Identification of the Leukemia Inhibitory Factor Cell Targets ... - BioOne

BIOLOGY OF REPRODUCTION 72, 602–611 (2005) Published online before print 10 November 2004. DOI 10.1095/biolreprod.104.034892

Identification of the Leukemia Inhibitory Factor Cell Targets Within the Rat Testis Isabelle Dorval-Coiffec,2 Jean-Guy Delcros,3 Harri Hakovirta,4,6 Jorma Toppari,5,6 Bernard Je´gou,2 and Claire Piquet-Pellorce1,2 INSERM U.625,2 Groupe d’Etude de la Reproduction chez l’homme et les mammife`res, Universite´ de Rennes I, 35042 Rennes cedex, France CNRS UMR6061,3 Ge´ne´tique et De´veloppement, Faculte´ de Me´decine, Universite´ de Rennes I, 35043 Rennes cedex, France Departments of Surgery4, Pediatrics,5 and Physiology6, University of Turku, FI-20014 Turku, Finland ABSTRACT

tiation, are still largely unknown. Several cytokines may be involved in the regulation of spermatogonial activity [3, 4]; these include the leukemia inhibitory factor (LIF), which presents a particular interest. LIF is a highly pleiotropic cytokine that belongs to a family of eight cytokines, including interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and interleukin 27 (IL-27), all referred as IL-6-related cytokines [5–7]. LIF action is mediated by its binding to a heterodimeric receptor (LIF-R) that includes the LIF-specific binding subunit (gp190) and the trans-membrane signal transducing subunit (gp130). This gp130 subunit is shared by all IL-6-related cytokines receptors and is essential for development and life; evidence of this is that deficient mice die by midgestation [8]. Because gp130 is fairly ubiquitous, the specificity of the binding and functional receptor complex formation depends on the expression of gp190, also called LIF-Ra [5]. Nevertheless, the LIF-Ra/gp190 subunit is also involved in several multimeric receptor complexes binding either OSM, CNTF, CT-1, or CLC. These cytokines using both gp190 and gp130 to transduce their signal are then called LIFrelated cytokines. LIF-Ra/gp190 subunit knockout mice die shortly after birth, suggesting its involvement in many physiological processes [9, 10]. IL-6-related cytokines exhibit an overlapping spectrum of action on many cell types [11] and specific activities that can be complementary, as exemplified in reproduction [12– 15]. Indeed, LIF-deficient mice fail to implant blastocysts [13, 16] and knocking out the ubiquitous IL-11Ra gene, which is specifically involved in IL-11 signaling, leads to a deficit in uterine decidualization reaction [14, 15]. In male reproduction, most of LIF-related cytokines have been detected in the testis, including IL-11 [17], OSM [18], IL-6 [19, 20], and LIF itself [21]. In vivo studies of mice overexpressing LIF first suggested that LIF was involved in the control of male reproduction. Indeed, mice engrafted with LIF-expressing FDCP-1 cells [22], transgenic male mice overexpressing LIF in T cells [23], and transgenic mice expressing LIF driven by the pituitary glycoprotein hormone a-subunit promoter [24] all display defective spermatogenesis. Those in vivo studies must be balanced by recent results by Molyneaux et al. showing that male mice whose gp130 was inactivated specifically in germ cells using a Cre-LoxP system were still fertile [25]. LIF is known to act on several types of stem cells originating from various organs. Indeed, LIF was first identified through its ability to maintain murine embryonic stem cells in their undifferentiated totipotent state, because it pro-

Leukemia inhibitory factor (LIF), a pleiotropic cytokine, is expressed in the rat testis and produced predominantly by peritubular myoid cells. The aims of this study were to characterize the testicular cell targets of LIF and to identify the role of LIF in the testis. The LIF receptor (LIF-R)/gp190 transcript was detected by reverse transcription-polymerase chain reaction (RTPCR) in the rat testis from Day 13.5 postcoitum until adulthood. Seven highly purified testicular cell populations, representative of the major testicular constituents, were studied at transcriptional and protein levels by, respectively, RT-PCR and flow cytometry with biotinylated-LIF. Spermatogonia and, to a lesser extent, the somatic cells, exhibited specific LIF-binding sites. These results were strengthened by in situ analysis, showing predominant LIF-R immunoreactivity in spermatogonia at all ages studied. In addition to the 190-kDa LIF-R, Western blot analysis revealed the presence of a 50- to 60-kDa C-terminal gp190 isoform. This truncated form, which is unable to bind LIF, was the only form expressed in meiotic germ cells, suggesting an original down-regulation process of LIF-R expression during spermatogenesis. Finally, we showed that LIF increased [3H]-thymidine incorporation in spermatogonia in microdissected, cultured seminiferous tubules. Taken together, our results strongly suggest that LIF has a role in the regulation of the spermatogonial cell compartment.

cytokines, meiosis, spermatogenesis, testis

INTRODUCTION

The production of spermatozoa is a dynamic and continuous process starting at puberty and lasting throughout adulthood [1, 2]. To that aim, the testis contains a limited stock of stem germ cells that are able to autorenew themselves, while some of them can be committed to a program of cell division followed by meiosis and terminal differentiation into spermatozoa. Correct control of the division and differentiation of spermatogonia, the diploid precursors of meiotic germ cells, is then essential for the maintenance of fertility. However, the mechanisms underlying this control, especially the balance between renewal and differenCorrespondence: Claire Piquet-Pellorce, INSERM U.522, Universite´ de Rennes 1, Hoˆpital de Pontchaillou, Rue Henri Le Guilloux, 35033 Rennes Cedex, France. FAX: 33 02 99 54 54 0137; e-mail: [email protected]

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Received: 25 August 2004. First decision: 13 September 2004. Accepted: 28 October 2004. Q 2005 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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motes their proliferation [26]. Besides its action on hematopoietic stem cells, LIF can also promote the proliferation of gonocytes [27] and murine primordial germ cells (PGCs) [28, 29] as well as their survival by preventing them from undergoing apoptosis [30]. Further evidence that LIF directly acts on PGCs was obtained from studies demonstrating that the LIF-R expressed by PGCs is required for their survival in vitro [31, 32]. Moreover, we recently demonstrated that LIF is expressed in the testis throughout gonadal ontogenesis and that large amounts of bioactive LIF are found in the testicular lymph, suggesting that this cytokine has a role in the testicular function beyond its involvement in PGC development in the embryo [21]. More precisely, we revealed that peritubular cells are the main LIF producer within testis. Peritubular cells lie outside the seminiferous epithelium at the interface with the interstitium; therefore, LIF should be available for both interstitial and tubular cell constituents, such as Leydig cells, Sertoli cells, and spermatogonia. These data prompted us to further investigate the LIF system by addressing the question of LIF targets and actions in the testis. Our strategy of investigation included the study of LIF-R expression in seven highly purified populations of testicular cells at both RNA and protein levels. These populations included the four somatic cell types and three germ cell populations that are representative of the major steps of the germ line development: diploid spermatogonia, primary spermatocytes with tetraploid DNA, and early haploid spermatids, which are the result of meiosis before their morphogenetic transformation into spermatozoa. We also used immunohistochemistry to investigate the modulation of LIF-R expression in the various seminiferous tubule stages and along ontogenesis. Furthermore, we used a fully characterized organotypic culture system allowing the maintenance of spermatogenesis in isolated seminiferous tubules to assay the LIF effect on germ cells. MATERIALS AND METHODS

Animals and Reagents Male Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest Saint Isle, France) and kept in sanitary conditions in accordance with the National Research Council (NRC) publication guide [33]. All investigations were conducted in accordance with French laws, which are in agreement with the NRC publication. Human chorionic gonadotropin (hCG) was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Dr. J.K. Heath (CRC, Birmingham University, U.K.) generously provided us with the pXmT2-mouse LIF expression vector. Mouse recombinant LIF (mr LIF) was prepared as a conditioned medium from Cos-7 cells that had previously been transfected with the pXmT2-mouse LIF expression vector [34]. Rabbit anti-murine/ human LIF-R/gp190 antibody (sc-659), the competitor peptide (sc-659P), rabbit anti-mouse gp130 (sc-656), and rabbit anti-human c-kit (sc-168) were purchased from Santa Cruz Biotechnology. The anti-CD163 antibody, alternatively called ED2, was obtained from Serotec. Secondary antibodies such as peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG were purchased from Jackson Laboratories (Immunotech, Beckman-Coulter).

Isolation and Culture of Sertoli Cells and Peritubular Cells Sertoli and peritubular cells were isolated from 20-day-old rats according to the method described by Skinner and Fritz [35] and modified by Toebosch et al. [36]. The isolated cells were then cultured for 4 to 7 days as described previously [21]. The resulting Sertoli cell preparations contained less than 1% peritubular cells and less than 0.1% germ cells. The peritubular cell populations were at least 96% enriched as assessed by alkaline phosphatase staining [37], and any contamination was detected

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by germ cells, Sertoli cells, Leydig cells, or macrophages (the remaining 4% of cells had a fibroblastic morphology). Both cell populations were recovered by use of trypsin and EDTA, and processed for RNA or protein extraction or for flow cytometry.

Isolation and Culture of Leydig Cells and Testicular Macrophages Highly enriched populations of Leydig cells and testicular macrophages were prepared from adult rat testes according to the procedure described by Klinefelter et al. [38]. More than 94% of the adherent cells in the macrophage-containing Percoll fraction were identified as macrophages as shown by nonspecific esterase-positive staining [39], anti-CD163 specific labeling [39, 40], and negative 3b-hydroxysteroid dehydrogenase (3bHSD) staining. The adherent Leydig cells were 94% pure as assessed by 3b-HSD staining [41]. The main contaminant was testicular macrophages (,4%). Both cell types then were collected and processed for either RNA or protein extraction or for flow cytometry. Alternatively, adherent Leydig cells were cultured further to assess testosterone production.

Germ Cell Preparation and Culture Testes from 90-day-old rats were trypsinized, differentially sedimented, and fractionated by centrifugal elutriation according to the method described by Meistrich et al. [42], leading to the recovery of highly enriched populations of primary pachytene spermatocytes (PS, $95% purity) and early spermatids (ES, $90% purity) [43]. The cell pellets were then directly processed for RNA or protein extraction or for flow cytometry. Spermatogonia were purified from 9-day-old rat testes using the method originally described by Bellve´ et al. [44] and modified by Dym et al. [45]. Briefly, the decapsulated testes were sequentially dissociated with various enzymes before sedimentation on a 2%–4% BSA gradient. A final differential adhesion step removed the few remaining contaminating somatic cells and enabled us to obtain a highly enriched population of spermatogonia (purity $94%, based on morphological criteria and c-kit membrane marker expression). These cells were then collected and processed for RNA or protein extraction or for flow cytometry.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis Total RNAs were extracted from the whole testis and from the different cell preparations by use of guanidium thiocyanate, followed by either centrifugation on a cesium chloride cushion or phenol extraction [46]. Complementary DNAs were prepared from 10 mg of RNA using 200 ng of random hexanucleotides (Amersham Pharmacia Biotech) and 200 U of Moloney murine leukemia virus-reverse transcriptase (M-MLV-RT) (Promega) according to the manufacturer’s instructions. A negative control was performed in parallel for each sample using the same reaction mixture without M-MLV-RT to check for genomic DNA contamination. Actin was amplified as a control for RNA quality, quantity, and good reverse transcription (data not shown). Polymerase chain reaction (PCR) was carried out as recommended by Perkin-Elmer starting with 100 ng of cDNA as a template. The sequence of the oligonucleotide primers used to amplify rat LIF-R/gp-190 were as follows: sense 148, 59-GAGGATGACTCCAAGC CTGC-39; antisense 673, 59-ATCTTTACCACTCAGCACTGTGTTG-39. These primers amplified a 526-base pair (bp) LIF-R/gp190 fragment (30 cycles, annealing temperature 648C), which was directly sequenced and shown to be 100% identical to the published rat LIF-R/gp190 sequence section, including the encoding nucleotides 148 to 673 [47]. Alternatively, another couple of oligonucleotide primers was used to detect rat LIF-R/ gp190 transcript: sense 2731, 59-AGTCTTCTCGCAGACACTCC39; antisense 3230, 59-GGACCTTGGGGAATCTGGGG39, which amplifies a DNA fragment homologous to the section that encompasses the nucleotides 2731 to 3230 encoding part of the intracellular region of the gp190. RNA samples obtained from three to eight independent cell preparations were analyzed for each cell type.

Detection of the LIF Receptor by Flow Cytometry LIF-binding sites were identified on the various testicular cell types by use of the fluorokine LIF receptor detection kit (reference NFLF0, including human recombinant LIF) according to the manufacturer’s instructions (R&D Systems). Briefly, 2 3 105 cells from each cell population were incubated for 1 h in the presence of either a control biotinylated protein or biotinylated human recombinant (hr) LIF (biot-LIF), or, as a specificity

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control, biot-LIF premixed with an anti-hr LIF antiserum. The streptavidin-FITC complex was added and the incubation was continued for a further 30 min. After extensive washing, cells were fixed with 3% paraformaldehyde and analyzed using a Coulter Epics Elite Flow Cytometer (Coultronics) equipped with an argon ion laser (excitation wavelength 488 nm). Debris and aggregates were excluded from the analysis on the basis of the forward-angle and the side-angle light scatters. Immunofluorescence histograms were generated by use of the immuno-4 analysis software (Coulter Corp.), which allows the subtraction of a control histogram (cells incubated with a control biotinylated protein or with the anti-LIF antiserum) from a test histogram (cells incubated with bio-LIF). A minimum of three independent cell preparations of each testicular cell population were analyzed. We have successfully used this biotinylated ligand approach to analyze the expression of SCF-R/c-kit on spermatogonia, a known marker of such cell population [45, 48]. These data, which are not shown here, validated both the method and the quality of the cell preparation. Another set of experiments was carried out with the following antibodies: rabbit anti-mouse/human LIF-R antibody (sc-659), rabbit antimouse gp130 (sc-656), and rabbit anti-human c-kit (sc-168). These three sera are directed against epitopes mapping at the carboxy terminus of these transmembrane receptors that correspond to their intracellular domains. Cells were then fixed and permeabilized using the appropriated reagents obtained from Becton Dickinson before proceeding to incubation with the primary antibody (2 mg/ml) for 1 h. Cells were then washed twice, and FITC-conjugated anti-rabbit IgG (10 mg/ml) was added for 30 min. After final washes, labeled cells were analyzed using the FacsCalibur Flow Cytometer (Becton Dickinson) equipped with an argon ion laser (excitation wavelength 488 nm). Debris and aggregates were excluded from the analysis on the basis of the forward-angle and the side-angle light scatters.

Protein Extraction and Western Blot Analysis Crushed frozen testis or isolated cell pellets were homogenized in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) using a syringe fitted with a 20gauge needle. After 30 min of centrifugation at 15 000 3 g (48C), the lysates were snap-frozen in liquid nitrogen and then kept at 2808C until use. Proteins from whole testis or purified cells (20 mg) were resolved on a 7.5% SDS-polyacrylamide gel (100 V, 2.5 h) and electrophoretically transferred (30 mA, 2 h) onto polyvinylidene difluoride membranes using Tris/glycine buffer (10 mM Tris-HCl, 96 mM glycine, 10% methanol pH 8.3). After an overnight incubation in blocking buffer (20 mM Tris-HCl, 150 mM NaCl; 5% skim milk, 0.1% Tween-20, pH 7.6), the membranes were first incubated with anti-LIF-R (0.1 mg/ml) for 3 h and then with peroxidase-conjugated secondary antibody (0.16 mg/ml) for 1 h in the same buffer. After washing, the labeling was detected by an enhanced chemiluminescence system according to the manufacturer’s instructions (ECL plus, Pharmacia). A control competition experiment was performed with anti-LIF-R that had been preincubated for 30 min with an excess of LIF-R peptide epitope (10 mg/ml).

Immunohistochemistry Analysis Testes were removed from 18.5-day postcoitum (dpc) fetuses or from 9-, 20-, or 90-day-old rats and fixed overnight in Bouin-Hollande solution before being dehydrated and embedded in paraffin wax. Sections (5 mm) were rehydrated and heated in an antigen-retrieval solution for 15 min as described by Cuevas et al. [49]. Endogeneous peroxidase was quenched by incubation with 3% H2O2 for 5 min, and nonspecific sites were saturated overnight with saline Tris buffer (20 mM Tris-HCl, 150 mM NaCl) supplemented with 5% skim milk. Sections were then incubated with antiLIF-R (2 mg/ml) for 3 h. After a second 1-h incubation with peroxidaseconjugated anti-rabbit IgG, bound primary antibodies were revealed by DAB chromogen (3,39-diaminobenzidine tetrahydrochloride; Sigma-Aldrich, France). The cells were counterstained with Masson hemalun and posttreated with saturated lithium carbonate. Dehydrated sections were then mounted in Eukitt (Labonord, France). Control experiments were performed either with rabbit IgG (2 mg/ml) or with anti-LIF-R that had been preincubated for 30 min with LIF-R peptide epitope (100 mg/ml).

Incorporation of [3H]-Thymidine by Isolated Seminiferous Tubule Segments Seminiferous tubule segments (2 mm long) were isolated under a transilluminating stereomicroscope, from 2- to 3-mo-old rat testes [50]. Stages I, V, VIIa, VIII–IX, and XII of the seminiferous epithelial cycle were

selected because they contain representative phases of mitotic and meiotic DNA synthesis [50]. They were identified by their specific transillumination patterns [51] and subsequently microdissected. Staged tubule segments were transferred into 10 ml of PBS in 96-well culture plates and then incubated at 348C in a 100-ml final volume of Hams F12-Dulbecco modified Eagle medium supplemented or not supplemented with 50 or 500 HILDA U/ml murine recombinant LIF for 24, 48, or 72 h (one HILDA unit corresponds to the amount of LIF inducing half-maximal proliferation of DA1-a cells [52]. During the last 4 h of culture, tubules were pulse-labeled by adding 20 kBq of [3H]-thymidine (185 Gbq/mmol; Amersham) and harvested on filter discs that were individually counted. Four separate experiments for each time point were assayed with four replicate samples. To count the proliferating cells, we performed autoradiography on squashed labeled tubule segments as previously described [53]. Cells were counted after 2 days of exposure and counterstaining with hematoxylin.

RESULTS

Detection of LIF-R Transcripts in the Rat Testis

The LIF-Ra/gp190 transcript was identified by RT-PCR in whole adult rat testis extracts (Fig. 1). When its ontogeny of expression was studied, LIFR mRNA was found at all ages investigated (Fig. 1A): 13.5 dpc (undifferentiated gonad), 16.5 dpc (gonocyte proliferation), 19.5 dpc and new born rats (gonocyte quiescence), 9 days postpartum (dpp) (spermatogonia differentiation), 20 dpp (meiosis onset), and 90 dpp (adult). Seven highly purified testicular cell populations were studied for LIF-Ra mRNA presence using the same RTPCR approach. These cell populations (purity 90%–98%) were representative of all the major cell partners involved in testicular function. They consisted of four somatic cell types (Sertoli cells, peritubular myoid cells, Leydig cells, and macrophages) and three germ cell populations corresponding to the major stages of germ line development (spermatogonia, primary spermatocytes, and early spermatids). Results shown in Figure 1B indicate that spermatogonia and the four purified somatic cell types express LIFRa mRNA. In contrast, no signal was detected in spermatocytes or spermatids. Identification of the Cellular Targets of LIF Within the Testis

To identify the putative LIF target cells, we searched for functional binding sites on testicular cells using a flow cytometry approach that we specifically developed, in which each of the purified testicular cell population was analyzed for its ability to bind biotinylated LIF (biot-LIF). As illustrated in Figure 2, the intensity of fluorescence of the somatic cells and spermatogonia was greatly increased compared with that of controls, when cells were incubated in the presence of biot-LIF plus streptavidin-FITC, showing that biot-LIF bound to these cells. The specificity of this binding was confirmed by carrying out neutralization procedures in each experiment involving the addition of protein A-purified anti-LIF antiserum to biot-LIF. The corresponding fluorescence profiles did not differ significantly from those of the controls, indicating that the binding of biot-LIF to the cells had been specifically blocked in the presence of anti-LIF. No differences in fluorescence profiles were consistently observed when primary spermatocytes or early spermatids were incubated with the control protein, with biot-LIF, or with biot-LIF plus anti-LIF antiserum. The results depicted in Figure 2 clearly show a homogenous LIF-R expression for each positive cell population (i.e., Leydig cells, Sertoli cells, macrophages, peritubular cells, and spermatogonia). The staining of each cell population


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was highly reproducible, as demonstrated by the mean percentage of labeled cells. To provide an indication of the number of binding sites per cell, the ratio between the average fluorescence intensity of cells with LIF, and the average fluorescence intensity of cells without LIF was calculated (Table in Fig. 2). The results suggested that spermatogonia are the testicular cell type that expresses the highest level of LIF-R even though all the cell populations assayed were not obtained from animals of the same age. The flow cytometry results fully agree with the RT-PCR results for LIF-R/gp190 (Fig. 1B). Taken together, these data demonstrate that spermatogonia, and to a lesser extent the four somatic testicular cell types, express functional LIF-R and that meiotic and postmeiotic germ cells (spermatocytes and spermatids) do not. Identification of a Truncated LIF-Ra Isoform on Germ Cells Undergoing Meiosis

Because the functional LIF-R is heterodimeric, we used flow cytometry to compare the abilities of anti-LIF-Ra and anti-gp130 antibodies, and of biot-LIF to label testicular cells. Spermatogonia, Sertoli cells, peritubular cells, Leydig cells, and macrophages were equally able to bind biot-LIF, anti-LIF-Ra, and anti-gp130. Surprisingly, spermatocytes and spermatids were labeled by anti-LIF-Ra but were unable to bind biot-LIF, as shown in Figure 3A. The anti-LIF-Ra antibody is a polyclonal IgG directed against a peptide epitope derived from the intracellular domain of the receptor; thus, these apparently contradictory results suggest that germ cells undergoing meiosis may express a truncated form of LIF-R devoid of its extracellular functional binding sites. This hypothesis was validated by a Western blot approach (Fig. 3B). Indeed, a 50–60 kDa LIF-Ra was revealed by the anti-LIF-Ra antibody in primary spermatocyte and spermatid extracts in the absence of the full-length LIF-Ra/gp190 form. Whole testis extracts exhibited both the 190 kDA and the 50–60 kDa forms, as did somatic cells and spermatogonia. Competition experiment performed with an excess of the peptide epitope demonstrated the specificity of both 50– 60 kDa and 190 kDa signals. To further support the hypothesis of a gp190 truncated form production by meiotic and postmeiotic germ cells, RT-PCR analyses of gp190 encoding mRNA were performed with two different couples of oligonucleotide primers designed to amplify a sequence encoding either a section of the extracellular domain or a section of the intracellular domain. As shown in Figure 3C, only the couple complementary to the intracellular domain encoding sequence (2731/3230) provided a signal in meiotic and postmeiotic germ cells, when both primer couples were efficient in whole testis extract. This result suggests that the meiotic and postmeiotic germ cells express a gp190 encoding mRNA truncated of its 59 end that could lead to the expression of a truncated LIF-R form devoid of its extracellular ligand binding domain. Expression Profile of LIF-R in the Male Gonad In Vivo

We used an immunohistochemistry approach with antiLIF-Ra to study the in situ expression of LIF-R and the ways in which it is regulated during the establishment of male reproductive function and in different stages of the seminiferous epithelium in the adult. Specific immunolabeling was observed in Sertoli cells at all ages studied (Fig. 4, a–c), although the signal was consistently higher in the adult testis (Fig. 4, c and h). With regard to peritubular cells, no labeling was observed at any stages or ages. The

FIG. 1. Identification of LIF-R transcripts in the testis and during testicular ontogenesis. Gel electrophoresis analysis of the RT-PCR products obtained using primers specific for LIF-R (couple sense 148, antisense 673; product size 526 base pairs). The control amplification performed with no DNA (0) is shown beside the molecular weight markers (M). A) Total RNA from the whole testis obtained on Days 13.5, 16.5, and 19.5 postcoitum newborns; and on Days 9, 20, 45, and 90 postpartum was amplified either following RT (1) or not following RT (2). B) Total RNA from pachytene spermatocytes (PS), early spermatids (ES), Sertoli cells (Sert.), peritubular cells (Peri.), Leydig cells (Ley.), testicular macrophages (Ma.), spermatogonia (Gonia), and whole adult testis (AT) was amplified either following RT (1) or not following RT (2). The results presented are representative of at least three independent RNA preparations for each age or cell type. The actin control amplification for each sample showed comparable signals.

interstitium was heterogeneously labeled at all ages studied (Fig. 4, a–c, f, and i). It is noteworthy that anti-LIF-Ra immunoreactivity was strong in spermatogonia throughout the entire period of postnatal testicular development. This labeling was more intense than Sertoli cell labeling, as shown by the signal persisting in spermatogonia even when the antibody concentration was decreased, whereas this signal was no longer detected in Sertoli cells (Fig. 4, e and g [0.5 mg/ml] versus Fig. 4, d and f [2 mg/ml]). An intense signal in gonocytes/ spermatogonia was also observed in 18 dpc fetuses and 7 dpp rat testes (data not shown). The most immature germ cells present in the 13.5 dpc fetal gonad have already been shown to express LIF-R by several groups [31, 32]. Differences in signal intensity were clearly noticed between spermatogonia subtypes (Fig. 4h); however, LIF-R expres-

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FIG. 2. Identification of LIF-binding sites on testicular cells. A typical fluorescence histogram is presented for each highly purified testicular cell population. Results are expressed as the number of cells for each fluorescence intensity leading to three profiles depending on whether the cells had been incubated with the biotinylated-control protein, biotinylated-LIF (biot-LIF), or biot-LIF plus anti-LIF antiserum. Cellular autofluorescence and results obtained when the cells were incubated in the presence of biot-LIF plus anti-LIF antiserum mostly overlap and thus are indicated as ‘‘controls.’’ The percentage 6 SEM of LIFR-positive cells in each cell population is indicated in the table along with the number of independent experiments performed within brackets. The ratio of the fluorescence intensity of the labeled cells to the fluorescence intensity of the unlabeled cells is also indicated (*P , 0.02, when values obtained from spermatogonia were compared with those obtained from either somatic cell type using a Student t-test).

sion did not appear to be modulated according to the seminiferous epithelial stage in the adult in any cell type. Our results indicate that the expression of LIF-R is not specific to primordial germ cells, but is a genuine property of gonocytes and spermatogonia that is maintained throughout testis development until adulthood. The specificity of the signal was demonstrated by appropriate controls, including rabbit IgG (which gave no signals), and competition with an LIF-R peptide immunogen (Fig. 4, j–n). The nonspecific signal observed in Figure 4, l and n, was associated with differentiating spermatids in the acrosomes, which are often unspecifically labeled. This, together with general Sertoli cell staining, made it difficult to analyze germ cells undergoing meiosis; however, meiotic and postmeiotic germ cells did not display any clear immunoreactivity.

The immunohistochemistry data were consistent with the flow cytometry data, with the exception of peritubular cells, which were labeled with biot-LIF but were not immunoreactive in situ. This can be explained by a greater sensitivity of the flow cytometric approach, or by a possible upregulation of the LIF-R expression in peritubular cells during the culture necessary to their recovery with a complete enrichment, or both. However, both techniques demonstrate that LIF-R was more highly expressed in spermatogonia than in somatic cells, strongly suggesting that spermatogonia are the major targets of LIF within the testis. Action of LIF on Spermatogonia

We analyzed the effects of LIF on stage-specific DNA synthesis by measuring the incorporation of [3H]-thymidine


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FIG. 3. Meiotic and postmeiotic germ cells expressed only a truncated LIF-R isoform. A) Comparison of the fluorescence histogram obtained with biotLIF and anti-LIF-R on premeiotic and meiotic germ cells by flow cytometry. Each control fluorescence histogram is included (i.e., obtained with a control biotinylated protein, or when preincubated with anti-LIF antibody and obtained when incubated with a control rabbit serum). The results presented are representative of three independent experiments performed for each cell type. B) Autoradiograph of a Western blot of total protein extracts (20 mg) obtained from whole adult rat testis or from different purified testicular cells using a polyclonal antibody directed against the c-terminal end of gp190 (0.1 mg/ml). The results presented are representative of those obtained with two or three independent protein preparations for each cell type. C) Gel electrophoresis analysis of the RT-PCR products obtained using two different couples of primers specific for LIF-R/gp190, sense148/antisense673 and sense2731/antisense3230, which are, respectively, complementary of an extracellular and an intracellular section of the encoding sequence for gp190. The control amplification performed with no DNA (0) is shown beside the molecular weight markers (M). Total RNA from pachytene spermatocytes, early spermatids, and whole adult testis was amplified either following (1) or not following (2) reverse transcription. The results presented are representative of at least three independent RNA preparations for each cell type. The actin control amplification for each sample showed comparable signals.

into cultured segments of seminiferous tubules. Such an organotypic culture system has been well characterized by researchers working with M. Parvinen and J. Toppari [50, 54]. In the described culture conditions, seminiferous tubules can be maintained over 3 days and exhibit functional meiosis during this period; the chosen stages develop through most of the stages of the seminiferous epithelium during culture. The incorporation of [3H]-thymidine into such an organotypic culture system can only be related to germ cell DNA synthesis. As shown in Figure 5A, LIF had no effect on stage-specific [3H]-thymidine incorporation in segments incubated for 24 h or 48 h. In contrast, when cultured for 72 h, LIF increased DNA synthesis in tubule

fragments that were at stages VIII–IX at the start of culture in a dose-dependent manner (Fig. 5A). Autoradiography of the cellular constituents of the labeled tubule fragments confirmed that LIF significantly increased the number of labeled germ cells at these stages (Fig. 5B). The labeled germ cells shown to be spermatogonia as stages VIII–IX developed to stage XII after 72 h when spermatogonia were the only cells incorporating [3H]-thymidine [50]. DISCUSSION

LIF is produced locally in the testis, its main source being peritubular cells. LIF is thus available to both inter-

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FIG. 4. Immunohistochemistry analysis of LIF-R expression in rat testes before and after puberty. Photographs of immunohistochemistry experiments performed on the testes of 9- (a, d, e), 20- (b, f, g), and 90- (c, h, l) day-old rats using a rabbit anti-LIF-R c-terminal antibody and revealed using antirabbit IgG coupled to peroxidase and DAB. The results obtained with two different doses of antibody are shown [i.e., 2 mg/ml (a–d, f, h, and i) and 0.5 mg/ml (e, g)]. Control experiments using the polyclonal antibody preincubated with peptide epitope (100 mg/ml) and 9-, 20-, and 90-day-old rats are shown at the bottom (j–n). Magnification 3200 (a–c, j, k, and m); 3400 (d–i, l, and n). The results are representative of similar experiments performed at least on three different embedded testes for each age.

stitial and tubular cells [21]. We then investigated LIF testicular cell targets by studying the LIF-R expression profile. Jenab and Morris [55] and Mauduit et al. [56] have already pointed out LIF-R expression in the testis by using, respectively, RT-PCR in rat and immunohistochemistry in boar. However, our study examined the LIF-R expression profile at both RNA and protein levels in all rat testicular cell populations using several complementary approaches.

With regard to spermatogonia, our results demonstrate that these cells constitute the major target of LIF within the rat testis. Indeed, both in vitro binding analysis by flow cytometry and immunohistochemistry revealed strong signals associated to spermatogonia at all ages studied. However, quantitative analysis could not be performed on adult testicular cells; our flow cytometric data demonstrated that 9-day-old spermatogonia exhibit LIF-R to a 3-fold higher


609 FIG. 5. Action of LIF on stage-specific DNA synthesis in seminiferous tubules. A) Incorporation of [3H]-thymidine into segments of seminiferous tubules at stages V, VII, VIII–IX, XII, and I of the seminiferous epithelium cycle cultured for either 24, 48, or 72 h in the absence or presence of 50 or 500 HILDA Units/ml of LIF. The results are expressed as the number of counts per minute per 2 mm of tubule and represent the mean 1 SEM of four independent experiments (*P , 0.05, **P , 0.01 when compared with the control using a Student t-test). B) Number of labeled cells per 2 mm of tubule (1 SEM; n 5 3) identified by autoradiography of squashed tubules (stages VIII–IX) that had been cultured in the absence or in the presence of 500 HILDA Units/ml of LIF for 72 h (*P , 0.05 using a Student t-test).

level than 20-day-old and adult somatic cells. Moreover, using an organotypic culture system of staged adult seminiferous tubule segments, we provided evidence that spermatogonia incorporate increased amounts of thymidine when exposed to LIF. This functional assay reveals that LIF stimulates either proliferation or survival of spermatogonia. Because these cells are highly affected by apoptosis in vivo [57], LIF may be a physiological regulator of their survival. Thus, our data contribute to the identification of the network of factors that control the spermatogonial cell compartment, which are still largely unknown despite their major interest. Indeed, the correct regulation of spermatogonial mitosis and, more specifically, of the balance between differentiation and proliferation allowing renewal of the stem cell stock, is essential for the maintenance of spermatozoa production throughout life. The importance of LIF for the precursor germ cells is also supported by the specific regulation of gp190/LIF-R expression in the germ line. Our results demonstrate that spermatogonia are able to bind to LIF, whereas meiotic germ cells are not. This desensitization process during differentiation seems to be strictly controlled, and this control probably occurs at the transcriptional level. Indeed, RTPCR experiments using primers specific for the 59 end of the transcript encoding the extracellular binding domain showed that the full-length gp190 mRNA was absent from

meiotic germ cells. This result is in total agreement with the results of flow cytometry binding studies using biotLIF. However, Western blot and flow cytometry experiments using an anti-c-terminus LIF-R antibody demonstrated that a truncated gp190 isoform was persistently expressed in meiotic and postmeiotic germ cells (50–60 kDa gp190 isoform). Taken together, our results suggest that functional LIF-R expression is down-regulated in germ cells when they enter meiosis, probably due to the induction of an alternative transcript lacking a major part of its 59 end encoding an LIF-R/gp190 isoform that is unable to bind to its ligand. Indeed, RT-PCR amplification of the 39 end of the LIF-R mRNA encoding the intracellular domain of the receptor was successful in spermatid and spermatocyte extracts. Further studies are required to characterize this transcript and its regulation. Such a transcriptional regulation process, leading to the expression of a truncated receptor deprived of its ligand binding ability, has already been demonstrated for another cytokine receptor, the protooncogene c-kit, the ligand of which is the stem cell factor (SCF) [58]. Indeed, when the full-length c-kit isoform is expressed in spermatogonia and in spermatocytes, an alternative transcript, called tr-kit, encoding the intracellular domain, is found in haploid cells throughout spermiogenesis [59, 60]. In addition to LIF action on spermatogonia, our study

610


provides evidence for the somatic cells to be targets of LIF in the testis. LIF has already been shown to promote Sertoli cell survival in a coculture system that includes gonocytes [27] and to stimulate the transcription of the c-fos gene in primary Sertoli cell cultures [55]. Our finding that LIF-R is present on Sertoli cells suggests that these actions are mediated through the direct binding of the cytokine to Sertoli cell receptors. In this context, it is noteworthy that LIF has recently been shown to activate the Jak/stat signal transduction pathway in Sertoli cells in culture, which is a characteristic of this cytokine family [55]. We also demonstrated that LIF-R was expressed on primary cultured peritubular cells, but this finding was not confirmed by immunohistochemistry, possibly because this technique is less sensitive than flow cytometry and RT-PCR. Alternatively, peritubular cells might have been activated when cultured, and then might not be in the same physiological state as in the normal testis. However, when we assessed the ability of LIF to modulate peritubular cell alkaline phosphatase activity and proliferation in vitro, we found that neither of these properties was affected by LIF. Further experiments are required to identify the regulatory actions of LIF on this cell population. We previously demonstrated that LIF bioactivity was high in testicular lymph, indicating that the cytokine was available for interstitial cells [21]. Indeed, we showed that testicular macrophages and Leydig cells express LIF-R. LIF inhibition of the hCG-induced testosterone production in porcine Leydig cells was recently found by Mauduit et al., pointing to a first paracrine role for LIF on Leydig cell function [56]. Of interest, LIF is also a neurotrophic factor, the absence of which in vivo leads to specific, sexually dimorphic alterations in the glial phenotype [61]. LIF and its receptor are both expressed in the adrenal cortex [62] and are both involved in the gonado-hypothalamo-pituitary axis [24]. LIF may thus be both a central and a local regulator of testosterone production. Several other cytokines are also produced by various testicular cell types [4, 63]. LIF might modulate the production of some of these cytokines, as described in many cellular models [64], and therefore be involved in the local regulatory cytokine network. Moreover, cytokines related to LIF have redundant actions on various cell types [11] and then probably exhibit overlapping activities in the testis. This could explain why male fertility is not altered in such cytokine-knockout mice [13, 65–68], whereas severe lethal phenotypes were observed in mice lacking the receptor subunit [8–10]. Thus, further studies on a few LIF-related cytokines and their receptors such as IL-11, OSM, CT-1, and CNTF are required to improve our understanding of the role of LIF in the male reproductive function. In conclusion, our study highlights LIF involvement in the testicular physiology and emphasizes its pleiotropic action in the male gonad targeting spermatogonia as well as Sertoli cells and Leydig cells. A similar pleiotropic role has been ascribed to SCF [58], a cytokine also known for its action on various types of precursor cells. Detailed investigations by several teams in the past 15 yr have clearly demonstrated the role of SCF in germ cell regulation at different stages of development and also in Leydig cell survival and steroidogenesis [58]. Another parallel between LIF and SCF can be drawn from these studies: the original regulation of their receptor expression in the germ line along with differentiation.

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