From Ancient to Emerging Infections: The Odyssey of Viruses in the Male Genital Tract.

The male genital tract (MGT) is the target of a number of viral infections that can have deleterious consequences at the individual, offspring, and population levels. These consequences include infertility, cancers of male organs, transmission to the embryo/fetal development abnormalities, and sexual dissemination of major viral pathogens such as human immunodeficiency virus (HIV) and hepatitis B virus. Lately, two emerging viruses, Zika and Ebola, have additionally revealed that the human MGT can constitute a reservoir for viruses cleared from peripheral circulation by the immune system, leading to their sexual transmission by cured men. This represents a concern for future epidemics and further underlines the need for a better understanding of the interplay between viruses and the MGT. We review here how viruses, from ancient viruses that integrated the germline during evolution through old viruses (e.g., papillomaviruses originating from Neanderthals) and more modern sexually transmitted infections (e.g., simian zoonotic HIV) to emerging viruses (e.g., Ebola and Zika) take advantage of genital tract colonization for horizontal dissemination, viral persistence, vertical transmission, and endogenization. The MGT immune responses to viruses and the impact of these infections are discussed. We summarize the latest data regarding the sources of viruses in semen and the complex role of this body fluid in sexual transmission. Finally, we introduce key animal findings that are relevant for our understanding of viral infection and persistence in the human MGT and suggest future research directions.

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.

Forty-four novel protein-coding loci discovered using a proteomics informed by transcriptomics (PIT) approach in rat male germ cells.

Spermatogenesis is a complex process, dependent upon the successive activation and/or repression of thousands of gene products, and ends with the production of haploid male gametes. RNA sequencing of male germ cells in the rat identified thousands of novel testicular unannotated transcripts (TUTs). Although such RNAs are usually annotated as long noncoding RNAs (lncRNAs), it is possible that some of these TUTs code for protein. To test this possibility, we used a "proteomics informed by transcriptomics" (PIT) strategy combining RNA sequencing data with shotgun proteomics analyses of spermatocytes and spermatids in the rat. Among 3559 TUTs and 506 lncRNAs found in meiotic and postmeiotic germ cells, 44 encoded at least one peptide. We showed that these novel high-confidence protein-coding loci exhibit several genomic features intermediate between those of lncRNAs and mRNAs. We experimentally validated the testicular expression pattern of two of these novel protein-coding gene candidates, both highly conserved in mammals: one for a vesicle-associated membrane protein we named VAMP-9, and the other for an enolase domain-containing protein. This study confirms the potential of PIT approaches for the discovery of protein-coding transcripts initially thought to be untranslated or unknown transcripts. Our results contribute to the understanding of spermatogenesis by characterizing two novel proteins, implicated by their strong expression in germ cells. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD000872.

Revisiting rat spermatogenesis with MALDI imaging at 20-microm resolution.

Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (MS) at high definition thus calls for technological developments that were established by a number of small steps. These included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, an increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with imaging MS. Currently, a performance level of 20-µm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2-16-kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is among the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20-µm image resolution level, different stages of germ cell development in testicular seminiferous tubules; to provide a molecular correlate for its well established stage-specific classification; and to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.

Loss of Dicer in Sertoli cells has a major impact on the testicular proteome of mice.

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.

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.

Sertoli cell Dicer is essential for spermatogenesis in mice.

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

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.

Comparison of the effect of semen from HIV-infected and uninfected men on CD4+ T-cell infection.

OBJECTIVES: Semen composition is influenced by HIV-1 infection, yet the impact of semen components on HIV infection of primary target cells has only been studied in samples from HIV-uninfected donors. DESIGN: We compared the effect of seminal plasma (SP) from chronically HIV-infected (SP+) versus uninfected donors (SP-) on HIV-1 infection of peripheral blood mononuclear cells (PBMCs) and CD4 T cells. METHODS: Primary cells were infected with HIV-1 in the presence of SP+ or SP- and analyzed for infection level, metabolic activity, HIV receptor expression, proliferation and activation. SP+ and SP- were compared for infection-enhancing peptides, cytokines and prostaglandin E2 levels. RESULTS: SP- efficiently enhanced HIV-1 R5 infection of CD4 T cells, whereas SP+ enhancing activity was significantly reduced. RANTES (CCL5) concentrations were elevated in SP+ relative to SP-, whereas the concentrations of infectivity-enhancing peptides [semen-derived enhancer of viral infection (SEVI), SEM1, SEM2] were similar. CCR5 membrane expression levels were reduced on CD4 T cells shortly postexposure to SP+ compared with SP- and correlated to R5-tropic HIV-1 infection levels, and CCR5 ligands' concentrations in semen. SP+ and SP- displayed similar enhancing activity on PBMC infection by X4-tropic HIV-1. Addition/depletion of RANTES (regulated on activation, normal T-cell expressed and secreted) from SPs modulated their effect on PBMC infection by R5-tropic HIV-1. CONCLUSION: Semen from HIV-infected donors exhibits a significantly reduced enhancing potential on CD4 T-cell infection by R5-tropic HIV-1 when compared with semen from uninfected donors. Our data indicate that elevated seminal concentrations of RANTES in HIV-infected men can influence the ability of semen to enhance infection.

Production of the chemokines monocyte chemotactic protein-1, regulated on activation normal T cell expressed and secreted protein, growth-related oncogene, and interferon-gamma-inducible protein-10 is induced by the Sendai virus in human and rat testicular cells.

Several viruses infect the testis, inducing inflammation, which may lead to infertility. In this study we investigated the production in rat and human testicular cells exposed to the Sendai virus of several chemokines that play a major role in inflammatory processes. Exposure of rat testicular macrophages and Sertoli, Leydig, and peritubular cells to the Sendai virus led to the production of mRNA and protein for monocyte chemotactic protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted protein, growth-related oncogene-alpha, and interferon-gamma-inducible protein-10. In rat peritubular cells exposed to the Sendai virus, MCP-1 production was time and dose dependent. In contrast, rat germ cells did not produce these chemokines. Chemokine synthesis was detected in human Leydig cells exposed to the Sendai virus, but not in human total germ cells, suggesting that rats and humans display similar responses in terms of chemokine production. MCP-1, regulated on activation normal T cell expressed and secreted protein, growth-related oncogene-alpha, and interferon-gamma-inducible protein-10 have been reported to be chemoattractants for a large variety of leukocytes. The ability of the Sendai virus to induce chemokine production in somatic cells (mostly peritubular and Leydig cells) may therefore increase the recruitment of leukocytes to sites of infection.

An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice.

Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.

The chromatin remodeling factor Mi-2alpha acts as a novel co-activator for human c-Myb.

The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodeling factor Mi-2alpha as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2alpha and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2alpha, previously studied as a subunit in the NuRD co-repressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2alpha in c-Myb-expressing K562 cells, led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2alpha were compared, the Myb-Mi-2alpha co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2alpha. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2alpha, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4-tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2alpha transactivational co-operation, as did co-transfection with p300.

Identification, molecular cloning, and cellular distribution of the rat homolog of minichromosome maintenance protein 7 (MCM7) in the rat testis.

As part of a program to decipher the rat testicular proteome, we studied spermatogonia and identified numerous proteins including the human homolog of the Minichromosome Maintenance Protein 7 (MCM7). MCM7 has been implicated in DNA replication in various species, but had not been detected in the testis. Here we describe the cellular distribution of MCM7 transcripts and protein, and their testicular ontogenetic expression. The full-length coding region of the rat MCM7 was also characterized. Northern blot analyses showed that MCM7 transcripts are more abundant in the testis than other organs and confirmed the presence of the 2.4 kb MCM7 transcript at all ages studied. Interestingly, two additional transcripts of 3.2 and 1.6 kb were found from 26 days post partum onwards, when spermatocytes and spermatids accumulate within the tubules. This was confirmed in isolated cell types: the three MCM7 transcripts were observed in meiotic and post-meiotic germ cells. The 3.2 kb isoform has an extended 5' untranslated region (UTR) and the 1.6 kb transcript is the result of alternative splicing of five exons. Western blot and immunohistochemistry experiments evidenced abundant MCM7 in proliferating gonocytes and Sertoli cells in the fetal testis. In the adult testis, an intense signal was observed in spermatogonia and primary spermatocytes. We conclude that the Mcm7 is one example of genes that are differently transcribed and translated in somatic and spermatogenetic cells in mammals. Further work is required to determine the roles of MCM7 in spermatogonia and germ lineage.

Study of the HIV-1 receptors CD4, CXCR4, CCR5 and CCR3 in the human and rat testis.

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.

New insights into the rat spermatogonial proteome: identification of 156 additional proteins.

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.

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.