SARS-CoV-2 replicates in the human testis with slow kinetics and has no major deleterious effects ex vivo.

IMPORTANCE: SARS-CoV-2 is a new virus responsible for the Covid-19 pandemic. Although SARS-CoV-2 primarily affects the lungs, other organs are infected. Alterations of testosteronemia and spermatozoa motility in infected men have raised questions about testicular infection, along with high level in the testis of ACE2, the main receptor used by SARS-CoV-2 to enter host cells. Using an organotypic culture of human testis, we found that SARS-CoV-2 replicated with slow kinetics in the testis. The virus first targeted testosterone-producing Leydig cells and then germ-cell nursing Sertoli cells. After a peak followed by the upregulation of antiviral effectors, viral replication in the testis decreased and did not induce any major damage to the tissue. Altogether, our data show that SARS-CoV-2 replicates in the human testis to a limited extent and suggest that testicular damages in infected patients are more likely to result from systemic infection and inflammation than from viral replication in the testis.

[The human zona pellucida: New genetic findings]. / La zone pellucide - Aspects génétiques et infertilité.

The Zona Pellucida (ZP) is an ovarian specialized extracellular coat surrounding the oocyte. In human, ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. It regulates sperm binding to the oocyte during fertilization. After fertilization, ZP prevents polyspermia and is important for the protection of the developing embryo and oviductal transport avoiding ectopic implantation. According to the development of sequencing techniques, many mutations have been described in infertile patients. The aim of this review is to synthesize mutations in genes encoding ZP glycoproteins described in humans and their effects on female fertility.

Title: La zone pellucide - Aspects génétiques et infertilité. Abstract: La zone pellucide (ZP) est une matrice extracellulaire spécifique enveloppant l'ovocyte. Elle régule la liaison des spermatozoïdes à l'ovocyte lors de la fécondation. Après la fécondation, la zone pellucide prévient la polyspermie en modifiant sa conformation. La zone pellucide est importante pour la protection de l'embryon pré-implantatoire en développement lors de son trajet oviductal en évitant l'implantation ectopique. Suite au développement des techniques génétiques et du séquençage du génome, de nombreuses mutations ont été récemment décrites chez des patientes infertiles. Après avoir présenté la structure et les fonctions des glycoprotéines ZP constituant la zone pellucide, nous discutons dans cette revue de l'impact des mutations mises en évidence dans les gènes codant ces glycoprotéines sur la fertilité féminine.

Human Testicular Germ Cells, a Reservoir for Zika Virus, Lack Antiviral Response Upon Zika or Poly(I:C) Exposure.

Zika virus (ZIKV) is an emerging teratogenic arbovirus that persists in semen and is sexually transmitted. We previously demonstrated that ZIKV infects the human testis and persists in testicular germ cells (TGCs) for several months after patients' recovery. To decipher the mechanisms underlying prolonged ZIKV replication in TGCs, we compared the innate immune response of human testis explants and isolated TGCs to ZIKV and to Poly(I:C), a viral RNA analog. Our results demonstrate the weak innate responses of human testis to both ZIKV and Poly(I:C) as compared with other tissues or species. TGCs failed to up-regulate antiviral effectors and type I IFN upon ZIKV or Poly(I:C) stimulation, which might be due to a tight control of PRR signaling, as evidenced by the absence of activation of the downstream effector IRF3 and elevated expression of repressors. Importantly, exogenous IFNß boosted the innate immunity of TGCs and inhibited ZIKV replication in the testis ex vivo, raising hopes for the prevention of ZIKV infection and persistence in this organ.

Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV.

Viruses have colonized the germ line of our ancestors on several occasions during evolution, leading to the integration in the human genome of viral sequences from over 30 retroviral groups and a few nonretroviruses. Among the recently emerged viruses infecting humans, several target the testis (e.g., human immunodeficiency virus [HIV], Zika virus, and Ebola virus). Here, we aimed to investigate whether human testicular germ cells (TGCs) can support integration by HIV, a contemporary retrovirus that started to spread in the human population during the last century. We report that albeit alternative receptors enabled HIV-1 binding to TGCs, HIV virions failed to infect TGCs in vitro Nevertheless, exposure of TGCs to infected lymphocytes, naturally present in the testis from HIV+ men, led to HIV-1 entry, integration, and early protein expression. Similarly, cell-associated infection or bypassing viral entry led to HIV-1 integration in a spermatogonial cell line. Using DNAscope, HIV-1 and simian immunodeficiency virus (SIV) DNA were detected within a few TGCs in the testis from one infected patient, one rhesus macaque, and one African green monkey in vivo Molecular landscape analysis revealed that early TGCs were enriched in HIV early cofactors up to integration and had overall low antiviral defenses compared with testicular macrophages and Sertoli cells. In conclusion, our study reveals that TGCs can support the entry and integration of HIV upon cell-associated infection. This could represent a way for this contemporary virus to integrate into our germ line and become endogenous in the future, as happened during human evolution for a number of viruses.IMPORTANCE Viruses have colonized the host germ line on many occasions during evolution to eventually become endogenous. Here, we aimed at investigating whether human testicular germ cells (TGCs) can support such viral invasion by studying HIV interactions with TGCs in vitro Our results indicate that isolated primary TGCs express alternative HIV-1 receptors, allowing virion binding but not entry. However, HIV-1 entered and integrated into TGCs upon cell-associated infection and produced low levels of viral proteins. In vivo, HIV-1 and SIV DNA was detected in a few TGCs. Molecular landscape analysis showed that TGCs have overall weak antiviral defenses. Altogether, our results indicate that human TGCs can support HIV-1 early replication, including integration, suggesting potential for endogenization in future generations.

New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing.

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/ß-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.

Minimal change nephrotic syndrome in patients infected with human immunodeficiency virus: a retrospective study of 8 cases.

BACKGROUND: Human immunodeficiency virus (HIV) is associated with diverse glomerular diseases. Characteristics of minimal change nephrotic syndrome (MCNS) in this setting have been little studied, and the specific features of this uncommon association remain to be determined. METHODS: We conduct a retrospective study. Clinical, biological and pathological characteristics of patients with MCNS and HIV infection were assessed. We evaluated HIV infection by in situ hybridization and CMIP expression by immunochemistry on kidney biopsies and compared it to HIV-associated nephropathy (HIVAN) and idiopathic MCNS. RESULTS: Eight patients were identifies. In all but one of these cases, MCNS occurred after HIV diagnosis (mean of 9.5 years). Acute kidney injury was detected in three cases. Mean CD4+ lymphocyte count was 733/mm3 and three patients had a detectable HIV viral load. In situ hybridization for HIV-1 RNA detection yielded a positive signal in a few tubular cells in the renal parenchyma in two of four patients with HIV infection associated with MCNS. Podocytes of these patients presented strong positive immunostaining for CMIP (4/4). Three patients suffered steroid-dependent nephrotic syndrome, and another two patients had at least one relapse. Rituximab treatment was initiated in four cases. After a median follow-up of 20 months, all patients were in remission (complete in 5 cases). CONCLUSIONS: In patients with MCNS occurring in a context of HIV infection, podocyte injury seems to be associated with CMIP induction rather than renal HIV infection but further studies are needed to determine the molecular link between these two conditions.

Zika virus infects human testicular tissue and germ cells.

Zika virus (ZIKV) is a teratogenic mosquito-borne flavivirus that can be sexually transmitted from man to woman. The finding of high viral loads and prolonged viral shedding in semen suggests that ZIKV replicates within the human male genital tract, but its target organs are unknown. Using ex vivo infection of organotypic cultures, we demonstrated here that ZIKV replicates in human testicular tissue and infects a broad range of cell types, including germ cells, which we also identified as infected in semen from ZIKV-infected donors. ZIKV had no major deleterious effect on the morphology and hormonal production of the human testis explants. Infection induced a broad antiviral response but no IFN upregulation and minimal proinflammatory response in testis explants, with no cytopathic effect. Finally, we studied ZIKV infection in mouse testis and compared it to human infection. This study provides key insights into how ZIKV may persist in semen and alter semen parameters, as well as a valuable tool for testing antiviral agents.

Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs.

The sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the human immunodeficiency virus (HIV)/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV-infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. In this study, we used single-genome amplification, amplicon sequencing (env gene), and phylogenetic analyses to compare the genetic structures of simian immunodeficiency virus (SIV) populations across all the male genital organs and blood in intravenously inoculated cynomolgus macaques in the chronic stage of infection. Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles, and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens, and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or proinflammatory cytokine mRNA levels. These data indicate that rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.IMPORTANCE Semen is instrumental for the dissemination of viruses through sexual contacts. Worryingly, a number of systemic viruses, such as HIV, can persist in this body fluid in the absence of viremia. The local source(s) of virus in semen, however, remains unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single-genome amplification, amplicon sequencing (env gene), and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them (i.e., from epididymis, vas deferens, and seminal vesicles) displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract.

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection.

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4+ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4+ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4+ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4+ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.

A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.

The kidney as a reservoir for HIV-1 after renal transplantation.

Since the recent publication of data showing favorable outcomes for patients with HIV-1 and ESRD, kidney transplantation has become a therapeutic option in this population. However, reports have documented unexplained reduced allograft survival in these patients. We hypothesized that the unrecognized infection of the transplanted kidney by HIV-1 can compromise long-term allograft function. Using electron microscopy and molecular biology, we examined protocol renal transplant biopsies from 19 recipients with HIV-1 who did not have detectable levels of plasma HIV-1 RNA at transplantation. We found that HIV-1 infected the kidney allograft in 68% of these patients. Notably, HIV-1 infection was detected in either podocytes predominately (38% of recipients) or tubular cells only (62% of recipients). Podocyte infection associated with podocyte apoptosis and loss of differentiation markers as well as a faster decline in allograft function compared with tubular cell infection. In allografts with tubular cell infection, epithelial cells of the proximal convoluted tubules frequently contained abnormal mitochondria, and both patients who developed features of subclinical acute cellular rejection had allografts with tubular cell infection. Finally, we provide a novel noninvasive test for determining HIV-1 infection of the kidney allograft by measuring HIV-1 DNA and RNA levels in patients' urine. In conclusion, HIV-1 can infect kidney allografts after transplantation despite undetectable viremia, and this infection might influence graft outcome.

Impact of short-term HAART initiated during the chronic stage or shortly post-exposure on SIV infection of male genital organs.

BACKGROUND: The male genital tract is suspected to constitute a viral sanctuary as persistent HIV shedding is found in the semen of a subset of HIV-infected men receiving effective antiretroviral therapy (HAART). The origin of this persistent shedding is currently unknown. Phylogenetic studies indicated that HIV in semen from untreated men arises from local sources and/or passive diffusion from the blood. We previously demonstrated in human and macaque low levels and localized infection of several semen-producing organs by HIV/SIV. Using a macaque model, this study investigates the impact of short term HAART (2-4 weeks) initiated either during the asymptomatic chronic stage or 4 h post-intravenous inoculation of SIVmac251 on the infection of male genital organs. METHODOLOGY/PRINCIPAL FINDINGS: Short term HAART during the chronic stage decreased blood viral load. No major impact of HAART was observed on SIV DNA levels in male genital organs using a sensitive nested PCR assay. Using in situ hybridization, SIV RNA+ cells were detected in all male genital tract organs from untreated and treated animals with undetectable blood viral load following HAART. Infected CD68+ myeloid cells and CD3+ T lymphocytes were detected pre- and post-HAART. In contrast, short term HAART initiated 4 h post-SIV exposure led to a drastic decrease of the male genital tissues infection, although it failed to prevent systemic infection. In both cases, HAART tended to decrease the number of CD3+ T cells in the male organs. CONCLUSIONS: Our results indicate that the established infection of male genital organs is not greatly impacted by short term HAART, whereas the same treatment during pre-acute phase of the infection efficiently impairs viral dissemination to the male genital tract. Further investigations are now needed to determine whether infection of male genital organs is responsible for long term persistent HIV shedding in semen despite HAART.

Excess type I interferon signaling in the mouse seminiferous tubules leads to germ cell loss and sterility.

Type I (α and ß) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/ß overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNß (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNß RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNß production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNß challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.

Seminal expression of NY-ESO-1 and MAGE-A4 as markers for the testicular cancer.

Testicular germ cell tumours (TGCTs) are the most common malignancies in Caucasian young men and their incidence has increased over the past decades. However, a non-invasive test allowing an early diagnosis of TGCT often proves inaccurate. We have previously shown that two Cancer-Testis Antigens (CTA), namely MAGE-A4 and NY-ESO-1, were expressed by TGCT. As exfoliation of carcinoma in situ (CIS) cells or tumour germ cells from testis into seminal fluid can occur, here we studied the expression of the 2 CTA in semen smears of patients with testicular cancer in comparison with healthy men. Using semen smears from healthy controls (n = 65) and patients diagnosed for testicular tumour (n = 57) and immunological staining, we observed expression of MAGE-A4 and NY-ESO-1 proteins in seminal fluid exfoliated cells. We found a highly statistically significant difference in the ratios of stained cells to the total number of round cells between testicular cancer patients and healthy controls. Multivariable analysis, including sperm parameters and immunostaining on sperm smears, shows the improvement. This technique can provide towards testicular cancer diagnosis when it is included in the current testing regime. However, the fact that expression of these markers was not restricted to foetal germ cells led to detection in the semen of a number of healthy subjects. Although the detection of these CTA could be useful to characterize the sub-type of individual TGCTs better, we stress here that the false positive rate precludes the exclusive employment of these CTA for the early detection of testicular neoplasia.

Infection of semen-producing organs by SIV during the acute and chronic stages of the disease.

BACKGROUND: Although indirect evidence suggests the male genital tract as a possible source of persistent HIV shedding in semen during antiretroviral therapy, this phenomenon is poorly understood due to the difficulty of sampling semen-producing organs in HIV+ asymptomatic individuals. METHODOLOGY/PRINCIPAL FINDINGS: Using a range of molecular and cell biological techniques, this study investigates SIV infection within reproductive organs of macaques during the acute and chronic stages of the disease. We demonstrate for the first time the presence of SIV in the testes, epididymides, prostate and seminal vesicles as early as 14 days post-inoculation. This infection persists throughout the chronic stage and positively correlates with blood viremia. The prostate and seminal vesicles appear to be the most efficiently infected reproductive organs, followed by the epididymides and testes. Within the male genital tract, mostly T lymphocytes and a small number of germ cells harbour SIV antigens and RNA. In contrast to the other organs studied, the testis does not display an immune response to the infection. Testosteronemia is transiently increased during the early phase of the infection but spermatogenesis remains unaffected. CONCLUSIONS/SIGNIFICANCE: The present study reveals that SIV infection of the macaque male genital tract is an early event and that semen-producing organs display differential infection levels and immune responses. These results help elucidate the origin of HIV in semen and constitute an essential base to improving the design of antiretroviral therapies to eradicate virus from semen.

Human prostate supports more efficient replication of HIV-1 R5 than X4 strains ex vivo.

BACKGROUND: In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH) was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ. RESULTS: The overall prostate characteristics were retained for 21/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4IIIB strain and a primary R5X4 strain showed less efficient replication in this organ. CONCLUSION: The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.

Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro.

Semen represents the main vector for human immunodeficiency virus (HIV) dissemination worldwide and has been shown to harbor replication-competent virus despite otherwise effective highly active anti-retroviral therapy, which achieves undetectable viral load in plasma. Despite this, the origin of seminal HIV particles remains unclear, as does the question of whether the male genital tract organs contribute virus to semen. Here we investigated the presence of HIV receptors within the human testis using immunohistochemistry and quantitative real-time polymerase chain reaction. We also analyzed the infectivity of a dual tropic HIV-1 strain in an organotypic culture, as well as the impact of viral exposure on testosterone production. Our study establishes that CXCR4+, CCR5+, CD4+, and DC-SIGN+ cells are present within the interstitial tissue of human testis and that these molecules persist throughout our organotypic culture. Our data also reveal that the human testis is permissive to HIV-1 and supports productive infection, leaving testosterone production apparently unaffected. Infected cells appeared to be testicular macrophages located within the interstitial tissue. That the testis itself represents a potential source of virus in semen could play a role in preventing viral eradication from semen because this organ constitutes a pharmacological sanctuary for many current antiretrovirals.

Molecular cloning of several rat ABC transporters including a new ABC transporter, Abcb8, and their expression in rat testis.

Several members of the ABC transporter superfamily play an important role in testicular physiology and defence against anticancer drugs. Using a reverse transcription-polymerase chain reaction strategy with degenerate primers and rat testis RNA as template, we have looked for the presence of other members of this superfamily. Of the six partial cDNA found, five corresponded to ABC transporters already known -Mdr1b, Mrp1, Tapl/Abcb9, Umat/Abcb6 and Sur2/Abcc9- and one presented a strong homology with mouse and human ABCB8. Using a 5' and 3' RACE approach, we cloned the full-length cDNA and found that the predicted protein presented 92% and 80% homology with the mouse and human proteins respectively. Strong expression of rat abcb8 was found in heart, brain and testis when compared with liver, lung and spleen. In the testis, rat abcb8 was expressed both in the somatic Sertoli cells and peritubular cells and in the germline (spermatogonia and pachytene spermatocytes). Furthermore, Umat/Abcb6 was very highly expressed in the testis (high amounts in meiotic pachytene spermatocytes and low amount in post-meiotic early spermatids). In conclusion, we confirm the presence of several ABC transporters in the testis and also provide evidence of the presence of Abcb8 in the organ.

Expression of fractalkine in the rat testis: molecular cloning of a novel alternative transcript of its gene that is differentially regulated by pro-inflammatory cytokines.

The testis is a very complex organ in which cellular communications and interactions are central to spermatogenesis and where inflammatory processes can lead to sterility. Fractalkine (CX3CL1) is a chemokine involved in cell-cell interactions and in leukocyte chemoattraction. It has been reported to be expressed in testis, but its cellular expression and function in this organ has not been described. In this study we report constitutive expression of fractalkine in the testis. Expression is higher in Leydig cells than Sertoli cells, spermatogonia, pachytene spermatocytes and elongated spermatids. In both, Sertoli cells stimulated by interleukin-1beta and tumour necrosis factor alpha, and in Leydig cells, two forms of fractalkine mRNA were observed: the previously described transcript of 3.7 kb and a novel transcript of 4.2 kb. The 4.2 kb transcript has a 5' elongation and is differentially regulated. To investigate fractalkine function in testis, we abolished Leydig cell expression of fractalkine by specific destruction of this cell type using ethylene dimethane sulphonate. The absence of fractalkine expression in Leydig cells did not seem to affect the fractalkine expression by other testicular cells. In addition, the destruction of testicular macrophages by Cl2MDP (chlodronate) did not seem to affect Leydig cell expression of fractalkine. We conclude that Leydig cell expression of fractalkine could be preferentially involved in inflammation in interstitial space whereas fractalkine expressed by germ cells may participate in the cellular interactions between germ cells and other seminiferous tubule cell types.

The cancer-testis gene, NY-ESO-1, is expressed in normal fetal and adult testes and in spermatocytic seminomas and testicular carcinoma in situ.

Cancer/testis genes are potential targets for therapeutic genetic and immunologic approaches, and are highly expressed in a large variety of human cancers. However, they are not expressed in normal tissues, with the exception of the testis. The NY-ESO-1 gene is the most recently identified member of the cancer/testis family and its product is one of the most immunogenic tumor antigens. We used immunohistochemistry to investigate the expression of NY-ESO-1 in healthy human prenatal and adult testes and in 59 human testicular tumors of different subtypes. We found that NY-ESO-1 was expressed from 18 weeks until birth in human fetal testes. In the adult testis, NY-ESO-1 was strongly expressed in spermatogonia and in primary spermatocytes, but not in post-meiotic cells or in testicular somatic cells. NY-ESO-1 was not expressed in the Sertoli cells, Leydig cells, classical seminomas, or nonseminomatous germ cells in the 59 testicular tumors. In contrast, NY-ESO-1 was expressed both in carcinomas in situ, which are the earliest stage of testicular tumors (7 of 15 cases), and in spermatocytic seminomas, which are believed to be derived from spermatogonia or primary spermatocytes (8 of 16 cases). We conclude that NY-ESO-1 is a marker that can be used to follow the early progression of testicular tumorigenesis when the tumors present a similar pattern of expression to the cells from which they originated, although the later tumors cease to express NY-ESO-1.