Zika Virus Infection of Sertoli Cells Alters Protein Expression Involved in Activated Immune and Antiviral Response Pathways, Carbohydrate Metabolism and Cardiovascular Disease.

Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain-Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral RNA has been detected in semen over a year after the onset of initial symptoms, but the mode of long-term persistence is not yet understood. ZIKV can proliferate in human Sertoli cells (HSerC) for several weeks in vitro, suggesting that it might be a reservoir for persistent ZIKV infection. This study determined proteomic changes in HSerC during ZIKV infections by TMT-mass spectrometry analysis. Levels of 4416 unique Sertoli cell proteins were significantly altered at 3, 5, and 7 days after ZIKV infection. The significantly altered proteins include enzymes, transcription regulators, transporters, kinases, peptidases, transmembrane receptors, cytokines, ion channels, and growth factors. Many of these proteins are involved in pathways associated with antiviral response, antigen presentation, and immune cell activation. Several immune response pathway proteins were significantly activated during infection, e.g., interferon signaling, T cell receptor signaling, IL-8 signaling, and Th1 signaling. The altered protein levels were linked to predicted activation of immune response in HSerC, which was predicted to suppress ZIKV infection. ZIKV infection also affected the levels of critical regulators of gluconeogenesis and glycolysis pathways such as phosphoglycerate mutase, phosphoglycerate kinase, and enolase. Interestingly, many significantly altered proteins were associated with cardiac hypertrophy, which may induce heart failure in infected patients. In summary, our research contributes to a better understanding of ZIKV replication dynamics and infection in Sertoli cells.

Uropathogenic Escherichia coli Infection Compromises the Blood-Testis Barrier by Disturbing mTORC1-mTORC2 Balance.

The structural and functional destruction of the blood-testis barrier (BTB) following uropathogenic E. coli (UPEC) infection may be a critical component of the pathologic progress of orchitis. Recent findings indicate that the mammalian target of the rapamycin (mTOR)-signaling pathway is implicated in the regulation of BTB assembly and restructuring. To explore the mechanisms underlying BTB damage induced by UPEC infection, we analyzed BTB integrity and the involvement of the mTOR-signaling pathway using in vivo and in vitro UPEC-infection models. We initially confirmed that soluble virulent factors secreted from UPEC trigger a stress response in Sertoli cells and disturb adjacent cell junctions via down-regulation of junctional proteins, including occludin, zonula occludens-1 (ZO-1), F-actin, connexin-43 (CX-43), ß-catenin, and N-cadherin. The BTB was ultimately disrupted in UPEC-infected rat testes, and blood samples from UPEC-induced orchitis in these animals were positive for anti-sperm antibodies. Furthermore, we herein also demonstrated that mTOR complex 1 (mTORC1) over-activation and mTORC2 suppression contributed to the disturbance in the balance between BTB "opening" and "closing." More importantly, rapamycin (a specific mTORC1 inhibitor) significantly restored the expression of cell-junction proteins and exerted a protective effect on the BTB during UPEC infection. We further confirmed that short-term treatment with rapamycin did not aggravate spermatogenic degeneration in infected rats. Collectively, this study showed an association between abnormal activation of the mTOR-signaling pathway and BTB impairment during UPEC-induced orchitis, which may provide new insights into a potential treatment strategy for testicular infection.

T Lymphocytes and Testicular Immunity: A New Insight into Immune Regulation in Testes.

The immune privilege of the testes is necessary to prevent immune attacks to gamete-specific antigens and paternal major histocompatibility complex (MHC) antigens, allowing for normal spermatogenesis. However, infection and inflammation of the male genital tract can break the immune tolerance and represent a significant cause of male infertility. Different T cell subsets have been identified in mammalian testes, which may be involved in the maintenance of immune tolerance and pathogenic immune responses in testicular infection and inflammation. We reviewed the evidence in the published literature on different T subtypes (regulatory T cells, helper T cells, cytotoxic T cells, γδ T cells, and natural killer T cells) in human and animal testes that support their regulatory roles in infertility and the orchitis pathology. While many in vitro studies have indicated the regulation potential of functional T cell subsets and their possible interaction with Sertoli cells, Leydig cells, and spermatogenesis, both under physiological and pathological processes, there have been no in situ studies to date. Nevertheless, the normal distribution and function of T cell subsets are essential for the immune privilege of the testes and intact spermatogenesis, and T cell-mediated immune response drives testicular inflammation. The distinct function of different T cell subsets in testicular homeostasis and the orchitis pathology suggests a considerable potential of targeting specific T cell subsets for therapies targeting chronic orchitis and immune infertility.

Zika virus dysregulates human Sertoli cell proteins involved in spermatogenesis with little effect on tight junctions.

Zika virus (ZIKV), a neglected tropical disease until its re-emergence in 2007, causes microcephaly in infants and Guillain-Barré syndrome in adults. Its re-emergence and spread to more than 80 countries led the World Health Organization in 2016 to declare a Public Health Emergency. ZIKV is mainly transmitted by mosquitos, but can persist in infected human male semen for prolonged periods and may be sexually transmitted. Testicular Sertoli cells support ZIKV replication and may be a reservoir for persistent ZIKV infection. Electrical impedance analyses indicated ZIKV infection rapidly disrupted Vero cell monolayers but had little effect upon human Sertoli cells (HSerC). We determined ZIKV-induced proteomic changes in HSerC using an aptamer-based multiplexed technique (SOMAscan) targeting >1300 human proteins. ZIKV infection caused differential expression of 299 proteins during three different time points, including 5 days after infection. Dysregulated proteins are involved in different bio-functions, including cell death and survival, cell cycle, maintenance of cellular function, cell signaling, cellular assembly, morphology, movement, molecular transport, and immune response. Many signaling pathways important for maintenance of HSerC function and spermatogenesis were highly dysregulated. These included IL-6, IGF1, EGF, NF-κB, PPAR, ERK/MAPK, and growth hormone signaling. Down-regulation of the PPAR signaling pathway might impact cellular energy supplies. Upstream molecule analysis also indicated microRNAs involved in germ cell development were downregulated by infection. Overall, this study leads to a better understanding of Sertoli cellular mechanisms used by ZIKV during persistent infection and possible ZIKV impacts on spermatogenesis.

Food-grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation, germ cells sloughing in seminiferous tubules and blood-testis barrier disruption in mice.

Food-grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood-testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood-testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood-testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood-testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.

Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy?

Sertoli cells (SeC) are responsible for the immunoprivileged status of the testis thanks to which allogeneic or xenogeneic engraftments can survive without pharmacological immune suppression if co-injected with SeC. This peculiar ability of SeC is dependent on secretion of a plethora of factors including maturation factors, hormones, growth factors, cytokines and immunomodulatory factors. The anti-inflammatory and trophic properties of SeC have been largely exploited in several experimental models of diseases, diabetes being the most studied. Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive pathology in which lack of functional dystrophin leads to progressive muscle degeneration culminating in loss of locomotion and premature death. Despite a huge effort to find a cure, DMD patients are currently treated with anti-inflammatory steroids. Recently, encapsulated porcine SeC (MC-SeC) have been injected ip in the absence of immunosuppression in an animal model of DMD resulting in reduction of muscle inflammation and amelioration of muscle morphology and functionality, thus opening an additional avenue in the treatment of DMD. The novel protocol is endowed with the advantage of being potentially applicable to all the cohort of DMD patients regardless of the mutation. This mini-review addresses several issues linked to the possible use of MC-SeC injected ip in dystrophic people.

Cytokines and male infertility.

Many male infertility cases have no apparent cause, being characterized as idiopathic. Both inflammation and obesity have long been associated with infertility. On one hand, inflammation, such as orchitis and male accessory gland infections (MAGIs), are regulated by inflammatory cytokines. The latter are also produced in the testis by Leydig and Sertoli cells, being associated with gap junctional communication at the blood-testis barrier. Furthermore, they regulate spermatogenesis through cell interaction, Toll-like receptors and production of reactive oxygen species. Additionally, they affect testosterone production, acting at many levels of the pituitary - gonadal axis. Any imbalance in their production may result in infertility. On the other hand, obesity has also been associated with infertility. Adipokines, cytokines produced by white adipose tissue, regulate the lipid and glucose metabolism and the inflammatory system. Recent data on leptin show that it regulates reproduction by adjusting hypothalamus - pituitary - gonadal axis at both the central and peripheral levels. In this regard, resistin, visfatin and the GH secretagogue peptic hormone ghrelin affect spermatogenesis, whereas data on adiponectin are rather scarce. In conclusion, inflammatory cytokines and adipokines seem to have a pivotal role in the regulation of spermatogenesis; any imbalance in this stable environment may lead to infertility. Nevertheless, further studies are needed to clarify their exact role.

Microcystin-leucine arginine (MC-LR) induced inflammatory response in bovine sertoli cell via TLR4/NF-kB signaling pathway.

Sertoli cells were treated with 0, 20, 40, 60 and 80 µg/L of MC-LR to investigate its toxic effects, mechanism of action and immune response of the cells. Our results revealed that treatment containing 20 µg/L of MC-LR was non-toxic to the cells. Treatments containing 40, 60 and 80 µg/L of MC-LR reduced the cell viability, induced nuclear morphological changes and downregulated the blood-testis barrier constituent proteins within 48 h after treatment. The toll-like receptor 4 (TLR4) and nuclear factor-kappaB (NF-kB) were activated and significantly (P < 0.05) upregulated in cells treated with 40, 60 and 80 µg/L of MC-LR compared to the control. The pro-inflammatory cytokines were upregulated within 48 h after treatment. However commencing from 72 h, upregulation of anti-inflammatory cytokines and expression of blood-testis barrier constituent proteins was observed. This study indicates that MC-LR induced inflammatory response in bovine Sertoli cell via activation of TLR4/NF-kB signaling pathway.

Characterization of inter-Sertoli cell tight and gap junctions in the testis of turtle: Protect the developing germ cells from an immune response.

It is conceivable that early developing germ cells must across the basal to the luminal region of seminiferous tubules (STs) during spermatogenesis is associated with extensive restructuring of junctional complex. However, very limited information is documented about these junctional complexes in reptiles. In the present study we have determined the localization of inter-Sertoli cell tight junctions (TJ's), protein CLDN11 and gap junction protein Cx43 during spermatogenesis in the testis. In early spermatogenesis, weak immunoreactivity of CLDN11and focal localization of Cx43 was observed around the Sertoli cell in the luminal region, but completely delaminated from the basal compartment of STs. In late spermatogenesis, strong focal to linear localization of CLDN11and Cx43 was detected at the points of contact between two Sertoli cells and around the early stages of primary spermatocytes in the basal compartment of STs. In late spermatogenesis, localization of CLDN11and Cx43 was drastically reduced and seen only around Sertoli cells and spermatogonia near the basal lamina. However, transmission electron microscopy revealed that inter-Sertoli cell tight junctions were present within the basal compartment of STs, leaving the spermatogonia and early primary spermatocytes in the basal region during mid spermatogenesis. Gap junctions were observed between Sertoli cells, and Sertoli cells with spermatogonia and primary spermatocytes throughout spermatogenesis. Moreover, adherens and hemidesmosomes junctions were observed during spermatogenesis. The above findings collectively suggest that the intensity and localization of TJ's and gap junctions vary according to the spermatogenetic stages that might be protected the developing germ cells from own immune response.

Immunoprofiles of human Sertoli cells infected with Zika virus reveals unique insights into host-pathogen crosstalk.

Confirmed reports of Zika virus (ZIKV) in seminal fluid months after clearance of viremia suggests that ZIKV can establish persistent infection in the seminiferous tubules, an immune privileged site of the testis. The seminiferous tubule epithelium is mainly composed of Sertoli cells that function to nourish and protect developing germ cells. We recently demonstrated that primary human Sertoli cells (hSeC) were highly susceptible to ZIKV as compared to dengue virus without causing cell death and thus may act as a reservoir for ZIKV in the testes. However, the cellular and immune responses of hSeC to infection with ZIKV or any other virus are not yet characterized. Using genome-wide RNA-seq to compare immunoprofiles of hSeC, we show that the most prominent response to ZIKV at early stage of infection was suppression of cell growth and proliferation functional pathways. Peak virus replication was associated with induction of multiple antiviral defense pathways. Unique ZIKV-associated signatures included dysregulation of germ cell-Sertoli cell junction signaling. This study demonstrates that hSeC are capable of signaling through canonical pro-inflammatory pathways and provides insights into unique cell-type-specific response induced by ZIKV in association with viral persistence in the testes.

Role of Sertoli Cell Proteins in Immunomodulation.

BACKGROUND: Sertoli cell, over the past 30 years, have been elevated from simple mechanical elements to the rank of a "sentinel" in spermatogenesis. By delivering potent immunomodulatory and trophic proteins, Sertoli cells are unique cell type with a pivotal role in maintaining testis immune privilege and the immune-protection of the antigenic germ cells. CONCLUSIONS: The findings from SC transplantation studies utilizing experimental animal models of disease, demonstrate the presence of the same immuno-modulation properties and mechanisms at tissue and organ sites far from testis. The complex pathways that generate and maintain the immune tolerance involve the production of several immunomodulatory or immune-related proteins such as cytokines, chemokines, growth factors, mediators of the inflammation, complement inhibitors or adhesion molecules. A better definition and understanding of these Sertoli cell proteins and the mechanisms of immunoprotection should help to elucidate their role in the spermatogenic process. The demonstration of their capabilities in transplantation experiments suggests that Sertoli cells may be good candidates in cell therapy for a number of cell-mediated chronic diseases.

Distribution of CD68-, CD8-, MHCI- and MHCII-positive cells in the bull and ram testis and epididymis.

The mammalian testis possesses a special immunological environment because of its properties of remarkable immune privilege and effective local innate immunity. The testicular immune privilege protects immunogenic germ cells from systemic immune attack, and local innate immunity is important in preventing testicular microbial infections. Thus, this study aimed to immunohistochemically demonstrate the distribution and localization of CD68-, CD8-, MHCI- and MHCII-positive immune cells in the testes and epididymes. Negative immunoreactivity was detected in the seminiferous tubule epithelium and peritubular myoid cells of the testes upon staining in CD68, CD8 and MHC Class I. Positive CD68 immunoreaction was determined in the Sertoli cells and some Leydig cells. The detection of positive cells for CD8 clearly indicated the presence of lymphocytes. Furthermore, the staining with MHCI intensity was ascertained to vary from weak to moderate in the Sertoli and Leydig cells and connective tissue cells. MHCII-positive immunoreactivity was determined in myoid cells and Leydig cells in the interstitial area. The epithelium of the epididymis showed positive staining for CD68 and CD8, but the stroma displayed a rather weak staining. In the ram epididymis, neither intraepithelial nor interstitial positive reaction was observed for MHCI. In the epididymis, the basal cells displayed a stronger staining for MHCII. In conclusion, these cells not only contribute to local immunity through their direct effects on the quality of fertility in males, but also contribute either directly or indirectly to immune privilege by minimizing the development of both autoimmune reactions and potentially harmful risks.

Rethinking Phagocytes: Clues from the Retina and Testes.

Specialized phagocytes are a newly appreciated classification of phagocyte that currently encompasses Sertoli cells (SCs) of the testes and the retinal pigment epithelial cells (RPE) of the retina. While these cells support very different tissues, they have a striking degree of similarity both as phagocytes and in ways that go beyond cell clearance. The clearance of apoptotic germ cells, cell debris, and used photoreceptor outer segments are critical functions of these cells, and the unique nature of their clearance events make specialized phagocytes uniquely suited for studying the larger implications of cell clearance in vivo. The shared functions of specialized phagocytes could provide novel insights into how phagocytosis impacts tissue homeostasis and immune modulation. In this review, we examine the remarkable similarities between SCs and RPE as specialized phagocytes and the physiological effects of cell clearance within a tissue.

Zika Virus Infects Human Sertoli Cells and Modulates the Integrity of the In Vitro Blood-Testis Barrier Model.

Confirmed reports of Zika virus (ZIKV) in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune-privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell (SC) barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV compared to the closely related dengue virus and induced the expression of alpha interferon (IFN-α), key cytokines, and cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and intracellular adhesion molecule 1 [ICAM-1]). Furthermore, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with a higher efficiency than in the blood-brain barrier model. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decreases in SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin, and JAM-A; however, exposure of SCs to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of the ZO-1 protein, which correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoon development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and to test the ability of anti-ZIKV drugs to cross the barrier.IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, which has not been reported for other flaviviruses. To be able to sexually transmit for months after the clearance of viremia, ZIKV must establish infection in the seminiferous tubules, the site of spermatozoon development. However, little is known about the cell types that support ZIKV infection in the human testis. Currently, there are no models to study mechanisms of virus persistence in the seminiferous tubules. We provide evidence that ZIKV infection of human Sertoli cells, which are an important component of the seminiferous tubules, is robust and induces a strong antiviral response. The use of an in vitro Sertoli cell barrier to describe how ZIKV or inflammatory mediators derived from ZIKV-infected macrophages compromise barrier integrity will enable studies to explore the interactions of other testicular cells with Sertoli cells and to test novel antivirals for clearing testicular ZIKV infection.

Microcystin-leucine arginine exhibits immunomodulatory roles in testicular cells resulting in orchitis.

Microcystin-leucine arginine (MC-LR) causes testicular inflammation and hinders spermatogenesis. However, the molecular mechanisms underlying the immune responses to MC-LR in the testis have not been elucidated in detail. In this study, we show that MC-LR induced immune responses in Sertoli cells (SC), germ cells (GC), and Leydig cells (LC) via activating phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa B (NF-κB), resulting in the production of pro-inflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and chemokine (C-X-C motif) ligand 10 (CXCL10). The observed effects were attributed to reduced activity of protein phosphatases 2A (PP2A) as a result of binding of MC-LR to the catalytic subunit of PP2A in SC and GC. By contrast, innate immune responses were triggered by Toll-like receptor 2 (TLR2) in LC because MC-LR could not enter into the LC and subsequently inhibit the PP2A activity. PI3K/AKT/NF-κB were also activated in SC, GC, and LC in vivo, with the enrichment of TNF-α, IL-6, MCP-1, and CXCL10 in the testis. Following chronic exposure, MC-LR-treated mice exhibited decreased sperm counts and abnormal sperm morphology. Our data demonstrate that MC-LR can activate innate immune responses in testicular cells, which provides novel insights to explore the mechanism associated with MC-LR-induced orchitis.

Sertoli Cells Are Susceptible to ZIKV Infection in Mouse Testis.

Flaviviruses including Dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are global health problems that caused several serious diseases such as fever, hemorrhagic fever, and encephalitis in the past century. Recently, Zika virus (ZIKV) which spreads from Asia to American and causes millions of infections emerges as a new dangerous member of the genus of Flavivirus. Unlike other well-known flaviviruses, ZIKV can be transmitted sexually and infect testes in murine models. Its impacts on sperm functions, and the exact susceptible cells, however, are not entirely clear. To investigate these issues, we infected interferon α/ß and γ receptors deficient AG6 mice with ZIKV and examined the outcomes of infection using an assortment of physiological, histopathological, immunological, and virological techniques. We found that infected mice displayed signs of reproductive system disorder, altered androgen levels in serum, and high viral load in semen and testes. Additionally, histopathological examinations revealed marked atrophy of seminiferous tubules and significant reduction in lumen size. Notably, these were accompanied by positive staining of ZIKV antigens on sertoli cells, detection of viral particles and vacuole changes within cytoplasm of sertoli cells. The susceptibility of sertoli cells to ZIKV was further validated in vitro study using cell lines. Importantly, the disruption of tight junctions within testis and altered sperm morphology were also observed in ZIKV infected mice. It is well-known that tight junctions formed by adjacent sertoli cells are major component of blood testis barrier, which plays important roles in maintenance of microenvironment for spermagenesis in testis. Taken together, these results demonstrate that sertoli cells are susceptible to ZIKV infection, which results in the disruption of tight junctions in testis and causes abnormal spermatogenesis in mice. These results also imply that long-term impact of ZIKV infection on human male reproductive system requires close monitoring.

Diversity and Versatility of Phagocytosis: Roles in Innate Immunity, Tissue Remodeling, and Homeostasis.

Phagocytosis, a critical early event in the microbicidal response of neutrophils, is now appreciated to serve multiple functions in a variety of cell types. Professional phagocytes play a central role in innate immunity by eliminating pathogenic bacteria, fungi and malignant cells, and contribute to adaptive immunity by presenting antigens to lymphocytes. In addition, phagocytes play a part in tissue remodeling and maintain overall homeostasis by disposing of apoptotic cells, a task shared by non-professional phagocytes, often of epithelial origin. This functional versatility is supported by a vast array of receptors capable of recognizing a striking variety of foreign and endogenous ligands. Here we present an abbreviated overview of the different types of phagocytes, their varied modes of signaling and particle engulfment, and the multiple physiological roles of phagocytosis.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Mono-(2-ethylhexyl) phthalate-induced Sertoli cell injury stimulates the production of pro-inflammatory cytokines in Fischer 344 rats.

Exposure of rodents to the Sertoli cell (SC) toxicant mono-(2-ethylhexyl) phthalate (MEHP) has been reported to trigger an infiltration of macrophages into the testis in an age- and species-dependent manner. Here we challenge the hypothesis that the peripubertal rat-specific infiltration of macrophages after MEHP exposure is due, in part, to an increase in SC-specific inflammatory cytokine expression. To rule out that germ cell(GC) apoptosis itself is responsible for macrophage recruitment, rats were exposed to a direct GC toxicant, methoxyacetic acid (MAA), but no infiltration of macrophages was observed. Next, mRNA levels of inflammatory cytokines were evaluated after MEHP exposure. IL-1α, IL-6, and MCP-1 expression were increased in vivo and correlated with macrophage infiltration in a species-specific manner. Additionally, IL-6 and MCP-1 expression was increased in SC-GC co-cultures and ASC-17D SCs. These results indicate that MEHP-injury in pubertal rats specifically stimulates secretion of pro-inflammatory cytokines and alters the immune microenvironment.

Toxic effects of TiO2 nanoparticles in primary cultured rat sertoli cells are mediated via a dysregulated Ca2+ /PKC/p38 MAPK/NF-κB cascade.

Although numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) can be accumulated in various animal organs and can cause toxicity, there is currently only limited data regarding reproductive toxicity especially on the toxic mechanisms of TiO2 NPs in Sertoli cells. In order to investigate the mechanism of reproductive toxicity, primary cultured rat Sertoli cells were exposed to 5, 15, or 30 µg/mL TiO2 NPs for 24 h, and TiO2 NPs internalization, expression of PKC (p-PKC) and p38 MAPK (p-p38 MAPK) as well as calcium homeostasis were examined. Our findings demonstrated that TiO2 NPs crossed the membrane into the cytoplasm or nucleus, and significantly suppressed cell viability of primary cultured rat Sertoli cells in a concentration-dependent manner. Furthermore, immunological dysfunction caused by TiO2 NPs was involved in the increased expression of NF-κB, TNF-α, and IL-1ß, and decreased IκB expression. TiO2 NPs significantly decreased Ca2+ -ATPase and Ca2+ /Mg2+ -ATPase activity and enhanced intracellular Ca2+ levels, and up-regulated the expression of p-PKC and p-p38 MAPK in a dose-dependent manner in primary cultured rat Sertoli cells. Taken together, these findings indicate that TiO2 NPs may induce immunological dysfunction of primary cultured rat Sertoli cells by stimulating the Ca2+ /PKC/p38 MAPK cascade, which triggers NF-κB activation and ultimately induces the expression of inflammatory cytokines in primary cultured rat Sertoli cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1374-1382, 2017.