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Endocrinology Vol. 141, No. 10 3821-3831
Copyright © 2000 by The Endocrine Society

ARTICLES

A Novel Messenger Ribonucleic Acid Homologous to Human MAGE-D Is Strongly Expressed in Rat Sertoli Cells and Weakly in Leydig Cells and Is Regulated by Follitropin, Lutropin, and Prolactin1

Benoit Hennuy, Eric Reiter, Anne Cornet, Marc Bruyninx, Marc Daukandt, Paule Houssa, Viet-Ha N'Guyen, Jean Closset and Georges Hennen

Biochemistry and Laboratory of Endocrinology (B.H., A.C., M.B., M.D., P.H., V.-H.N., J.C., G.H.), Institute of Pathology B23, University of Liège, B-4000 Liège, Belgium; Institut National de la Recherche Agronomique (INRA) (E.R.); Station de Physiologie de la Reproduction des Mammifères Domestiques, URA CNRS 1291, 37380 Nouzilly, France

Address all correspondence and requests for reprints to: Dr. J. Closset, Biochemistry and Laboratory of Endocrinology, Institute of Pathology B23, Avenue de l’Hôpital 3, University of Liège, B-4000 Liège, Belgium. E-mail: jclosset{at}ulg.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have cloned a novel complementary DNA whose expression was decreased in rat Sertoli cell cultures after treatment with FSH. This complementary DNA encodes a protein of 570 amino acids and shares 92% homology with the human MAGE-D protein. In contrast to other MAGE genes (A, B, or C), we have shown that MAGE-D expression was ubiquitous in healthy rat tissues. In the seminiferous tubules, the MAGE-D was expressed in Sertoli cells but not in germ cells as demonstrated by RT-PCR and in situ hybridization, whereas for the other MAGE genes, expression has been shown to be restricted to germ cells. Interestingly, MAGE-D was also detected for the first time in the female gonad by Northern blotting. In MLTC-1 cells (mouse Leydig tumor cell line-1), LH and PRL stimulated MAGE-D expression. Using hypophysectomized rats, it was confirmed that FSH decreased MAGE-D expression, whereas LH and PRL increased MAGE-D messenger RNA level in the whole testis most probably through a direct action on Leydig cells. As MAGE-D is present in both the seminiferous compartment and interstitium and hormonally regulated in each, it is possible that it has specific functions in each compartment during the development and the maintenance of the testis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNTIL RECENTLY, the human MAGE genes were thought to be expressed in a wide variety of tumors but not in normal cells, except in the male germ cells, placental cells, and possibly for some of these genes, in cells of the developing embryo (1, 2, 3, 4). The MAGE genes previously described in the literature, encode tumor-specific antigens associated with the major histocompatibility complex I (MHC I) and recognized by cytolytic T lymphocytes (5). However, the function of the intact MAGE proteins in the testis is unknown (1). The MAGE protein sequences are original and share homology with that of necdin, a nuclear protein expressed in virtually all postmitotic neurons in the central and peripheral nervous system (1, 6, 7). Necdin is a growth suppressor controlling the cell growth during the brain development (8, 9, 10) and involved in the Prader-Willi syndrome (11, 12, 13).

The MAGE gene sequences all show a main open reading frame in the last exon, encoding a putative protein of about 300 amino acids. The human MAGE genes are located in different regions of the X chromosome and are organized in three loci named MAGE-A (12 genes, Xq28 region), MAGE-B (4 genes, Xp21.3 region) and MAGE-C (1 gene, Xq26 region). Mouse MAGE genes homologous to their human counterpart were recently described with a similar organization on the mouse X chromosome (14).

Recently, a fourth human MAGE gene representing a new locus was isolated and named MAGE-D. This gene was located in the Xp11.23 region (15). The MAGE-D gene displays a very different structure from the other MAGE genes, with 13 exons and an ORF spread over exon 2 to 12. This ORF encodes a putative 574 amino acids MAGE-D protein. Unlike the other MAGE-family members, MAGE-D is expressed not only in several tumor cell lines but also ubiquituously in normal human tissues including testis (16).

The mammalian testis consists of seminiferous tubules containing the Sertoli cells providing structural and nutritional support for the developing germ cells. The interstitium between the tubules contains the Leydig cells responsible for steroidogenesis (17).

Most Leydig cell functions are controlled by lutropin (LH), which increases the production of androgens by this cell type. A second gonadotropin, FSH, acts on Sertoli cells by promoting stages I through VIII of spermatogenesis and by stimulating the production of several paracrine or autocrine regulatory factors targeting the germ cells, peritubular/myoid cells, and Leydig cells (18, 19). The responsiveness of Sertoli cells to FSH is maximal during postnatal development and the prepubertal period. It then decreases until adulthood, although it varies through the seminiferous epithelium cycle (20). It is well established that among these locally secreted Sertoli cell factors, lactate, activin/inhibin, androgen binding protein (ABP), transferrin, plasminogen activator (tPA), anti-Müllerian hormone (AMH), and insulin-like growth factor I (IGF-I) are responsible for some but not for all FSH mediated actions.

To discover new FSH-regulated Sertoli cell factors, we applied a recently described differential cloning method [suppression subtractive hybridization (SSH)] (21) to FSH-treated primary rat Sertoli cell cultures. This enabled us to isolate a new rat complementary DNA (cDNA) displaying FSH-down regulated expression and high homology with the human MAGE-D cDNA.

As the MAGE-A, -B, and -C gene expression in the testis seems to be restricted to germ cells, we examined rat MAGE-D expression in all testicular cell types. We also analyzed its regulation by FSH and other pituitary hormones. Our results suggest that MAGE-D could play a very different role from MAGE-A, -B, and -C, in the testis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormone preparations
Porcine FSH (follitropin; pFSH) was purified according to Closset and Hennen (22, 23) with minor modifications. The last step was an immunoaffinity chromatography using anti-LH antibodies immobilized on Affigel 10 (Bio-Rad Laboratories, Inc.). The immunological potency of the preparation was 160 times higher than that of the reference preparation of NIH-FSH-P1.

Human LH (hLH) was purified according to Closset and Hennen (23) with additional chromatographies on immobilized purified antihuman thyroid-stimulating hormone (hTSH) and anti-hFSH coated columns. The biopotency of the hLH preparation was 8500 U/mg (International Standard 68/40) as measured in specific radioreceptor assays.

Human PRL (hPRL) was purified in our laboratory according to Hodgkinson and Lowry (24). The immunological potency of this preparation was 39 IU/mg, (International Standard 75/504). The biological activity of the hormone was assessed by means of a RRA using pregnant mammary gland receptor. It was found to equal 34 IU/mg in terms of International Standard 57/08. The specific biological activity of comparable hPRL preparations has been evaluated by studying their effects on testicular function in immature rats (25, 26).

Cross-contaminations of each pituitary hormone preparation were measured using specific RIAs and radio-receptor assays. On the basis of these analytical methods, they were found to be less than 0.001% by weight for each hormone.

Animals and treatments
Male Wistar rats were hypophysectomized at 19 day-old (IFFA-Credo, Lyon, France). They were randomly divided into several groups (n = 10), and they received the first injection 2 days postoperatively. They were daily treated sc with saline 0.9% or with 5, 25, or 75 µg of pFSH, 30 µg of hLH, 30 µg, of hPRL or 4 mg/kg of testosterone propionate for 7 days. Rats had free access to food and water and they were maintained on a cycle of 12 h light, 12 h dark. Twenty-four hours after the last injection, rats were killed, and the testes were removed, decapsulated, and frozen in liquid nitrogen; they were stored at -70 C until use. Tissues were randomly divided into two independent groups for each experimental condition and analyzed independently.

Cell culture
TM4 (testicular mouse) and MLTC-1 cell lines were purchased from the ATCC. The TM4 cell line was cultured in a 1:1 mixture of Ham’s F12K medium and DMEM supplemented with 4.5 g/liter glucose, 1.2 g/liter sodium bicarbonate, and 15 mM HEPES, 5% horse serum, 2.5% FBS, streptomycin sulfate 100 µg/ml, penicillin G 100 IU/ml. The MLTC-1 cell line was cultured in RPMI-1640 medium (Life Technologies, Inc., Gent, Belgium) supplemented with 10% FBS, streptomycin sulfate 100 µg/ml, and penicillin G 100 IU/ml. The cells were treated as described in the legends.

Primary Sertoli cell culture
Sertoli cell-enriched cultures were prepared from 19 day-old Wistar rats (Iffa-Credo, Lyon, France) following the method of Welsh and Wiebe (27) with modifications (28). Briefly, 40 testes were removed and decapsulated. They were transferred to 20 ml of medium A (PBS Dulbecco’s without Ca2+ and Mg2+ and containing streptomycin sulfate 100 µg/ml, penicillin G 100 IU/ml, geomycine 25 µg/ml, and glucose 1.5 g/ml) supplemented with trypsin (2 mg/ml) and DNase I (10 µg/ml). The mixture was incubated for 20 min at 32 C in a shaking water bath (120 osc/min). At the end of the incubation, the mixture was transferred into a conical tube, 10 ml of medium A was used to rinse the tube and pooled with the mixture. The mixture was left to sediment for 5 min at unit gravity. The supernatant containing interstitial cells was discarded and 20 ml of medium A were added. These washing/sedimentation cycles were repeated three times. Seminiferous tubules were next resuspended in 20 ml of medium A supplemented with collagenase A (1 mg/ml) and incubated as described above for 50 min. The mixture of tubular fragments was transferred into a conical tube and the recipient was rinsed with 10 ml of medium A. After sedimentation of the tubular fragments, the supernatant was discarded and the washing/sedimentation cycle was repeated three times. Next, tubular fragments were resuspended in medium A containing hyaluronidase (1 mg/ml) and incubated for 30 min as described above. Three cycles of washing sedimentation were carried out and finally, the tubular fragments were resuspended in 20 ml of RPMI medium, which was composed by RPMI-1640 supplemented with 22.5 mM HEPES pH 7.4, 5.5 mM L-glutamine, streptomycin sulfate 100 µg/ml, penicillin G 100 IU/ml, and gentamycin 25 µg/ml. The tubular fragments were mechanically disrupted by 10 thorough strokes through a syringe with a needle 18G1/2. Separated cells were recovered by centrifugation at 250x g for 10 min and the pellet was resuspended in 20 ml of RPMI medium. The cells were passed through a nylon screen (pore size: 100 µm) which was rinsed with 10 ml of fresh RPMI medium. The cells were counted and plated at a density of 3.5 x 105 cells/cm2 in culture flasks (Nunc, Roskilde, Denmark). After 72 h of culture at 32 C in a humidified incubator gassed with 5% CO2, the medium was removed and the attached cells were washed twice with RPMI medium. Cultured Sertoli cells were kept for an additional 24 h in RPMI medium before treatment with or without FSH at 50 ng/ml for 6 h.

RNA isolation and Northern blotting
Total RNA was prepared from different sources following the method of Chomczinski and Sacchi (29). Poly (A)+ RNA was extracted with oligo deoxy-thymidine coupled with magnetic particles [PolyA Tract messenger RNA (mRNA) isolation system Iv) (Promega Corp., Madison, WI].

Total RNA was separated by electrophoresis in 1% agarose-2% formaldehyde gels in MOPS buffer (pH 7.0) and blotted on reinforced nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by means of the Vacugene apparatus (Amersham Pharmacia Biotech, Aylesbury, UK). 28S and 18S ribosomal RNAs were used as size indicators. After baking at 80 C in a vacuum oven for 2 h, filters were hybridized at 42 C with 32P-labeled cDNA probes in SSC 5x, Denhardt’s 1x, dextran sulfate 10%, formamide 50% containing 100 µg/ml of salmon sperm DNA for 16 h (30). Quantitations were performed with the ImageQuant software (Molecular Dynamics, Inc.).

The data presented are means of the results obtained from at least two different pools of tissues or cell culture experiments. One-way ANOVA was applied to the data. Simultaneous confidence intervals were estimated to determine possible differences between groups (by Scheffé’s test). Results were considered significant at the 5% level.

Suppression subtractive hybridization (SSH) and cloning of the full-length of rat MAGE-D cDNA
The procedure derived from Diatchenko et al. without modifications (21) was used. Briefly, 2 µg of polyadenylated RNA from FSH-treated Sertoli cells were used to synthesize "driver" cDNAs although 2 µg of polyadenylated RNA from untreated cells were used to synthesize "tester" cDNAs.

Products from the second PCR after SSH reaction were inserted into pGEM-Z using a T/A cloning kit (Promega Corp.). Fragments of cDNAs were [32P]-labeled by random priming using the DNA labeling bead (-dCTP) kit (Amersham Pharmacia Biotech) and used as probes for Northern blots loaded with 10 µg of total RNA from FSH treated and untreated Sertoli cell culture. A fragment of 530 bp showing decreased expression after FSH treatment was isolated, sequenced, and used as probe to screen a commercially available testis cDNA library (Stratagene, La Jolla, CA). The clones containing the full-length cDNA were sequenced using the T7 and T3 primers, and a series of specific internal primers.

Database searching was performed using the FASTA and Blast programs at BEN (Belgian EMBNet Node) computers and at Blast server: http://www.ncbi.nlm.nih.gov/BLAST/.

RT-PCR amplification of rat MAGE-D
The sense primer (PR1) at position 1091 (5'-AAATGCTGAGAGATATCATCC-3') and antisense primer (PR2) at position 1701 (5'-TCAAACTCAATGTCATCCCAGC-3) were used. RT reaction (45 min at 41 C) was carried out using total RNAs extracted from Sertoli cells, Leydig cells, round spermatids (RS), and pachytene spermatocytes (PS). Leydig cells were prepared by Percoll gradient according to (31), RS and PS were prepared by centrifugal elutriation (32, 33). The RT-PCR reactions also contained (50 µl final volume) 200 U of MMLV-RT, 10 nM of antisense primer, 8 mM DTT, 400 µM of each dNTP, 3.5 mM MgCl2, and 5 µl of 10x Taq PCR buffer (500 mM KCl, 200 mM Tris-HCl, pH 8.3, 1 mg/ml gelatin). After completion of the RT reaction, the temperature was raised to 95 C for 30 sec, then equilibrated to 80 C. PCR was initiated by adding 50 µl of a mix containing 1.25 U of Taq polymerase, 200 nM of sense and antisense primers, 1.5 mM MgCl2, and 5 µl of 10x Taq PCR buffer. The samples were processed for 20, 25, or 30 cycles (95 C for 30 sec, 56 C for 30 sec, 72 C for 1 min) in the presence of 5 µCi of {alpha} (32)[P]-dCTP, the last of which had an elongation time of 10 min. Samples were electrophoresed through a 2% low melting point agarose gel, and autoradiographed.

In situ hybridization
In situ hybridization was performed according to the protocol described by Arce et al. (34). Ten-micrometer-thick sections prepared from adult rat testis were fixed in Davidson reagent (33% methanol, 11% acetic acid, 22% formalin) for 24 h. Testes were dehydrated by increased alcohol series followed by 100% toluol, embedded in toluol-Paraplast (1, 1) at 37 C and finally in Paraplast at 58 C for 16 h. Paraffin was removed from sections before use by successive treatments with 100% xylol, with decreasing alcohol concentrations and the sections were washed with water. The subsequent steps were previously described (30).

Generation of rat MAGE-D antibody and Western blotting
From the putative deduced amino acid sequence of rat MAGE-D, we generated a synthetic peptide named Pep1 (NH2-PDWQNLRPSPNLRSS-COOH) corresponding to residues 229–243 (Neosystem, Strasbourg, France). The peptide (1 mg) was coupled to 6 mg of human thyroglobulin and used to raise polyclonal antirat MAGE-D rabbit antibody, as described by Staros and co-workers (35). The antibody was immunopurified on an affinity column of peptide coupled to activated Sepharose column (Amersham Pharmacia Biotech) according to the protocol of the manufacturer.

Protein homogenates were extracted from about 3 x 107 Sertoli cells in culture. This was done by sonicating the samples for 30 sec in 1 ml of ice-cold 50 mM Tris, pH 7.4, in the presence of protease inhibitors (50 U Trasylol (Bayer Corp., Leverkusen, Germany) and 10 mg of phenylmethylsulfonyl fluoride). Proteins were denaturated by adding 100 µl of 10% SDS and ß-mercaptoethanol to a final concentration of 0.2 M. The homogenates were centrifuged at 15,000x g for 10 min at 4 C. Supernatants were collected and 90 µg of proteins were size-fractionated by electrophoresis on a homogenous 12% polyacrylamide gel. Western blotting was performed according to the standard protocol described by the manufacturer of the apparatus (Bio-Rad Laboratories, Inc. Hercules, CA). Immunochemical detection of rat MAGE-D gene product was carried out with a polyclonal goat antirabbit IgG coupled to horseradish peroxidase, and 4-chloro-1-naphtol as substrate following the supplier’s instructions (Sigma-Aldrich Corp.).

Preparation of subcellular enriched fractions
The method was described previously according to Rickwood et al. (36). Sertoli cell cultures were washed twice with ice-cold buffer 0.25 M sucrose, 10 mM triethanolamine-acetate, pH 7.6. They were scraped with a rubber-policeman and centrifuged 10 min at 250x g at 4 C. The cells were homogenized in the washing buffer supplemented with 1 mM EDTA pH 7.6, using a tight-fitting Dounce apparatus and the homogenate was centrifuged at 600x g for 10 min at 4 C. The pellet was resuspended in the same medium, homogenized, and centrifuged as described to obtain a crude nuclear fraction. The supernatants were pooled and centrifuged for 20 min at 27, 000x g at 4 C to obtain the membrane-enriched fraction (pellet) and cytoplasm-organelle-enriched fraction (supernatant). The pellet of crude nuclei was resuspended in 9 vol of 2.2 M sucrose, 1 mM MgCl2, 10 mM Tris-HCl pH 7.4, homogenized, and centrifuged at 70, 000x g for 80 min at 4 C. The supernatant was discarded and the pellet containing the nuclei-enriched fraction was conserved. The enriched fractions were solubilized in denaturing medium containing 10% SDS and 0.2 M ß-mercaptoethanol for Western blotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequencing of rat MAGE-D
Using the SSH technique with control and FSH-treated Sertoli-cell mRNAs, we isolated a 3'end cDNA fragment (530 bp) showing decreased expression after FSH treatment. This cDNA fragment shared significant homology with several EST sequences in the databases. To obtain a full-length cDNA, we screened a testis cDNA library (Stratagene, La Jolla, CA) using the isolated cDNA fragment as a probe. The largest cDNA isolated represented an mRNA of 2150 bp. Complete sequencing and further database comparisons revealed that this cDNA was highly similar to the recently described human MAGE-D1 gene. We concluded that this novel rat cDNA corresponded to the product of the rat MAGE-D gene.

Sequence analysis (Fig. 1Go) revealed an open reading frame (ORF) starting at position 209, ending at position 1918, and encoding a putative 570-residue protein with a relative molecular mass of 60.6 kDa. The 3' untranslated region was 232 bp long with a polyadenylation site at position 2109. Two putative sites of complex glycosylation were present, one at position 97, and one at position 103. These were conserved in the human sequence. Cysteinyl residues were present at positions 220, 309, and 493. Of these, only cysteine 309 was conserved in the human sequence. As already observed in human MAGE-D, the rat sequence showed several imperfect hexameric repeats in the N-terminal half between residues 88 and 228; the structural motif corresponding to these repeats is unknown.



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  		Figure 1. Sequence of the complete rat MAGE-D cDNA. The different regions of the sequences are indicated as follows: nucleic acids in capital letters, open reading frame; underlined amino acids, the repetitive portion of rat MAGE-D; boldface amino acids, portion with high degree of homology to other human MAGE genes and necdin; boxed amino acids, sequence of peptide against which the antibody is directed; dashed arrows and boldface nucleic acids indicate primers or their complementary sequences whose orientation was given by overlining. Two replicate sequences were performed on both strands.

 
The putative rat MAGE-D protein shared 92% homology with its human counterpart but lacked 4 amino acids present at the start of the human sequence (Fig. 2Go). The C-terminal region spanning residues 349 to 479 had strong homology with other MAGE-family members (between 45% and 54% according to the considered sequence) and with necdin. No known specific motifs (signatures) were found in the rat MAGE-D amino acid sequence.



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  		Figure 2. The alignment of the entire sequences of rat and human MAGE-D. Only the nonconserved amino acids are shown for the respective sequences.

 
Expression of rat MAGE-D
Using two primers located respectively at position 413, (5'-CTTCCGGGAACTGGAGGT-3') named PP1 and at position 1012, (5'-GCATCAAGTACTTGACCAGCTT-3') named PP2, we generated a PCR probe corresponding to a region sharing no homology with the other MAGE-family genes. The transcript identified by means of this probe was similar to that revealed with the previously isolated 530-bp probe. Its size was near 2.5 kb, indicating that the isolated clone represented the full-length (or nearly full-length) cDNA of rat MAGE-D.

Northern analysis showed that MAGE-D was expressed to various degrees in a broad range of rat organs brain, thymus, spleen, liver, kidney, lung, heart, prostate, seminal vesicles, epididymis, and for the first time in the ovary (Fig. 3Go). Rat MAGE-D expression was also detected in several rat and mouse cell lines: lymphoma, mastocytoma, teratocarcinoma, melanoma, prostatic cell lines, and Sertoli and Leydig cell lines (Table 1Go).



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  		Figure 3. Northern blot analysis of MAGE-D mRNA level in different organs of the rats. Ten micrograms of total RNA extracted from brain (Br), thymus (Thy), spleen (Sp), liver (Li), kidney (Ki), lung (Lu), heart (Ht), prostate (Pr), seminal vesicle (SV), epididymis (Ep), and ovary (Ov) of rat were separated by electrophoresis and hybridized with 32P-labeled MAGE-D probe. 18 S ribosomal RNA hybridization was used as loading control.

 

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  		Table 1. List of rat or mouse cell lines in which expression of MAGE-D was assessed by Northern blot analysis of total RNA

 
Cellular and subcellular localization of MAGE-D expression
As the testis contains many different cell lineages, we investigated expression of rat MAGE-D in Sertoli cells, Leydig cells, round spermatids, and pachytene spermatocytes. Our RT-PCR results showed that high-level expression of the rat MAGE-D gene was restricted to Sertoli cells. Very low-level expression was also detected in Leydig cells but not in round spermatids or pachytene spermatocytes (Fig. 4Go, A and B).



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  		Figure 4. Cellular localization of MAGE-D expression in purified testicular cells. A, RT-PCR reactions were performed in the presence of [{alpha}-32P]dCTP and were stopped after 20, 25, or 30 cycles. Expected sizes of each amplified product are indicated on the left. +, positive control; Leydig or Leyd., Percoll gradient isolated Leydig cells; Sertoli or Sert., cultured Sertoli cells; RS, round spermatids purified by elutriation; PS, pachytene spermatocytes purified by elutriation. B, Quantitation of blot are expressed in arbitrary absorbance unit (AU) and normalization of results was relative to the value measured in ß-actin amplification. C, In situ hybridization of MAGE-D mRNA in rat testis 10-µm-thick sections with a digoxygenin-UTP labeled MAGE-D antisense riboprobe. Visualization of bound-DIG probe was performed using anti-DIG-alkaline phosphatase coupled antibody and a AP-catalyzed color reaction with NBT/BCIP as substrate. Sections were countercolored with eosin. Magnification 1000x. D, Negative control of in situ hybridization where the sense probe was used. Magnification 1000x.

 
An antisense probe for rat MAGE-D was labeled with DIG-UTP and allowed to hybridize with the material on adult testis slides. There was marked labeling at the edge of the seminiferous tubules where the Sertoli-cell nuclei were located (Fig. 4CGo). In addition, there was diffuse filamentous labeling going from the edges of the tubules toward the center, indicating that there was also specific though less marked staining of the Sertoli cell cytoplasm. Neither the germ cells nor the interstitium were specifically labeled. With the sense probe (negative control), no cells were labeled (Fig. 4DGo). These results confirmed those obtained by RT-PCR analysis of isolated different cell lineages.

MAGE genes share significant homology with the nuclear protein necdin. To obtain information regarding the role of the MAGE-D protein in Sertoli cells, we studied its subcellular localization by Western blotting. A polyclonal antibody was raised against a synthetic peptide matching the MAGE-D specific sequence (residues 229–243). Rabbit antiserum was prepared and used to probe Western blots of subcellular-enriched fractions prepared from Sertoli cells. A major protein was immunodetected around 60 kDa both in the nuclei- and cytoplasm-enriched fractions (Fig. 5Go), this was consistent with the predicted molecular mass of MAGE-D protein. Minor bands of lower molecular weight were also detected. These bands might represent degradation products generated during the subcellular fractionation or post translational modifications since only one transcript of 2.5 kb was revealed by Northern blotting.



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  		Figure 5. Western blot analysis of MAGE-D gene product in subcellular enriched fractions prepared from Sertoli cells in culture. Ninety micrograms of protein extract from nuclei-enriched fraction (NF), membrane-enriched fraction (MF), and cytoplasm-organelle-enriched fraction (CF) were size fractionated on 12% polyacrylamide gel and probed with anti-MAGE-D polyclonal antibody. Size of MAGE-D gene product was determined by comparison with the migration of 14.3–220 kDa Rainbow colored protein molecular weight marker (Amersham Pharmacia Biotech).

 
Hormonal regulation of rat MAGE-D
Northern blot analysis was used to study expression of MAGE-D in cultured immature rat Sertoli cells exposed for 6 h to of various doses of pFSH. FSH-treated cultures (from 100 ng/ml to 0.1 ng/ml doses) showed a lower MAGE-D mRNA level than the untreated control culture. Surprisingly, at the dose of 1000 ng/ml of FSH, the mRNA level was similar to that of the control. The lower doses decreased MAGE-D mRNA level in a reverse dose-response curve. A similar regulation was observed when cells were treated with increasing doses of dbcAMP (Fig. 6AGo).



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  		Figure 6. Northern blot analysis of hormonal regulation of MAGE-D expression. A, Sertoli cells in culture were treated for 6 h with various doses of FSH (1000–0.1 ng/ml) or dbcAMP (1–0.01 mM). Total RNA (10 µg per lane) was extracted and analyzed for MAGE-D expression. 18 S ribosomal RNA hybridization was used as loading control. The Northern blots were quantified using the ImageQuant software, the values were corrected according to the amount of 18S rRNA and are presented in arbitrary units (AU) (the results for control condition were taken as 100). The data represents the mean ± SEM of two individual experiments. *, Significantly different from the result for control at the 5% level (by Scheffé’s test). B, Ten hypox rats were treated daily for 7 days with various doses of pFSH (5, 25, or 75 µg), or 30 µg of hLH or 30 µg of hPRL or 4 mg/kg testosterone propionate. Total RNA (10 µg per lane) was extracted and analyzed for MAGE-D expression. 18 S ribosomal RNA hybridization was used as loading control. Quantitation of Northern blots and statistical analysis of results were performed as described. C, TM4 cells were cultured as described in Materials and Methods and treated for 6 h with dbcAMP (0.1 mM and 0.01 mM) and by forskolin (1 µM and 10 µM). Total RNA was extracted, and analyzed for MAGE-D expression. 18 S ribosomal RNA hybridization was used as loading control. Quantitation of Northern blots and statistical analysis of results were performed as described. D, Total RNA was extracted from testes of rats aged of 5 days (5d), 10 days (10d), 20 days (20d), and 60 days (60d) and was analyzed for MAGE-D expression. 18 S ribosomal RNA hybridization was used as loading control. Quantitation of Northern blots and statistical analysis of results were performed as described.

 
The relevance of this regulation was then assessed in an in vivo model. Immature hypophysectomized (hypox) rats were treated with several doses of FSH, LH, or PRL and MAGE-D expression was measured. These experiments confirmed an expression-decreasing effect of FSH (0.5-fold) in the rat testis but the effect was not dose-related. On the contrary, LH and PRL were found to increase expression of MAGE-D in the whole testis of hypox rats (2-fold and 2.2-fold, respectively; Fig. 6BGoa). A negative regulation of MAGE-D expression was also demonstrated by treatments with both dbcAMP and forskolin on the TM4 cell line in culture (Fig. 6CGo). The decrease was of 10 and 5 times for cells treated dbcAMP 0.1 mM and 0.01 mM, respectively; forskolin decreased the MAGE-D mRNA level by a factor of 7 independently of the doses used. In hypox-treated rats, testosterone had no effect on MAGE-D expression (Fig. 6BGob), although the expression of ABP was stimulated by this treatment.

Mammalian testicular development during puberty is characterized by increasing concentrations of FSH in the bloodstream. To study the regulation of rat MAGE-D expression during this physiological process, whole testes from rats aged 5, 10, 20, and 60 days were collected and analyzed by Northern blotting (Fig. 6DGo). The signal intensity was found to decrease markedly during the ontogenesis of the rat testis, a period when there is a decrease in the Sertoli cell-germ cell ratio.

As we detected a slight expression by RT-PCR of MAGE-D in Leydig cells and by Northern blotting in the MLTC-1 cell line, in addition as LH and PRL were able to increase the expression of MAGE-D in the whole testis of hypox rats, the direct effects of LH and PRL on MLTC-1 cells were assessed. These two hormones stimulated the expression of MAGE-D and the maximal increase was observed with LH at 100 ng/ml (4.8-fold) and with PRL at 25 ng/ml (9.2-fold) after 6 h of treatment (Fig. 7Go).



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  		Figure 7. Northern blot analysis of effects of hLH and hPRL on MAGE-D expression in MLTC-1 cells. Total RNA was extracted from MLTC-1 cells treated for 6 h with various doses of hLH or hPRL (25 ng/ml, 100 ng/ml) and analyzed for MAGE-D expression. 18 S ribosomal RNA hybridization was used as loading control. The Northern blots were quantified using the ImageQuant software, and the values were corrected according to the amount of 18S rRNA and are presented in arbitrary units (AU) (the results for control condition were taken as 100). The data represents the mean ± SEM of two individual experiments. *, Significantly different from the result for control at the 5% level (by Scheffé’s test).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have cloned and characterized a new cDNA from cultured rat Sertoli cells treated by FSH. This cDNA is homologous to the recently described human MAGE-D cDNA.

The amino acid sequences of the rat and human MAGE-D proteins show a high degree of conservation. This is in agreement with the observations of Pold (15), who compared mouse EST’s available in databases with the corresponding human sequences and suggested that the MAGE proteins could be highly conserved among mammals. Our results confirm the presence in the N-terminal portion of the sequence of a repetitive region unlike any known motif, whose function is unknown and which is apparently characteristic of the MAGE-D protein. As already described for the human sequence, a portion of the rat protein also displays high homology with the other human MAGE-family proteins.

Expression of the rat MAGE-D gene, like that of its human counterpart, would appear to be ubiquitous. Indeed, we detected expression in all tissues studied, particularly those of the uro-genital tract (testis, epididymis, seminal vesicles, prostate). We also observed MAGE-D expression in the ovaries. This is the first time the expression of any MAGE gene has been detected in this organ. Neither MAGE-A nor MAGE-B nor MAGE-C were expressed there (4, 3, 37). We also found MAGE-D to be expressed in several rat and mouse tumor cell lines.

Expression of the MAGE-A, -B, and -C genes is not detected in healthy tissues except in the testis, where expression is restricted to the germinal cells (15). For MAGE-D, our RT-PCR and in situ hybridization data showed, on the contrary, that expression occurred mainly in the Sertoli cells, even if a weak signal was also detected also in the Leydig cells. These various results suggest that MAGE-D could have a function in both the male and female gonads, very different from the function of the other MAGE proteins.

Based on alignment of amino acid sequences, MAGE-D appears more closely related to necdin than MAGE-A, -B, and -C (16). Necdin is a nuclear protein with cell growth suppressing properties expressed in postmitotically differentiated neurons. It is possible that MAGE-D in Sertoli cells was implicated in the control of Sertoli cell proliferation arrest. Indeed, it is known that Sertoli cells stop dividing mitotically from 15 days postpartum even though circulating FSH levels continue to increase. As FSH decreases the expression of MAGE-D, a putative role of MAGE-D protein in Sertoli cells might be those of a growth promoting factor. If this is true, the MAGE-D function could oppose that of necdin. Nevertheless, the putative role of MAGE-D as a transcription factor or a growth promoter requires further investigation.

The testis is under the control of both pituitary and steroid hormones. There are no published data concerning the regulation of the MAGE genes in the testis by these hormones. In our experiments, we have shown that FSH decreased MAGE-D expression in primary Sertoli-cell cultures, but it was seen that the effect of FSH on MAGE-D expression decreased as the FSH dose increased. This could be due to various signaling regulatory processes occurring at high doses of FSH during the 6 h of treatment (38, 39, 40, 41). However, because dbcAMP likewise decreases MAGE-D expression in a dose-dependent manner in Sertoli cell cultures, the FSH dose-effects should be preferentially attributable to molecular mechanisms occurring after cAMP production and involving cAMP responsive element binding protein system (CREB, CREM, ICER) (42, 43). Such negative effects of dbcAMP and forskolin on MAGE-D expression were also confirmed in the TM4 cell line. Our results thus suggest that MAGE-D is involved in a cascade of events responding quickly to FSH stimulation. FSH-decreased MAGE-D gene expression was also confirmed in hypox rats treated for 7 days with different concentrations of pFSH. That the effect is not dose-dependent in this case is surprising. This could reflect the fact that hypox rats are a model where testicular regression is the main phenomenon (44). Although the number of Sertoli cells remains quite constant during the treatment, regression of the other cell lines may imply paracrine regulations within the organ, modifying the action of FSH on its target cells (45).

In mammals, bloodstream levels of FSH increase during puberty. During this process, the decreased MAGE-D expression reflects rather the decreasing Sertoli cells to germ cells ratio than a negative regulatory effect of FSH on the MAGE-D expression in the seminiferous tubules. Indeed, it is well established that the increase in testicular weight observed at puberty is due mainly to an increase in the number of germinal cells (46, 47). Taken together, these results are consistent with the view that MAGE-D expression is restricted to the Sertoli cells in the seminiferous tubules.

Comparing the magnitude of MAGE-D gene expression with those of other negatively regulated genes was difficult because only a few genes have been reported to display decreased expression after FSH treatment (48, 39). However, the inhibition factor observed for MAGE-D is of the same order as the stimulation factors observed for other genes with this hormone (49, 50).

We detected slight expression of MAGE-D in Leydig cells and in the MLTC-1 cell line where we studied the direct effects of LH and PRL on MAGE-D expression. It is interesting to note that these hormones, well known to trigger different second messenger pathways, are both able to stimulate the expression of MAGE-D in MLTC-1 cells. These results would suggest that the MAGE-D promoter contains regulatory elements specific for these two hormones.

In hypophysectomized rats treated with LH or PRL, accordingly, the MAGE-D level rises in the whole testis, possibly due to regulation in the Leydig cells. Yet given the preferential expression of MAGE-D in Sertoli cells, we cannot exclude a paracrine action of Leydig-cell factors on the Sertoli cells, causing increased MAGE-D expression by the latter. Moreover, we cannot totally exclude a direct action of PRL on Sertoli cells because the presence of PRL receptor in Sertoli cells has been recently reported (51). Because the main action of LH and PRL on MLTC-1 cells is to stimulate steroidogenesis, we have checked that neither testosterone (1 and 0.1 µM) nor estradiol (1 and 0.1 µM) can stimulate MAGE-D expression in cultured Sertoli cells during 6 h of treatment. Moreover, the lack of effect of testosterone on MAGE-D expression was confirmed by treatment of hypox rats with this hormone. The effect might be caused by other LH- and PRL-dependent paracrine factors. This hypothesis was checked by treatments of Sertoli cells with culture medium conditioned by LH- and PRL-stimulated MLTC-1 cells. In our experimental conditions, we did not observe any variation of MAGE-D expression in Sertoli cells (data not shown). These results clearly indicated that MAGE-D expression in Sertoli cells was not indirectly regulated by protein paracrine factors produced in the interstitium after LH or PRL stimulation. The increased expression of MAGE-D in the testes of hypox rats treated by these hormones appeared essentially due either to the direct effect of LH and PRL on interstitial cells or to regulatory factors involving germ cells or peritubular cells.

In conclusion, it is probable that MAGE-D plays different biological roles during the differentiation and the development of the testis and serves to maintain function in the mature organ. MAGE-D belongs to a family of genes for which the other members are expressed only in the germinal cells. Therefore, it would be interesting to study the roles of all the members of the family in the reproductive function of the male. In addition, similar studies should be carried out in females as we have shown significant expression of MAGE-D in the ovary.


   Acknowledgments
 
We are grateful to Dr. J. Poncin for sequencing reactions. We thank Dr. P. Durand (Lyon, France) for sorting rat germ cells.


   Footnotes
 
1 This work has been supported by personal grants from the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture, the Fondation L. Fredericq, the Fonds National de la Recherche Scientifique (Convention No. 3.4551.91). The nucleotide sequence for rat MAGE-D cDNA has been deposited in the EMBL bank under EMBL Accesssion number AJ33038. Back

Received February 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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