Molecular Human Reproduction, Vol. 8, No. 5, 419-425,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Study of the HIV-1 receptors CD4, CXCR4, CCR5 and CCR3 in the human and rat testis*
Germ-Inserm U.435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France
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Abstract |
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Sexual transmission of HIV is one of the main routes of transmission of AIDS. Despite the fact that the virus has been found in the semen and germ cells of patients with HIV, little is known about how the virus infects the cells of the genital tract. We studied the cellular distribution of CD4, a receptor necessary for HIV infection, and the major HIV co-receptors CCR3, CCR5 and CXCR4 in the rat and human testis. We used RT–PCR, Northern blotting and immunohistochemistry to demonstrate that CCR3 is absent from the testes of both species, whereas CCR5 and CXCR4 are present on the resident testicular macrophages in the interstitial space but not in the germ cell line. All of the human testicular macrophages expressed the markers CD45 and MAC387 and most also expressed CD4. Thus, our data suggest that macrophages in the testis may be infected by HIV and that these macrophages may be a site of early viral localization and a potential HIV reservoir. This may in turn alter the activity of Leydig cells and subsequently affect spermatogenesis.
CCR5/CD4/CXCR4/macrophages/testis
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Introduction |
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The testis is a potential target for HIV-1, as AIDS and asymptomatic seropositive patients are known to suffer from serious testicular affections such as orchitis, oligo- or azoospermia and some germ cell tumours (Sellmeyer and Grunfeld, 1996
; Dejucq and Jégou, 2001). The testis is also a potential reservoir for the virus, as tissue-specific HIV-1 quasispecies have been identified in the testis (Van't Wout et al., 1998). Furthermore, the viral load in the blood has been found to decrease in patients given anti-retroviral therapy, whereas the viral load in sperm remains high (Zhang et al., 1998) and HIV particles have been found in the semen of these patients (Zhang et al., 1998). Finally, the testis is the cellular source of spermatozoa which are a component of the semen that is one of the main transmission vectors of HIV-1. HIV has been found in the mononuclear cell fraction of semen and in the cell-free seminal fluid of seropositive men (Ho et al., 1984; Zagury et al., 1984, 1985; Borzy et al., 1988). HIV-1 has been reported to be present in the spermatozoa themselves (Baccetti et al., 1994, 1998) and in the germ cells within the seminiferous tubules of the testis (Muciaccia et al., 1998; Shevchuk et al., 1998), although the latter observation remains controversial (Pudney et al., 1998; Quayle et al., 1998).
It is important to determine which mechanism HIV uses to penetrate the male genital tract and the precise cellular targets of this virus. To enter a target cell, HIV-1 requires CD4 (Maddon et al., 1986) and other co-receptors (Berger et al., 1999). Among the latter, the chemokine receptors CXCR4, CCR5 and CCR3, which belong to the family of G-protein coupled receptors with seven membrane-spanning domains, have been identified as the principal HIV-1 co-receptors. CXCR4 and CCR5 are involved in the entrance of T-cell tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 isolates respectively (Samson et al., 1996; Berger et al., 1999) and CCR3 is mainly involved in the entry of M-tropic HIV-1 strains into the microglia of the central nervous system (He et al., 1997). It is still not clear whether CD4 is expressed in the germ line (Gil et al., 1995; Kim et al., 1999), but it is known that this antigen is present on the surface of lymphocytes and monocytes in semen (Gobert et al., 1990). Although Kim et al. have previously reported that CXCR4 and CCR5 are not found on the surface of sperm (Kim et al., 1999), no studies have investigated the expression of CXCR4, CCR5 and CCR3 within the testis.
We have analysed the cellular distribution of CD4 and the HIV-1 co-factors CXCR4, CCR5 and CCR3 in the testis to elucidate the cellular and molecular systems involved in the interaction between HIV and the testis.
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Materials and methods |
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Human tissues and animals
Five normal testis specimens were obtained from patients who underwent orchidectomy to treat prostate tumours at the CHU Ponchaillou Hospital, Rennes, France. The clinical status of the patients revealed no reproductive abnormalities or testicular infections, but did reveal a prostate tumour. This study was approved by local ethics committees and informed consent was obtained from the patients. Male Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest-Saint-Isle, France).
Preparation of Sertoli cells, peritubular cells, Leydig cells, resident macrophages and germ cells
Sertoli cells (enrichment >98%) and peritubular cells (enrichment >99%) were isolated from 20-day-old Sprague-Dawley rats as described previously (Skinner and Fritz, 1985; Toebosch et al., 1989). Leydig cells (purity >98%) and testicular resident macrophages (purity >94%) (Piquet-Pellorce et al., 2000) were isolated from 90-day-old rats according to a previously described method (Klinefelter et al., 1987). Spermatogonia were prepared from 9-day-old Sprague-Dawley rats according to the method of Bellvé et al. (Bellvé et al., 1977). A mixed population of germ cells was prepared from adult rat testes as previously described (Pineau et al., 1993), pachytene spermatocytes and round spermatids (purity >90%) were prepared by centrifugal elutriation (Pineau et al., 1993).
RNA analyses
After harvesting, the isolated cells were dried and stored at –20°C until RNA extraction. Total RNA was isolated by use of the RNeasy kit (Qiagen GmbH, Les Ulis, France).
For Northern blotting, total RNA samples (10 µg per lane) were separated on a 1% agarose gel and transferred onto a Hybond N membrane (Amersham, Les Ulis, France) as previously described (Sambrook et al., 1989). The blots were prehybridized for at least 6 h at 42°C and hybridized overnight with the probes for CD4, CXCR4, CCR5 and ß-actin obtained from a PCR product and 
32P-labelled by random priming (Feinberg and Vogelstein, 1983). The hybridization solution used contained 50% formamide, 10% dextran sulphate and 1% sodium dodecyl sulphate (SDS). The Northern blot membranes were washed for 40 min at 42°C in a solution containing 2xsaline sodium citrate (SSC) and 0.05% SDS and for 30 min at 65°C in a solution containing 0.1xSSC and 0.1% SDS. They were exposed to an X-ray film in the presence of an intensifying screen at –80°C.
For RT–PCR, 4 µg of total RNA was treated with 5 IU of RNase free DNase I (Promega, Charbonnières, France), in the presence of 40 IU/µl RNasin (Promega) for 30 min at 37°C. Reverse transcription was performed by use of the superscript pre-amplification system kit (Gibco Life Technologies, Cergy-Pontoise, France). The cDNA samples were amplified using the following forward primers: 5'-TCACTTGGGTGGTGGCTG-3', 5'-TCACTTGGGTGGCTG-3', 5'-GACTACCTCATGAAGACT-3', 5'-TGAGAAGCATGACGGA CAAG-3', 5'-GACCTGCTCTTCCTCGTCAC-3' and 5'-TCAGGGAAAGAAAGTGGTGC-3' and the following reverse primers: 5'-TCAG-CTTTCAAAGACCCAATC-3', 5'-GTCTCTGAGAACCCTACT-3', 5'-TTGCTGATCCACATCTTG-3', 5'-TGGAGTGTGACAGCTTGGAG-3', 5'-TCATGCAGCAGTGGGAGTAG-3' and 5'-AAGAAGGAGCCCTGATTTCC-3' for rat CCR5 (Genbank accession number Y12009), human CCR5 (Genbank accession number NM000579), hCXCR4 (Genbank accession number AF052572), human CCR3 (Genbank accession number NM001837), human CD4 (Genbank accession number X87579) and ß-actin (Genbank accession number J00691) respectively. The PCR conditions were: 93°C for 3 min, followed by 35 cycles of 93°C for 1 min, 54°C (for ß-actin) or 60°C (for the other transcripts) for 1 min and 72°C for 2 min. The amplified products (512 bp for ß-actin, 411 bp for human CCR5, 483 bp for human CXCR4, 652 bp for human CCR3 and 138 bp for human CD4) were analysed electrophoretically on 1% agarose gels containing ethidium bromide. The 754 bp rCCR5 product was additionally transferred onto a Hybond N membrane (Amersham). Probes were 32P-labelled by random priming (Feinberg and Vogelstein, 1983) and used for blot hybridization.
The nucleotide sequences of both strands of each PCR product were determined with the dye nucleotide cycle sequencing technique and an 373A DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Immunohistochemistry
Human testes were dissected out, fixed in 4% formaldehyde solution, dehydrated through increasing grades of alcohol and embedded in paraffin wax. Tissue sections (6 µm) were deparaffinized and rehydrated. Rat testes were dissected out and frozen in isopentane cooled in liquid nitrogen. The tissue samples were cut into sections (6 µm) at –20°C using in a Microm cryostat HM 560 (Walldorf, Germany). The sections were fixed for 10 min in acetone at –20°C and permeabilized for 2 min in methanol at –20°C. The sections (except those used to label ED1, a marker for newly infiltrated circulating macrophages, and ED2, a marker of resident macrophages from non-lymphoid tissues) were incubated in an antigen-retrieval solution (10 mmol/l citrate, pH 6) for 15 min as previously described (Cuevas et al., 1994) and then washed in 0.05 mol/l Tris-buffered saline (TBS, pH 7.6). The sections were incubated for 5 min in 3% H2O2 to block endogenous peroxidase activity, rinsed in TBS and incubated twice for 10 min in TBS supplemented with 1% bovine serum albumin (BSA) (or 3% BSA for the sections used to stain CXCR4) to block the non-specific sites. The tissue sections were incubated overnight at 4°C with either the first antibody or with IgG as a negative control. All other steps were performed at room temperature.
After two washes with TBS, the sections were incubated for 30 min with a biotinylated second antibody, rinsed again with TBS and then incubated with peroxidase-conjugated streptavidin (1:500 dilution; Dako, Trappes, France) for 30 min. The cryostat sections used to stain CXCR4 were then incubated with the goat anti-rabbit EnVision-HRP-enzyme conjugate (Dako) for 1 h. After a final wash with TBS, the sections were incubated with either amino-ethyl-carbazole substrate for 30 min or 3,3' di-amino-benzidine substrate (both Dako) for 5–10 min to reveal the specific staining. The antibodies, dilutions and IgG controls used are summarized in Table I. The nuclei were counterstained with a Hemalun Masson solution. The sections were photographed by use of an Olympus AX60TF microscope with monochromatic objectives (Olympus, Paris, France), coupled to a digital macro camera (Kigamo, Metis, France). The stained positive cells were analysed in up to 30 different microscopic fields. Co-localization was carried out on adjacent histological sections.
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Results |
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CXCR4, CCR5, CCR3 and CD4 mRNA expression in the human and rat testis
The possible presence of CXCR4, CCR5, CCR3 and CD4 mRNA in the testis was investigated by RT–PCR and Northern blot. RT–PCR products of the predicted sizes for CCR5, CXCR4 and CD4 were found in the whole human testis, but no products corresponding to CCR3 were found (Figure 1A
). Due to the rarity of human testicular tissue, the rat was used as a source of Sertoli cells, Leydig cells, peritubular cells, testicular resident macrophages and total germ cells.
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Northern blotting analysis showed that CXCR4 and CD4 were not constitutively produced in Sertoli, Leydig, peritubular or total germ cells, but a 1.9 kb transcript corresponding to CXCR4 and a 3.5 kb transcript corresponding to CD4 were detected in resident testicular macrophages (Figure 1B
). In contrast, CCR5 mRNA was not detected by Northern blotting in any testicular cell type (data not shown). We therefore used an RT–PCR approach to determine whether the absence of CCR5 mRNA was due to the low expression level of the gene, and found that there is no CCR5 mRNA in peritubular, Sertoli, Leydig or germ cells. However, the predicted 754 bp band was present in resident testicular macrophages (Figure 1C).
Immunohistolocalization of CXCR4 and CCR5 within the human testis
The IgG controls, used for detection of non-specific immunostaining, were always negative (data not shown). No positive staining was ever observed in peritubular cells or seminiferous tubules from human adults (Figure 2), whereas specific staining for CXCR4 (Figure 2A,C,E) and CCR5 (Figure 2G,I,K) was observed in a few interstitial cells. No positive staining for CCR3 was observed in any of the testis sections (data not shown), thus confirming the results of the RT–PCR and Northern blot analyses described above. CXCR4- and CCR5-positive cells both presented the morphological characteristics of macrophages (Hedger, 1997).
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To determine the precise nature of the cells stained in the interstitial tissue, we cut several serial sections, on which we found both CCR5- and CXCR4-positive cells and the CD45 antigen, which is a marker for all leukocytes including macrophages (Donovan and Koretzky, 1993
). We also found the CD68 antigen, a marker for monocytes, macrophages, dendritic cells, neutrophiles, basophiles and large lymphocytes (Pulford et al., 1990; Bukovsky et al., 2001) and the Mac387 antigen, a marker for a subset of reactive/infiltrating monocytes/macrophages (Flavell et al., 1987). We demonstrated that combinations of these antigens could be found in the same sections: CXCR4 and CD45 in Figure 2A versus 2B, CCR5 and CD45 in Figure 2G versus 2H, CCR5 and CD68 in Figure 2I versus 2J, CXCR4 and Mac387 in Figure 2E versus 2F, and CCR5 and Mac387 in Figure 2K versus 2L. However, except for the co-localization of CXCR4 and Mac387 and of CCR5 and Mac387, co-localization was never complete. In fact, by quantifying serial histological sections, we demonstrated that fewer interstitial cells were positive for CXCR4 and CCR5 than for CD45 and CD68: 89% of the CXCR4-positive cells were also CD45-positive, 82% of the CCR5-positive cells were also CD45-positive, 14% of the CXCR4-positive cells were also CD68-positive, and 37% of the CCR5-positive cells were also CD68-positive.
To characterize the CXCR4- and CCR5-positive testicular macrophages further, we co-localized CCR5- and CXCR4-positive cells with the CD4 antigen, which is present in T cells and monocytes/macrophages: 53% of the CXCR4-positive cells also expressed CD4 (Figure 3A versus 3B) and 66% of the CCR5-positive cells also expressed CD4 (Figure 3C versus 3D). Importantly, 
100% of CXCR4-positive cells were also CCR5-positive (Figure 3E versus 3F).
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Immunohistolocalization of CXCR4 within the rat testis
The IgG controls used for detection of non-specific immunostaining were always negative (data not shown). In the adult rat, no positive staining for CXCR4 was observed in the peritubular cells or within the seminiferous tubules (Figure 4A,B
), but CXCR4 was found to be localized in the interstitial tissue. The CXCR4-positive cells had the morphologic characteristics of rat testicular macrophages, with an indented or convoluted nucleus containing distinct heterochromatin and a granular cytoplasm (Hedger, 1997). To confirm this observation, the rat macrophages were characterized with ED1 and ED2 antibodies, as the corresponding antigens are known to be markers for newly infiltrated circulating macrophages and resident macrophages from non-lymphoid tissues respectively (Dijkstra et al., 1985). We found that CXCR4 and these antigens were frequently co-localized (Figure 4A versus 4B for ED1 and Figure 4C versus 4D for ED2). More precisely, we found that most CXCR4-positive cells were also ED2-positive (61.9 ± 8.6%), whereas less than half were ED1-positive (44.3 ± 4.7%).
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We could not study the cellular distribution of CCR5 in the rat testis as an anti-rat CCR5 antibody is not commercially available.
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Discussion |
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There are a number of arguments favouring the hypothesis that the seminiferous tubules of the testis are a target of HIV-1 (Baccetti et al., 1994
; Nuovo et al., 1994; Muciaccia et al., 1998; Shevchuk et al., 1998). One of the classical approaches that can be used to investigate whether HIV can contaminate a particular cell type is to seek their specific receptors or co-factors. Among these receptors, CD4, CXCR4, CCR3 and CCR5 are the most likely candidates (Berger et al., 1999).
Our results showed that there is no CXCR4, CCR5 or CCR3 protein within the seminiferous tubules in humans. There was also no mRNA or protein for CXCR4, CCR5 or CCR3 in highly pure mitotic, meiotic or post-meiotic germ cells in the rat. The lack of CCR5 and CXCR4 in sperm had been previously reported (Kim et al., 1999). Nevertheless, this does not rule out the possibility that HIV particles may enter the different constituents of the germ cell lineage as other alternative receptors have not yet been investigated.
Whether or not HIV is present in the seminiferous tubules, a number of arguments suggest that the testis in general is a target for HIV. Men often have increased testosteronaemia during the early stages of HIV infection (Merenich et al., 1990; Christeff et al., 1992), whereas men with AIDS often have lower concentrations of circulating testosterone (Dobs et al., 1988; Raffi et al., 1991; Schurmeyer et al., 1997). This suggests either a direct effect on Leydig or interstitial cells or a dysfunction of the endocrine regulation system. Thus, the testis is a likely site of early viral localization and a potential HIV-1 reservoir (Coombs et al., 1998; Zhang et al., 1998).
In contrast to the situation in seminiferous tubule cells, our immunolabelling results showed that CCR5 and CXCR4, but not CCR3, are only found in the interstitial cells that are macrophages. A few of the CXCR4- and CCR5-positive cells did not express the leukocyte marker CD45. This is probably either due to the loss of some cells from the serial sections or to differences in the sensitivity of the different antibodies used. We found that all of the cells that expressed CXCR4 and CCR5 also expressed the Mac387 antigen and most also expressed the CD45 antigen. Only a few of these cells also expressed CD68. All of these cells can be considered to be leukocytes. In fact, as they all expressed the Mac387 macrophage marker they can all be considered to be testicular macrophages. Interestingly, Mac387-positive macrophages accumulate in paracortical areas during intense primary simian immunodeficiency virus (SIV) infection in monkeys and are involved in the intense primary SIV production. This strongly suggests that testicular CCR5-, CXCR4- and Mac387-positive macrophages participate in HIV-1 production during primary HIV infection (Otani et al., 1999). This is further substantiated by our observations that the majority of the testicular CCR5-, CXCR4- and Mac387-positive macrophages expressed the CD4 antigen, and must therefore have all of the molecular equipment required to be infected with HIV-1 (Berger et al., 1999). As the HIV-2 virus uses a CD4-independent pathway, which only involves CXCR4 (Endres et al., 1996) and CCR5 (Reeves et al., 1999), to infect its host cells, it is possible that the CCR5- and CXCR4-positive resident macrophages can be infected by both T-tropic and M-tropic strains of HIV-1, or by HIV-2 strains.
Given that CD4 and the HIV co-factors were only found in resident testicular macrophages, these cells might be an HIV reservoir. This hypothesis is further supported by the fact that macrophages have already been shown to constitute a potential cellular reservoir in other tissues (Martin and Bandres, 1999; Worgall et al., 1999; Brodie, 2000) and that testicular macrophages have been shown to be HIV infected cells (Pudney and Anderson, 1991). Macrophages would provide HIV with a favourable environment due to the so-called `immunological privilege' situation in the testis (Head and Billingham, 1985). Thus, HIV could fail to elicit an immunological response during infection.
We also showed that in rats, as in humans, the only cells that express CXCR4 are the interstitial leukocytes. This therefore shows strict evolutionary conservation. Moreover, our results show that the CXCR4-positive cells are mainly the interstitial macrophages expressing the ED1 and/or ED2 antigens, while the other CXCR4-positive cells, which are ED1- and ED2-negative, are most probably lymphocytes.
The chemokine receptors CXCR4 and CCR5, observed at the membrane surface of testicular macrophages, may also be involved in the activation of resident testicular macrophages and in the recruitment of newly infiltrated circulating macrophages during orchitis. Indeed, we have previously shown that the testicular somatic cells contribute to chemokine production during a testicular inflammation (Aubry et al., 2000a,b)
In conclusion, we have shown that the HIV-1 receptors CD4, CXCR4 and CCR5, but not CCR3, are found in the human and rat testis. These receptors, which play a major role in the cellular entry of HIV-1, are only found in human testicular macrophages that express the Mac387 marker and some of which also express CD4. Therefore, the testicular macrophages may be infected with T-tropic and M-tropic strains of HIV-1 and potentially constitute a reservoir for HIV.
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Acknowledgements |
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This work was supported by INSERM, the Ministère de l'Education Nationale de la Recherche et de la Technologie, the Fondation pour la Recherche Medicale (FRM), the Association pour la Recherche sur le Cancer (ARC), the Ligue nationale contre le cancer, the Région Bretagne and the Fondation Langlois.
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Notes |
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1 To whom correspondence should be addressed. E-mail: michel.samson{at}rennes.inserm.fr
* *Preliminary reports of this work were presented at the 11th Workshop on Molecular and Cellular Endocrinology of the Testis, Saint-Malo, France, 13–17 May, 2000
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Submitted on October 2, 2001; accepted on February 13, 2002.
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