| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
Institut National pour la Santé et la Recherche Médicale INSERM U-407,2 Faculté de Médecine Lyon-Sud, Oullins Cedex, France
Institut de Physiologie,3 Université de Lausanne, Lausanne, Switzerland
Department of Biochemistry,4 St. Jude Children's Research Hospital, Memphis, Tennessee 38105
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and TGFß also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFß and TNF, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell–germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.
cytokines, follicle-stimulating hormone, gametogenesis, male reproductive tract, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In most cells, a family of specific MCTs (monocarboxylate transporters), named for their characteristic substrate specificity for short-chain monocarboxylates [6], are largely responsible for the transport of L-lactate and other monocarboxylates across the plasma membrane. Several isoforms of MCT have been characterized, and their tissue distribution has been described. The first MCT, designated MCT1, was cloned from Chinese hamster ovary cells. It is a membrane-bound protein with 12 predicted transmembrane regions. MCT1 homologues were later cloned and sequenced from mouse, rat, and human and found to be highly conserved between species [7–10]. A second isoform of MCT, termed MCT2, was cloned from a Syrian hamster liver library and has approximately 60% sequence homology with MCT1 [7]. Jackson et al. [11] reported the rat MCT2 sequence and presented evidence that MCT2 distribution in hamster is quite different from that in rats and mice. Price et al. [12] have reported the identification of new human MCT homologues in the database of expression sequence tags as well as the cloning and sequencing of four new full-length MCT-like sequences from human cDNA libraries, which they have named MCT3, MCT4, MCT5, and MCT6. More recently, Lin et al. [13] reported the cloning of the human monocarboxylate transporter MCT2, which exhibits a high affinity for the transport of pyruvate, suggesting that it is a primary pyruvate transporter in man. Direct demonstration of proton-linked lactate and pyruvate transport has been shown for mammalian MCT1–MCT4. For MCT1 and MCT2, further detailed analyses of substrate and inhibitor kinetics were described following heterologous expression in Xenopus laevis oocytes. In this study, the authors have shown that the uptake of lactate strongly increased with decreasing pH and that monocarboxylate transport via MCT2 could be inhibited by -cyano-4-hydroxy-cinnamate, anion-channel inhibitors, and flavoids [14]. Although various tissues expressed MCTs, little if anything is known about their expression and hormonal regulation during mouse testis development.
The aim of the present study was to characterize the expression and the potential regulation of MCT2 expression throughout spermatogenesis. Our results support evidence for a developmentally regulated and germ cell–specific expression of MCT2 under the control of gonadotropins and testicular growth factors.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
OF-1 mice and 35-day-old hypophysectomized Sprague-Dawley rats were obtained from IFFA CREDDO (Lyon, France). White spotting (W/Wv) mice were generously provided by I. Adham (Institut für Humangenetik, Gottingen, Germany). Ovine FSH (oFSH-19-SIAFP, lot AFP4 117A) was a gift from Dr A. F. Parlow (NHPP, Torrance, CA). Dulbecco's modified Eagle's medium (DMEM)/Ham F-12 medium (1:1) was obtained from Life Technologies (Eragny, France). Collagenase/dispase, Sal I, positively charged nylon membrane, CPD Star, Dig RNA labeling kit, blocking reagent, and anti-DIG alkaline phosphatase (AP) antibody were obtained from Roche Molecular Dynamics (Meylan, France), and human recombinant Tumor Necrosis Factor alpha (TNF) was obtained from Pepro Tech (Canton, MA). Testosterone, insulin, transferrin, -tocopherol, Hepes (4-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), anti-actin antibody, and deoxyribonuclease type I (Dnase I) were purchased from Sigma Chemical Co. (St. Louis, MO). TRIzol Reagent was purchased from Life Technologies (Cergy Pontoise, France). Radioactive [-32P]UTP (3000 Ci/mmole) and [-32P]dCTP (3000 Ci/mmole) were obtained from ICN (ICN Pharmaceuticals, Orsay, France). The mouse MCT2 antisens and sens cDNAs and the polyclonal rabbit MCT2 antibodies were obtained from P.J. Magistretti (Université de Lausanne, Switzerland). The antibody diluant and the biotinylated anti-rabbit secondary antibody were obtained from DAKO (Copenhagen, Denmark). The Bam HI/Eco RI double digest 18 S cDNA probe was a gift of Ann Ferguson (University of Texas Medical Branch at Galveston, TX). LH was obtained from Organon (Eragny, France) and FSH from Serono (London, United Kingdom). All studies on animals were conducted in accordance with current regulation and standards approved by the INSERM animal care committee.
Animal Treatments
Sprague-Dawley rats hypophysectomized at 35 days of age were obtained from Iffa Credo (Lyon, France). Treatments began just after the hypophysectomy. Groups of at least three rats were treated once daily at 0900–1000 h by a subcutaneous injection for 2 days with one of the following: 0.5-ml vehicle; 75 UI FSH or 200 UI LH. Rats were killed at the end of treatment by CO2 inhalation.
Isolation of Different Types of Testicular Germ Cells
Testes were obtained from 7–8-wk-old mice. Cell suspensions were prepared by trypsinization and purified using an elutriation approach. Each testis was stripped of its tunica albuginea and chopped into 1–2-mm sections with a razor blade. The tissue was incubated in PBS containing 0.1% glucose, 0.1 mg/ml trypsin, and 0.02 mg/ml of Dnase I at 32°C with constant stirring for 20 min. Trypsin was inhibited by the action of fetal calf serum (FCS) added to a concentration of 8%. The suspension was filtered through a 37-mm nylon screen into an iced test tube. The elutriator rotor was driven by a Beckman (Palo Alto, CA) J 6-MC preparative centrifuge. A peristaltic pump was used to move the buffer (0.5% bovine serum albumin in PBS at pH 7.2) through the system. The flow rate of the buffer is measured before each elutriation experiment, and the length of time required to collect each sample was recorded. The elutriation of testicular germ cells was performed at a centrifuge-well temperature setting of 4°C. The assembled rotor and the reservoir of buffer were cooled to 4°C, and the entire system was filled with buffer before the cell suspension was added to the mixing chamber. Between 3 x 108 and 3 x 109 cells in 10–70 ml of buffer were injected into the mixing chamber. The first collected fraction was the one that flowed through the separation chamber during the loading period (usually 10 min). Its volume was 200 ml. The second and all subsequent fractions were 150 ml. Cell purity was determined by morphological examination using phase-contrast optics, and cell death was monitored by trypan blue exclusion.
Seminiferous Tubule Purification
Albuginea from the testes of 30-day-old mice were removed, and testicular tissue was mechanically dispersed with forceps in DMEM/F12 medium containing 0.05 mg/ml of DNase I and further subjected to collagenase dissociation. The testicular tissue was incubated with 0.5 mg/ml collagenase/dispase, 0.05 mg/ml DNase I, and 2% FCS in DMEM/F12 medium, 40–60 min, at 32°C, through mild stirring. At the end of the enzymatic dissociation, testicular cells were submitted to gravity sedimentation (3–5 min). The seminiferous tubules were recovered from the pellet and washed three times by gravity sedimentation with DMEM/F12 medium, then centrifuged 10 min at 200 x g. Seminiferous tubules (1–2 mm) were plated in 6-cm Petri dishes and cultured for 48 h at 32°C in a humidified atmosphere of 5% CO2, 95% air in DMEM/Ham F-12 medium (1:1) containing sodium bicarbonate (1.2 mg/ml), and 15 mM Hepes and gentamicin (20 µg/ml). This medium was supplemented with insulin (2 µg/ml), transferrin (5 µg/ml), and -tocopherol (10 µg/ml). No significant germ cell degeneration was observed under these conditions as monitored by blue trypan exclusion assay.
RNA Isolation and Northern Blotting Analysis
Total RNA was isolated from mouse testes using TRIzol reagent as described elsewhere [15]. For Northern blot analysis, about 20 µg total RNA from each point was separated by electrophoresis on a denaturating 1.2% agarose gel containing 2 M formaldehyde and subsequently transferred onto nitrocellulose membrane (Hybond-C extra, Amersham, Orsay, France) for use with radioactive probe or nylon membrane (Roche) for nonradioactive probes. MCT2 probe was labeled with 50 µCi of [-32P]UTP using a Riboprobe combination system kit (Promega, Charbonnières, France). 18S cDNA probe was labeled with [-32P]dCTP as described recently [15]. Hybridization was performed overnight at 65°C with MCT2 riboprobe in 50% formamide, 5x SSPE (0.9 M NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4), 5x Denhardt solution (1 g Ficoll, 1 g polyvinylpyrrolidone, 1 g/L BSA), 1% SDS, and 100 µg/ml yeast tRNA. Filters were then washed twice with 2x SSC/0.1% SDS at 65°C for 15 min and one with 0.1x SSC/0.1% SDS at 65°C for 15 min. Filters were exposed to Kodak X-OMAT films (Eastman Kodak, Rochester, NY) for 1–2 days at -70°C. The nonradioactive probes (MCT2 and 18S DIG-labeled riboprobes) were transcribed in vitro using T7 polymerase according to the manufacturer's recommendations. The nylon membranes were prehybridized 1 h at 68°C and hybridized with DIG-labeled probe (MCT2: 100 ng/ml or 18S: 20 ng/ml) overnight at 68° C in the same conditions as radioactive probes. Afterward, membranes were washed as described previously, equilibrated for 1 min in buffer 1 (maleic acid 100 mM, NaCl 150 mM, pH 7.5), and blocked 30 min in buffer 1 containing 1% blocking reagent. The antibody (anti-DIG AP) was diluted (1:10 000) in blocking solution and incubated for 30 min with the filters. The membranes were washed twice in buffer 1 (15 min, RT) and equilibrated in detection buffer (Tris 100 mM, NaCl 100 mM, pH 9.5). Detection was performed with CPD Star chemiluminescent substrate solution at a 1:100 dilution in detection buffer (5 min, at RT). Filters were autoradiographed 15 min. After stripping the filters in formamide 80%, Tris-HCl pH 8 50 mM, SDS 1%, 2 x 30 min at 75°C, they were hybridized with an 18S RNA DIG-labeled probe as a loading control. Intensities of autoradiographic bands were quantified by densitometric scanning using the Intelligent Quantifier Software (Bio-Image, Cheshire, UK).
In Situ Hybridization
The same MCT2 RNA probe was used for northern blotting and in situ hybridization. Paraffin sections of 4% PAF-fixed testis were cut onto silanized slides. The samples were deparaffinized and rehydrated in PBS. The sections were permeabilized by incubating 8 min at room temperature with proteinase K 1/25° (DAKO, Trappes, France) in Tris 0.05 M pH 7.5. The sections were washed 2 x 5 min in SSC x 2, then prehybridized 2 h at 58°C in hybridization buffer (formamide 50%, SSC x 2, Denhardt x 5, tRNA 50 µg/ml, salmon sperm DNA 250 µg/ml, dextran 2.5%). The sections were incubated overnight at 58°C in the hybridization buffer containing 200 ng/ml of sense or antisense MCT2 DIG-labeled RNA probe (see the Northern Blotting Analysis section). At the end of incubation the sections were washed successively in SSC x 2 at 37°C 2 x 15 min, SSC x 1 at 37°C 2 x 15 min. The sections were incubated with RNAse A 20 µg/ml 30 min at 37°C, and then the sections were washed 2 x 30 min in SSC x 0.1/formamide 50%. Immunodetection of the hybridized probes was performed at room temperature by using the same buffers as described in the Northern Blotting Analysis section. The slides were incubated 5 min in buffer 1, then 30 min in buffer 2. After incubation of the section with anti-DIG antibody (1/500°) 30 min at 37°C, the sections were washed twice 10 min in buffer 1. The sections were then incubated 5 min in buffer 3 before incubation 1 h at 37°C with the NBT/BCIP chromogen (Roche, Meylan, France). The slides were mounted in aqueous mounting medium.
Western Blotting Analysis
Fresh whole testes or testicular cell protein extracts were prepared by direct addition of 5 volumes of cold lysis buffer to the samples. Lysis buffer consisted of 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, and 50 mM NaF and was supplemented immediately prior to use with a cocktail of protease inhibitors (ICN, France). The protein concentration of the tissue lysates was determined using the BioRad (Marnes-la-Coquette, France) protein assay. Protein samples were resolved by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Hybond C) at 100 V for 60 min using 25 mM Tris, 185 mM glycine (pH 8.3) containing 20% methanol. The membrane was blocked by soaking in 1x TBS, 0.05% Tween 20, and 5% nonfat dried milk for 1 h; incubated with the 1:5000 diluted primary antibody overnight at 4°C; washed three times (15 min) with TBST (1x TBS, 0.05% Tween 20); incubated with 1:2500 diluted secondary antibody (horseradish peroxydase-conjugated goat anti-Ig G rabbit, CovalAb, Lyon, France) for 1 h; and washed three times with TBST. Bound antibodies were detected by chemiluminescence using a CovalAb kit. Membranes were stripped 30 min at 70°C in Tris-HCl 62.5 mM (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol. The membranes were then washed twice in TBS and blocked in TBS/5% nonfat dried milk before incubation with the anti-actin antibody (1:500) to check the protein loading.
Immunohistochemistry
Paraffin sections of Bouin-fixed testis were cut onto silanized slides. The samples were deparaffinized (xylene 2 x 5 min) and rehydrated in graded ethanol (100° 2 x 1 min; 95° 2 x 1 min; 70° 2 x 1 min) and in PBS. Unmasking antigen procedure was done by incubating the slides in pH 6 citrate buffer (0.01 M) for 40 min at 97°C and leaving them in the hot buffer for 20 min at room temperature. The Basic DAB Detection Kit Ventana was then used as recommended by the manufacturer at 37°C in automated Nexes module (Ventana Medical System, Tucson, AZ). Briefly, after endogenous peroxidases were quenched in 3% H2O2 solution for 4 min, the MCT2 mouse primary antibody was diluted 1:6000 in antibody diluent and incubated for 32 min. Biotinylated anti-rabbit secondary antibody was applied for 8 min, and after washing avidin-horseradish peroxidase complex was applied for 8 min. DAB was used as peroxidase chromogen for 10 min. Between each step, the slides were washed three times with PBS x 1. Sections were then briefly counterstained with Harris hematoxylin, dehydrated, and mounted in mounting medium. For negative control slides, the primary antibody was replaced either by the antibody diluent or by nonimmune rabbit serum.
Electron Microscopy
Small fragments of fresh mouse testes were fixed in 0.1 M cacodylate buffer (pH 7.6) containing 4% paraformaldehyde and 0.05% glutaraldehyde for 20 min at 4°C. After blocking of aldehyde residues with 0.05% NaBH4 and 0.1% glycine in phosphate buffer for 10 min, nonspecific staining was minimized with phosphate buffer containing 0.5% acetylated bovine serum albumin and 0.1% gelatin (PBG buffer) for 1 h at room temperature. Tissues were then incubated overnight with MCT2 antibody diluted 1:5000 in PBG buffer at 4°C in a moist chamber. PBG buffer alone or rabbit preimmune serum instead of primary antibody was used as a control.
After washing in PBG buffer, tissues were incubated in 0.8-µm gold particles conjugated F(ab')2 goat-anti-rabbit IgG (Aurion, Wageningen, Netherlands) diluted 1:80 in PBG buffer for 5 hours at 37°C in a moist chamber. After washing, tissues were fixed in 2.5% glutaraldehyde, postfixed in 0.5% OsO4, and carefully washed in distilled water. A silver enhancement was then performed (Aurion) for 25 min in the dark and at 26°C. Tissues were rinsed in distilled water and then contrasted with 0.5% uranyl acetate in 50% ethanol for 20 min at 4°C. Testes fragments were then classically dehydrated in graded ethanol and in propylene oxide, embedded in Epon, and polymerized for 3 days at 60°C. Ultra thin sections were then picked up on nickel grids and observed after a brief lead citrate staining with an Elmiskop 102 Siemens (Munich, Germany) electron microscope at 80 kV.
Data Analysis
All experimental data are presented as the mean ± SD of triplicate determinations within each treatment group. All experiments reported here were repeated at least three times. A representative experiment of each series is presented.
For statistical analyses, one-way ANOVA (analysis of variance) was performed to determine whether there were differences between all groups (P < 0.05), and then Bonferroni/Dunn posttest was performed to determine the significance of the differences between pairs of groups. A P-value below 0.05 was considered significant. The statistical tests were performed on StatView software version 5.0 (SAS Institute Inc, SAS Campus Drive, Cary, NC) on a Macintosh computer.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To examine the developmental pattern of MCT2 mRNA levels during mouse spermatogenesis, total RNAs from prepubertal to sexually mature mice testes were analyzed by Northern blot. We detected two transcripts of MCT2 of approximately 2.0 and 2.4 kb. The 2.4-kb transcript was first detected in the testis at Day 18 of postnatal (pn) development. The 2.0-kb transcript was observed at Day 25 of pn development. The amounts of MCT2 mRNA increased progressively with postnatal development (Fig. 1A).
|
To confirm the specific MCT2 expression in the germ cell compartment, a different approach was used. First, use was made of germ cell-deficient mice such as c-Kit (i.e., stem cell factor receptor)-deficient mutant mice (W/Wv) that are devoid of meiotic and postmeiotic (i.e., spermatids) germ cells but have the normal complement of somatic cell types, including Leydig cells, Sertoli cells, and peritubular myoid cells (reviewed in [16]). No MCT2 transcripts were detected in testes from W/Wv mice, whereas comparable amounts of MCT2 transcripts were detected in testes from wild-type adult mice as well as from the Olt and Qk mutant mice, whose testes are only devoid in spermatozoa (Fig. 1B). Second, in situ hybridization experiments, in adult (90 days) testes, show the presence of the MCT2 mRNA in spermatocytes and round spermatids, but no expression of MCT2 was detected in somatic cells or in elongating spermatids (Fig. 2, A and B). The sens MCT2 probe used at the same concentration induced no staining (Fig. 2C). Together, the results in Figures 1 and 2 suggest that expression of MCT2 initiates at the stage of spermatocyte formation and continues through spermatid formation.
|
Next, we addressed if MCT2 mRNA was translated into protein in germ cells. Total proteins from purified populations of pachytene spermatocytes, and round spermatids were analyzed by Western blotting (Fig. 3A). We detected a 40-kDa protein that was described in the rat testis as MCT2 [11]. It was abundantly expressed in round spermatids but not in pachytene spermatocytes. Finally, the developmental expression pattern of MCT2 protein shows that MCT2 was detected as early as 24 days pn and was expressed at high levels from the 32-day pn (Fig. 3B).
|
Immunolocalization of MCT2 within the Testis
To further determine the cellular localization and to establish the stage at which MCT2 is translated into protein within the seminiferous tubules, an immunohistochemistry approach was performed on testis sections from prepubertal and sexually mature mice. MCT2 protein (Fig. 4, a–c) was not expressed in testis from 18, 21, and 23 days pn. At 25 days pn, a few seminiferous tubules show a positive staining for MCT2, specifically in the elongating spermatids (Fig. 4d). By 60 days pn (Fig. 4, e–g), a strong MCT2 staining was observed overlying the adluminal region of the seminiferous epithelium except in tubules at stages IX–X, suggesting a stage-specific expression of MCT2 (Fig. 4g). MCT2 appeared specifically localized to the distal part of sperm tails (Fig. 4f). MCT2 was not detected in spermatogonia or in somatic cells, including Leydig cells, Sertoli cells, and peritubular myoid cells.
|
In order to gain insight into the subcellular localization of MCT2 protein, immunolocalization was performed using electron microscopy. Silver-enhanced gold particles were detected at the outer face of the cell membrane of spermatid tails (Fig. 5). They were localized at the distal side of the principal piece and at the terminal piece (Fig. 5a). Immune label was found neither in the middle piece nor in the proximal side of the principal piece (Fig. 5b).
|
All together, these observations indicate that MCT2 protein is localized to elongated spermatids as it appears in 25-day-old mice, 7 days after the appearance of MCT2 mRNA.
In Vivo and In Vitro Regulation of MCT2
The ability of hormones to control germ cell development prompted us to examine their role in the regulation of MCT2 mRNA expression using surgically hypophysectomized rats treated or not with different hormones including LH and FSH. This model was used because it is possible to perform surgical hypophysectomy more easily in rats than in mice. In total RNA isolated from 35-day-old hypophysectomized and sham-hypophysectomized rat testes, two MCT2 transcripts (2.0 and 2.4 kb) were detected by Northern blotting analysis as was evident in mouse (Fig. 6). Two days after hypophysectomy, the levels of MCT2 (2.4 kb) transcript exhibited an 8-fold increase when compared to intact animals (Fig. 6). Hypophysectomized rats treated with LH, FSH, or both exhibited significantly reduced levels of MCT2 mRNA, comparable to those observed in untreated intact rats.
|
To further delineate the molecular target of hormone withdrawal through hypophysectomy on MCT2 expression, we used, as an in vitro model, mouse seminiferous tubules cultured in the presence of hormones or testicular growth factors. In a first set of experiments, we added to the culture testosterone or FSH. Both hormones reduced the MCT2 mRNA (2.4 and 2.0 kb) levels. The data in Figure 7 (histograms) show the similar changes in MCT2 2.4 and 2.0-kb transcript levels. Indeed, addition of testosterone showed an inhibition of MCT2 mRNA levels in a dose-dependent manner with a maximal inhibitory dose of 55.5 ng/ml (P < 0.0001) (Fig. 7A). FSH inhibited MCT2 mRNA levels at doses as low as 2.2 ng/ml (P < 0.0004) with a maximal effect obtained with 6.6 ng/ml (P < 0.0001; Fig. 7B).
|
In a second set of experiments, we tested the action of local testicular growth factors, TNF and TGFß, known to regulate lactate metabolism in the testis [15, 17]. The data in Figure 7 show similar changes in MCT2 transcript (2.4 and 2.0 kb) levels. TGFß inhibited significantly (P < 0.0003) MCT2 mRNA levels at 0.37 ng/ml, and a maximal effect was obtained at 10 ng/ml (P < 0.0001; Fig. 7C). TNF inhibited MCT2 mRNA levels with a significant (P < 0.0048; Fig. 7D) and maximal effect at 6.6 ng/ml.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The developmental expression of MCT2 was examined in terms of both mRNA and protein levels in postnatal mouse testes. Two different MCT2 mRNA transcript sizes (2.0 and 2.4 kb) were detected in the adult animals. These MCT2 transcripts are developmentally regulated during the postnatal mouse testis development since the 2.4 kb was detected from the 18th pn day, whereas the 2.0-kb transcript was detected (although at lower intensity) from Postnatal Day 25. The meaning of the appearance of these two transcripts specifically in terms of their translation into active proteins during testis development remains to be clarified. The data from the ontogeny study could suggest that MCT2 mRNA appeared in postmeiotic germ cells, as MCT2 mRNA was first detected at Day 18 pn, when spermatocytes begin to differentiate into round spermatids. Indeed, this expression pattern of the MCT2 gene is concomitant with the temporal appearance of haploid round spermatids in the seminiferous epithelium of mouse (approximately 1%, 4%, and 10% of total spermatogenic cells differentiate into the round spermatids at the 18th, 20th, and 22nd pn day, respectively [1]). However, in situ hybridization experiments in adult testis suggested the presence of MCT2 mRNA in spermatocytes and round spermatids. The possibility exists that MCT2 mRNA is first expressed in postmeiotic (round spermatids) germs cells at 18 days pn and that in adult testes the expression of MCT2 mRNA appears in spermatocytes (induced by the presence of spermatids?). At the present time, we do not know which of the two transcripts (2.4 and 2.0 kb) is translated into the 40-kDa MCT2 protein. However, it seems possible that the 2.4-kb transcript is the mRNA that is translated into MCT2 protein. Indeed, based on the data from other laboratories, 2.3- and 2.0-kb MCT2 transcripts were detected in the rat testis, whereas only a 2.3-kb transcript was observed in the liver and brain. Western blotting experiments in the testis, brain, and liver revealed in these tissues a 40-kDa band for MCT2 protein [11, 18]. These results suggest that the 2.3/2.4-kb MCT2 transcript could be translated into the 40-kDa MCT2 protein. Moreover, we reported here that the MCT2 gene is transcribed in spermatocytes and round spermatids and that the protein is translated later in elongated spermatids. Indeed, 1) MCT2 protein was detected as early as Day 24 by Western blotting and Day 25 by immunohistochemistry, and 2) MCT2 protein is present in purified spermatids but not in pachytene spermatocytes. The apparent delay between the appearance of MCT2 transcripts around Day 18 and the initial detection of the protein at Day 24 suggest that MCT2 could be subject to translational regulation. It is possible that MCT2 protein is synthesized from mRNA that was synthesized several days earlier and stored in a translationally inactive state. The synthesis and the storage of mRNAs prior to their translation is a necessity during spermatogenesis, as global transcription ceases several days prior to the completion of spermatid differentiation. For example, transcription of the protamine 1 and 2 gene expression and the transition protein 1 and 2 genes is detected only in early spermatids, at which time these mRNAs accumulate to very high levels. Translation of these mRNAs occurs several days later (reviewed in [19]).
The concept that Sertoli cells metabolize glucose to lactate to be used by postmeiotic germ cells arose because of the capability of Sertoli cells to produce high amounts of lactate and the efficient use of lactate and pyruvate but not of glucose by germ cells. In this context, the specific expression of MCT2 in the postmeiotic germ cells is consistent with the exogenous lactate-dependent metabolism of these cells. Similar examples of metabolic cooperation involving lactate as an exchangeable energy metabolite exist, particularly in the brain and in the retina. Previous observations have suggested that MCT2 is expressed in tissues, such as the liver or heart, which mainly consume lactate, whereas MCT1 is expressed, for example, in the muscle, which is believed to preferentially release lactate [20]. This differential cellular distribution of MCT1 and MCT2 was also observed in the brain [18, 21, 22], where astrocytes may supply neurons with lactate under conditions of increased neuronal energy demand (reviewed in [23]). Similarly, during ocular development, lactate produced in the Müller cells by glycolysis is transported out of the cells and is used by the photoreceptor cells to fuel oxidative phosphorylation [24]. A differential distribution of MCT1 and MCT3 has been reported in the retina [24, 25].
Several studies have indicated that the expression of genes coding proteins involved in lactate production in somatic and germ cells (LDH A) and those for the use (LDH C) and the transport of lactate in postmeiotic germ cells (MCT2) increase in the developing mouse testis, specifically with the appearance of lactate-consuming haploid germ cells. Indeed, using highly purified isolated rat testicular cells, Bajpai et al. [26] reported that the dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilize lactate, pyruvate, or both while retaining a better ability to utilize glucose. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes than did the testicular germ cells [26]. In a different study, it has been reported that in cryptorchid testes, spermatids and spermatocytes are lost between 3 and 8 days, while in cryptorchid testes supplemented with lactate, elongated spermatids persist in a few seminiferous tubules at Day 15. The authors suggested that lactate supplementation supports spermatocyte development and delays the degeneration of spermatids [27]. Indeed, lactate could be one of the survival factors produced by Sertoli cells for germ cells since lactate effectively inhibits germ cell apoptosis in the human testis in a dose-dependent manner [28].
One could argue that among the reasons differentiated spermatids express MCT2 in their tails is that the advanced spermatogenic cells have specialized to utilize exogenous lactate for ATP production and that these cells have largely or completely given up the capacity to metabolize other energy-yielding substrates. Another explanation might be that postmeiotic germ cells show biosynthetic activity that preferentially takes place around the nucleus and in the midpiece where the endoplasmic reticulum and the Golgi apparatus are concentrated. Biosynthesis activity requires both energy and carbohydrates, which may be produced by glycolysis.
Concerning the regulation of the MCTs expression in vivo, there are, to our knowledge two reports. The first one is related to the work of Jackson et al. [11], who did not detect any consistent increase in either MCT1 or MCT2 mRNAs and proteins in the liver after starvation of rats or hamsters. The second report originates from Leino et al. [29], who have shown that MCT1 is more abundant (15–25-fold) by using a quantitative electron microscopic immunogold study in brains from 17-day-old suckling rats when compared to those from adult rats. It is reported in the present study that testicular MCT2 expression is also under the control of the endocrine/paracrine systems. We report herein that MCT2 expression is under the hormonal control because MCT2 mRNA levels increased dramatically 48 h after hypophysectomy (8-fold increase) as compared to that in sham-operated animals. The observed decrease in testicular MCT2 mRNA after administration of exogenous FSH or LH in hypophysectomized rats indicates that the decrease in gonadotropin levels triggers an increase in MCT2 mRNA amounts, while the presence of the hormones results in the reduction of the transporter amounts. Such an observation was also confirmed in vitro based on the culture of seminiferous tubules. Indeed, in this in vitro model, FSH or testosterone treatment resulted in the decrease of MCT2 mRNA levels. Since FSH and androgen (testosterone) receptors are expressed in Sertoli cells, we hypothesize that the hormonal action on MCT2 expression in spermatids is indirect. Two types of intermediates might be involved in these interactions between Sertoli cells and germ cells: 1) local regulatory factors originating from Sertoli cells under hormonal control that act through specific receptors expressed in germ cells (reviewed in [30, 31]) and 2) lactate itself, which is produced in Sertoli cells under FSH control. Although these potential Sertoli cell factors that regulate MCT2 expression remain to be identified, local growth factors and cytokines could appear as interesting candidates. Among testicular local factors, we identified TNF and TGFß as negative regulators of MCT2 expression in the testis, suggesting that they could act as potential hormonal relay on germ cells. As MCT2 in haploid germ cells is supposed to be involved in the uptake of lactate and as both MCT2 expression and lactate production are under hormonal control, we suggest the existence of a short regulatory loop between MCT2 and lactate amounts to ensure appropriate levels of lactate for germ cells. It is reasonable to suggest that the decrease in lactate levels may trigger, as a compensatory mechanism, an increase in MCT2 levels resulting in a maximal uptake of the energy substrate into germ cells. Conversely, the increase in lactate levels will induce a decrease in MCT2 levels to prevent an overaccumulation of lactate in the cells. Although such a hypothesis remains to be proved, the data related to the hormonal regulation of MCT2 presented here are compatible with it. Indeed, the increase in rat testicular MCT2 levels following the withdrawal of the gonadotropins (known to stimulate lactate production) might be related to the decrease in lactate levels that occurs after hypophysectomy. Conversely, the decrease in MCT2 levels both in vivo after the administration of the hormones, specifically FSH, which enhances lactate production [32, 33] to the hypophysectomized rats, and in vitro in cultures of seminiferous tubules incubated with FSH or testosterone remains compatible with the proposed hypothesis. Similarly, the decrease in MCT2 expression induced by TNF and TGFß could be related to the stimulatory effects exerted by the two factors on lactate production in Sertoli cells [17, 33].
In summary, we report herein a developmentally regulated expression pattern of MCT2 mRNA and protein in the mouse testis. The expression of MCT2 in the mouse testis during development is specifically localized to the differentiated germ cells. The presence of MCT2 in the principal piece of the elongated spermatids is in accordance with a high energy demand of these cells. MCT2 expression is shown to be also under the hormonal (FSH and testosterone) and local (TNF and TGFß) control. Together, these data and previous observations support that both the production and transport of lactate is a metabolic process occurring in the context of the interactions between Sertoli cells and germ cells and that such a process is dependent on the endocrine/paracrine systems controlling the testis function.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
5 Correspondence: Mohamed Benahmed, INSERM U-407, Faculté de Médecine Lyon-Sud, BP 12, F-69921 Oullins Cedex. France. FAX: (+33) 4 78 86 31 16; benahmed{at}grisn.univ-lyon1.fr
Received: 7 August 2002.
First decision: 5 September 2002.
Accepted: 8 May 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Jansen, M. Pantaleon, and P. L Kaye Characterization and Regulation of Monocarboxylate Cotransporters Slc16a7 and Slc16a3 in Preimplantation Mouse Embryos Biol Reprod, July 1, 2008; 79(1): 84 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Benbrahim-Tallaa, B. Siddeek, A. Bozec, V. Tronchon, A. Florin, C. Friry, E. Tabone, C. Mauduit, and M. Benahmed Alterations of Sertoli cell activity in the long-term testicular germ cell death process induced by fetal androgen disruption J. Endocrinol., January 1, 2008; 196(1): 21 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. X. L. Zhang, T. R. Searcy, Y. Wu, D. Gozal, and Y. Wang Alternative promoter usage and alternative splicing contribute to mRNA heterogeneity of mouse monocarboxylate transporter 2 Physiol Genomics, December 19, 2007; 32(1): 95 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M Schuh, B. Hille, and D. F Babcock Adenosine and Catecholamine Agonists Speed the Flagellar Beat of Mammalian Sperm by a Non-Receptor-Mediated Mechanism Biol Reprod, December 1, 2007; 77(6): 960 - 969. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Enoki, Y. Yoshida, J. Lally, H. Hatta, and A. Bonen Testosterone increases lactate transport, monocarboxylate transporter (MCT) 1 and MCT4 in rat skeletal muscle J. Physiol., November 15, 2006; 577(1): 433 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. El Chami, F. Ikhlef, K. Kaszas, S. Yakoub, E. Tabone, B. Siddeek, S. Cunha, C. Beaudoin, L. Morel, M. Benahmed, et al. Androgen-dependent apoptosis in male germ cells is regulated through the proto-oncoprotein Cbl J. Cell Biol., November 21, 2005; 171(4): 651 - 661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J Meachem, S. M Ruwanpura, J. Ziolkowski, J. M Ague, M. K Skinner, and K. L Loveland Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation J. Endocrinol., September 1, 2005; 186(3): 429 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Florin, M. Maire, A. Bozec, A. Hellani, S. Chater, R. Bars, F. Chuzel, and M. Benahmed Androgens and Postmeiotic Germ Cells Regulate Claudin-11 Expression in Rat Sertoli Cells Endocrinology, March 1, 2005; 146(3): 1532 - 1540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Brauchi, M. C. Rauch, I. E. Alfaro, C. Cea, I. I. Concha, D. J. Benos, and J. G. Reyes Kinetics, molecular basis, and differentiation of L-lactate transport in spermatogenic cells Am J Physiol Cell Physiol, March 1, 2005; 288(3): C523 - C534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Miki, W. Qu, E. H. Goulding, W. D. Willis, D. O. Bunch, L. F. Strader, S. D. Perreault, E. M. Eddy, and D. A. O'Brien Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility PNAS, November 23, 2004; 101(47): 16501 - 16506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Coles, J. Litt, H. Hatta, and A. Bonen Exercise rapidly increases expression of the monocarboxylate transporters MCT1 and MCT4 in rat muscle J. Physiol., November 15, 2004; 561(1): 253 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Pellerin and P. J. Magistretti Neuroenergetics: Calling Upon Astrocytes to Satisfy Hungry Neurons Neuroscientist, February 1, 2004; 10(1): 53 - 62. [Abstract] [PDF]
|
|
| ||||||||||
