chronic treatment (i.e. 3 or 7 days) in our model. Under these conditions different LXR agonists (T0901317 and GW3925) did not induce StAR expression. It is also worthy of note that in contrast to what was observed in adrenal glands (32), LXR beta is the dominant LXR isoform in luteinized granulosa cells, and only LXR beta mRNA levels not LXR alpha mRNA levels were markedly induced (by up to 6-fold) after hCG treatment. These two points might account for the lack of any effect of LXR agonists on StAR gene expression in our model. They also indicate that the impact of LXR agonists on steroidogenic pathways in adrenal and granulosa cells might be tightly dependent on the relative abundance of LXR alpha and LXR beta isoforms that differ from one tissue to the other. As shown in the present study, LXR beta is the major functional LXR isoform in luteinized granulosa cells, and as such it should be considered in future studies as the first candidate in triggering the effects of LXR agonists in this model. Interestingly, isolated LXR beta deficiency was recently reported to be associated with hypofertility, producing the accumulation of cholesterol in the testes of male mice and dysfunctional oocytes in female mice (Steffensen et al., 2006). Although female mice with isolated LXR alpha deficiency are also hypofertile, the phenotype was less clear than for female counterparts with isolated LXR beta deficiency. In all cases, combined LXR beta/LXR alpha deficiency was associated with a magnification of related phenotypes as compared to isolated LXR beta or LXR alpha deficiencies, suggesting that both isoforms may contribute significantly and synergistically to reproductive biology.
In a recent study, Nilsson et al. (2006) reported that treatment of cultured adrenal cells by LXR agonists reduced steroid hormone production. In addition, Cummins et al. (2006) showed that LXR deficiency induced adrenomegaly, cholesterol accumulation and excessive steroidogenesis in adrenal glands. Due to the toxic effect of free cholesterol at high levels, it was suggested in the latter study that LXR provided a safety valve to prevent free cholesterol accumulation in the adrenal gland. Our results suggest that LXR might also function similarly to prevent cholesterol accumulation in luteinized granulosa cells. Indeed, the effect of LXR agonists progressively increased along the luteinization phase in our model, in particular with greater effect after 7 days than after 3 days of treatment despite an earlier peak of progesterone production. Thus, LXR may contribute to the regression of progesterone secretion by the corpus luteum at the end of the cycle, rather than to the direct regulation of progesterone production in an earlier phase. Again, as recently suggested with adrenal cells (Cummins et al., 2006), LXR may act as a compensatory and protective factor by preventing excessive accumulation of cholesterol in luteal cells, thus preserving their normal function. It is likely that the increase in LXR beta expression during luteinization is preventive, but does not significantly reduce progesterone synthesis in the absence of pharmacological activation.
In conclusion, our study identified LXRs as new actors contributing to the regulation of cholesterol homeostasis in luteinized granulosa cells. Overall, cellular cholesterol depletion accounted primarily for the LXR-mediated decrease in progesterone secretion. Indeed, high levels of progesterone production by luteinized granulosa cells is known to be tightly dependent on cholesterol supply, and lipoproteins are known to markedly enhance progesterone production when added to granulosa cells in culture (Brannian and Stouffer, 1993; Azhar et al., 1998; Azhar and Reaven, 2002). In other words, these observations indicate that beyond cholesterol homeostasis and reverse cholesterol transport, synthetic LXR agonists may have an unexpected effect on reproductive biology through their ability to modify steroid hormone production. Further experiments will be necessary to evaluate the effects of LXR agonist on progesterone production in vivo.
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Acknowledgements |
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In memory of Dr Jean Desgres who shared his helpful expertise concerning progesterone measurement in cell culture medium. The authors also wish to thank Philip Bastable for editing the manuscript and Anne Athias (plateau technique lipidomique IFR 100) and Stephanie Lemaire-Ewing for excellent technical assistance. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Conseil Régional de Bourgogne, the Fondation de France, the CHU Dijon and ASGOD.
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Submitted on December 28, 2006; resubmitted on February 27, 2007; accepted on March 5, 2007.
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