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BOR - Papers in Press, published online ahead of print March 5, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.064881
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BIOLOGY OF REPRODUCTION 79, 17–25 (2008)
DOI: 10.1095/biolreprod.107.064881
© 2008 by the Society for the Study of Reproduction, Inc.

Pituitary; 

Disruption of Lipid Rafts Induces Gonadotropin Release in Ovine Pituitary and LbetaT2 Gonadotroph Cells1

E Robin , J Cognié , F Foulon-Gauze , J Fontaine , and X Cayla 2

UMR Physiologie de la Reproduction et des Comportements, INRA/CNRS/Université Tours/Haras Nationaux, 37380 Nouzilly, France

ABSTRACT

In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.

exocytosis, follicle-stimulating hormone, lipid rafts, luteinizing hormone, pituitary

INTRODUCTION

In vertebrates, the gonadotroph cells of the anterior pituitary gland have a key function in the hormonal control of reproduction. They integrate the activity of the hypothalamic neuroendocrine signal, GnRH, with feedback information from the gonads, leading to regulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion. The gonadotropins, LH and FSH, stimulate sex hormone production and gametogenesis. The hypothalamic hormone, GnRH, acts on the gonadotroph cell through its seven-transmembrane receptor to induce varied complex signaling pathways, depending on the frequency of its delivery [1–3] and the abundance of the receptor at the cell surface [4]. Gonadotropins belong to a family of structurally related glycoprotein hormones, which are composed of two distinct noncovalently associated subunits with a common alpha subunit and a hormone-specific beta LH or beta FSH subunit [5]. It is well known that the GnRH receptor, depending of the frequency of GnRH pulse delivery, differentially controls the synthesis of mRNA for the CGA ({alpha}), LHB (LHβ), and FSHB (FSHβ) subunits, as well as gonadotropin storage patterns [6–8].

The cholesterol- and glycosphingolipid-rich membrane microdomains, also referred to as lipid rafts, have been shown recently to be critical for GnRH signaling in gonadotroph cells [9, 10]. These microdomains exhibit a liquid-ordered structure and are resistant to nonionic detergent; they are also named detergent-resistant membranes (DRMs) or detergent-insoluble, glycosphingolipid-enriched membranes (DIGs) [11, 12]. Compared with the nonraft bulk plasma membranes, two main characteristics of lipid raft microdomains are their higher viscosity and their specific protein composition. Indeed, it has been proposed that the functional property of lipid rafts is to spatially concentrate specific sets of proteins in order to promote processes occurring at the membrane, including signal transduction [13, 14], endocytosis, or exocytosis [15, 16]. Additionally, lipid rafts are used by certain viruses and bacteria to invade their host cells [17, 18].

Since cholesterol determines lipid raft stability, any alteration in cholesterol content modifies its physical and biological properties. Indeed, many studies have shown that lipid rafts are disrupted by cholesterol depletion and that lipid raft components are released into the bulk of the plasma membrane [19–21]. Methyl-beta-cyclodextrin (MβCD) is a derivative of a cyclic oligomer of glucose with a lipophilic property [22], used to extract cholesterol from membranes. To date, a large body of data has been obtained in various models reporting defects in exocytosis following MβCD-mediated cholesterol depletion. For example: 1) In a PC12 neuronal-derived cell line and in its neurite-related synaptosomes, treatment with MβCD impaired exocytosis [23, 24]. In rat brain synaptosomes, glutamate/aspartate release was severely reduced by MβCD-induced cholesterol depletion [25–27]. Finally, at the crayfish neuromuscular junction, MβCD blocked evoked synaptic transmission, suggesting a failure of transmitter release [28]. 2) In exocrine cells, such as natural killer (NK) cells, neutrophil promyelocytic cells (PLB-985 cells), and rat basophil leukemia cells (RBL-2H3 cells), MβCD-induced cholesterol depletion inhibited primary granule exocytosis [29–32]. In epithelial alveolar type II cells, secretagogue-stimulated secretion of lung surfactant was reduced by MβCD [33]. Similarly, in the epithelial-derived Madin-Darby canine kidney cells (MDCK cells), apical exocytosis was largely blocked by cholesterol depletion [34–36]. 3) In endocrine cells, MβCD treatment of the pancreatic β-cell model, MIN6 β cells, inhibited KCl-induced insulin release [37]. Surprisingly, few studies have reported opposite effects of MβCD-mediated lipid raft disruption in pancreatic cells. An increase in exocytosis was measured in both pancreatic β cells [38, 39] and {alpha} cells by MβCD treatment [40].

In the present study, we addressed the contribution of lipid rafts to GnRH-induced gonadotropin release, first in LβT2 murine gonadotroph cells and second in ovine pituitary cells. We found that cholesterol depletion in LβT2 cells by MβCD did not inhibit the GnRH-induced LH release, but it unexpectedly caused a rapid and substantial release of LH in the absence of GnRH treatment. These effects were confirmed in primary culture of ovine pituitary cells, where MβCD induced LH release from a pool of LH larger than that released by GnRH, and that was independent of protein synthesis. Furthermore, in ovine pituitary cells, MβCD treatment induced FSH secretion. Importantly, these data were confirmed by in vivo studies, because following MβCD injection into the pituitary gland of anaesthetized sheep, a peak of LH release was detected in the blood.

MATERIALS AND METHODS

Hormones, Antibodies and Reagents

Ovine LH (oLH; CY1072 and CY1083) and the antibody to oLH (number 534) were gifts from Dr. Y. Combarnous and Dr. C. Taragnat (INRA, Nouzilly, France), respectively. Purified ovine FSH was provided by the National Institutes of Health (NIH RP2). The monoclonal antibody directed against ovine FSHB subunit was a gift from Dr. Henderson [41]. Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody used for the ELISA and Western blot determinations was from Bio-Rad (Marne la Coquette, France). Methyl-β-cyclodextrin, apo-transferrin, albumin from bovine serum (BSA), L-ascorbic acid, cycloheximide, Dulbecco modified Eagle medium (DMEM; D5796), filipinIII, and DMEM modified (DMEMm; D2902) were all from Sigma-Aldrich. The GnRH (Stimu-LH) was from Ferring Pharmacological Products (Gentilly, France). Collagenase A and DNase I were from Roche Diagnostics (Meylan, France), and the antibiotic-antimycotic treatment (10 000 U/ml penicillin G sodium, 10 000 µg/ml streptomycin sulphate, and 25 µg/ml amphotericin B) and the fetal calf serum (FCS) were from Gibco/Fisher (Cergy Pontoise, France).

Animals, Primary Cell Cultures, and Surgical Procedures

Experiments were conducted using adult female and male Ile-de-France sheep, which were kept under normal husbandry conditions at INRA (Nouzilly, France). All experiments were performed in accordance with local animal welfare regulations (authorization number A38801 from the French Ministry of Agriculture; Agreement of Direction of Veterinary Services C37-175-2).

Sheep pituitary cell cultures. Freshly collected pituitary glands were washed twice in 0.9% NaCl, finely sliced in culture medium (DMEMm with 1% antibiotic-antimycotic, 100 µM L-ascorbic acid, and 5 µg/ml apo-transferrin) supplemented with collagenase A (0.4 mg/ml) and DNase I (25 µg/ml), and then incubated for 1 h in a shaking water bath at 37°C, followed by manual dispersion through different sizes of needles. Cells were then centrifuged twice at 100 x g for 5 min, and the pellet of was resuspended in culture medium supplemented with 10% fetal bovine serum (FBS). Cells (106 cells/ml of culture medium) were maintained at 37°C under 5% CO2 in a humidified atmosphere for 4–16 h before use in experiments. Cell culture media were collected sequentially and stored at –20°C. For the analysis of intracellular LH, cells were washed with PBS and stored at –20°C until lysis.

In vivo pituitary injection. Anesthesia of sheep was induced with atropine and barbiturics and maintained following intubation with isoflurane (2.5%–4%). A 20-gauge catheter was introduced into the jugular vein in order to collect venous blood samples. Access to the pituitary gland was by a surgical parapharyngeal route. The sphenoidal bone was drilled facing the sella turcica. Through this opening, intrapituitary injections of vehicle (0.9% NaCl) or MβCD in saline (3 x 60 µl) were slowly (30 sec/injection) carried out using a 30-gauge needle (0.3 mm). Subcutaneous tissue and skin were sutured to close the wound, and the animals were given antibiotics (Intramycine ND, benzylpenicillin, and dihydrostreptomycin), anti-inflammatory (Diurizone ND, dexamethason, and hydrochlorothiazide), and analgesic (Finadyne ND, flunixin meglumin) support. Blood samples were collected from the jugular catheter (2 ml with intervals varying from 5 to 15 min). The blood was centrifuged and the plasma recovered and stored at –20°C. At the end of the experiment, animals were killed by an intravenous injection of barbiturates.

LβT2 Cell Culture and Cell Treatment

LβT2 cells, generously provided by Dr. P. Mellon (La Jolla, CA), were grown in cell culture medium (DMEM supplemented with 2 mM L-glutamine, 10 mM Hepes, 1% antibiotic-antimycotic) and 10% FCS, at 5% CO2 at 37°C in a humidified environment. Before treatment, the cells were maintained in cell line culture medium containing 0.1% FCS for 16 h. They were then collected by scraping and were washed in serum-free cell line culture medium and incubated with the indicated reagents.

In kinetic experiments, cells (LβT2 cells or pituitary ovine cells) were incubated in suspension (106/ml) and then, at the indicated times, homogeneous samples containing cells and medium were taken so that the cell concentration was maintained for subsequent prelevments in the kinetic analysis. The supernatants were stored at –20°C.

Staining LβT2 cells for the visualization of lipid rafts. Monolayers of LβT2 cells were incubated for 30 min at 37°C with 6 µg/ml FITC-labeled cholera toxin-B subunit (Sigma) in a humidified atmosphere with 5% CO2. Cells were washed twice with PBS and then incubated with serum-free cell line culture medium before microscopic observation. Images were taken every 30 sec using Metamorph software (Roper Scientific, Evry, France) on a Leica microscope inversed (Leica Microsystems, Reuil-Malmaison, France).

Gonadotropin Measurement by ELISA

The concentrations of ovine gonadotropins (using ovine standards) in blood plasma and of murine LH (using a rat LH standard) in LβT2 culture media were determined using double-antibody ELISA for all experiments, as previously described in Faure et al. [42] and Dupont et al. [43], respectively, with a sensitivity 0.1 ng/ml for ovine and rat LH and 0.4 ng/ml for ovine FSH.

SDS-PAGE and Western Blot

Cell culture medium was concentrated by trichloroacetic acid protein precipitation: 80 µl trichloroacetic acid was added to 400 µl medium, frozen for 2 h at –80°C, and then centrifuged for 20 min at 14 000 x g. The protein pellet was washed twice with 500 µl acetone. Pellet was then solubilized in Laemmli buffer, with or without 0.1% β-mercaptoethanol. Samples were either reduced (heated for 5 min at 100°C) or not reduced (and not heated) and resolved by electrophoresis on 12% SDS-PAGE, electroblotted on nitrocellulose membranes (BA83S; Schleicher & Schuell). Nitrocellulose membranes were washed in Tris-buffered saline (TBS; pH 7.4) containing 0.1% Tween-20 (TBS-T; 0.1%) and blocked with 3% (w/v) dry milk and 3% (w/v) BSA in TBS-T 0.1% for 30 min. They were then incubated overnight at 4°C with anti-oLH antibody (1:1000), washed 3 x 5 min with TBS-T 0.1%. Membranes were then incubated for 1 h in the presence of peroxidase-conjugated secondary antibody (dilution 1:2000), washed 3 x 5 min with TBS-T 0.1%, then 5 min with TBS-T 0.01%, and then 5 min with TBS. Immunoreactive bands were visualized by chemiluminescence with the ECL kit (Perkin Elmer, Courtaboeuf, France) as described by the manufacturer.

Statistical Analysis

Data are presented as means ± SEMs. The data were analyzed using a one-way ANOVA followed, where appropriate by Bonferroni multiple-comparison posthoc test or by a two-way ANOVA in kinetic study for MβCD vs. MβCD + cholesterol. The data for in vivo experiments were analyzed individually within animals using the unpaired t-test. All analyses were computed using Graphpad Prism Statistical Software (GraphPad, San Diego, CA).

RESULTS

MβCD Induces LH Secretion in LβT2 Cells

In order to study the involvement of lipid rafts in GnRH-induced LH secretion, we used the cholesterol-sequestering agent MβCD and measured LH release in LβT2 cells. Surprisingly, an increase in LH secretion was detected when 10 mM MβCD was added to GnRH, compared with GnRH alone (Fig. 1A). Furthermore, in the absence of GnRH, depletion of membrane cholesterol with MβCD induced LH release in a dose-dependent manner (Fig. 1B). At a dose of 2.5 mM, MβCD released similar amounts of LH as 10 nM GnRH, whereas 5 and 10 mM MβCD induced significantly higher amounts of LH within 5 min of treatment (Fig. 1B). We then followed up the analysis mainly using 10 mM MβCD, a condition reported specific for lipid raft disruption [44]. Since cholera toxin-B subunit has a very high affinity for the lipid raft component, ganglioside M1 (GM1), we used cholera toxin-B subunit fused to FITC (CTx-FITC) in living LβT2 cells. Although CTx-FITC-labeled cells had relatively continuous membrane staining, the adition of MβCD immediately and subtly affected membrane staining at 10 mM but had a very pronounced effect at 40 mM, indicating that MβCD treatment induced very rapid disorganization of lipid rafts in LβT2 cells (Fig. 2).


Figure 01
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  		FIG. 1 MβCD treatment of LβT2 cells induces LH release. A) LβT2 cells were incubated at 37°C for 60 min in medium alone (control) or supplemented with either 10 nM GnRH, or in the presence of 10 nM GnRH plus 10 mM of MβCD (MbCD + GnRH). Data are the mean ± SEM of four independent experiments. ***P ≤ 0.001 compared with control; *P ≤ 0.05 GnRH vs. GnRH+ MβCD. B) LβT2 cells were incubated at 37°C for 130 min in medium alone (control) or supplemented with either 10 nM GnRH or in the presence of the indicated concentrations of MβCD (MbCD). Samples of culture medium were collected sequentially, and LH release was measured by ELISA. Data are the mean ± SEM for three independent experiments.


Figure 02
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  		FIG. 2 Effect of MβCD treatment on FITC-labeled cholera toxin-B-stained lipid rafts in LβT2 cells. Lipid rafts in LβT2 cells were stained using FITC-labeled cholera toxin-B. Images were taken every 30 sec using Metamorph software on a Leica microscope inversed (objective immersion x40). At 15 sec before the acquisition of image number 11 (marked with *), MβCD was added to the medium to give a final concentration of 40 mM. Bar = 10 µm.

MβCD Induces LH Secretion in Ovine Pituitary Cells

To assess whether MβCD-mediated LH secretion was associated not only with transformed cells, namely LβT2, we evaluated the effect of MβCD on primary ovine pituitary cells, thus mimicking in vivo conditions more closely. As seen in Figure 3, MβCD did not prevent LH release in the presence of GnRH and, furthermore, induced a substantial LH secretion when used alone on ovine pituitary cells. Kinetic studies of LH secretion induced by MβCD in ovine pituitary cells showed similar patterns to those observed with LβT2 cells, albeit with the characteristic of higher LH released under all conditions (data not shown). To control for the effect of MβCD on cholesterol, we combined cholesterol with MβCD prior to cell exposure. When MβCD was preloaded with cholesterol, its ability to induce LH release was impaired (Fig. 4). Furthermore, when some cholesterol was added to the cell culture medium during MβCD treatment, the MβCD effect was immediately attenuated (data not shown).


Figure 03
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  		FIG. 3 MβCD treatment of ovine pituitary cells induces LH release. Ovine pituitary cells were incubated at 37°C for 30 min in medium alone (control; n = 26) or supplemented with either 10 nM GnRH (n = 26) or 10 mM MβCD plus 10 nM GnRH (MbCD + GnRH; n = 6) or 10 mM MβCD (MbCD; n = 13) or 5 µg/ml filipin III (n = 8). Luteinizing hormone released into the cell culture medium was measured by ELISA. ***P ≤ 0.001 compared with control; ns, P > 0.05 MβCD + GnRH vs. MβCD.


Figure 04
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  		FIG. 4 The effect of MβCD is dependent on its cholesterol-sequestering ability. Ovine pituitary cells were incubated at 37°C either in medium alone (control) or supplemented with either 10 mM MβCD (MbCD) or in cholesterol-preloaded MβCD (MbCD + cholesterol; cholesterol 1 mg/ml in MβCD 10 mM). Samples of culture media were collected sequentially, and LH release was measured by ELISA. Data are the mean ± SEM for three independent experiments. ***P ≤ 0.001 compared with MβCD.

To eliminate the possibility of a specific effect limited to MβCD, we tested filipin III, another compound that disrupts lipid rafts. Filipin is a polyene antibiotic with antifungal properties that binds selectively to cholesterol, forming complexes in the plasma membrane that sequester cholesterol and induce structural disorder [45, 46]. Despite using different mechanisms to sequester cholesterol in membranes, MβCD and filipin III had similar effects on ovine pituitary cells, and filipin III induced LH release in the absence of GnRH (Fig. 3).

MβCD-Treated Cell Recovery

To rule out the possibility of nonspecific effects of MβCD treatment implying critical cell damage, we have evaluated cell viability 24 h after MβCD treatment. Ovine pituitary cells treated the first day (D0) with vehicle or MβCD were then incubated overnight in medium containing 10% FCS in order to reconstitute cholesterol cell content. The next day (D1), viability was estimated using the Trypan blue exclusion test (Fig. 5A). A small decrease in the number of live cells was observed in all groups, and they were not significantly different from each other (Fig. 5A). In these experiments, the gonadotroph cell number, among the pituitary cells, has not been evaluated. To eliminate the possibility of a toxic effect of the MβCD specifically on gonadotroph cells, we evaluated their functional recovery. We measured LH release following control, GnRH, or MβCD treatment on D1. There were no significant differences for LH release on D1 for similar treatment, regardless of the cell treatment done on D0 (Fig. 5B). Altogether, these results suggest a limited toxic effect of MβCD on cells under our experimental conditions.


Figure 05
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  		FIG. 5 Recovery of ovine pituitary cells after MβCD treatment. A) Day 0, 106 ovine pituitary cells were incubated 30 min at 37°C in medium alone (control) or supplemented with either 10 nM GnRH or 10 mM MβCD (MbCD) and briefly washed with PBS. The cells were then maintained overnight in medium containing 10% FCS. The following day (Day 1), the proportion of living cells was determined using Trypan blue. Data are the mean ± SEM of three independent experiments. ns, P > 0.05 compared with control. B) Ovine pituitary cells were treated on Day 0 (D0) as described above and incubated the following day (Day 1) for 30 min in medium alone (control) or supplemented with either 10 nM GnRH or 10 mM MβCD (MbCD). Luteinizing hormone release into the culture medium was measured by ELISA. Data are the mean ± SEM of six independent experiments. Data were normalized by log transformation prior to statistical analysis. Bars with different letters are significantly different; P ≤ 0.01 for a vs. b and P ≤ 0.001 for a vs. c and b vs. c.

MβCD-Released LH Pool Is Larger than GnRH-Released Pool

We next compared the features of LH release induced by MβCD or GnRH, because MβCD released higher amounts of LH. To do this, we tested whether protein synthesis was important for either of these two patterns of LH release, even though MβCD-mediated LH release occurred within minutes. The use of cycloheximide, an inhibitor of protein synthesis, clearly indicated that the LH release within 30 min of treatment does not require protein synthesis (data not shown). In addition, we designed experiments with sequential treatments. Primary culture of ovine pituitary cells was first pretreated or not for 15 min with GnRH, washed briefly, then incubated in control medium with GnRH or MβCD. Under these conditions, GnRH-pretreated cells still released substantial amounts of LH when stimulated with MβCD, but not when restimulated with GnRH (Fig. 6). In the latter case, LH release to restimulation did not exceed basal levels. These results suggest the existence of a specific pool of LH that can be released by MβCD but that is not accessible by GnRH stimulation. We next assessed the possibility of a pool of LH selectively sensitive to MβCD. Therefore, following MβCD or GnRH treatments of ovine pituitary cells, we analyzed intracellular LH and secreted LH by SDS-PAGE and Western blot using anti-LHB antibodies (Fig. 7). The levels of intracellular LH and secreted LH detected by Western blotting were consistent with the levels of secreted LH detected by ELISA in the cell culture supernatant. However, the electrophoretic mobility patterns for LH secreted in response to GnRH or MβCD were not different. This would have suggested potential differences in the physical state (maturity) of LH released by GnRH compared with MβCD. Altogether, these data suggest that a large MβCD-releasable pool of LH contains within itself a smaller GnRH-releasable pool of LH.


Figure 06
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  		FIG. 6 Effect of GnRH pretreatment on LH release induced by GnRH or MβCD treatment. Ovine pituitary cells were incubated at 37°C for 15 min in medium alone (without pretreatment) or with 10 nM GnRH (pretreatment GnRH). Next, cells were briefly washed with PBS, and then each sample of cells was divided into three pools. The cells were then incubated in 1) medium alone or supplemented with either 2) 10 nM GnRH or 3) 10 mM MβCD (MbCD). Samples of culture medium were collected sequentially, and LH release was measured by ELISA. Data are the mean ± SEM for three independent experiments.


Figure 07
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  		FIG. 7 Western blot analysis of secreted and intracellular LH from ovine pituitary cells. Cells were incubated in medium alone (C) or supplemented with either 10 nM GnRH (G) or 10 mM MβCD (M) for 30 min. The cells were washed with PBS and frozen. An amount of 25 µg protein from culture medium (Medium) or from the cell lysate was subjected to 12% SDS-PAGE in reduced (R) or nonreduced (NR) conditions. Western blots were performed with an anti-ovine LH beta subunit antibody (1:1000).

MβCD Induces FSH Secretion in Primary Culture of Ovine Pituitary Cells

We next investigated the possible effect of MβCD on FSH release, since some FSH is associated in LH secretory granules [8]. Thirty minutes of MβCD treatment induced a substantial release of FSH in primary cultures of ovine pituitary cells. In contrast, we did not detect any GnRH-induced FSH release under the same conditions (Fig. 8).


Figure 08
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  		FIG. 8 MβCD treatment in ovine pituitary cells induces FSH release. Ovine pituitary cells were incubated at 37°C for 30 min in medium alone (control) or supplemented with either 10 nM GnRH or 10 mM MβCD (MbCD). Follicle-stimulating hormone release into the cell culture medium was measured by ELISA. Data are the mean ± SEM of nine independent experiments. **P ≤ 0.01 for MβCD compared with control.

MβCD Induces a Release of LH In Vivo

Since MβCD had a direct effect on gonadotropin release by cultured primary cells in vitro, we investigated whether the same effects were observed in vivo. To test whether MβCD induced LH release in vivo, we injected MβCD directly into the pituitary gland of anesthetized sheep. When the control vehicle, physiological saline, or MβCD up to 40 mM was injected, we did not detect any significant change in plasma LH (Fig. 9, A and B). When MβCD was injected at 80 mM or more, a peak of plasma LH was detected (Fig. 9, A and B). In five independent in vivo experiments, MβCD induced a 16.3 ± 3.83-fold (mean ± SEM) increase in plasma LH, compared with the basal level. The maximal concentration of plasma LH was reached about 30 min after MβCD injection, and the plasma LH returned to basal levels less than 10 h later (Fig. 9, A and B). The release of LH induced by intrapituitary injections of MβCD was observed in male (IF4 and IF5) and female (IF1, IF2, and IF3) sheep, although the levels of hormone release varied between sex (Fig. 9C).


Figure 09
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  		FIG. 9 In vivo effect of pituitary injection of MβCD on plasma gonadotropin release. Vehicle (V) or MβCD (M) was injected in three aliquots of 60 µl into the pituitary of anesthetized Ile de France ewes (IF1, IF2, and IF3) or rams (IF4 and IF5). A) M20, M40, and M80 indicate MβCD concentrations of 20 mM, 40 mM, and 80 mM, respectively. B) M indicates an MβCD concentration of 160 mM. Plasma was collected as indicated, and LH (open circles) or FSH (dark circles) was measured by ELISA. C) Open bars and dark bars indicate the mean ± SEM of basal plasma LH and plasma LH following MβCD injection, respectively. The number of sample is indicated (sample size). D) Open bars indicate the mean ± SEM of basal plasma FSH; the dark bars indicate the FSH after MβCD injection. The number of samples is indicated (sample size). ***P ≤ 0.001; **P ≤ 0.01; and *P ≤ 0.05; ns, P > 0.05 for gonadotropin concentration in plasma after pituitary injection of MβCD compared with gonadotropin concentration in plasma before pituitary injection; ne, only one sample for IF5.

MβCD Effect on FSH Release In Vivo

Ovine pituitary cells in culture released FSH (3-fold increase) when stimulated for 30 min by treatment with MβCD, but not when stimulated with GnRH. In experiments conducted with anaesthetized ewes (IF1, IF2, and IF3), the intrapituitary injection of MβCD led to a significant increase in plasma FSH (Fig. 9D). In contrast, in the males IF4 and IF5, we did not detect any significant increase in plasma FSH following intrapituitary injection of MβCD (Fig. 9D), despite the finding that LH plasma concentration was increased by this treatment (Fig. 9C).

DISCUSSION

The vast majority of published data reports that the cholesterol-sequestering agent MβCD markedly impairs exocytosis for different secretory products and cellular models [23–37]. However, some studies report contradictory results because they show the opposite effect of lipid raft disorganization [38–40]. In this study, in order to better understand the cellular mechanisms underlying LH and FSH secretion, we have studied the functional relationships between lipid raft structure and the secretion of gonadotropins.

We first investigated the effect of depletion of cholesterol from the plasma membrane on LH release in the LβT2 mouse-derived gonadotroph cell line. The MβCD cholesterol-sequestering agent was used in conditions affecting principally the lipid raft content of cholesterol [44]. We found that MβCD induced rapid and substantial LH release in the absence of GnRH. To verify the physiological relevance of these observations, we examined LH secretion in primary cultures of ovine pituitary cells, and we obtained the same result using MβCD, indicating that LH release induced by the disruption of lipids was not restricted to a particular cell line. Furthermore, filipin III, another cholesterol agent that sequesters cholesterol in membranes, but by a mechanism different from MβCD [46], also induced LH release in primary pituitary cell culture. Altogether, these results suggest a critical role for lipid rafts in the mechanism of LH secretion.

Increased exocytosis following disorganization of lipid rafts has been observed in several systems. In isolated endocrine cells from the pancreas, insulin secretion by the β cell [38] and its sequential exocytosis from the β cell of intact pancreatic islets [39], as well as glucagon secretion by pancreatic {alpha} cells [40], were all increased by treatment with MβCD. The secretion of mast cells induced by the Ca2+ ionophore A23187 was increased by MβCD [47] and, finally, in spermatozoa, specific acrosomal exocytosis was promoted by a short incubation with MβCD [48].

Our study has shown that when MβCD was preloaded with cholesterol or when cholesterol was added to MβCD during incubation, the stimulation of LH release was inhibited. For the following reasons, we believe that LH release is not caused by a nonspecific degradation of the plasma membrane. First, cell viability 24 h after manipulation was similar in MβCD-treated or nontreated cells, as assessed by the Trypan blue dye exclusion test. Second, when cells were treated with MβCD and subsequently maintained in culture medium for 24 h, they responded like control cells for GnRH- or MβCD-mediated LH release the second day. These data indicate full recovery not only of the GnRH-releasable LH pool, but also of the MβCD-releasable pool and membrane cholesterol, as indicated by total MβCD responsiveness. Third, when MβCD-treated LβT2 cells were stained with the FITC-labeled B subunit of cholera toxin, the GM1 staining that had been strongly affected by MβCD treatment became similar to a control pattern 24 h after treatment (Robin et al., unpublished observation).

We observed a larger release of LH with MβCD treatment than with GnRH stimulation. Furthermore, GnRH-pretreated cells were still able to release LH in response to MβCD treatment immediately following GnRH pretreatment. In contrast, GnRH treatment immediately following GnRH pretreatment of the same cells did not release more LH. These data suggest that MβCD, in contrast to GnRH, is not susceptible to the desensitization of gonadotrophs induced by GnRH. This could explain partly the observed differences in the magnitude of LH release following treatment with MβCD and GnRH. The use of cycloheximide allowed us to eliminate active protein synthesis as an explanation of the difference between GnRH- and MβCD-mediated LH release. Our data suggest the existence of two pools of LH in the cultured pituitary cell population, an immediately releasable pool mobilized by GnRH or MβCD and a less-readily releasable pool that can only be mobilized by MβCD. This interpretation agrees with the "two-LH pool" hypothesis; a "readily releasable pool" located at or near the cell surface that is responsible for the LH secretion following GnRH treatment, and a second, less "releasable pool" that requires either mobilization from deep within the cell or further processing prior to release [49–51]. One explanation for the two pools is that the physical properties of their LHs are not the same. However, if the maturation states of LH are different for the GnRH-releasable pool and for the specific MβCD-releasable pool, they are not associated with major changes in electrophoretic mobility determined under reduced or nonreduced conditions. Further work is required to precisely determine the physical nature of the LH in these two pools; for example, studies of the glycosylation patterns of LH in the two pools. We cannot exclude the possibility that the differences in response to GnRH and MβCD were caused by the presence of a population of gonadotrophs lacking the GnRH receptor [2, 52, 53]. In which case, treatment with MβCD treatment would stimulate LH release from a population of incompetent cells unable to respond to GnRH stimulation.

When MβCD was injected into the pituitary gland of anesthetized sheep, a rapid and obvious peak of LH was detected in blood. The concentration of MβCD needed to induce detectable LH release was higher than for in vitro treatment of cultured pituitary cells. In addition, we have observed that in the presence of serum, in vitro LH release from isolated pituitary cells required higher doses of MβCD (data not shown). The potential influence of tissular context in the animal and limited diffusion of the injected liquid MβCD into the pituitary gland both suggest that a higher dose of MβCD would be needed to induce LH release in vivo.

The release of FSH into the cell culture medium from MβCD-treated pituitary cells was significantly higher than the almost undetectable FSH release induced by GnRH treatment. Consistently in ewes, intrapituitary injection of MβCD induced clear FSH release, whereas the two males did not release any FSH following intrapituitary injection. In gonadotrophs, FSH storage is limited because of constitutive secretion of the hormone [2, 7, 42, 54]. The FSH released by MβCD would correspond to the LH granule-associated FSH. Following MβCD treatment, the release of FSH was easily detectable in the supernatant from primary cultures of ovine pituitary cells and in the plasma of female sheep. In males, LH release after MβCD injection was about a hundred times lower than in females (0.4 and 1 ng/ml for sheep IF4 and IF5, compared with 70, 220, and 210 ng/ml for sheep IF1, IF2, and IF3, respectively). In male sheep, LH-associated FSH release may exist, but because of dilution, it was not detectable in the blood of male animals.

Lipid rafts are membrane microdomains that are platforms for the initation of signal transduction because of the numerous associated cell signaling proteins within the complex, including G-protein-coupled receptors, insulin receptors, kinases, channel proteins, docking proteins, scaffolding proteins and SNARE proteins, which are collectively important for exocytosis, neurotransmitter, and hormone actions [13, 14]. Recently, the GnRH receptor has been constitutively localized in lipid rafts, and it colocalized in these domains with several proteins required for the GnRH-induced signaling cascade, such as GNAQ (G{alpha}Q/11), RAF1 (C-raf), and the MAPKs (also termed ERKs) [9, 10]. In these publications, the authors have demonstrated localization of the GnRH-R within lipid rafts. When they pretreated cells with MβCD for 1 h, GnRH-R signaling was impaired, and this is the opposite of our observation that gonadotropin release was induced by MβCD. It is important to remember that we did not pretreat the cells, but that we analyzed the immediate effect of MβCD treatment. This key procedural point may explain the seemingly opposite effects of MβCD on LH release and GnRH-induced intracellular signaling mechanisms in gonadotrophs. When cells were preincubated with MβCD, washed, and then incubated in the presence of GnRH or MβCD again, no or very little LH release was detected, respectively. In contrast, when cells were treated with MβCD without pretreatment, LH release was easily detected. It seems reasonable to suggest that pretreatment by MβCD fully depleted the releasable GnRH-only pool of LH and some of the MβCD-releasable pool of LH.

In natural killer cells [29], promyelocytic PLB-985 cells [30], and mast cells [31], MβCD-impaired exocytosis was related to defective initiation of the signaling cascade. This may also apply to gonadotrophs following sequestration of cholesterol from the lipid raft by MβCD. Another possibility implicates downstream events directly related to the regulation of exocytosis. The activation of voltage-gated Ca2+ or K+ channels has been observed during GnRH-stimulated LH release [55, 56]. In addition, the voltage gate-dependent channels, KCND1/3 (Kv4.1/4.3) KCNB1 (Kv2.1), and CACNA1C (Cav1.2), have been localized in lipid rafts, and the perturbation of exocytosis by MβCD could be related to cholesterol deprivation-induced channel delocalization [32, 38, 40]. In rat basophilic leukemia cells, the calcium influx from extracellular medium through the store-operated calcium channel is inhibited by MβCD [32]. Additional research is required to establish the nature of the link between MβCD-induced LH release and ion channels in gonadotrophs.

Exocytosis, the fusion of the secretory vesicle with the plasma membrane, is the final step of regulated hormone secretion. Before the secretory vesicles, which are processed at the trans-Golgi network, fuse with the plasma membrane, they undergo several modifications, such as translocation (transport to the plasma membrane), docking (attachment to the plasma membrane), and final maturation, which prepare them for final fusion [57]. Regulated exocytosis is controlled and mediated by SNAREs (soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor), which have a crucial role in intracellular membrane fusion [58]. Recently, evidence has been provided to show that SNAREs accumulate in lipid raft microdomains of the plasma membrane [23, 24, 59–61]. SNARE proteins are a vital component of the intracellular fusion process within the secretory pathways of eukaryotic cells. Target SNARE, SNAP25 (synapatosome-associated protein 25), SNAP23, and syntaxin in the plasma membrane, and vesicular SNARE, synaptobrevin 2, or VAMP2 (vesicle-associated membrane protein-2) in membranes of vesicles, are the most studied of the SNARE proteins involved in regulated exocytosis. In gonadotrophs, the role of SNARE has been established recently; Ca2+-induced LH secretion was decreased after treatment with SNAP25 antibodies in permeabilized cells [62], and GnRH inhibited SNAP25 expression [63]. In several models, MβCD treatment induced delocalization of SNARE, associated either with the inhibition of exocytosis [24, 33, 61] or the enhancement of exocytosis [38, 40, 64]. Interestingly, recent studies on SNAP25 and SNAP23 in PC12 neuronal-derived cells implicate lipid rafts as negative regulators of neuronal exocytosis [59, 65]. Our present studies showing gonadotropin-induced release by treatment with MβCD suggest an inhibitory role of lipid rafts in gonadotropin exocytosis from pituitary cells.

ACKNOWLEDGMENTS

The authors wish to thank Dr. R. Scaramuzzi, Dr. I. Tardieux, and Dr. J.C. Thiery for their help and constructive comments on the manuscript; Dr. C. Taragnat, Dr. Y. Combarnous, and the members of Hypophyse laboratory for helpful discussions; and the members of the Service Hôpital-Abattoir for their expert assistance.

FOOTNOTES

1Supported by Conseil régional Région Centre/Institut National de la Recherche Agronomique. E.R. is supported by a PhD fellowship from Institut National de la Recherche Agronomique/Région Centre. Back

Correspondence: 2FAX: 33 24 742 7743; e-mail: cayla{at}tours.inra.fr

Received: 7 August 2007.

First decision: 21 September 2007.

Accepted: 28 February 2008.

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