Zika virus infection damages the testes in mice.

Infection of pregnant women with Zika virus (ZIKV) can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually. Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was caused by a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

Porcine reproductive and respiratory syndrome virus infects the reproductive system of male piglets and impairs development of the blood-testis barrier.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and ß-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.

Infectious bronchitis virus in testicles and venereal transmission.

Even though males represent only 8%-12% of the birds of a breeder flock, their role in infectious bronchitis virus (IBV) dissemination is largely unknown. We first assessed the effect of IBV replication in the chicken testes. Ten-week-old males were inoculated with Arkansas (Ark) or Massachusetts (Mass) IBV virulent strains. Seven days postinoculation (DPI) IBV RNA was detected in testicles of 100% of M41- and in 96% of Ark-infected males. Marginal nonsynonymous variation was detected in spike (S) gene of the predominant population of IBV replicating in the testes compared to the S gene of the predominant population of viruses prior to inoculation. IBV M41 and Ark were detected in spermatogonia and Sertoli cells of testicles of infected roosters by immunofluorescence, without evident histopathological changes. We next assessed venereal transmission of IBV by artificially inseminating 54-wk-old hens either with semen from IBV-infected roosters or with IBV suspended in naïve semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.

The testis and epididymis are productively infected by SIV and SHIV in juvenile macaques during the post-acute stage of infection.

BACKGROUND: Little is known about the progression and pathogenesis of HIV-1 infection within the male genital tract (MGT), particularly during the early stages of infection. RESULTS: To study HIV pathogenesis in the testis and epididymis, 12 juvenile monkeys (Macacca nemestrina, 4-4.5 years old) were infected with Simian Immunodeficiency Virus mac 251 (SIVmac251) (n = 6) or Simian/Human Immunodeficiency Virus (SHIVmn229) (n = 6). Testes and epididymides were collected and examined by light microscopy and electron microscopy, at weeks 11-13 (SHIV) and 23 (SIV) following infection. Differences were found in the maturation status of the MGT of the monkeys, ranging from prepubertal (lacking post-meiotic germ cells) to post-pubertal (having mature sperm in the epididymal duct). Variable levels of viral RNA were identified in the lymph node, epididymis and testis following infection with both SHIVmn229 and SIVmac251. Viral protein was detected via immunofluorescence histochemistry using specific antibodies to SIV (anti-gp41) and HIV-1 (capsid/p24) protein. SIV and SHIV infected macrophages, potentially dendritic cells and T cells in the testicular interstitial tissue were identified by co-localisation studies using antibodies to CD68, DC-SIGN, alphabetaTCR. Infection of spermatogonia, but not more mature spermatogenic cells, was also observed. Leukocytic infiltrates were observed within the epididymal stroma of the infected animals. CONCLUSION: These data show that the testis and epididymis of juvenile macaques are a target for SIV and SHIV during the post-acute stage of infection and represent a potential model for studying HIV-1 pathogenesis and its effect on spermatogenesis and the MGT in general.

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.

Production of the antiviral proteins 2'5'oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia.

We report an in vitro analysis of the spatial pattern of production of three antiviral proteins (2'5'oligoadenylate synthetase, 2'5'AS; double-stranded RNA-activated protein kinase, PKR; and Mx protein, Mx) in the rat testis, in basal conditions and following stimulation with interferon (IFN) or Sendai virus. The two major constituents of interstitial tissue--Leydig cells and macrophages--constitutively produce 2'5' oligoadenylate synthetase (2'5'AS), PKR and Mx. Production of an isoform of 2'5'AS was induced following Leydig cells stimulation by the Sendai virus. The most immature germ cells, spermatogonia, were devoid of 2'5'AS whatever the type of stimulation, whereas IFN treatment induced Mx production and increased PKR production in this cell type. IFN stimulation strongly increased PKR production in all three cell types. This new set of data extends our previous investigations and demonstrates that the testis possesses an anti-viral defense system involving IFNs and IFN-induced anti-viral proteins.

Vertical transmission of gill-associated virus (GAV) in the black tiger prawn Penaeus monodon.

Chronic gill-associated virus (GAV) infection is endemic in Penaeus monodon broodstock captured from north-east Queensland in Australia and in farmed shrimp produced from these. We investigated the role of vertical transmission in perpetuating the high prevalence of these chronic GAV infections. Reverse transcription (RT)-nested PCR detected GAV in spermatophores and mature ovarian tissue from broodstock and in fertilized eggs and nauplii spawned from wild-fertilized females. In laboratory-reared P. monodon (> 12 mo old) that had a high mortality rate, RT-nested PCR detected GAV in male spermatophores at levels significantly higher than that detected in the lymphoid organ. By transmission electron microscopy (TEM), GAV virions were detected in spermatophore seminal fluid, but not sperm cells. Histological evidence of hypertrophied cell foci (spheroids) and TEM observation of GAV nucleocapsids and virions in spheroid cells was also found in 100% of lymphoid organs of approximately 1.2 g juvenile P. monodon reared in the laboratory from postlarvae collected from commercial hatcheries. Sequence analysis of PCR amplicons from parental P. monodon and fertilized eggs of artificially inseminated broodstock indicated that GAV associated with eggs can originate from both the male and female parents. Although the female GAV genotype was predominant in eggs, there was some dependence on infection levels in the male and female shrimp as indicated by RT-PCR. RT-nested PCR data on GAV levels in eggs, nauplii, protozoea and PL5 progeny of the artificial matings suggests that vertically transmitted virus is most probably associated with the egg surface.

Detection of viral RNA by electron microscopic in situ hybridization (ISH-EM) in the germinal epithelium of mice infected with encephalomyocarditis (EMC) virus.

Electron microscopic in situ hybridization (ISH-EM) was first applied to the detection of viral RNA in the germinal epithelium of mice inoculated i.p. with 10(5) plaque-forming units/mouse of the D variant of encephalomyocarditis virus (EMC-D). Signals of viral RNA were first detected in a small number of Sertoli cells showing mild degeneration at 2 days post inoculation, and 2 days later, they were also detected in germinal cells and spermatogonia when Sertoli cells showed prominent degeneration. The results clearly demonstrated that the first site of viral attack in the germinal epithelium was Sertoli cell in the case of EMC-D-induced mouse orchitis.

Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar.

The objective of this study was to determine the pathogenesis of experimental infection with a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the sites of viral replication and apoptosis in male gonads from infected boars for a period of 21 days after intranasal inoculation. Microscopically, hypospermatogenesis and abundant germ cell depletion and death were observed in the testes. Such germ cell death occurs by apoptosis, as determined by a characteristic histological patterns and evidence of massive DNA fragment detected in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction. PRRSV was detected in the testicular tissue of infected boars only. Viral nucleic acid was localized in spermatogonia, spermatocytes and spermatids but not in the vesicular and bulbourethral gland. In serial sections, PRRSV-positive cells did not co-localized with apoptotic cells. TUNEL-positive apoptotic cells were more numerous than PRRSV-positive cells in testicular sections. The present study demonstrated that type 1 PRRSV infects the spermatogonia and their progeny, and induces apoptosis in these germ cells.

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells.

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Localization of classical swine fever virus in male gonads during subclinical infection.

In an experiment using ten boars, the distribution of classical swine fever virus (CSFV) was determined in the male reproductive tract by in situ hybridization over a period of 120 days after intranasal inoculation. CSFV was detected in the testicular tissue of infected boars. Viral nucleic acid was localized to spermatogonia, spermatocytes and spermatids but was not detected in the epithelia of the prostate, epididymis or bulbourethral gland. Sections from control, CSFV-negative, pigs showed no hybridization signals for CSFV. The demonstration that CSFV infects the spermatogonia (and their progeny) suggests that this may serve as a primary reservoir for the venereal spread of CSFV.

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo.

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.

Determinants of retroviral-mediated gene delivery to mouse spermatogonia.

Spermatogonia represent a new route to transgenesis in mice and potentially in some commercially important domesticated animals. In addition, these cells are also a potential target for viral integration in patients receiving somatic cell gene therapy. But the factors influencing retroviral transduction into spermatogonia are not well understood. Because retroviral transduction is affected in part by the proliferative status of the host cell, we developed an improved cell culture system in which spermatogonia survive and proliferate for several days. We used this system to test the ability of a variety of murine and avian retroviruses to infect spermatogonia. We investigated the factors influencing retroviral transduction of spermatogonia, including the proliferative status of the infected cell, the type of viral envelope, the type of retroviral long terminal repeat, and the method of viral delivery. Here we show that many of the widely used retroviral vector systems can be used to successfully transduce spermatogonia at high efficiency. Moreover, we show that retroviral delivery of MDM2, the major downregulator of p53, promotes spermatogonial survival in culture, suggesting that p53 plays a role in regulating spermatogonial apoptosis induced by growth factor deprivation. These results further demonstrate the usefulness of this novel system of targeting substances of interest to the testis. These data have important implications for improving animal transgenesis and for understanding the risks associated with somatic cell gene therapy.