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Ludwig Institute for Cancer Research (M.M., K.S.), Stockholm Branch, Karolinska Institutet, SE-17177 Stockholm, Sweden; Pediatric Endocrinology Unit (C.P.), Astrid Lindgren Children’s Hospital, Karolinska Hospital, SE-171 76 Stockholm, Sweden; and Swedish Governmental Agency for Innovation Systems (K.N.), VINNOVA, SE-10158 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Kerstin Sollerbrant, Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Box 240, SE-17177, Stockholm, Sweden. E-mail: kerstin.sollerbrant{at}licr.ki.se.
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Introduction |
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Transendothelial and transepithelial migration of leukocytes requires recruitment of leukocytes from the circulation to the site of inflammation, attachment to the surface of the endothelium/epithelium, and migration through intercellular junctions (1). Regulated transendothelial and transepithelial migration is required for a proper immune response and cell homing after transplantation. Uncontrolled transendothelial and transepithelial migration can result in chronic inflammation and metastasis of malignant cells.
Migration of germ cells in the mammalian testis is a prerequisite both for proper testis morphogenesis during development and for spermatogenesis in the adult organ. Malfunction of germ cell migration results in infertility. During the first week after birth, rodent germ stem cells, the gonocytes, migrate from a central position in the developing seminiferous epithelium to the basement membrane, where they are called spermatogonia (2, 3). Spermatogonia become trapped at the basal side when the tight and adherens junctions form the blood-testis barrier (BTB) between adjacent Sertoli cells (SCs) around 2–3 wk after birth. In the adult, the BTB divides the epithelium in a basal and adluminal compartment harboring germ cells of specific developmental stages (4). In the basal compartment, spermatogonia proliferate and transform into preleptotene spermatocytes. Preleptotene/leptotene cells then migrate from the basal to the adluminal compartment, a process that involves extensive restructuring of the BTB (5). The integrity of the BTB is maintained during cell transit to limit paracellular diffusion and prevent the immune system from recognizing autoantigens on germ cells in the haploid phase of spermatogenesis. In the adluminal compartment, meiosis is completed by the formation of spermatids, followed by spermatid differentiation, and release of spermatozoa in the lumen of the seminiferous tubule.
Many cell surface proteins such as Igs and integrins appear to be involved in both leukocyte and germ cell trafficking. Of particular interest are proteins belonging to the cortical thymocyte marker of the Xenopus (CTX) family of Ig proteins. These proteins harbor an ectodomain consisting of one V-type and one C-type Ig domain, a single transmembrane region and an intracellular tail that often terminate in a PDZ-1-domain-binding motif (6). CTX proteins are found in cellular junctions such as tight junctions, although other localizations have also been reported. Many of the family members are able to form both homophilic and heterophilic interactions with other proteins. The cellular receptor for two unrelated viruses, the coxsackievirus and adenovirus receptor (CAR), was one of the first CTX members to be cloned (7, 8, 9). CAR is expressed in tight junctions of most epithelial cells in vivo (10) but has also been found in other types of specialized junctions as well as in the acrosome region of germ cells in the testis (11, 12). CAR protein expression is regulated by signals that control proliferation and differentiation, as well as by inflammatory responses (13, 14, 15, 16, 17, 18). CAR was recently found to play an essential role in heart development because a targeted disruption of CAR was lethal due to heart failure (19, 20, 21). Although ubiquitous overexpression of a tailless CAR protein has no obvious phenotype, overexpression of CAR from a muscle-specific promoter resulted in a severe myopathic phenotype (22, 23). The molecular mechanism(s) behind these CAR-mediated phenotypes remain unknown. CAR has been found to form a functional complex with junctional adhesion molecule-like (JAML), another CTX family member (24). JAML is expressed on leukocytes, and the interaction between CAR and JAML regulate transepithelial migration of the leukocytes. Junction adhesion molecules (JAMs) -A, -B and -C form a subgroup of proteins within the CTX family. JAMs appear to both regulate transendothelial migration of leukocytes to sites of inflammation and mediate the establishment of cell polarity (25). JAM-A, -B and -C are all expressed in the testis, but only JAM-C has had an essential role in spermatogenesis (26). JAM-C was also recently shown to localize to desmosomes and regulate neutrophil transepithelial migration (27). JAM-C interacts with both JAM-B and CAR (12, 26). Finally, endothelial cell-selective adhesion molecule, an endothelial tight junction protein, was reported to function in leukocyte extravasation (28).
The aim of this study was to investigate CAR expression in perinatal, postnatal, and adult testis. Based on the known function of CTX proteins in transepithelial migration, particular emphasis was put on analyzing CAR expression in cells involved in migration across the BTB. Here, we show that high levels of CAR are expressed in the developing perinatal and early postnatal testis. CAR levels then decline, and CAR is not, in contrast to other epithelium, a general component of the tight junctions in the adult testis seminiferous epithelium. Instead, CAR is expressed in a stage-dependent manner in germ cells in transit from the basal to the adluminal compartment. We investigate the presence and subcellular localization of other CTX proteins previously shown to be involved in the passage of cells across endothelial and epithelial barriers. We also analyze the effect of FSH on CAR expression in primary SCs. Finally, we present evidence for the existence of an intermediate compartment occupied by CAR-expressing, migrating germ cells, surrounded by occludin-containing structures.
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Materials and Methods |
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Antibodies
Rabbit, polyclonal anti-CAR antibodies RP 291 and IG1 have previously been described (12). Rat serum toward CAR (CARic) were produced by ImmunoPrecise Antibodies Ltd. in Victoria, Canada, by immunizing rats with the intracellular part of mouse CAR fused to glutathione-S-transferase. The specificity of the sera was confirmed by transfection of mouse CAR cDNA into mammalian cells, and analysis of CAR protein by Western blotting and immunofluorescence experiments (Fig. 1B
) (data not shown). Monoclonal rat anti-occludin (MOC37) was a kind gift from Dr. M. Furuse, Kobe University, Kobe, Japan. Rabbit anti-claudin-3 was purchased from Invitrogen (no. 34-1700; Invitrogen Corp., Carlsbad, CA). Monoclonal rat antibodies toward JAM-A, JAM-B, and JAM-C were a kind gift from Professor Beat Imhof, University of Geneva School of Medicine, Geneva, Switzerland. Animal sources and references are summarized in Table 1.
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and see Fig. 6) or 20 µm (Fig. 5) in thickness, transferred onto Superfrost slides (Menzel-Glàser, Braunschweig, Germany), dried for 24 h in room temperature, and then fixed in either ice-cold absolute ethanol for 20 min or methanol/acetone 1:1 for 10 min. Sections were then rehydrated in 1x PBS, blocked and incubated for 1 h in blocking buffer [0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.5% blocking reagent (Tyramide Signal Amplification Biotin System Kit; PerkinElmer Life And Analytical Sciences, Inc., Waltham, MA), 10% goat serum, and 4% BSA], and incubated with primary antibody overnight at 4 C in a humidified chamber. Sections were then washed in 0.1 M Tris-HCl, 0.15 M NaCl, and 0.05% Tween 20, and incubated for 1 h with 0.3 µg/ml 4',6-diamidino-2-phenylindole (DAPI) and secondary antibodies goat antirabbit Alexa Fluor 488 and donkey antirat Alexa Fluor 594 (Molecular Probes, Inc., Eugene, OR) at a dilution of 1:300. Stained sections were analyzed in a Leica confocal fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Each channel was scanned separately to avoid bleed through. Immunofluorescence experiments performed with pre-immune sera or without primary antibodies did not show any specific stainings (data not shown).
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Bromodeoxyuridine (BrdU) incorporation
BrdU incorporation and analysis were performed using the cell proliferation kit from GE Healthcare, RPN20 (Piscataway, NJ). Briefly, mice were injected ip with 1 ml BrdU/100 g mouse and killed 2 h later. Testis was dissected and processed as described previously. Immunofluorescence was performed according to the manufacturer’s protocol with a mouse anti-BrdU monoclonal antibody provided with the kit and a goat antimouse Alexa Fluor 594 secondary antibody from Molecular Probes.
Microdissection and staging of the seminiferous tubule
Testis from 60-d-old male Sprague Dawley rats was dissected and placed in PBS without calcium and magnesium (Life Technologies), and microdissected with the aid of a transilluminating stereomicroscope. The spermatogenic stages of the seminiferous epithelium were identified with an accuracy of ± one stage, as described previously (33, 34). One-millimeter segments of the epithelium in each of the 14 different stages were transferred individually in 10 µl PBS onto SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany) and squashed carefully against a coverslip. The squash preparations were subsequently frozen in liquid nitrogen, followed by removal of the coverslips, and fixation for 20 min in absolute ethanol.
Protein extraction and Western blot
Cell extracts were prepared from untransfected and transfected 293 cells as described previously (35). Transfection was done in 10-cm dishes using 5 µg of a mouse CAR cDNA expressing plasmid. Testis tissue was homogenized in lysis buffer [50 mM Tris-HCl (pH 7.5), 137 mM NaCl, 0.5% Triton X-100, and EDTA-free 1 x complete protease inhibitor from Roche Molecular Biochemicals] on ice, sonicated, and incubated on ice for 15 min before centrifugation at maximum speed in an Eppendorf centrifuge (Hamburg, Germany) at +4 C for 10 min. The lysate was removed and run on a 10% SDS-PAGE gel under reducing conditions, and transferred to PROTRAN nitrocellulose transfer membrane (Schleicher & Schuell, Dassel, Germany). Western blotting was performed with CAR-specific antibodies as indicated.
RNA extraction and RT- PCR
Total RNA was isolated from mouse testis and SCs using the RNeasy mini kit (QIAGEN, Inc., Valencia, CA), and an on-column DNase digestion was performed during RNA purification according to the manufacturers protocol with the RNase-Free DNase set (QIAGEN). cDNA was obtained using the iScript cDNA synthesis kit according to instructions (Bio-Rad Laboratories, Hercules, CA). RT-PCR was performed with JAML-specific mouse and rat primers (Table 2), and PCR products were analyzed on a 2% agarose gel. Quantitative RT-PCR was performed with CAR-specific mouse and rat primers (Table 2) using iQ SYBR Green Supermix (Bio-Rad Laboratories). Primers were validated according to the manufacturer’s instructions. Based on the comparative cycle threshold value method, gene expression levels were calculated, and β-actin was used as a control gene. Testis sample from mouse postnatal d 2 and SCs in the absence of FSH were set to 100%. Changes in CAR expression are presented with SD values. Statistical analysis was performed with the Student’s t test.
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. All antibodies specifically recognized mouse CAR protein as shown in Western analysis and indirect immunofluorescence of cells transfected with mouse CAR cDNA (Fig. 1B) (data not shown).
To analyze CAR expression in perinatal and postnatal mouse testis, RP291 was used in indirect immunofluorescence experiments (Fig. 2A). DAPI was included in the magnification to the right to visualize nuclei. Strong CAR staining was seen already at embryonic d 18.5 in the developing cords and expression maintained high at d 2 after birth. At these time points, the gonocytes, which have large pale blue nuclei located in the middle of the cords, are easily distinguishable from the smaller SC nuclei. Both Sertoli and germ cells appear to express CAR.
CAR expression remains high during the first week after birth. At d 12, when migration of gonocytes is completed, CAR staining appears weaker. Between d 14 and 18, the BTB is formed close to the basal membrane, creating a basal and adluminal compartment within the seminiferous epithelium. Because CAR has been expressed in most epithelial tight junctions in vivo colocalizing with the tight junction marker occludin (10), we analyzed whether CAR was present also in the BTB. Therefore, CAR was costained with occludin to visualize the BTB at d 17 and 21 (Fig. 2B). Surprisingly, although CAR-positive cells were now confined to the region of the developing BTB, CAR only partially overlapped with occludin. All tubules showed similar basal CAR staining at d 17, although the intensity of the staining varied between tubules. Interestingly, at d 21 only a subset of tubules expressed CAR in the basal region, and the intensity of the staining varied from strong to completely negative. This staining pattern was maintained in the adult testis as described in Fig. 3A. Similar stainings were also seen with the CAR-specific IG1 antibody (data not shown). To determine the identity of the CAR-positive cells, testis sections from d 21 were carefully analyzed by immunofluorescence in which the IG1 antibody and DAPI were used to visualize CAR and nuclei, respectively (Fig. 2C). The magnified image reveals that CAR expression is now confined to germ cells, which have round, strongly DAPI-stained nuclei. SCs, which have irregular, pale nuclei with conspicuous nucleolus, no longer express CAR. Based on these results, we conclude that in testis, in contrast to other tissues, CAR is not a general component of the tight junctions.
Expression of CAR was also examined by quantitative RT-PCR (Fig. 2D). CAR mRNA was successively down-regulated, and at d 21, only around 30% CAR mRNA remained. Thus, the RNA expression profile correlated with the indirect immunofluorescence stainings shown in Fig. 2, A and B. Quantitative PCR was not performed on adult tissue because CAR is expressed in the developing spermatids at this time point (12).
To analyze the size of the CAR protein expressed in testis, a Western analysis was performed on homogenates from a 2-d-old testis (Fig. 2E). A specific band of 46 kDa was identified as expected.
The stainings shown in Fig. 2A indicate that CAR was expressed in both germ and SCs in perinatal and postnatal testis. To investigate this further, SCs were isolated from a 15-d-old rat testis, grown in vitro and incubated with or without FSH for 24 h. RNA extraction followed by quantitative RT-PCR analysis revealed a 2-fold induction of CAR mRNA after FSH stimulation (Fig. 2F). Indirect immunofluorescence experiments using a CAR-specific antibody confirmed that FSH treatment induced also the amount of CAR protein in the isolated SCs (data not shown). FSH receptor is expressed on SCs, and FSH promotes SC mitosis (37). Therefore, these results indicate that CAR is indeed expressed in SCs and that CAR is a previously undescribed downstream target of FSH.
Together, these results show that CAR is highly expressed in both germ and SCs throughout the developing seminiferous epithelium of the perinatal and early postnatal testis. Expression then rapidly declines, SCs lose their CAR expression, and only germ cells express CAR at d 21.
CAR is expressed in migratory germ cells in the adult testis
The experiments presented in Fig. 2B showed that CAR was expressed only in a subset of tubules at d 21. A careful examination of many cross-sectioned tubules using the RP291 antibody confirmed this expression pattern also in the adult testis, indicating that CAR expression was stage dependent (Fig. 3A). Costaining with occludin, which stained the BTB in all tubules, indicated that CAR was expressed close to but did not coincide with the BTB in one of the four tubules visible in the image. Based on size, morphology, and location of the DAPI-stained nuclei, we estimate the CAR-positive tubule shown in this image to be from stage VII (4). In general, tubules from stages VII and VIII showed strongest CAR staining, but weaker CAR staining was also found in tubules from other stages (data not shown). Similar stainings were also seen with the CAR-specific IG1 and CARic antibodies (data not shown).
At d 21, CAR was expressed in basal germ cells, and no expression was found in SCs (Fig. 2C). To analyze whether the situation was the same in adult testis, CAR-positive cells were analyzed as described for Fig. 2C using the RP291 antibody and DAPI (Fig. 3B). This experiment clearly demonstrated that also in the adult testis only basal germ cells, not SCs, expressed CAR.
The fact that strong CAR staining was seen in basal germ cells from stages VII and VIII indicated that it was the preleptotene cells that expressed CAR. To investigate this, a BrdU-labeling experiment was performed. As shown in Fig. 3C, BrdU-positive cells were also CAR positive, showing that CAR was indeed expressed in cells undergoing DNA synthesis. DAPI was included in the experiment to visualize nuclei. Not all BrdU-positive cells in the testis sections were CAR positive, indicating that some replicating basal germ cells did not express CAR (data not shown).
To analyze more closely the stage-dependent expression of CAR, we took advantage of the well-established staging system in the rat (4). Testis was microdissected using a transilluminating stereomicroscope, and the spermatogenic stage of each segment was identified with an accuracy of ± one stage as described (33, 34). Segments of seminiferous epithelium were transferred onto slides, squashed against a coverslip, fixed in ethanol, and analyzed by indirect immunofluorescence using the CAR-specific antibody RP291 (Fig. 3D). Germ cells were identified based on size and morphology of the DAPI-stained nuclei according to Ref. 4 . We found CAR expressed in germ cells in a stage-dependent manner. Strong CAR staining was seen in B spermatogonia and preleptotene cells in stages V and VII, respectively. In addition, leptotene cells from stage IX were CAR positive, although staining appeared weaker. No CAR staining was seen in germ cells from stage I or XI.
From these experiments we conclude that CAR expression is stage dependent and confined to the migrating germ cells, suggesting that CAR may be involved in transepithelial migration across the BTB.
CAR and claudin-3 target the same migrating germ cells
The claudin family consists of integral membrane proteins important for tight junction formation and function. Claudin-3 was recently expressed in adult mouse SCs in a stage-dependent manner with pronounced expression in stage VIII (38). Interestingly, claudin-3 appeared to associate transiently with newly formed tight junctions at the time germ cells move across the BTB. Indirect immunofluorescence analysis of claudin-3 in sections of adult testis revealed that the localization of claudin-3 in the tubules was strikingly similar to CAR when the tight junction protein JAM-A was used to visualize the BTB (Fig. 4A). Importantly, costaining of CAR and claudin-3 showed that the same migratory cells were indeed targeted by the two proteins (Fig. 4B). CAR and claudin-3 appeared not to colocalize completely, indicating that resolution was enough to distinguish the two cytoplasmic antibodies in germ and SCs, respectively.
We conclude that CAR and claudin-3 show similar expression patterns in the seminiferous epithelium, indicating that both proteins might be involved in transepithelial migration of germ cells during spermatogenesis.
Migrating germs cells are localized in an intermediate compartment during transit across the BTB
The BTB differs form other tight junctions in that it has a dynamic structure that periodically must break down to allow germ cells to migrate from the basal to the adluminal compartment. The mechanism of transmigration, including maintenance of barrier properties, has only recently begun to unveil. Although the presence of an intermediate compartment occupied by germ cells in transit has been suggested (39), previous studies have failed to demonstrate more than one tight junction structure per SC (40, 41)
A careful examination of sections from adult testis costained with antibodies toward CAR and occludin revealed migrating cells positioned between two occludin-containing junctions (Fig. 5). Not all CAR-positive cells in the testis were found in this putative intermediate compartment. Instead, the presence of this compartment was a rare event, and we estimate that we find this compartment in approximately one to two tubules per adult tissue section. Based on the fact that CAR staining in these cells is rather weak, results from the microdissected squash experiments, and the lack of late, elongating spermatids, we estimate the stage of these tubules to be IX. The infrequency of the intermediate compartment suggests a limited time frame for its existence, which might explain why it has not been found previously. The boxed area in Fig. 5A was selected for a three-dimensional image stack analysis (Fig. 5B). As revealed by the y-z plane image, the migratory cells were surrounded by a luminal and a basal occludin-containing structure.
This experiment indicates that barrier integrity during germ cell transit across the BTB is maintained by the existence of an intermediate, occludin-based compartment occupied by migrating germ cells.
Expression of JAM proteins in the testis
Proteins belonging to the JAM family are important for migration of leukocytes across endothelial and epithelial barriers. Because several of the JAMs are expressed in the testis, we studied the distribution of JAMs during migration of germ cells across the BTB. Therefore, indirect immunofluorescence analyses using specific antibodies toward CAR and JAM-A and -B were performed (Fig. 6A). JAM-A and JAM-B are known components of the BTB, and are expressed in all stages of the spermatogenic cycle (26) (and data not shown). Figure 6A shows that JAM-A and JAM-B partially colocalized with CAR during germ cell transit. However, attempts to coimmunoprecipitate CAR with JAM-A or JAM-B from testis homogenate or cell cultures failed, and we were not able to demonstrate an interaction between these proteins (data not shown). JAM-C was not found in the BTB, as was also previously reported (26) (data not shown).
JAML is mainly expressed on leukocytes and has previously been shown to form a functional complex with CAR, important for transepithelial migration (24, 42). Whether JAML is expressed also on endothelial or epithelial cells has not yet been investigated. To analyze if JAML was expressed in the testis, an RT-PCR was performed with JAML-specific primers on RNA purified from isolated rat SCs and from adult mouse testis perfused with PBS. Interestingly, JAML expression was found in both isolated SCs (rat JAML, Fig. 6B, lane 1) and in the testis (mouse JAML, Fig. 6B, lane 3), indicating that JAML might be a component of the seminiferous epithelium. No bands were detected when reverse transcriptase was omitted from the RT-PCR reaction (Fig. 6B, lanes 2 and 4).
We conclude that CAR partially colocalizes with JAM-A and JAM-B, but not with JAM-C, during germ cell transit, and that JAML is expressed in the testis and isolated SCs. Thus, JAM proteins may participate in migration of germ cells across the BTB.
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In the immature testis, CAR-positive cells were found throughout the developing tubules. However, by the time the BTB is formed, the CAR-expressing cells were relocalized to the basal side of the tubule. Surprisingly, CAR did not colocalize with the tight junction marker occludin, and, therefore, CAR appears not to be a component of the tight junctions in the BTB. This finding was unexpected because CAR is found in the tight junctions of most epithelia in vivo (10). The seminiferous epithelium of the testis differs from epithelium in other organs in that it periodically goes through an extensive reorganization of the BTB to accommodate the migration of germ cells across the BTB (5). The testis-specific expression pattern of CAR might reflect a different function for CAR in the testis compared with epithelia in other organs.
The most intriguing finding described in this paper is the stage-dependent expression of CAR in migrating germ cells in the adult testis. The specific expression of CAR in these cells suggests that CAR is involved in the transmigration of germ cells across the BTB, an event that is poorly understood at the molecular level. CAR was also recently reported to be important for transepithelial migration of leukocytes (24). Together, these findings support the hypothesis that CAR is a cell surface molecule involved in migration of cells across tight junctions. CAR and claudin-3 might contribute to formation of an adhesive "tunnel" that surrounds the germ cells as they migrate across the BTB, as has been suggested for JAM-A in transendothelial migration (46). A tight seal between the germ cell and the SC might help to keep the epithelium intact. Transmigration requires the creation of a microenvironment that facilitates the extensive restructuring of the BTB. Cytokines appear to play an important role because they are able to change transiently steady-state levels or subcellular distribution of junction proteins, such as occludin, ZO-1, N cadherin JAM-A, and CAR (15, 18, 47, 48). Testosterone was recently shown to regulate the expression of claudin-3 in SCs. As shown in this paper, claudin-3 encodes a transient component of newly formed tight junctions and is coexpressed with CAR during germ cell migration across the BTB (38). However, we have no indications that CAR expression is testosterone dependent (data not shown).
During transendothelial and transepithelial migration of leukocytes, cell surface receptors on migrating leukocytes act in concert with cell surface proteins on the endothelium/epithelium. Most likely, a similar situation exists when germ cells migrate across the seminiferous epithelium. This raises the possibility that CAR has a coreceptor expressed on the SC surface. There are several possible candidates. The extracellular domain of CAR has previously been found to mediate homophilic interactions between CAR molecules on neighboring cells (49). However, although we found CAR expressed in SCs during postnatal development, we have no evidence that CAR is expressed in SCs in the adult. Therefore, we do not believe that CAR itself is the homophilic partner. CAR also forms heterophilic interactions with JAM-C and JAML, two other cell surface proteins from the CTX family (12, 24). JAML is a very interesting candidate for a coreceptor because CAR and JAML were previously shown to form a functional complex important for transepithelial migration of leukocytes (24). Although JAML was previously expressed mainly on leukocytes, no detailed analysis of JAML expression in endothelial or epithelial tissues has been undertaken (42). Here, we report for the first time that JAML is expressed in a nonhematopoietic tissue in the testis as well as in isolated SCs, at least at the RNA level. Although we cannot exclude a contamination of remaining leukocytes in the perfused testis, the results indicate that JAML is expressed in the seminiferous epithelium. Analysis of the localization of JAML in the testis as well as investigation of a possible interaction with CAR will be interesting but require production of suitable antibodies toward JAML. Colocalization of CAR and JAM-A, -B, and -C during germ cell transmigration was also investigated. We find JAM-A and JAM-B, but not JAM-C, in the BTB as previously reported (26). JAM-A and JAM-B did partially colocalize with CAR, but no protein complex between CAR and these proteins could be demonstrated (data not shown). Although not investigated here, also other CTX members such as BT-IgSF, JAM4, and CLMP are expressed in the testis (50, 51, 52). However, these proteins have so far not been implicated in cell migration across tight junctions. The counter receptor might also be an unrelated protein that has not previously been shown to form heterophilic interactions with CAR.
A long-standing question has been how epithelial integrity can be maintained during germ cell migration across the BTB. The intermediate compartment theory predicts the existence of a unique compartment occupied by germ cells in transit from the basal to adluminal compartment (39). However, attempts to identify more than one occludin-containing junction per SC have failed (40, 41). By using CAR as a marker to identify migrating cells, indirect immunofluorescence experiments revealed the presence of migrating germ cells in close association with two different occludin-containing junctions. Not all CAR-positive migrating germ cells were enclosed in this compartment, indicating that it only briefly existed, explaining why it has not been detected previously. We do not find occludin staining completely surrounding each separate germ cell, and, therefore, we hypothesize that germ cells migrate as a clone with intact cytoplasmic bridges across the BTB. These results strengthen the intermediate compartment theory and suggest that epithelial integrity is maintained by the formation of new tight junctions at the basal side of the migrating germ cells, before the original tight junction is broken down to allow the germ cells to continue their migration toward the lumen of the seminiferous tubule. However, immunoelectron microscopy or dual beam microscopy is necessary to analyze this putative intermediate compartment in more detail.
We suggest that CAR is expressed in migratory germ cells, probably in the plasma membrane. However, the subcellular localization of CAR appears to change at the onset of spermiogenesis, since we have previously shown that CAR is expressed in the Golgi-derived acrosome of germ cells (12). This relocalization of CAR, together with the fact that CAR is found in complexes with JAM-C and tubulin, indicates that CAR is also involved in cell differentiation in the later phases of spermatogenesis (12, 53).
CAR is emerging as a multifunctional protein. In the testis, CAR might be involved in several important processes, including germ cell migration, tight junction formation, and spermatid differentiation. Considering that CAR expression may be regulated by inflammatory signaling, it may also be involved in the pathogenesis of inflammation-induced testicular damage. Because targeted disruption of CAR is embryonic lethal due to heart failure, the analysis of CAR function in the testis requires a testis-specific conditional knockout of CAR.
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Acknowledgments |
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Disclosure Statement: The authors have nothing to declare.
First Published Online August 9, 2007
Abbreviations: BrdU, Bromodeoxyuridine; BTB, blood-testis barrier; CAR, coxsackievirus and adenovirus receptor; CTX, cortical thymocyte marker of the Xenopus; DAPI, 4',6-diamidino-2-phenylindole; JAM, junction adhesion molecule; JAML, junctional adhesion molecule-like; SC, Sertoli cell.
Received March 20, 2007.
Accepted for publication August 1, 2007.
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