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Molecular Endocrinology 21 (5): 1014-1027
Copyright © 2007 by The Endocrine Society

Multiple Roles of the Nuclear Receptors for Oxysterols Liver X Receptor to Maintain Male Fertility

David H. Volle, Kévin Mouzat, Rajesha Duggavathi, Bénazir Siddeek, Pierre Déchelotte, Benoît Sion, Georges Veyssière, Mohamed Benahmed and Jean-Marc A. Lobaccaro

Physiologie Comparée et Endocrinologie Moléculaire (D.H.V., K.M., G.V., J.-M.A.L.) and Research Center for Human Nutrition, Unité Mixte de Recherche, Centre National de la Recherche Scientifique (CNRS) 6547, 63177 Aubière Cedex, France; Institut de Génétique et de Biologie Moléculaire et Cellulaire (D.H.V., R.D.), CNRS/Institut National de la Santé et de la Recherche Médicale (INSERM)/Université Louis Pasteur, 67400 Illkirch, France; Centre Hospitalier Universitaire Clermont-Ferrand (P.D.), Service d’Anatomie Pathologique, Hôtel Dieu, 63058 Clermont-Ferrand, France; Laboratoire de Biologie du Développement et de la Reproduction (B.Sio.), Equipe d’Accueil 975 Université d’Auvergne, 63001 Clermont-Ferrand, France; and INSERM Unité 407 (B.Sid., M.B.), Faculté de Médecine Lyon-Sud, 69921 Oullins Cedex, France

Address all correspondence and requests for reprints to: Jean-Marc A. Lobaccaro, Unité Mixte de Recherche Centre National de la Recherche Scientifique-Université Blaise Pascal 6547, 24 avenue des Landais, 63177 Aubière Cedex, France. E-mail: j-marc.lobaccaro{at}univ-bpclermont.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oxysterol nuclear receptors liver X receptor (LXR){alpha} and LXRß are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXR{alpha}, LXRß, and both, of which only the lxr{alpha};ß–/– mice are infertile by 5 months of age. We demonstrate that LXR{alpha}-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRß-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxr{alpha};ß–/– mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxr{alpha};ß–/– mice, with a higher accumulation of all-trans retinoid receptor {alpha}, all-trans retinoid receptor ß, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxr{alpha};ß–/– mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE TWO MAJOR functions of the testis include testosterone production and spermatogenesis. Testosterone is essential for normal development of the genital tract in males during fetal life and then for the establishment of puberty and maintenance of male sexual characteristics (1), as well as for spermatogenesis (2, 3). The spermatogenesis requires a continuous supply of differentiating spermatogonia involving a delicate balance between mitosis and meiosis (4). Both androgen synthesis and reproductive capacity are also tightly linked and regulated by various hormones including LH and FSH. These gonadotropins act, respectively, on the Leydig cells that secrete testosterone and the Sertoli cells that provide structural and nutritional support for the developing germ cells (4, 5, 6). Furthermore, the studies using antiandrogens have shown that androgens from the Leydig cells are involved in the regulation of germ cells apoptosis (7, 8).

The liver X receptors, LXR{alpha} (NR1H3) and LXRß (NR1H2), belong to a subclass of nuclear receptors that form obligate heterodimers with retinoid X receptors (RXRs) and are activated upon binding, by their ligands, a class of naturally occurring oxysterols (9). In the absence of ligands, the RXR/LXR heterodimer constitutively binds to specific sequences on the promoters of the target genes and interacts with corepressors (10, 11). During the last few years, LXRs have been shown to act as major sensors of intracellular concentrations of sterols (12). The development of lxr-knockout (lxr–/–) mice has helped to elucidate the roles of LXRs in various tissues (13) that are far beyond the simple regulation of cholesterol and fatty acid homeostasis (14). Identified target genes indicate that LXRs, in addition to lipid homeostasis, are also involved in glucose homeostasis, immunity, skin development, and brain functions (15).

Even though lipid metabolism was severely affected in Sertoli cells of the LXRß-deficient mice, they did not present any infertility. However, we and others have recently shown that mice deficient of both LXRs are completely infertile by around 5 months of age (16, 17), which suggests that both LXRs could have redundant functions in the testis, such that only the absence of both the isoforms causes infertility.

The aim of the present study was to further identify the physiological roles of both LXRs in testis using LXR-deficient mouse models. Here, we show specific cell-type expression patterns of lxr{alpha} and lxrß and define complementary and/or redundant roles of both isoforms in testicular physiology. We also demonstrate that LXR{alpha} and LXRß are involved, respectively, in germ cell apoptosis and proliferation. Additionally, they regulate testosterone synthesis, retinoid metabolism, and lipid metabolism in response to a specific agonist in vivo. Combination of all these altered pathways in lxr–/– mice explains the infertility observed, even though the alteration in lipid metabolism seems to have a central role leading to the phenotype. Taken together, our results show novel roles for RXR/LXR{alpha} and RXR/LXRß to maintain testicular integrity and reproductive functions in male.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Male Mice Deficient of Both LXR{alpha} and LXRß Develop Infertility Associated with a Complete Loss of Germ Cells
To explore the mechanisms underpinning the infertility in lxr{alpha};ß–/– mice, male mice of various lxr genotypes (n = 4–5) were bred with three to four wild-type fertile females, and the presence of vaginal plugs was monitored daily. Consistent with previous reports (17, 18), no infertility was observed in lxr{alpha}–/– and lxrß–/– males up to 14 months of age (Fig. 1Go, A–C). In contrast, infertility was observed in lxr{alpha};ß–/– mice (16, 17) by the age of 5 months, clearly suggesting some complementary and/or redundant functions between the LXR isoforms. Up to 5 months of age, lxr{alpha};ß–/– males showed no alteration in their reproductive capacities compared with wild-type males (Fig. 1Go, A–C). When lxr{alpha};ß–/– males older than 5 months were used, there was a significant decrease in the number of vaginal plugs and only 55% of the females became pregnant (Fig. 1Go, A and B). The number of pups per litter dramatically fell when 5- to 7-month-old lxr{alpha};ß–/– males were used. Finally, lxr{alpha};ß–/– males older than 7 months were unable to impregnate the females, demonstrating complete infertility (Fig. 1BGo). Altogether, these data suggested alterations in the male fertility starting around 5 months of age. In addition, a significant decline in the testis weight was observed at 9 to 10 months of age only in the lxr{alpha};ß–/– (Fig. 1DGo), when the testis showed, based upon histological examination, complete loss of germ cells.


Figure 1
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  		Fig. 1. Fertily of lxr–/– Mice

A, Number of matings per male. Pairing between wild-type females and males from various genotypes was monitored daily for the presence of a vaginal plug to determine whether mating occurred. B, Percentage of deliveries. After 19–20 d, efficacy of mating was visually inspected by the female delivery. C, Number of pups per litter. D, Testis weight of the wild-type and LXR knockout mice compared with the total body weight. Number of mice for each experiment, n = 4–5. All the histograms are indicated as mean ± SEM. One-way ANOVA: *, P < 0.05.

 
A close observation of the testis histology showed that no major alterations were observed in the testes of lxr{alpha}–/– mice up to 14 months of age, whereas lxrß–/– mice only presented some vacuoles in the Sertoli cells at up to 12 months of age (data not shown). Dramatic changes in histological characteristics could be observed with aging in lxr{alpha};ß–/– mice (Fig. 2AGo, f–j) when compared with the wild-type controls (Fig. 2AGo, a–e). In lxr{alpha};ß–/– mice, histologically visible alterations appeared around 3.5 months of age, characterized by larger interstitial/Leydig cells and the presence of vacuoles in Sertoli cells (Fig. 2AGog). At 5.5 months, 20–30% of the lxr{alpha};ß–/– testis presented abnormal tubules (Fig. 2AGoh) with cell aggregates in the center of the tubules without any spermatozoa. These aggregates seemed to be germ cells and not Sertoli cells, based on the absence of the androgen receptor immunostaining (Fig. 2BGo). With aging, the percentage of abnormal tubules increased and by 10 months most of the tubules were totally empty without any spermatozoa (Fig. 2AGoi), by which time the mice were already completely infertile. It could be noticed that the numerous vacuoles were still present in the Sertoli cells. By 12 months, the testis morphology was completely disorganized, as shown by the presence of signs indicating that cholesterol crystals occurred (angular-shaped vacuoles), calcifications, as well as necrosis of both interstitial tissue and seminiferous tubules, and macrophage invasion (Fig. 2AGoj). Oil red O staining showed that vacuoles in Sertoli cells of lxrß–/– and lxr{alpha};ß–/– correspond to lipids (data not shown). Biochemical analyses of the lipids showed that the accumulation was mainly due to cholesteryl esters (Fig. 2CGo). Consistent with a previous report (17), accumulation of cholesteryl esters was higher in lxr{alpha};ß–/– than in lxrß–/– mice (data not shown).


Figure 2
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  		Fig. 2. Pathology of the Testes at Various Ages and Expression of the LXRs in the Testis

A, Hematoxylin-Eosin-Safran staining from wild-type (a–e) and lxr{alpha};ß–/– (f–j) mice at 2.5, 3.5, 5.5, 10, and 12 months of age. Original magnification, x100. Arrows indicate the vacuoles (v) within the Sertoli cells. lh, Leydig hyperplasia; cc, crystal of cholesterol; c, calcification; n, necrosis. Some tubules are surrounded by a black dashed line. B, Immunostaining of the AR in testis of wild-type and lxr{alpha};ß–/– mice at 10 months of age. Counterstaining was done with hematoxylin. LC, Leydig cells; SC, Sertoli cells; Vac, vacuole. C, Biochemical analyses of the cholesterol content (mean ± SEM, n = 5–6) of wild-type and lxr{alpha};ß–/– mice at 1, 2, and 6 months. One-way ANOVA: *, P < 0.05 compared with the wild-type mice. D, Relative levels of lxr{alpha} and lxrß in Leydig, Sertoli cells, spermatocytes (cytes), and spermatids (tides). Histograms are expressed as mean ± SEM (n = 5–6). 18S was used as internal control.

 
LXR{alpha} and LXRß Are Differentially Expressed in Various Cell Types of the Testis
Altogether the previous data suggested that both LXRs isoforms are involved in the maintenance of testicular functions and morphological integrity. To identify the putative roles of LXR{alpha} and LXRß, we analyzed their respective expression patterns in different testicular cell types, which were selectively purified. The mRNA expression pattern of lhcgr and fshr, encoding the LH-chorionic gonadotropin (CG) and FSH receptors, respectively, was used to ensure the purity of the cell populations obtained. As shown in Fig. 2DGo, lxr{alpha} is highly expressed in Leydig and germ cells (spermatocytes and spermatids). Conversely, lxrß is expressed in Sertoli cells and at a lower level in germ cells but not in interstitial cells. These results suggested that LXR isoforms might have some specific functions in Sertoli and Leydig cells and redundant functions in germ cells as suggested by the coexpression in germ cells. This hypothesis was consistent with the fact that germ cell loss was only found in lxr{alpha};ß–/– males.

LXR{alpha} and LXRß Are Involved in Apoptosis and Proliferation, Respectively, in Mouse Testis
Many studies have demonstrated that testis integrity and spermatogenesis are controlled by a balance between proliferation and apoptosis (19). The mRNA of cyclinA1, one of the most important cyclins in postmitotic germ cells (20), was less accumulated in lxr{alpha};ß–/– than in wild-type testis at 2.5 months (P < 0.07), whereas no significant change was observed for cyclinE, cmyc, and cmyb (Fig. 3AGo). The mRNA expression of cyclinA1 was further decreased at 4.5 months (Fig. 3AGo). Quantification of proliferation using Ki67 immunochemistry (Fig. 3BGo) showed that the number of proliferating cells was significantly lower in lxrß–/– and lxr{alpha};ß–/– mice compared with the wild-type (decreased by 27% and 43%, respectively; P < 0.05), whereas the lxr{alpha}–/– mice showed a tendency to have 21% more Ki67-positive cells (P = 0.07). Consistent with this fact, mRNA accumulation of cyclinA1 was significantly lower in lxrß–/– and lxr{alpha};ß–/– mice (Fig. 3CGo), suggesting that LXRß is involved in germ cell proliferation.


Figure 3
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  		Fig. 3. Proliferation and Apoptosis Status in Testes of Wild-Type and lxr–/– Mice

A, Relative levels of mRNA of genes encoding proteins involved in cellular proliferation (mean ± SEM). Number of mice per experiment, n = 3–5. B, Quantification of proliferation and apoptosis in wild-type and knockout mice. Ki67 staining and TUNEL were done as described in Materials and Methods. Mice were 3.5 months old. n = 4–6 per group. One-way ANOVA: *, P < 0.05. C, Proliferation status in wild-type and the various lxr knockout testes. Cyclins A1 and E, cmyc, cmyb, and AMH were studied by qPCR as described. Mice (n = 3–6) were 3.5 months old. One-way ANOVA: *, P < 0.05. 18S was used as an internal control. D, Apoptosis status in wild-type and knockout testes. Bcl-2, bcl-XL, cflip, Bad, bax, TNF{alpha}, IKK, and AR were studied by qPCR as described. Mice (n = 3–6) were 3.5 months old. One-way ANOVA: *, P < 0.05. 18S was used as an internal control. E, mRNA analysis of TNF{alpha}, Bad, and AR in wild-type and the various lxr knockout testes. Mice (n = 3–6) were 3.5 months old. One-way ANOVA: *, P < 0.05. 18S was used as an internal control. F, Significant Western blot of Bcl-2 and BAX proteins from two wild-type (4.5 months) and two lxr{alpha};ß–/– (4.5 months) mice.

 
With respect to the apoptotic pathway, significantly higher mRNA expression of several genes including TNF{alpha}, the essential regulatory subunit NF-{kappa}B essential modulator/inhibitor of {kappa}B kinase (IKK){gamma} of the IKK complex (21), and Bad (Bcl2-antagonist of cell death) and lower mRNA accumulation of androgen receptor (AR) were observed in lxr{alpha};ß–/– compared with the wild-type mice (Fig. 3DGo). Interestingly, all these alterations in mRNA expression were amplified by 4.5 months of age in lxr{alpha};ß–/– mice. Moreover, the mRNA expression of the proapoptotic gene Bax (Bcl2-associated X protein) was higher and that of the antiapoptotic gene Bcl2 (B-cell CLL/lymphoma 2) was decreased in lxr{alpha};ß–/– compared with the wild type. These results were confirmed at the protein level by Western blot analysis (Fig. 3FGo). Study of apoptosis using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay revealed that lxr{alpha}–/– and lxr{alpha};ß–/– mice had significantly higher number of apoptotic cells compared with wild-type mice, whereas a slightly but not significantly decreased number of apoptotic cells (P = 0.09) was observed in lxrß–/– mice (Fig. 3BGo). Consistent with these data, mRNA expression analyses showed a higher accumulation of the proapoptotic gene Bad as well as TNF{alpha} and a lower expression of the AR in 3.5-month-old lxr{alpha}–/– and lxr{alpha};ß–/– mice (Fig. 3Go, D and E). However, no alteration in the AR protein level was observed in these mice (data not shown). It appears thus that LXR{alpha} might be involved in the regulation of the apoptosis in the testis.

It is interesting to note that in the lxrß–/– mice the lower proliferation rate is counterparted by a tendency to a lower apoptosis (Fig. 3BGo). In contrast, in lxr{alpha}–/– mice the higher apoptosis rate observed was associated with a tendency of a higher proliferation rate. The respective proliferation vs. apoptosis ratio is not statistically different in lxrß–/– (11.6 ± 1.3) and lxr{alpha}–/– (6.3 ± 0.7) compared with wild-type mice (9.2 ± 1.4). In contrast, this proliferation/apoptosis ratio is statistically different in lxr{alpha};ß–/– mice (2.2 ± 0.4, P < 0.05 compared with the wild-type mice).

The treatment of mice with a LXR synthetic agonist, T0901317 (T1317), for 12 h did not alter the mRNA expression of the genes involved in proliferation or apoptosis, suggesting that these genes may not be the direct LXR targets (data not shown). Supporting this, the analysis of the 5' flanking region of these different genes with GEMSLauncher (Genomatix Software, München, Germany) did not identify any direct repeat 4 (DR4) classical LXR-response element.

Testicular Testosterone Level Is Altered in Mice Lacking LXR{alpha}
Because androgens have clearly been associated to the regulation of germ-cell apoptosis in testis, we checked whether the increased apoptosis observed in mice lacking LXR{alpha} could be linked to an altered level of androgens. Testosterone production was significantly lower in lxr{alpha}–/– and lxr{alpha};ß–/– compared with their respective wild-type controls, whereas testosterone levels did not differ between the lxrß–/– and the wild-type mice (Fig. 4AGo). Gene expression analysis showed that the level of type 1 3ß-hydroxysteroid dehydrogenase isomerase (3ßhsdI) was significantly decreased in lxr{alpha}–/– and lxr{alpha};ß–/– mice (Fig. 4BGo), whereas the levels of steroidogenic acute regulatory protein (StAR) and the cytochromes 11a1 (cyp11a1) and 17 (cyp17) transcripts remained unchanged. It is interesting to note that androgen-target organs such as seminal vesicles and vas deferens did not show any significant change in weight (data not shown), suggesting that the existing testosterone levels in lxr{alpha}–/– and lxr{alpha};ß–/– mice were enough for the maintenance of the secondary male sexual characteristics.


Figure 4
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  		Fig. 4. Endocrine Parameters of the lxr–/– Mice

A, Intratesticular levels of testosterone (mean ± SEM, nmol/mg tissue) in 2.5-month-old wild-type, lxr{alpha}–/–, lxrß–/– and lxr{alpha};ß–/– mice, stimulated or not with 5 IU hCG. The fold inductions were obtained from the unstimulated animals of the same genotype. Number of mice per experiment, n = 9–19. nd, Not determined. B, Basal relative expression of genes encoding steroidogenic enzymes. Number of mice per group, n = 3–6. C, Plasma LH levels (ng/ml) obtained from 3-month-old mice of the four genotypes. D, Relative expression of ßlh in the pituitary (n = 6). E, Effect of T1317 on testosterone production by testes in 2.5-month-old wild-type, lxr{alpha}–/–, lxrß–/–, and lxr{alpha};ß–/– mice. Number of mice per experiment, n = 10. F, Relative levels of steroidogenic genes in testes from various genotypes gavaged vehicle or T1317 as measured by qPCR. Number of mice per experiment, n = 4–6. In all experiments, one-way ANOVA: *, P < 0.05. For the expression analysis, 18S was used as an internal control. Histograms are indicated as mean ± SEM. G, Significant Western blot of StAR and ß-actin from two wild-type and two lxr{alpha};ß–/– (4.5 months old) mice.

 
To study whether these low levels of testosterone production in mice lacking LXR{alpha} could be due to a central defect, plasma LH concentration was measured. A significantly lower concentration of LH was found in both lxr{alpha}–/– and lxr{alpha};ß–/– mice compared with their controls (P < 0.05; Fig. 4CGo). These data were confirmed by a lower mRNA expression of the specific ß-chain of LH in pituitary of lxr{alpha}–/– and lxr{alpha};ß–/– mice (Fig. 4DGo). Additionally, the lxr{alpha}–/– and lxr{alpha};ß–/– mice were able to respond to human CG (hCG) challenge by increased production of testosterone similar to their wild-type controls. These data suggest that the lack of LXR{alpha} results in decreased testosterone concentrations due to central defects, whereas the sensitivity of Leydig cells to LH remains unaffected (Fig. 4AGo). In contrast, plasma concentrations and mRNA expression level for the FSH (data not shown) were not affected by the loss of LXR{alpha}.

LXR Agonist Increases Testosterone Secretion
To avoid the basal differences in testosterone concentrations among the different genotypes used in the experiment, mice from the four genotypes were treated with dexamethasone for 5 d as previously described (22). On the fifth day, the mice were gavaged with the synthetic LXR agonist T1317 or vehicle and killed 12 h later. T1317 induced a large increase in the intratesticular testosterone concentration of the wild-type mice (13.3-fold compared with the vehicle-gavaged mice); however, this response to T1317 was significantly lower in lxr{alpha}–/– (5.5-fold) and lxrß–/– (3.3-fold) mice (Fig. 4EGo). The increase in testosterone concentration was correlated with a higher mRNA expression of StAR and 3ßhsdI (Fig. 4FGo) in wild-type, lxr{alpha}–/–, and lxrß–/– mice treated with T1317, whereas no variations were observed in the lxr{alpha};ß–/– mice treated with T1317. Nonetheless, cyp11a1 and cyp17 were unchanged after the T1317 treatment. This effect of T1317 on StAR accumulation was confirmed at the protein level by Western blot analysis (Fig. 4GGo).

Lack of LXRs Altered Expression of the Genes Involved in Fatty Acid Metabolism
Because fatty acid metabolism is important for reproductive functions (23), we analyzed the expression pattern of the genes involved in this pathway. mRNA levels of srebp1c (sterol response element binding protein-1c) and fatty acid synthase (fas), encoding the sterol response element binding protein-1c and the fatty acid synthase, respectively, were decreased by 40% in the lxr{alpha};ß–/– mice compared to the wild-type mice (Fig. 5AGo; P < 0.05). In contrast, the level of scd1, encoding the stearoyl coA-desaturase 1, was increased by 2-fold in the lxr{alpha};ß–/– mice compared with the wild type (Fig. 5AGo; P < 0.05). No change in the mRNA expression was observed for scavenger receptor, class B type I, encoding the scavenger receptor B1, abca1 (ATP-binding cassette, sub-family A member 1), and scd2. Treatment with T1317 induced the expression of srebp1c and scd1 in the wild-type mice (Fig. 5BGo), whereas no variation in the expression of fas was observed (data not shown). Hence, whereas srebp1c and scd1 appear to be regulated by both LXRs, abca1 appeared to be mainly under the control of LXRß because no up-regulation with T1317 was observed in mice lacking this isoform (Fig. 5BGo). This is consistent with the specific expression of LXRß in the Sertoli cells (Fig. 2CGo) paralleled with the lipid vacuoles observed in lxrß–/– and lxr{alpha};ß–/– mice (data not shown). As both isoforms are expressed in the germ cells, it is possible that they also present similar lipid accumulation. To check this possibility, semi-thin sections of the testes were stained with azure 2 blue, by which lipids are stained yellow. Indeed, a lipid accumulation was observed in the germ cells of lxr{alpha};ß–/– mice (Fig. 5CGo). All these results suggested that, as already described in other tissues, LXRs might be involved in the regulation of lipid homeostasis in testis.


Figure 5
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  		Fig. 5. Analysis of the Lipid Metabolism in Testes of Wild-Type and LXR-Deficient Mice

A, Relative basal levels of mRNA of abca1, srb-I, srebp1c, fas, scd1, and scd2 in the testes from the various genotypes. Mice were 3.5 months old. B, Relative levels of abca1, srebp1c, and scd1 in testes from wild-type and lxr{alpha};ß–/– mice gavaged vehicle or T1317 as measured by qPCR. Number of mice per experiment, n = 5–6. C, Azure blue 2 staining of testis semi-thin sections. LXR-deficient mice present an abnormal accumulation of lipids (stained in yellow) in spermatids as indicated by arrows. Bar, 5 µm. L, Lumen of the tubule.

 
The Genes Involved in Retinoic Pathways Are Up-Regulated in LXR-Deficient Mice
The retinoid pathway has an important role in reproduction (24, 25). The mRNA expression of the all-trans retinoid receptor (RAR){alpha} and RARß was significantly higher in lxr{alpha};ß–/– mice (2.6- and 2.3-fold, respectively; Fig. 6AGo), whereas no change was observed for RAR{gamma}. These results suggested that the lack of LXRs modified the RAR-signaling pathway. To confirm this hypothesis, we next analyzed the expression of known RAR-target genes. The expression of dmc1 (meiosis-specific recombinase-1), scp3 (synaptonemal complex protein 3), and raldh-2 (retinoic aldehyde dehydrogenase 2), encoding the meiosis-specific recombinase, the synaptonemal complex protein 3, and the retinoic aldehyde dehydrogenase 2, respectively, was higher in lxr{alpha};ß–/– mice compared with wild-type mice. Because T1317 did not change the expression of these genes 12 h after treatment and no classical DR4 LXR response elements were found on their promoters, we concluded that they were not bona fide LXR target genes.


Figure 6
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  		Fig. 6. Analysis of the Retinoic Acid Signaling Pathway in Testes of Wild-Type and lxr{alpha};ß–/– Mice

A, Relative basal levels of mRNA of rar{alpha}, rarß, rar{gamma}, raldh-2, scp3, and dmc1 in the testes from the various genotypes. Mice were 3.5 months old. In all experiments, histograms are expressed as mean ± SEM. One-way ANOVA: *, P < 0.05; **, P < 0.01. For the expression analysis, 18S was used as an internal control. B, Relative basal levels of mRNA of scd1, srebp1c, cyclinA1, bad, bax, rar{alpha}, rarß, dmc1, scp3, StAR, cyp11a1, 3ßhsd, and cyp17 in the testes from the various genotypes. Mice were 1.0 months old (n = 3 for each group). In all experiments, histograms are expressed as mean ± SEM. One-way ANOVA: *, P < 0.05. For the expression analysis, 18S was used as an internal control.

 
Modifications in Lipid Metabolism Could Be the Primum Movens of the Infertility in lxr{alpha};ß–/– Males
We checked which alteration could be responsible for the severe infertility observed in the lxr{alpha};ß–/– males. In 1-month-old mice, the expression of the genes involved in apoptosis (Bad, Bax), proliferation (CyclinA1), the retinoic acid signaling pathway (rar{alpha}, rarß, dmc1, scp3), or steroidogenesis (StAR, cyp11a1, 3ßhsd, cyp17) (Fig. 6BGo) did not differ between the lxr{alpha};ß–/– and wild-type mice. Conversely, the expression of scd1 and srebp1c were basally modified in lxr{alpha};ß–/– compared with the wild-type mice (Fig. 6BGo). This was paralleled with a higher accumulation of cholesteryl esters in the testis of the same animals (P = 0.08) (Fig. 2CGo). It could thus be suggested that the alteration in lipid homeostasis is the first event in this long process of testis disorganization in the lxr{alpha};ß–/– mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this work, we provide evidence for the critical roles played by both LXR{alpha} and LXRß in the regulation of male reproductive functions. Using quantitative PCR (qPCR) on extracts enriched with different cell types of the testis, we showed that these nuclear receptors present a mirror-like profile of expression: the Leydig cells express LXR{alpha} and Sertoli cells LXRß, whereas the germ cells express both LXRs. Consistent with these differential expression patterns, we demonstrate a clear involvement of each LXR within the testis. LXR{alpha} controls basal testosterone synthesis and is involved in the control of the germ cells apoptosis. LXRß controls lipid metabolism of Sertoli cells by regulating cholesterol export and also germ-cell proliferation. Moreover, both LXRs together regulate ligand-induced steroidogenesis, fatty acid metabolism, and more surprisingly the retinoic acid signaling pathway in the testis. Our data show that the lack of LXR{alpha} and LXRß results in severe infertility due to the combined effect of the alterations in various pathways, of which the alteration of lipid metabolism appears to have a central effect in leading to this phenotype.

We recently demonstrated that LXR{alpha} regulates adrenal steroidogenesis (26). However, in contrast to adrenals, the lack of LXR{alpha} resulted in a lower secretion of testicular steroids. The lower testosterone levels observed in the lxr{alpha}–/– mice is probably due to the lower LH secretion, as these mice have reduced serum levels of LH and are still able to respond to hCG stimulation. It is well known that glucocorticoids inhibit the production of testosterone directly at the testicular level (27, 28) as well as through the inhibition of the LH secretion (29). Hence, the lower serum concentrations of testosterone and LH in lxr{alpha};ß–/– mice could be secondary to the high level of corticosterone observed in mice lacking LXR{alpha} (26). Treatment with the LXR synthetic agonist induced an increase in the testicular testosterone production. Because no modification of the lh-ß mRNA levels was observed in the pituitary of the wild-type animals after the T1317 treatment (data not shown), it could be hypothesized that LXR{alpha} was the main isoform regulating the testicular steroidogenesis because of its expression in the Leydig cells. However, the slight inductions observed in the single knockout mice led us to postulate that LXRß might also control steroidogenesis, probably through the expression of paracrine factors produced by Sertoli cells. Identification of these cellular interactions is currently under investigation.

Consistent with the expression of LXRß in Sertoli cells, we demonstrate that abca1 expression is specifically controlled by LXRß in response to a T1317 treatment, without any alteration of its basal expression. These results suggest that it might be the lack of abca1 up-regulation in response to endogenous LXR ligand rather than a decrease of its expression that is responsible for the lipid accumulations observed in the Sertoli cells, as demonstrated in different LXR knockout models (17, 18, 30). The major impact of LXRß in the Sertoli cells was also enlightened by the decrease of the basal anti-Müllerian hormone (AMH) mRNA in both lxrß–/– and lxr{alpha};ß–/– mice (Fig. 3CGo).

Spermatogenesis is maintained by a delicate balance between cell proliferation, cell differentiation, and cell death (19, 31). It has been hypothesized that alteration of these processes would result in spermatogenic impairment, and thus infertility. Interestingly, proliferation and apoptosis are altered in LXRß- and LXR{alpha}-deficient mice, respectively, whereas these mice did not show any fertility troubles. Indeed, in the lxrß–/– mice the lower proliferation rate was associated with a compensatory decline in apoptosis (Fig. 3BGo). In contrast, in lxr{alpha}–/– mice the higher apoptosis was associated with a compensatory increase in proliferation. These compensatory effects could thus be hypothesized to explain the normal fertility in the single LXR knockout mice. Interestingly, these adaptive processes have been suggested to be important during aging or infertility in humans (31). It is interesting to note that, in lxr{alpha};ß–/– mice, the combined effect of lack of both LXRs led to a dramatic decline in proliferation and increase in apoptosis. This could be one of the explanations for the complete loss of germ cells, thus leading to the infertility. Whether LXR{alpha} and LXRß could have some transcriptional effect, respectively, on several pro- and antiapoptotic and proliferation gene expressions is under investigation even though no classical DR4 LXR response elements were identified in the regulating sequences of these genes using a promoter analysis software.

Expression of both LXRs in the germ cells, even at different levels, is of interest. Indeed, this result could be consistent with the fact that only lxr{alpha};ß–/– mice show the loss of germ cells with aging. Besides, both LXRs showed redundancy in the control of retinoid pathway. Retinoic acids have been shown for many years to be important actors of testicular functions. Indeed, excess vitamin A leads to testicular lesions and spermatogenic disorders, whereas a vitamin A deficiency induces early cessation of spermatogenesis and adversely affects testosterone production (25). Our data show that the lack of LXR leads to an increase of RAR{alpha}, RARß, and RALDH-2 expressions, which should result in a more important retinoic acid signaling, as shown by the expression pattern of the known RAR-target genes, e.g. dmc1 and scp3, and could lead to spermatogenic disorders. Interestingly, lipid accumulation was already observed in Sertoli cells of rat with a hypervitaminosis A (32), suggesting some links between retinoids and lipids. How lack of LXRs could act on the retinoic acid signaling pathway remains to be understood.

We also demonstrate that both LXR isoforms show a redundancy in the control of lipid homeostasis. Indeed, scd1 and srebp1c were found to be regulated by both LXR isoforms in the testis in vivo. Noteworthy, fatty acid metabolism is essential for male reproduction and notably in spermatozoa structure (23). Indeed, testes have an active lipid metabolism that produces a rearrangement of fatty acid during spermatogenesis, and it has been suggested that troubles in fatty acid metabolism could lead to a defect of germ-cell differentiation and/or maturation (23) or a spermatozoa defect as we previously reported (16).

In conclusion, we provide important evidence for specific and common roles of LXR{alpha} and LXRß in the testis (Fig. 7Go). Both the isoforms are together involved in the regulation of testosterone production at both testicular and pituitary levels, whereas the germ cell apoptosis and proliferation are individually regulated by LXR{alpha} and LXRß, respectively. In addition, LXRs regulate lipid and retinoic acid metabolism through the transcriptional activation of enzymes and membrane transporters. More experiments will be necessary to elucidate the exact sequence of events and the relative importance and combination of the various physiological roles of LXRs in the testis, which will probably require the use of cell-specific knockout strategy.


Figure 7
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  		Fig. 7. Model for the Physiological Roles of the LXRs in the Testis

Testosterone production is regulated by LXR{alpha} at the testicular levels as well as at the pituitary by the control of LH secretion. LXRß expressed in the Sertoli cells regulates the lipid homeostasis by the induction of ABCA1 and might also control steroidogenesis through the induction of paracrine factors. In germ cells, LXR{alpha} and LXRß are involved in the processes of apoptosis and proliferation together with the regulation of the lipid homeostasis.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Lxr-knockout mice (lxr{alpha}–/–, lxrß–/–, and lxr{alpha};ß–/–) (30, 33) were maintained on a mixed strain background (C57BL/6:129Sv) and housed in a temperature-controlled room with 12-h light, 12-h dark cycles. Mice were fed ad libitum with water and Global-diet 2016S from Harlan (Gannat, France). Mice gavaged with 50 mg/kg T1317 (Sigma-Aldrich, L’Isle D’Abeau, France) or vehicle (methyl-cellulose) as described (30) were previously treated with dexamethasone (Sigma-Aldrich) for 5 d (75 µg twice a day, sc) to drop off the levels of testosterone production (22). Indeed dexamethasone treatment selectively inhibits serum {alpha}LH and ßLH mRNA responses to GnRH, but not the serum FSH or ßFSH mRNA responses (34). Some mice were injected with 5 IU hCG. Twelve hours later mice were killed by decapitation less than 1.5 h after the beginning of the light cycle and exsanguinated before organ harvest. To reduce the effect of the stress, the time between the capture of a mouse and its death was less than 1 min. All aspects of animal care were approved by the Regional Ethics Committee (authorization CE2-04).

Pathology Analyses
Testes from 2- to 12-month-old mice (n = 3 animals per group) were collected, fixed, and embedded in paraffin, and 4- to 6-µm-thick sections were prepared and stained with hematoxylin/eosin/safran. Lipid staining was performed on cryosections from 2- and 6-month-old males with 1,2 propanediol for 1 min and in Oil red O (Sigma-Aldrich) at 60 C for 7 min. Nuclei and cytoplasm were stained in Harris’ hematoxylin solution (Sigma-Aldrich) according to the manufacturer’s instructions. For semi-thin sections, chemicals were from Sigma-Aldrich and Agar Scientific (Saclay, France). Testes were fixed and post-fixed as previously described (35). After dehydration, organs were embedded in propylene oxide and epon epikote resin (vol/vol) overnight and in epon twice for 3 h. Resin polymerization was conducted at 60 C for 72 h. Semi-thin sections (0.8 µm) were cut with a diamond knife (Leica Ultracut S, Rueil-Malmaison, France), and stained with Azure 2 dye, which identifies lipids in yellow.

Immunochemistry of the AR
Paraffin sections of Bouin-fixed testis were sectioned at 5 µm. The sections were mounted on positively charged glass slides (Superfrost plus; Menzel-Glaser, Frelburg, Germany), deparaffinized, rehydrated, treated 20 min at 93–98 C in citric buffer (0.01 M, pH 6), rinsed in osmosed water (2 x 5 min), and washed (2 x 5 min) in Tris-buffered saline. Immunohistochemistry was conducted according to the manufacturer’s recommendations as described earlier (36). Slides were then counterstained with hematoxylin.

Lipid Measurements
Lipids were extracted as described previously (37). High-performance thin-layer chromatography plates (Silica gel 60; Merck) were used after being prewashed with a mixture of methanol/chloroform (1:1, vol/vol) followed by heating at 125 C for 5 min. Plates were developed with hexane, diethylether, and glacial acetic acid (80:20:2, vol/vol) and analyzed by densitometry (Sigma Scan Pro; Sigma-Aldrich) using standards as previously described (26).

Cell Purification
Sertoli and Leydig cells were isolated from 21-d-old mice as described (38). Briefly, decapsulated testes were submitted to collagenase-dispase dissociation (0.5 mg/ml, 20 min at 34 C) in DMEM/F12 medium containing desoxyribonuclease I (0.05 mg/ml) and to gravity sedimentation (5 min). Supernatants containing Leydig cells were centrifuged (200 x g, 10 min) and washed in PBS. Pellets containing the sedimented tubules were washed three times and the cells were dissociated with a collagenase-dispase treatment, as described above, until small clumps resulted. Sertoli cell pellets were then submitted to centrifugation (200 x g, 10 min). Spermatogenic cells were isolated from 12-wk-old mice testes by trypsinization. The resulting crude germ cell population (containing germ cells from all developmental steps) was submitted to a centrifugal elutriation using a rotor Beckman (Fullerton, CA) JE-6, as described previously (38, 39). Two fractions enriched at 80–85% with pachytene spermatocytes and early spermatids were harvested. After collection, the different cell populations were processed for RNA and protein extraction.

Western Blot Analysis
Protein extracts (30 µg) from whole testis were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Orsay, France). Membranes were incubated overnight at 4 C with primary polyclonal antibodies raised against Bax (1:1000; from Santa Cruz Biotechnology, Santa Cruz, CA), Bcl2 (1:1000; from Santa Cruz Biotechnology), or ß-actin (1:2000; from Santa Cruz Biotechnology) followed by a 1-h incubation with a peroxidase-conjugated antirabbit or antimouse IgG (1:10000; from Sigma).Peroxidase activity was detected using the Western Light System (PerkinElmer Life Sciences, Courtaboeuf, France).

TUNEL Analysis
Five-micrometer-thick paraffin-embedded sections were deparaffined with toluol followed by rehydratation. The slides of each group were incubated for 5 min in unmasking buffer (citrate acetate 1.8 mM, sodium citrate 8.2 mM, pH 6.0) at 86 C. Then the slides were incubated with 0.3 U/µl terminal deoxynucleotidyl transferase (Euromedex, Mundolsheim, France), 6.7 mM biotin-11-dUTP (Euromedex), and 26.7 mM dATP (Promega, Charbonnières, France) in terminal deoxynucleotidyl transferase buffer 1 h at 37 C. For the negative control, the enzyme was omitted from the system. Extravidin alkaline phosphatase conjugate (dilution 1:100; Sigma-Aldrich) was added onto the slides for 25 min. Sigmafast FastRed TR/Naphthol AS-MX (Sigma-Aldrich) was used as the substrate according to the manufacturer’s instructions. Counterstain was performed with Mayer’s hematoxylin solution (Sigma-Aldrich) for 30 sec. In each testis, at least 100 random seminiferous tubules were counted. Results are expressed as the number of TUNEL-positive cells per 1000 seminiferous tubules.

Ki67 Staining
Ten-micrometer cryosections of testis were fixed 10 min in 4% paraformaldehyde and washed three times for 10 min in 1x PBS. Cells were permeabilized with 0.1% Triton X-100 and 0.1% citrate solution in PBS for 2 min at 4 C. Slides were incubated with anti Ki67 1/500 (Tebu-bio, Le Perray en Yvelines, France) overnight at 4 C and then washed three times in 1x PBS. Slides were incubated for 1 h at room temperature with a goat antirabbit secondary antibody labeled with Alexa 488 (1/250; from Invitrogen Detection Technologies, Cergy-Pontoise, France). In each testis, at least 100 random seminiferous tubules were counted. Results are expressed as the number of Ki67-positive cells per 100 seminiferous tubules.

Endocrine Investigations
Testosterone concentration was measured on 200 µl of heparin-treated plasma or from frozen testis extracted with 10 vol of ethylacetate-isooctane (30:70, vol:vol). A commercial kit (ICN, Orsay, France) was used for the assays. LH values were determined on 200 µl of EDTA-treated plasma by the Core Services of the University of Virginia Center for Research and Reproduction.

qPCR
Testis and pituitary RNA was isolated using the RNeasy kit (Qiagen, Courtaboeuf, France). cDNA was synthesized from total RNA with the SuperScript II First-Strand Synthesis System (Life Technologies, Cergy-Pontoise, France) and random hexamer primers. The real-time PCR measurement of individual cDNAs was performed using SYBR green dye to measure duplex DNA formation with the Roche Lightcycler system. Primer sequences and typical cycle threshold values (observed in untreated wild-type mice) are reported in Table 1Go.


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  		Table 1. qPCR Primer Sequences and Typical Cycle Threshold (Ct) Values Observed in Untreated Wild-Type Mice

 
Statistical Analysis
For statistical analysis, one-way ANOVA was performed to determine whether there were differences between the various groups. A P value of 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We thank Jean-Paul Saru for his excellent technical help; Mrs. Christine Puchol and Sandrine Plantade for expert technical assistance in breeding the transgenic mice; Dr. D. J. Mangelsdorf (Howard Hughes Medical Institue, Dallas, TX) for consulting and providing the mice; Drs. C. L. Cummins (Dallas, TX), F. Caira, L. Morel, C. Beaudoin, and S. Baron (Unité Mixte de Recherche Centre National de la Recherche Scientifique 6547, Aubière, France) for critically reading the manuscript; and the members of the Chester’s lab for assistance in animal dissections and discussions.


   FOOTNOTES
 
This work was supported by grants from the Centre National de la Recherche Scientifique, the Université Blaise Pascal, the Fondation pour la Recherche Médicale INE2000-407031/1, and the Fondation BNP-Paribas. University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core is supported by National Institute of Child Health and Human Development (SCCPRR) Grants U54-HD28934. K.M. is the recipient of a doctoral fellowship from the Ministère de l’Education Nationale de la Recherche et de la Technologie. J.-M.A.L. is a Professor of the Université Blaise Pascal.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2007

Abbreviations: abca1, ATP-binding cassette, sub-family A member 1; AMH, anti-Müllerian hormone; AR, androgen receptor; bad, BCL2-antagonist of cell death; bax, Bcl2-associated X protein; CG, chorionic gonadotropin; cyp11a1, cytochrome 11A1 cholesterol side-chain cleavage; cyp17, cytochrome 17; dmc1, meiosis-specific recombinase-1; DR4, direct repeat 4; fas, fatty acid synthase; 3ßhsdI, type 1 3ß-hydroxysteroid dehydrogenase isomerase; IKK, inhibitor of {kappa}B kinase; LXR, liver X receptor; lxr–/–, lxr-deficient mouse; qPCR, quantitative PCR; RALDH-2, retinoic aldehyde dehydrogenase 2; RAR, all-trans retinoid receptor; RXR, 9-cis retinoid receptor; scd, encoding the stearoyl coA-desaturase; scp3, synaptonemal complex protein 3; srebp1c, sterol response element binding protein-1c; StAR, steroidogenic acute regulatory protein; T1317, LXR agonist T0901317; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling.

Received for publication July 7, 2006. Accepted for publication February 28, 2007.


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