Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.003558
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
67/6/1699    most recent
biolreprod.102.003558v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.
Agricola
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.
Biology of Reproduction 67, 1699-1707 (2002)
DOI: 10.1095/biolreprod.102.003558 © 2002 Society for the Study of Reproduction, Inc.

Testis

Multidrug Resistance Genes and P-Glycoprotein in the Testis of the Rat, Mouse, Guinea Pig, and Human1

Nathalie Melaine3,a, Marie-Odile Liénard3,a, Isabelle Dorvala, Claude Le Goascogneb, Hervé Lejeunec, and Bernard Jégou2,a

a GERM-INSERM U.435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France b INSERM U.488, Unité de Recherche des Stéroïdes et Système Nerveux, 94276 Le Kremlin-Bicêtre, France c INSERM-INRA U.418, Hôpital Debrousse, 69322 Lyon Cedex 05, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs. P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis. These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively. The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes. Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human. Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter. Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids. We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier. The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs. In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense.

Leydig cells, Sertoli cells, stress, testis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis takes place within the seminiferous tubules. These tubules are composed of peritubular and Sertoli cells and of the various generations of germ cells, from the mitotic spermatogonia to the meiotic spermatocytes and ultimately the haploid spermatids, which are the cells that metamorphose into spermatozoa. The interstitial tissue between the seminiferous tubules contains the blood and lymphatic vessels and various types of cells, with the testosterone-producing Leydig cells and the macrophages being the major constituents [1].

Commencing at the turn of the last century, structural and functional data demonstrating the existence of permeability barriers between the blood and the inner part of the seminiferous tubules have been accumulated, giving rise to the concept of the blood-testis barrier (BTB) [2]. Structurally, tight junctions between Sertoli cells divide the tubule epithelium into two compartments: a basal compartment containing spermatogonia and primary spermatocytes in the early stages of meiotic prophase and a luminal compartment containing the more advanced spermatocytes and spermatids [3, 4]. These junctions are involved in the polarization of the Sertoli cells, form the "Sertoli-cell barrier" (a key element of the BTB [5]), and are responsible for the generation of a luminal physico-chemical environment different from that of the systemic circulation [2, 6, 7]. In addition to the Sertoli-cell barrier, the concept of the BTB also encompasses the peritubular myoid cell layer in rodents and, more generally, the endothelial lining of the blood and lymphatic vessels in mammals [8].

Although it has been decades since the first experiments designed to study functional and structural aspects of the BTB were conducted, the molecular support and mechanisms underlying the function of this barrier are unknown. In contrast, over the same period of time very significant progress has been made in understanding the molecular support of the blood-brain barrier (BBB). Central to this context is the discovery of the multidrug resistance (MDR) genes encoding the P-glycoprotein (P-gp) in the endothelium of the brain capillaries [9, 10].

P-gp was first identified in multidrug-resistant cancer cells by Juliano and Ling [11]. It is encoded by the small family of MDR genes, conserved through evolution and so named because overexpression of MDR type I genes confers resistance to a wide variety of amphipathic chemotherapeutic drugs [12]. The MDR genes belong to a large family, the ABC transporters (ATP-binding cassette transporters). These molecules are responsible for the transport of many compounds, such as steroids, peptides, and drugs. The human gene that confers multidrug resistance is called MDR1, whereas in the mouse and rat there are two genes with this activity (mdr1 and mdr3 in the mouse, and mdr1a and mdr1b in the rat; Table 1). MDR expression and P-gp activity have been detected in the endothelial cells of the BBB in which they play a central role in drug efflux [13]. High levels of P-gp expression have also been found in a large variety of epithelial cell types with secretory/excretory functions [14, 15]. Early studies suggested that P-gp forms a protective mechanism to exclude ingested toxins from normal cells and excrete them from the body [14]. Another biological role has also been suggested for P-gp in the normal, physiological, polarized secretion of metabolites [14, 16].


View this table:
[in this window]
[in a new window]
  		TABLE 1. Nomenclature and classification of type I MDR genes in human, mouse and rat.a

It is paradoxical that so very little attention has been devoted to the study of the molecular basis of the drug efflux in the testis given that one of the most negative side effects of anticancer treatment is a high rate of gonad damage, often leading to long-term oligo- or azoospermia, and consequently permanent sterility [17, 18]. This is also surprising considering growing concern about the possible deleterious effects of xenobiotics on testicular function [19–21]. Apart from morphological reports indicating the presence of MDR mRNA and P-gp in the vascular endothelium of rat, mouse, and human testis [9, 22–24], nothing is known about the cellular topography of MDR gene expression and P-gp production in the testis. In this work, using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), P-gp functional tests, and Western blotting, we investigated MDR gene expression in the testis of the rat, guinea pig, mouse, and human. We reveal a remarkable conservation of the distribution of the P-gp and MDR type I genes among the different species studied: in the interstitial compartment, in addition to the cells of the endothelial capillaries, type I MDR genes and P-gp are expressed in resident macrophages and Leydig cells, whereas within the tubules they are expressed in somatic and haploid cells, but not in the mitotic and meiotic germ cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal and Human Biological Materials

Male Sprague-Dawley rats and Swiss mice were purchased from Elevage Janvier (Le Genest Saint Isle, France). Male Hartly guinea pigs were purchased from Iffa-Credo (L'Arbresle, France). Human testes were obtained from men undergoing therapeutic orchidectomy, without hormonal replacement, for metastatic carcinoma or from 20- to 40-yr-old men after recent cerebral death (protocol approved by the Ethical Committee of the University of Lyon, France).

Immunohistochemistry

The testes of all species studied were fixed in Bouin solution. Ages of animals used were 90 days for rats, 50 days for mice, and 70 days for guinea pigs. Testes were then dehydrated through a graded series of alcohol solution and embedded in paraffin wax. Paraffin sections (5 µm) were incubated in an antigen-retrieval solution (10 mM acetate, pH 6) for 15 min as described by Cuevas et al. [25]. Endogenous peroxidase was quenched by incubation with 3% H2O2 for 5 min. The sections were saturated by incubation for 10 min with tris-buffer saline (TBS) 1% BSA and were then incubated with the primary antibody in TBS 1% BSA for 2 h. Three antibodies directed against human P-gp were used: a goat polyclonal antibody C19 (sc1517; Santa Cruz Biotechnology, Santa Cruz, CA); a mouse monoclonal antibody C219 (Centocor, Malvern, PA); and a mouse monoclonal antibody JSB1 (Harlan Sera-Lab, Crawley Down, U.K.). These three antibodies are specific for MDR types I and II from the mouse, rat, and human (Table 2). The primary antibody was labeled using an avidin-biotin-peroxidase technique (DAKO, Trappes, France). The color reaction was developed using DAB chromogene (3–3'-diaminobenzidine tetrahydrochloride; Sigma-Aldrich, Saint-Quentin Fallavier, France). The cells were counterstained with hemalum de Masson (AlKO8S2 100 mg/ml, Hematein 2 mg/ml, acetic acid 2%), dehydrated, and mounted in mounting medium (Eukitt, Labonord, Villeneuve d'Ascq, France). The primary antibody was replaced by normal goat IgG or mouse IgG (DAKO) in control sections at the same concentration. To ascertain further the specificity of the immunolocalization of P-gp, control experiments were performed by preabsorbing the C19 antibody with a synthetic peptide (sc 1517 P; Santa Cruz) corresponding to the carboxy terminus of MDR1 of human origin.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Specificity of the antibodies used in immunohistochemistry experiments

Microdissection of the Mouse Fetal Gonads

Pregnant Swiss mice from 7.5 to 18.5 days postcoitum (dpc) were killed by cervical dislocation. The uterus was rapidly collected and the embryo separated and placed in cold phosphate-buffer saline (PBS). Fetuses were dissected out under a binocular microscope (SZH10; Olympus, Rungis, France). Until 10.5 dpc, the whole posterior part of the embryo body was dissected because the gonad could not be isolated. After 10.5 dpc, the gonad associated with mesonephroi was collected, and from 14.5 dpc the gonads could be dissected free of other structures. Before 13.5 dpc, as the sex of the gonads was not morphologically recognizable, PCR with primers for mouse SFY1 were used to identify male fetuses. From 13.5 dpc onward, sex was determined morphologically. Immediately after dissection, the fetal gonads were frozen in liquid nitrogen and kept at -80°C until use.

Cells and Cultures

Isolation and culture of Sertoli and peritubular cells Rat and mouse Sertoli and peritubular cells were prepared from 20-day-old and 15-day-old animals, respectively, as described by Toebosch et al. [26]. Human Sertoli cells were prepared as described by Lejeune et al. [27]. These cell types were then cultured in appropriate media at 32°C in a humidified atmosphere (5% CO2, 95% air). The rat and mouse Sertoli cell cultures were >=97% pure, and human Sertoli cell cultures were >=90% pure based on morphological criteria. Rat and mouse peritubular cells became confluent after 8 days of culture. The cells were treated once with trypsin and allowed to reach confluence again. The purity of peritubular cell culture was determined by alkaline phosphatase assay [28] and was shown to be 96%. Sertoli cells and the peritubular cells were used for RNA and protein extraction. Uptake studies were performed with confluent rat peritubular cells and Sertoli cells on Day 2 of culture.

Isolation and culture of rat and human Leydig cells and rat testicular macrophages Highly enriched Leydig cells and testicular macrophage populations were prepared from adult rat testes using the method of Klinefelter et al. [29]. This procedure involves testicular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll density gradient centrifugation. After centrifugation, the Percoll gradient was divided into a fraction lighter than 1.068 g/ml containing germ cells, macrophages, and damaged Leydig cells and a fraction heavier than 1.068 g/ml containing intact, steroidogenically active Leydig cells. At this stage, the purity of the Leydig cell preparation, determined by 3-ß-HSD assay, was >=94%. Contaminants were mainly testicular macrophages (<4%), peritubular cells ({approx}0.5%), and very few Sertoli and germ cells [30]. Testicular macrophages were incubated for 15 min in a medium supplemented with 10% fetal calf serum (FCS) and then washed thoroughly five times with PBS to remove contaminating cells. The testicular macrophage preparations were >=94% pure, as determined with the specific ED2 antibody. Testicular macrophages preparation were slightly contaminated by Leydig cells (1%) and peritubular cells (0.1%) [30]. Rat Leydig cells and testicular macrophages were cultured for 24 h in Ham F12/DMEM medium (1:1 v/v) and supplemented with gentamicin (50 µg/ml), 10% FCS, and 0.1% bovine albumin. The cells were then used for RNA and protein extraction and uptake studies. Human Leydig cells were prepared and cultured as described elsewhere [27] before being used for RNA extraction.

Isolation and culture of rat spermatogonia Testes of 9-day-old male Sprague-Dawley rats were excised and decapsulated. Seminiferous epithelial cells were dispersed by enzyme treatment and separated by the method described by Bellvé et al. [31], with the minor modifications introduced by Dym et al. [32]. Briefly, after enzymatic dissociation, the cells were separated by sedimentation velocity at unit gravity at 4°C using a 2%–4% BSA gradient in Ham F12/DMEM in an SP-120 chamber (STAPUT). After 2.5 h of sedimentation, 35 fractions were collected. Cell fractions 1–26 were pooled, washed, resuspended in medium supplemented with 10% FCS, and incubated for 2 h at 32°C in a humidified 5% CO2, 95% air atmosphere to eliminate contaminating somatic peritubular cells and Sertoli cells. Spermatogonia were >90% pure with less than 5% nongerm cell contaminants. Among the 5% of nongerm cells contaminants were mainly Sertoli cell fragments, but also a few peritubular and Leydig cells as determined using electronic microscopy [33]. The population enriched in spermatogonial cells was then collected and cultured for 14 h. After this period of time, the cells were either used for RNA and protein extraction or uptake studies.

Isolation of rat, mouse, and human postmitotic germ cells Postmitotic germ cell preparations were obtained from adult rat [34] and mouse testes [35] by mechanical dissociation. Equivalent cell preparations were collected from adult human testes according to a method including both mechanical and enzymatic dispersion [36]. These rat, mouse, and human cells were separated by centrifugal elutriation (Beckman JE5 rotor driven by a J2-21B centrifuge; Beckman Instruments, Fullerton, CA) into two populations: primary spermatocytes and early spermatids. In the rat, the cytoplasmic lobes of late spermatids were also prepared. This fraction was composed of a mixture of cytoplasmic fragments of step 10–18 spermatids [37] and of residual bodies that are the cytoplasmic lobes, which are in situ shed by the late spermatids at the time of spermiation and eventually phagocytosed by Sertoli cells. Flow rate and/or rotor speed were changed progressively, as described by Loir and Lanneau [35], Pineau et al. [34], and Guillaudeux et al. [36] for mouse, rat, and human germ cells, respectively. Cell viability was evaluated by the trypan blue exclusion test and was found to be at least 95%. Rat primary spermatocytes and rat and human early spermatids fractions were found to be about 90% pure [34, 36]. The fraction of rat late spermatid cytoplasmic fragments was found to be approximately 75%–85% pure. Mouse spermatocyte and early spermatid and human primary spermatocyte fractions were >=75% pure [35, 36]. The enriched germ cell fractions were used for RNA extraction, whereas rat germ cell fractions were used for RNA and protein extraction and uptake studies.

Total RNA Preparation and RT-PCR Analysis

RNA was extracted from cultured cells using guanidium thiocyanate followed by centrifugation on a cesium chloride solution [38]. RNA was quantified by absorbance at 260 nm. To analyze MDR gene expression in the various cell preparations, RT-PCR was performed as follows: first strand cDNA synthesis was performed as recommended by Promega (Madison, WI) using a 5-µg RNA template, 20 µg/ml hexanucleotides, and 200 U Moloney murine leukemia virus reverse transcriptase in the reaction medium (Tris-HCl-MgCl2) containing 0.5 mM of each deoxy-NTP, 10 mM dithiothreitol, and 40 U RNasin in a final volume of 20 µl. After incubation for 1 h at 37°C, the reaction volume was brought up to 100 µl. In parallel, a negative control was performed using the same reaction mixture but without reverse transcriptase to ensure the absence of genomic DNA amplification during PCR.

RT-PCR was carried out as recommended by Perkin-Elmer (Courtaboeuf, France) starting with 250 ng cDNA as a template (in equivalent RNA starting material). Amplification was carried out using 30–35 cycles (1 min at 94°C, 1 min at 55°C, and 2 min at 72°C) in a Trio-Thermoblock thermocycler (Biometra, Göllingen, Germany). Oligonucleotide primers were designed from the sequence data deposited in GenBank and were synthesized by Eurogentec (Seraing, Belgium). The sequences of the primers used for RT-PCR were the following for human MDR1: 5'-primer, GCT GGT TGC TGC TTA CAT TC; 3'-primer, GCT GAC AGT CCA AGA ACA GG [39]. For rat mdr1b, 5'-primer, GTG CTT ACC GTC TTC TTC TC; 3'-primer GCT TCG CTT TCT GTG TCC AA [40]. For mouse mdr1, 5'-primer, GGC ATT GCC TAC CTG TTG G; 3'-primer GCT TTC TGT GGA CAC TTC TG [41]. For mouse mdr3, 5'-primer, GAG CCA TGT TTG CCA AAC TG; 3'-primer GGA GAA AAG CTG CAC CCA TG [42]. As no cDNA sequence has been published for guinea pig mdr, primers from rat mdr1b were used to amplify guinea pig mdr. Testis was used as a positive control for rodent mdr mRNA expression. For human MDR1 mRNA expression, the HL60R and HL60S cell lines were used as positive and negative controls, respectively. RT-PCR products were run on a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide and viewed under UV light. For all the cDNAs, reverse transcription was checked by amplification of ß-actin mRNA (data not shown). RT-PCR products were checked by sequencing the amplified cDNA on an automatic sequencer using the ABI PRISM dye terminator kit (Perkin Elmer).

Determination of the P-gp Activity in Isolated Rat Testicular Cells

The intracellular concentrations of two well known P-gp substrates, doxorubicin hydrochloride and rhodamine 123 (Sigma-Aldrich) were determined as described by Schott et al. [43] with slight modifications. In brief, cultured rat testicular cells were exposed to doxorubicin (10 µg/ml) for 2 h, with or without verapamil (5 µg/ml), a substrate of P-gp that inhibits the transport of its substrate in a competitive manner [44]. The cells were washed with PBS, harvested, subjected to sonication, and the proteins were precipitated with 20% trichloroacetic acid. The acid-soluble fraction was then used to measure the intracellular concentrations of doxorubicin by fluorometry using an excitation wavelength of 485 nm and an emission wavelength of 590 nm. To determine the intracellular concentration of rhodamine 123, cells were incubated with 1 µg/ml of this dye for 2 h, with or without verapamil (5 µg/ml). They were then washed with PBS and harvested in 1%-acetic acid—50% ethanol. Rhodamine 123 concentrations were evaluated by fluorometry using an excitation wavelength of 495 nm and an emission wavelength of 530 nm [45]. In both doxorubicin and rhodamine 123 assays, total protein was determined with the BCA Reagent Protein Assay (Pierce, Rockford, IL). Two cell lines, HL60R and HL60S, were used as positive and negative controls, respectively, for the MDR phenotype. Preliminary studies using the MTT assay showed that doxorubicin, rhodamine, and verapamil were not cytotoxic at the concentrations used during the incubation period.

Western Blot Analysis

For protein extraction, rat Leydig cells, testicular macrophages, peritubular cells, Sertoli cells, spermatogonia, primary spermatocytes, and early spermatids and the HL60R cell line were pelleted and washed twice with PBS and then lysed in a Tris-HCl ice-cold buffer (10 mM Tris-HCl, pH 7.5; 5 mM magnesium acetate; 10% glycerol; 0.2 mM EDTA; 0.5 mM DTT; 1 mM PMSF) for 15 min. Testicular human samples were homogenized in Tris-HCl ice-cold buffer then sonicated 3 times during 5 sec. The lysates (50 µg of total proteins for rat testicular cells, 100 µg for human testes, and 20 µg of total proteins for HL60R cells) were electrophoresed in SDS on 7.5% polyacrylamide gels and transferred electrophoretically to a polyvinylidene difluoride membrane. The membrane was blocked overnight at 4°C in 5% BSA-TBS containing 0.05% Tween 20 (TBST). After washing with TBST, the membrane was incubated for 2 h at room temperature with the specific mouse monoclonal anti-P-gp C19, used for immunohistochemistry, at a working dilution of 1:10 000. Control experiments were performed by preabsorbing the C19 antibody with the synthetic peptide (20-fold). After washing, the membrane was incubated with anti-goat immunoglobulins horseradish peroxidase conjugated (Jackson Laboratory, Interchim, Asnieres, France) for 30 min at room temperature. The labeling was then detected by an enhanced chemiluminescence system according to the manufacturer's instructions (ECL Plus, Amersham, Les Ulis, France).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunolocalization of P-gp in the Rat, Mouse, Guinea Pig, and Human Testis

In a series of preliminary experiments, we found that the three antibodies directed against the P-gp types I and II (C219, JSB1, and C19) generated positive specific signals on human and rat testis sections. Two of these antibodies tested in the mouse (C19 and C219) and one (C219) tested in the guinea pig also gave specific immunolabeling. Only data produced by the two antibodies giving optimal reactions (C19 and C219) are presented here (Fig. 1). In the human testis, the C19 antibody specifically labeled interstitial cells and late spermatids (Fig. 1A). In addition to the late spermatids, the C219 also strongly labeled the endothelium of the testicular capillaries (Fig. 1A insert). In the mouse and rat testes, using the C19 antibody, strong immunolabeling was detected in the interstitial cells and the endothelium of capillaries, as well as in the late spermatids (Fig. 1, B and C, respectively). Immunoreactivity was abolished in all cases when the primary antibody was omitted or when the C19 antibody was preneutralized by the synthetic peptide corresponding to human MDR1 (data not shown). In the guinea pig testis, the labeling generated by the C219 antibody was detected in early and late spermatids (Fig. 1, D and E). A summary of the results obtained in the four species studied is presented in Table 3.



View larger version (97K):
[in this window]
[in a new window]
  		FIG. 1. Immunolocalization of P-gp types I and II in the human, rat, mouse, and guinea pig testis. P-gp was detected using two different antibodies: a goat polyclonal antibody C19 (A–C) and a mouse monoclonal antibody C219 (A insert, D, and E). A specific labeling was detected by avidin-biotin-peroxidase complex amplification combination. In human (A), mouse (B), and rat testes (C), immunoreactivity was detected in the Leydig cells (thick arrows) as well as in the endothelium of the capillaries (arrow heads in A–C and corresponding inserts). Elongated spermatids (arrows) were labeled in human (A), mouse (B), rat (C), and guinea pig testes (D), as well as in early spermatids (asterisks) in the guinea pig testes (E). A–C) Bar = 100 µm; D and E) bar = 50 µm. Different filters were used between pictures A–C and pictures D and E


View this table:
[in this window]
[in a new window]
  		TABLE 3. Summary of the immunohistochemistry results obtained on human, mouse, rat, and guinea pig testes sections using the C219, JSB1, and C19 anti-P-gp antibodies (Ab).a

MDR mRNA Expression in Isolated Testicular Cells from Various Mammalian Species

The RT-PCR technique was used here to ascertain that the negative results obtained for some cell types by immunohistochemistry (Fig. 1) or in the functional tests (Fig. 1A) were not due to very low levels of expression of the P-gp.

As expected, the human MDR1 mRNA was detected by the amplified fragments of 312 base pairs (bp) by RT-PCR in HL60R cells used as positive controls, but not in HL60S cells, the negative controls (Fig. 2A). The same fragments were observed for human Leydig cells and Sertoli cells, but not for primary spermatocytes and early spermatids (Fig. 2A). A 691-bp PCR product corresponding to rat mdr1b mRNA was detected in rat testes (Fig. 2B), as well as in rat testicular macrophages, Leydig cells, peritubular cells, Sertoli cells, and in the late spermatid cytoplasmic lobes (Fig. 2B). No signal was ever detected in spermatogonia, primary spermatocytes, or early spermatids (Fig. 2B). In the guinea pig testes, the rat mdr1b primers amplified a PCR product of the same size as in the rat (Fig. 2B). In the mouse, PCR products 1126 bp and 612 bp in size, corresponding to mdr1 and mdr3 mRNA, respectively, were obtained for the testis, peritubular, and Sertoli cells, whereas no signal was detected in primary spermatocytes and early spermatids (Fig. 2, C and D). Our ontogenic study in the mouse demonstrated that expression of mdr1 (Fig. 3A) and mdr3 genes (Fig. 3B) began at 13.5 days postcoitum and were maintained thereafter. All the RT-PCR products from the various species investigated were sequenced and the result compared with the GenBank database. In all cases, there was an exact correspondence with the MDR gene in the species studied (not shown).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 2. RT-PCR analysis of MDR mRNA expression in the testis and in isolated testicular cell types from various species. Testicular expression of human MDR1 (A), rat mdr1b (B), guinea pig mdr1 (B), mouse mdr1 (C), and mouse mdr3 (D) was analyzed by RT-PCR in the whole testis (Testis; rat, guinea pig, and mouse); in testicular macrophages (T. Mac; rat); Leydig cells (Leydig; human and rat); peritubular cells (Peritub; rat and mouse); Sertoli cells (Sertoli; human, rat, and mouse); spermatogonia (Gonia; rat); primary spermatocytes (PS; human, rat, and mouse); early spermatids (ES; human, rat, and mouse); and in the cytoplasmic lobes of late spermatids (CL; rat). For human, HL60R and HL60S cell lines were used as positive and negative controls, respectively. For rat, mouse, and guinea pig, the negative controls were performed using the same reaction mixture but without reverse transcriptase (RT-). Human primers were selected from a DNA sequence including an intron. In contrast, in rat and mouse for which only cDNA sequences have been published, negative controls were performed by omitting the reverse transcriptase. The results presented are representative of three independent experiments



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 3. Ontogenesis of mouse mdr1 and mdr3 expression. Expression of mouse mdr1 (A) and mdr3 (B) in male gonad from 7.5 days postcoitum (dpc) to birth. Adult liver was used as a positive control (liver+), whereas adult liver mRNA was treated without reverse transcriptase (liver-), and H2O was the negative control. The PCR products were the same size as that in Figure 2 and the results are representative of three independent experiments

P-gp Activity in Isolated Rat Testicular Cells

We investigated whether the expression of MDR detected in the different types of testicular cells was associated with an MDR phenotype by developing uptake assays using two classical P-gp substrates, doxorubicin and rhodamine, in isolated rat testicular cells (Fig. 4). The rat model was used here because it is possible to isolate large numbers of most testicular cell types from this species with high degrees of purity. In these two assays, the resistant HL60R cell line, used as a positive control, had a strong MDR phenotype, as shown by a higher level of xenobiotic uptake in the presence of verapamil, a P-gp inhibitor. In contrast, as expected, no difference in the cellular accumulation of doxorubicin and rhodamine was observed in the HL60S cell line if the cells were cultured in the presence or absence of the P-gp modulator, confirming that this cell type does not express an MDR phenotype (Fig. 4, A and B). In the doxorubicin assay, we found that testicular macrophages, peritubular cells, and Sertoli cells consistently presented significant MDR activities, whereas Leydig cells and the various types of germ cell types did not (Fig. 4A). The rhodamine uptake assay further established the lack of a germ cell MDR phenotype and the MDR activity of testicular macrophages and peritubular cells. Furthermore, in the rhodamine assay, unlike in the doxorubicin assay, Leydig cells showed resistance, whereas Sertoli cells did not (Fig. 4B).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4. Determination of the P-gp activity in isolated rat testicular cells. MDR activities were assessed in testicular macrophages (T. Mac), Leydig cells (Leydig), peritubular cells (Peritub), Sertoli cells (Sertoli), spermatogonia (Gonia), primary spermatocytes (PS), and early spermatids (ES) using a doxorubicin assay (A) and a rhodamine assay (B). A) the cells were incubated with doxorubicin hydrochloride (10 µg/ml, Doxo) or doxorubicin hydrochloride and the P-gp inhibitor verapamil (5 µg/ml; Doxo + Vera). B) the cells were incubated with rhodamine 123 (1 µg/ml; Rhod) or rhodamine 123 and verapamil (5 µg/ml; Rhod + Vera). In both assays, the data are expressed as the mean ± SEM of groups consisting of three different samples assayed three times in three independent experiments. Intracellular drug retention values (µg doxorubicin/µg protein or pg rhodamine 123/µg protein) were expressed as percentages of initial drug accumulation and were analyzed by a paired two-tailed Student t-test (not significant, ns: P > 0.05; *0.005 > P > 0.01; **0.01 > P > 0.001; ***P < 0.001)

Immunoblotting of P-gp

Using the C19 antibody, P-gp was evidenced in the HL60R control cells, as well as in rat Leydig cells, peritubular cells, macrophages, and Sertoli cells. No labeling was ever observed in the three germ cell populations investigated (Fig. 5A). The labeling corresponding to a protein of 175 kDa was totally abolished when the antibody was preneutralized with the synthetic peptide (Fig. 5A). Similarly, a specific 175-kDa signal was found in total human testes extracts (Fig. 5B).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 5. Western blot analysis of P-gp in isolated rat testicular cells and the human testis. The presence of P-gp was investigated by Western blot analysis using the C19 antibody and human HL60R cells used as the positive control. A protein of 175 kDa that was neutralized when the antibody was preincubated with the synthetic peptide (+Neut.) was detected in HL60R cells as well as in rat Leydig cells (Leydig), peritubular cells (Peritub), testicular macrophages (T. Mac), and Sertoli cells (Sertoli; A). No specific labeling was ever found in spermatogonia (Gonia), primary spermatocytes (PS), and early spermatids (ES; A). A similar specific 175-kDa protein P-gp was detected in the human testis (Human testis, B). The blots presented are representative of two (rat) and three (human) experiments using different cellular and tissue preparations


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Drug extrusion by P-gp present on the luminal pole of blood brain capillaries has been shown to constitute an essential element of the BBB. The strongest evidence in favor of a key role for P-gp in the brain antixenobiotic defense system is that homozygous disruption of the mdr3 gene in the mouse leads to the accumulation in the cerebral tissue of large amounts of several anticancer drugs such as vinblastine and of other medically important drugs such as ivermectin, cyclosporin A, ondansetron, and digoxin, leading to the death of the animal [46]. A greater accumulation of the anticancer drug vinblastine and other therapeutic drugs, loperamide and ondansetron, has also been observed in the testes of mice lacking the mdr3 gene than in normal mice [46, 47], thus demonstrating that this gene is also involved in drug extrusion in this organ.

The present study establishes the testicular distribution of expression of MDR type I genes and the P-gp phenotype in different mammalian species, including the human species, therefore clarifying the organization of the testicular antidrug defense system.

Immunohistochemistry showed P-gp to be present in the interstitial tissue in the rat, mouse, and human testis, in early spermatids in the guinea pig testis, and in late spermatids in the rat, mouse, guinea pig, and human testis. The presence of a P-gp in the rat and human testis, with a molecular weight of 175 kDa, was further established by Western blot analysis. RT-PCR analysis revealed that type I MDR genes are expressed in testicular macrophages (rat); in Leydig cells (rat and human); in Sertoli cells (rat, mouse, and human); in peritubular cells (rat and mouse); and in the late spermatid cytoplasmic lobes (rat). The differences between the RT-PCR data and the immunohistochemistry observations (no labeling with the latter approach in Sertoli cells and peritubular cells) are most probably due to the relatively low sensitivity of the immunochemistry technique. This is confirmed by the fact that our Western blot analysis ascertained the presence in the rat of P-gp in Leydig cells and testicular macrophages, but also in Sertoli and peritubular cells and its absence in spermatogonia, primary spermatocytes, and early spermatids.

Immunohistochemistry also demonstrated that high levels of P-gp synthesis occurred in the endothelial cells of the testicular microvessels in the rat, mouse, and human testis. An endothelial labeling has previously been described by Cordon-Cardo et al. [9] in the human and by Stewart et al. [24] in the rat testis endothelium. According to the latter authors, P-gp is located in the rat on both basal and luminal sides of the testicular endothelial cells, whereas it is present only on the luminal side in brain endothelial cells.

Using two classical uptake assays, we then demonstrated that testicular P-gp and MDR gene expressions were associated with an MDR phenotype in somatic testicular cells. Using the same assays, the absence of such phenotype was also evidenced in the mitotic and meiotic germ cells. Testicular macrophages and peritubular cells displayed MDR to both doxorubicin and rhodamine, whereas Sertoli cells extruded only doxorubicin and Leydig cells only rhodamine. This diversity in the MDR phenotype of two different types of cell has been encountered in cells from other organs [48], each individual MDR phenotype reflecting differences in the type of P-gp produced (i.e., P-gp/mdr1a or P-gp/mdr1b; [49]).

It has been suggested that P-gp is the primary differentiation factor of the BBB, with mdr3 mRNA expression commencing in the endothelial cells of mouse fetus neural tissue at 10.5 dpc [50]. Our RT-PCR analysis reveals that mouse mdr1 and mdr3 expression begins in the testis later than in the brain at 13.5 dpc, concomitantly to the apparition of the blood vessels and the structuration of the seminiferous cords [51]. This reflects the very early requirement for the testicular tissue to ensure its antidrug defense system.

The higher permeability of testicular microvessels to hydrophilic substances than of brain microvessels [23] probably indicates that capillary P-gp expression does not represent a strict defense system within the testis. The levels of P-gp and its activity in somatic cells in this study were much lower than those in the control cell lines HL60R. However, in the study reported here the isolated cells were primary cells and the amplitude of the drug efflux may be influenced to some extent by the method of their preparation and culture. It is noticeable also that levels of P-gp activity, similar to those found here in the testicular nonendothelial somatic cells, were reported for a number of other organs and in noncancerous cell types [52, 53] in which P-gp is thought to play a self-defense function [54]. Of note also is that a quantitative comparison between the intensity of the efflux observed with the different cell types is not really possible as the different cell categories were isolated using different techniques and cultured under very different conditions.

Several anticancer drugs such as doxorubicin, actinomycin D, and vinblastine have been shown to have potent toxic effects on spermatogonia in rodents [55]. This is explained by the results presented here, showing that spermatogonia do not express MDR genes. The lack of expression of the MDR phenotype by other types of germ cells is also consistent with the susceptibility of these cells to a large number of xenobiotics. The presence of MDR genes in haploid cells in all the species studied probably reflects the particular requirement for spermatozoa to be protected against xenobiotics that may be present in the rete testis, the epididymal tract, the seminal plasma, and the female genital tract.

On the basis of this study, a tentative summary of the present knowledge of the testicular antidrug defense system is presented in Figure 6. A remarkable conservation of the distribution of P-gp and MDR genes expression in the interstitial compartment and within the seminiferous tubules is revealed in the different species studied.



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 6. Schematic representation and summary of the topography of P-gp expression in mammalian testis. For hydrophobic drugs entering the testis via the interstitial blood capillaries, the first line of cellular efflux is the capillary endothelium itself as P-gp is localized on both the luminal and adluminal membrane of capillaries [24], whereas the testosterone-producing Leydig cells and the resident macrophages displayed a P-gp self-defense system. In the tubules, the mitotic and meiotic germ cells named spermatogonia and spermatocytes, respectively, are devoid of P-gp expression and are therefore under the protection of the tubular defense system composed by the peritubular cells (rat and mouse) and Sertoli cells. This system also probably ensures self-protection to these somatic cells. Although early spermatids expressed P-gp only in the guinea pig, in all species studied a strong expression of P-gp exists in late spermatids and, therefore, most likely in spermatozoa. The latter finding probably reflects the requirement for the sperm cells to be protected against the xenobiotics that may be present in the rete testis, in the epididymal tract, in the seminal plasma, or in the female genital tract

Recently, Winjhold et al. [56] showed that MRP1, a member of the ABC transporter family, prevents the entry of etoposide, a drug commonly used against testicular tumors, into the seminiferous epithelium. This suggests that the molecular framework of the BTB includes MDR genes plus one or several other members of the ABC transporter family. In addition to testicular cells' protection against various drugs, P-gp may also be involved in the constitution of the seminiferous tubule microenvironment, as it has been also described to be able to transport a large number of molecules [57] and involved in testicular steroid biosynthesis or transport [58, 59]. The latter hypothesis remains to be investigated.


   ACKNOWLEDGMENTS
 
We thank Dr. Charlotte Beau and Dr. Daniel Guerrier for help with the microdissection of mouse fetal gonads and Sophie Desmot for her very valuable contribution to the Western blot experiments.


   FOOTNOTES
 
1 This study was funded by INSERM, Ministère de l'Education Nationale de la Recherche et de la Technologie, Fondation Langlois, Ministère de l'Environnement et de l'Aménagement du Territoire. Back

2 Correspondence: Bernard Jégou, GERM-INSERM U.435, Université de Rennes I, Campus de Beaulieu, Avenue du Général Leclerc, 35042 Rennes Cedex, Bretagne, France. FAX: 33 223 23 50 55; bernard.jegou{at}rennes.inserm.fr Back

3 These authors contributed equally to this work Back

Received: 15 January 2002.

First decision: 5 April 2002.

Accepted: 18 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jégou B. The Sertoli-germ cell communication network. Int Rev Cytol 1993 147:25-96[Medline]
  2. Setchell BP. . In: Finn CA (ed.), The Mammalian Testis. London: Elek Books; 1978: 1–422.
  3. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 1970 3:308-326[Abstract]
  4. Russell LD. Form, dimension, and cytology of mammalian Sertoli cells. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993: 1–38.
  5. Byers S, Jégou B, MacCalman C, Blaschuk O. Sertoli cell adhesion molecules and the collective organization of the testis. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993: 461–476
  6. Waites GMH, Gladwell RT. Physiological significance of fluid secretion in the testis and the blood-testis barrier. Physiol Rev 1982 62:624-671[Free Full Text]
  7. Jégou B. The Sertoli cell. Bailliere's Clin Endocrinol Metab 1992; 6:273–311.
  8. Plöen L, Setchell BP. Blood-testis barriers revisited. A homage to Lennart Nicander. Int J Androl 1992 15:1-4[Medline]
  9. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A 1989 86:695-698[Abstract/Free Full Text]
  10. Jette L, Pouliot JF, Murphy GF, Beliveau R. Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein. Biochem J 1995 1:761-766
  11. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976 455:152-162[Medline]
  12. Juranka PF, Zastawny RL, Ling V. P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins. FASEB J 1989 3:2583-2592[Abstract]
  13. Schinkel AH, Smit JJ, van Tellingen O, Beijnen JH, Wagenaar E, van Deemter L, Mol CA, van der Valk MA, Robanus-Maandag EC, Riele HP, Berns AJM, Borst P. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 1994 77:491-502[CrossRef][Medline]
  14. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A 1987 84:7735-7738[Abstract/Free Full Text]
  15. Cordon-Cardo C, O'Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 1990 38:1277-1287[Abstract]
  16. Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA, van der Valk MA, Offerhaus GJA, Berns AJM, Borst P. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993 75:451-462[CrossRef][Medline]
  17. Viviani S, Santoro A, Ragni G, Bonfante V, Bestetti O, Bonadonna G. Gonadal toxicity after combination chemotherapy for Hodgkin's disease. Comparative results of MOPP vs ABVD. Eur J Cancer Clin Oncol 1985 21:601-605[CrossRef][Medline]
  18. Velez de la Calle JF, Jégou B. Protection by steroid contraceptives against procarbazine-induced sterility and genotoxicity in male rats. Cancer Res 1990 50:1308-1315[Abstract/Free Full Text]
  19. Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ Jr, Jégou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Muller J, Rajpert-De Meyts E, Scheike T, Sharpe R, Sumpter J, Skakkebaek NE. Male reproductive health and environmental xenoestrogens. Environ Health Perspect 1996 104:741-803
  20. Sharpe RM. Declining sperm counts in men. Is there an endocrine cause?. J Endocrinol 1993 136:357-360[Medline]
  21. Jégou B, Auger J, Multigner L, Pineau C, Thonneau P, Spira A, Jouannet P. The saga of the sperm count decrease in humans and wild and farm animals. In: Gagnon C (ed.), The Male Gamete. From Basic Science to Clinical Applications. Vienna: Cache River Press; 1999:445–454
  22. Croop JM, Raymond M, Haber D, Devault A, Arceci RJ, Gros P, Housman DE. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues. Mol Cell Biol 1989 9:1346-1350[Abstract/Free Full Text]
  23. Holash JA, Harik SI, Perry G, Stewart PA. Barrier properties of testis microvessels. Proc Natl Acad Sci U S A 1993 90:11069-11073[Abstract/Free Full Text]
  24. Stewart PA, Beliveau R, Rogers KA. Cellular localization of P-glycoprotein in brain versus gonadal capillaries. J Histochem Cytochem 1996 44:679-685[Abstract]
  25. Cuevas EC, Bateman AC, Wilkins BS, Johnson PA, Williams JH, Lee AH, Jones DB, Wright DH. Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J Clin Pathol 1994 47:448-452[Abstract/Free Full Text]
  26. Toebosch AM, Robertson DM, Klaij IA, de Jong FH, Grootegoed JA. Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone. J Endocrinol 1989 122:757-762[Abstract]
  27. Lejeune H, Skalli M, Sanchez P, Avallet O, Saez JM. Enhancement of testosterone secretion by normal adult human Leydig cells by co-culture with enriched preparations of normal adult human Sertoli cells. Int J Androl 1993 16:27-34[Medline]
  28. Chapin RE, Phelps JL, Miller BE, Gray TJ. Alkaline phosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture. J Androl 1987 8:155-161[Abstract/Free Full Text]
  29. Klinefelter GR, Hall PF, Ewing LL. Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 1987 36:769-783[Abstract]
  30. Dejucq N, Liénard MO, Guillaume E, Dorval I, Jégou B. Expression of interferons-alpha and -gamma in testicular interstitial tissue and spermatogonia of the rat. Endocrinology 1998 139:3081-3087[Abstract/Free Full Text]
  31. Bellvé AR, Millette CF, Bhatnagar YM, O'Brien DA. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem 1977 25:480-494[Medline]
  32. Dym M, Jia MC, Dirami G, Price JM, Rabin SJ, Mocchetti I, Ravindranath N. Expression of c-kit receptor and its autophosphorylation in immature rat type A spermatogonia. Biol Reprod 1995 52:8-19[Abstract]
  33. Guillaume E, Dupaix A, Moertz E, Courtens JL, Jégou B, Pineau C. Proteome analysis of spermatogonia: identification of a first set of 53 spermatogonial proteins. Proteome Electronic Publication 2000; DOI 10.1007/s102160000003.
  34. Pineau C, Syed V, Bardin CW, Jégou B, Cheng CY. Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells. J Androl 1993 14:87-98[Abstract/Free Full Text]
  35. Loir M, Lanneau M. A strategy for an improved separation of mammalian spermatids. Gamete Res 1982 6:179-188
  36. Guillaudeux T, Gomez E, Onno M, Drenou B, Segretain D, Alberti S, Lejeune H, Fauchet R, Jégou B, Le Bouteiller P. Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells. Biol Reprod 1996 55:99-110[Abstract]
  37. Meistrich ML, Longtin J, Brock WA, Grimes SR, Mace ML. Purification of rat spermatogenic cells and preliminary biochemical analysis of these cells. Biol Reprod 1981 25:1065-1077[Abstract]
  38. Glisin V, Crkvenjakov R, Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 1974 13:2633-2637[CrossRef][Medline]
  39. Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB. Internal duplication and homology with bacterial transport proteins in the MDR1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 1986 47:381-389[CrossRef][Medline]
  40. Silverman JA, Raunio H, Gant TW, Thorgeirsson SS. Cloning and characterization of a member of the rat multidrug resistance (mdr) gene family. Gene 1991 106:229-236[CrossRef][Medline]
  41. Gros P, Ben Neriah YB, Croop JM, Housman DE. Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 1986 323:728-731[CrossRef][Medline]
  42. Devault A, Gros P. Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities. Mol Cell Biol 1990 10:1652-1663[Abstract/Free Full Text]
  43. Schott B, Robert J. Comparative cytotoxicity, DNA synthesis inhibition and drug incorporation of eight anthracyclines in a model of doxorubicin-sensitive and -resistant rat glioblastoma cells. Biochem Pharmacol 1989 38:167-172[CrossRef][Medline]
  44. Ford JM, Hait WN. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol Rev 1990 42:155-199[Medline]
  45. Ludescher C, Thaler J, Drach D, Drach J, Spitaler M, Gattringer C, Huber H, Hofmann J. Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123. Br J Haematol 1992 82:161-168[Medline]
  46. Schinkel AH, Wagenaar E, Mol CA, van Deemter L. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 1996 97:2517-2524[Medline]
  47. Van Asperen J, Schinkel AH, Beijnen JH, Nooijen WJ, Borst P, van Telligen O. Altered pharmacokinetics of vinblastine in mdr1a P-glycoprotein-deficient mice. J Natl Cancer Inst 1996 88:994-999[Abstract/Free Full Text]
  48. Vander Bliek AM, Borst P. Multidrug resistance. In: Vande Woude GF, Klein G (eds.), Advances in Cancer Research. New York: Academic Press; 1988: 165–203
  49. Tang-Wai DF, Kajiji S, Di Capua F, de Graaf D, Roninson IB, Gros P. Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators. Biochemistry 1995 34:32-39[CrossRef][Medline]
  50. Qin Y, Sato TN. Mouse multidrug resistance 1a/3 gene is the earliest known endothelial cell differentiation marker during blood-brain barrier development. Dev Dyn 1995 202:172-180[Medline]
  51. Kaufman MH. The Atlas of Mouse Development. London: Academic Press; 1992: 1–512.
  52. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I. Expression of a multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci U S A 1987 84:265-269[Abstract/Free Full Text]
  53. Baas F, Borst P. The tissue dependent expression of hamster P-glycoprotein genes. FEBS Lett 1988 229:329-332[CrossRef][Medline]
  54. Borst P, Ouellette M. New mechanisms of drug resistance in parasitic protozoa. Annu Rev Microbiol 1995 49:427-460[CrossRef][Medline]
  55. Nolte T, Harleman JH, Jahn W. Histopathology of chemically induced testicular atrophy in rats. Exp Toxicol Pathol 1995 47:267-286[Medline]
  56. Wijnholds J, Scheffer GL, van der Valk M, van der Valk P, Beijnen JH, Scheper RJ, Borst P. Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage. J Exp Med 1998 188:797-808[Abstract/Free Full Text]
  57. Gupta S. P-glycoprotein expression and regulation. Age-related changes and potential effects on drug therapy. Drugs Aging 1995 7:19-29[Medline]
  58. Metherall JE, Li H, Waugh K. Role of multidrug resistance P-glycoproteins in cholesterol biosynthesis. J Biol Chem 1996 271:2634-2640[Abstract/Free Full Text]
  59. Ueda K, Okamura N, Hirai M, Tanigawara Y, Saeki T, Kioka N, Komano T, Hori R. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem 1992 267:24248-24252[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Pharmacol. Rev.Home page
D. D. Mruk, B. Silvestrini, and C. Y. Cheng
Anchoring Junctions As Drug Targets: Role in Contraceptive Development
Pharmacol. Rev., June 1, 2008; 60(2): 146 - 180.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
B. Lassalle, H. Bastos, J. P. Louis, L. Riou, J. Testart, B. Dutrillaux, P. Fouchet, and I. Allemand
`Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells
Development, January 15, 2004; 131(2): 479 - 487.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
67/6/1699    most recent
biolreprod.102.003558v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.
Agricola
Right arrow Articles by Melaine, N.
Right arrow Articles by Jégou, B.