C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis

Mumps virus (MuV) infection usually results in germ cell degeneration in the testis, which is an etiological factor for male infertility. However, the mechanisms by which MuV infection damages male germ cells remain unclear. The present study showed that C-X-C motif chemokine ligand 10 (CXCL10) is produced by mouse Sertoli cells in response to MuV infection, which induces germ cell apoptosis through the activation of caspase-3. CXC chemokine receptor 3 (CXCR3), a functional receptor of CXCL10, is constitutively expressed in male germ cells. Neutralizing antibodies against CXCR3 and an inhibitor of caspase-3 activation significantly inhibited CXCL10-induced male germ cell apoptosis. Furthermore, the tumor necrosis factor-α (TNF-α) upregulated CXCL10 production in Sertoli cells after MuV infection. The knockout of either CXCL10 or TNF-α reduced germ cell apoptosis in the co-cultures of germ cells and Sertoli cells in response to MuV infection. Local injection of MuV into the testes of mice confirmed the involvement of CXCL10 in germ cell apoptosis in vivo. These results provide novel insights into MuV-induced germ cell apoptosis in the testis.

Mumps is a contagious disease caused by the mumps virus (MuV) and is characterized by painful parotitis. Orchitis is the most common extra-parotid gland complication of mumps that affects up to 30% of mumps cases in post-pubertal men. 1 More than 50% of patients with bilateral mumps orchitis experience infertility. 2 Moreover, a major pathological manifestation of mumps orchitis is germ cell degeneration. 3 Mumps orchitis is associated with the presence of MuV in the testis, thereby suggesting that MuV should directly induce pathogenesis. 4 However, the mechanisms underlying MuVinduced male germ cell degeneration remain unknown.
Spermatogenesis and steroidogenesis are two major functions of the testis. Several inflammatory cytokines are involved in testis pathophysiology. 5 Interleukin 1 (IL-1), IL-6 and tumor necrosis factor-α (TNF-α) have important roles in regulating spermatogenesis under physiological conditions. 6 However, these cytokines can be upregulated and impair testicular functions under inflammatory conditions. 7 High levels of IL-1, IL-6 and TNF-α inhibit steroidogenesis in Leydig cells. [8][9][10] Moreover, TNF-α upregulation induced male germ cells apoptosis in an experimental autoimmune orchitis model. 11 We recently demonstrated that the C-X-C motif chemokine ligand 10 (CXCL10) expression is remarkably upregulated in Leydig and Sertoli cells in response to MuV infection, 12 but the effect of the increased CXCL10 on testicular function remains unknown.
CXCL10 was initially identified as an IFN-γ-inducible cytokine, 13 and functions by binding to CXC chemokine receptor 3 (CXCR3). 14 CXCL10 is a pleiotropic cytokine capable of exerting various functions, including chemotactic homing of leukocytes, induction of cell apoptosis and regulation of cell proliferation. 15 Moreover, CXCL10 is involved in the pathogenesis of various autoimmune and infectious diseases. 16,17 Notably, CXCL10 upregulation in simian immunodeficiency virus (SIV) encephalitis induces neuronal apoptosis. 18 The downregulation of CXCR3 expression reduced neuronal apoptosis in a mouse model of West Nile virus encephalitis. 19 Increased CXCL10 level in the cerebrospinal fluid of individuals infected with human immunodeficiency virus (HIV) has been associated with the progression of neuropsychiatric impairment. 20 These studies indicated that CXCL10 upregulation is involved in the pathogenesis of viral encephalitis.
CXCL10 is expressed in rat Leydig cells and can be upregulated by TNF-α and IFN-γ. 21 Sendai viral infection induces CXCL10 expression in rat testicular somatic cells, including testicular macrophages, Sertoli, Leydig and peritubular myoid cells. 22 By contrast, the Sendai virus does not induce CXCL10 expression in rat male germ cells. We recently demonstrated that MuV dramatically induces CXCL10 expression in mouse Leydig and Sertoli cells but not in germ cells. 12 We speculated that CXCL10 production by testicular somatic cells in response to viral infections might be detrimental to male germ cells. This study examined the role of MuV-induced CXCL10 production by mouse Sertoli cells in inducing male germ cell apoptosis.

Results
Expression of CXCR3 in testicular cells. CXCL10 is significantly produced by mouse Leydig and Sertoli cells in response to MuV infection. 12 To reveal the potential role of MuV-induced CXCL10 in the testis, we examined CXCR3 expression in major testicular cells. Leydig, Sertoli and germ cells were identified by staining with luteinizing hormone receptor (LHR), Wilms tumor nuclear protein 1 (WT1) and mouse VASA homolog (MVH), respectively ( Figure 1a). The purity of each cell types was 495%. Real-time quantitative RT-PCR (qRT-PCR) results showed that the CXCR3 mRNA level was considerably higher in male germ cells than in Leydig and Sertoli cells (Figure 1b). MuV infection did not significantly affect CXCR3 expression in testicular cells. Western blot analysis demonstrated that CXCR3 protein was abundantly detected in germ cells in the absence and presence of MuV ( Figure 1c). Moreover, CXCR3 protein was faintly detected in Leydig cells. By contrast, CXCR3 protein was not detected in Sertoli cells. , were isolated from 4-week-old C57BL/6J mice. Each cell type was identified by immunostaining for respective markers: LHR for LC, WT1 for SC and MVH for GC. Insets in the upper right corners of the images represent negative controls, in which the preimmune rabbit sera were used as the first antibodies. (b) CXCR3 mRNA. Cells were cultured in the absence (Ctrl) and presence of 10 7 PFU/ml MuV for 24 h. Total RNA was extracted from the testicular cells, and relative mRNA level of CXCR3 was determined using realtime qRT-PCR by normalizing to β-Actin. The lowest CXCR3 mRNA level in SC was set as '1'. The fold increases in LC and GC compared to SC were presented. (c) CXCR3 protein. Testicular cell lysates were subjected to western blot analysis to probe CXCR3 using specific antibodies. β-Actin was probed as loading controls. (d) CXCR3 distribution in the testis. Immunohistochemistry was performed to localize CXCR3 on the paraffin sections of the testis from 5-week-old C57BL/6J mice. The inset in the upper right corner of the image in the right panel represents negative control, in which the preimmune goat serum was used as the first antibody. Black arrows, black arrowheads, white arrows, white arrowheads and asterisk indicate round spermatids, SC, spermatogonia, spermatocytes and interstitial cells, respectively. (e) CXCR3 locations in GC and SC co-cultures. IF costaining with CXCR3 (green) and MVH (red) for GC and SC co-cultures. Scale bar = 20 μm. Images are the representatives of at least three experiments. Real-time qRT-PCR data are the means ± S.E.M. of three experiments Mumps virus damages male germ cells Q Jiang et al Immunohistochemistry ( Figure 1d) and immunofluorescence (IF) co-staining of CXCR3 and MVH ( Figure 1e) confirmed that CXCR3 protein was obviously located in male germ cells. A weak CXCR3 signal was observed in interstitial cells (asterisk) (Figure 1d), but not detected in Sertoli cells (Figure 1e).
CXCL10-induced germ cell apoptosis. Considering that CXCL10 induces neuronal apoptosis, 18  effects of CXCL10 on male germ cell apoptosis were quantitatively analyzed. Furthermore, flow cytometry analysis confirmed that apoptotic germ cells were significantly increased at 24 h in the presence of 5 ng/ml CXCL10 ( Figure 2c). Notably, both AO/EB staining and flow cytometry analysis showed comparable results, indicating that these two approaches are reliable in determining germ cell apoptosis.
Activation of caspase-3 in germ cell apoptosis. To further understand the mechanism by which CXCL10 induced germ cell apoptosis, we examined the activation of the caspase cascade, an important apoptotic pathway. 23 CXCL10 induced essential to activate caspase-3. We found that caspase-8 was significantly cleaved in germ cells by CXCL10 (Figure 3b). DEVD-fmk, an inhibitor of caspase-3 activation, efficiently inhibited caspase-3 cleavage at 8 h in the presence of CXCL10 (Figure 3c). Accordingly, DEVD-fmk significantly   Effect of MuV on spermatogenesis. To evaluate pathological consequences of MuV infection in male infertility/ subfertility, we examined the spermatogenesis status. Local injection of MuV remarkably reduced the seminiferous tubules with elongated spermatids in the lumen at 2 weeks after injection (Figure 8a, lower panels). However, the impaired spermatogenesis was observed 1 and 3 weeks after MuV injection. The spermatogenesis status was quantitatively analyzed (Figure 8b). Further, we examined the roles of TNF-α and CXCL10 in the MuV-mediated impairment of spermatogenesis. We showed that either Mumps virus damages male germ cells Q Jiang et al mutation of TNF-α or CXCL10 significantly increased the seminiferous tubules with elongated spermatids at 2 weeks after MuV injection (Figure 8c).

Discussion
MuV infection usually impairs spermatogenesis, but the underlying mechanisms remain to be clarified. This study demonstrated that the CXCL10 produced by Sertoli cells in response to MuV infection induces germ cell apoptosis and TNF-α upregulates CXCL10 production in an autocrine manner. These results provide novel insights into the mechanisms underlying MuV-impaired spermatogenesis. We recently found that mouse Leydig and Sertoli cells remarkably produced CXCL10 in response to MuV infection. 12 To determine the potential role of CXCL10 in the MuV-infected Figure 7 MuV-induced CXCL10 production and germ cell apoptosis in the testis. MuV (1 × 10 7 PFU) in 10 μl of PBS was injected into the testis of 5-week-old WT and CXCL10 − / − mice. The contralateral testis that was injected with an equal volume of PBS alone served as Ctrl. (a) MuV detection. MuV-NP in testicular lysates was detected by western blot analysis at 24 h after MuV injection. (b) CXCL10 level. The testis was lysed by grinding in PBS at 24 h after MuV injection. CXCL10 levels in the lysates were measured using ELISA. (c) Apoptosis of male germ cells. The testis of WT (left panels) and CXCL10 − / − (right panels) mice were injected with PBS or MuV. After 24 h, apoptotic germ cells in paraffin sections were detected using co-staining of TUNEL and IF with antibodies to MVH. Insets in upper right corners represent the higher resolution for dotted box areas (lower panels). (d) A time-dependent germ cell apoptosis. The testes of WTand CXCL10 − / − mice were injected with MuV for the indicated durations. Apoptotic germ cells were quantitatively analyzed based on the co-staining of TUNEL and IF. Apoptotic germ cell numbers per tubular section were presented. One hundred tubules per testis were counted. Images are the representatives of three mice. Scale bar = 40 μm. Data represent the means ± S.E.M. of three mice. *Po0.05 and **Po0.01 testes, we examined the CXCR3 distribution in testicular cells. CXCR3 was predominantly expressed in male germ cells, suggesting that CXCL10 acts on germ cells. Moreover, CXCL10 induced germ cell apoptosis in vitro. Flow cytometry is a common approach for quantitatively analyzing the apoptosis of suspended cells after labeling with Annexin V-FITC. In the present study, the apoptosis of male germ cells cultured alone was analyzed by flow cytometry. However, we determined germ cell apoptosis using AO/EB staining in cocultures of Sertoli and germ cells, because the germ cells firmly bound to Sertoli cells and could not be collected for flow cytometry. AO/EB staining approach has been used to detect cell apoptosis. 25 We found that both flow cytometry and AO/ EB staining gave comparable results, confirming that these two approaches are consistent for measuring male germ cell apoptosis.
To further understand the mechanisms underlying CXCL10induced male germ cell apoptosis, we examined the activation of caspase-3 in male germ cells. Caspase-3 activation, which is induced by caspase-8 activation, is a critical pathway in executing cell apoptosis. 26 We demonstrated that CXCL10 activated caspase-3 and caspase-8 in male germ cells. Notably, DEVD-fmk, a caspase-3 inhibitor, 27 protected germ cells from CXCL10-induced apoptosis. These observations suggested that CXCL10 induces germ cell apoptosis via the activation of caspase cascades. In accordance with the in vitro results, we confirmed the association between CXCL10 upregulation and germ cell apoptosis in the testes after MuV infection in vivo. We recently found that MuV infection suppresses testosterone synthesis in mouse Leydig cells. 12 However, whether the inhibition of testosterone synthesis contributes to MuV-impaired spermatogenesis remains to be clarified.
We further analyzed spermatogenesis status in response to MuV injection. The ratio of the seminiferous tubules containing elongated spermatids was significantly reduced at 2 weeks after local MuV injection in WT mice; however, this phenotype was recovered at 3 weeks. We did not observe permanent impairment of spermatogenesis in vivo after MuV infection. These results agreed with the previous observations that mice are resistant to MuV infection. 28 Several aspects may be responsible for the resistance to MuV in mice: (1) mice adopt efficient antiviral ability. A previous study showed that murine Leydig cells exhibit higher efficient antiviral response than their human counterparts. 29 Accordingly, various viral infections lead to orchitis in human beings, but natural viral orchitis has not been observed in murine animals. 30 (2) MuV does not efficiently proliferate in mice after infection in vivo. MuV was significantly removed from the testis several days after local injection (data not shown). Therefore, MuV only transiently affects the mouse testis. (3) Although the detrimental effect of few virus, such as Zika virus, on the mouse testis has been investigated, the testicular damage only occurred in mice lacking interferon signaling. [31][32][33] These studies indicated that the antiviral system in mice inhibits virus-mediated testicular damage. Therefore, whether MuV infection permanently disrupts spermatogenesis in mice lacking interferon signaling is worthy of determination.
CXCL10 expression can be upregulated by bacterial and parasitic infections. [34][35][36] In particular, various viral infections, such as rhinovirus, respiratory syncytial virus, hepatitis virus and Ebola virus, induce CXCL10 expression. [37][38][39][40] These studies suggested that CXCL10 might be involved in the pathogenesis of different infectious diseases. CXCL10 facilitates the recruitment of CXCR3-positive immune cells, including macrophages, dendritic cells and activated T lymphocytes, to infected sites, which has an important role in initiating inflammatory responses against the invading microbial pathogens. 41,42 Moreover, several studies have shown that CXCL10 upregulation induces cell apoptosis in certain viral infectious diseases. CXCL10 induces neuronal apoptosis in SIV and West Nile virus encephalitis. 18,19 In addition, it promotes cancer cell apoptosis in human papillomavirus-associated cervical carcinoma. 43 We recently demonstrated that MuV significantly induces CXCL10 expression in mouse testicular somatic cells. 12 The present study showed that MuV-induced CXCL10 in Sertoli cells triggered the apoptosis of male germ cells in a paracrine manner. Whether CXCL10 upregulation facilitates the development of orchitis by recruiting leukocytes to the testes after MuV infection requires clarification in vivo. Notably, Zika virus infection induces CXCL10 production in mouse testicular somatic cells and leads to leukocyte infiltration in the testes, resulting in orchitis. 31,32 These results suggest that CXCL10 production might be involved in the pathogenesis of the testes after MuV and Zika virus infection.
Understanding the mechanisms by which MuV induces CXCL10 production in the testes would be helpful for the development of therapeutic interventions in MuV orchitis. The present study shows that TNF-α induces CXCL10 expression in an autocrine manner in Sertoli cells after MuV infection. This observation corresponds to a previous report, which showed that TNF-α upregulates CXCL10 expression in rat Leydig cells. 21 However, CXCL10 expression was not completely abolished in TNF-α − / − cells, suggesting that TNF-α-independent mechanisms should be also involved in MuV-induced CXCL10 expression in Sertoli cells. Although IFN-γ induces CXCL10 expression, 13 this mechanism cannot be involved in MuV-induced CXCL10 production, because MuV did not induce IFN-γ expression in co-cultures of Sertoli and germ cells (data not shown). Moreover, CXCL10 mRNA was significantly upregulated as early as 6 h after MuV infection, when TNF-α was not detected at the protein level. These observations suggested that MuV may also directly induce CXCL10 expression independently of TNF-α and IFN-γ in Sertoli cells. In support of this speculation, an early study showed that HIV envelope glycoprotein gp120 induces CXCL10 expression independent of IFN-γ in the mouse brain. 44 Whether MuV envelope glycoprotein directly induces CXCL10 expression in Sertoli cells is worthy of clarification.
In addition to Sertoli cells, Leydig cells in the testicular interstitial spaces also produce CXCL10 in response to MuV infection. 12 Increased CXCL10 levels in the testicular interstitial spaces may facilitate the migration of leukocytes into the testis, which remains to be tested. By contrast, CXCL10 produced by Leydig cells should not induce the apoptosis of germ cells behind the blood-testis barrier. Therefore, MuVinduced CXCL10 production by Sertoli and Leydig cells may play different roles in the pathogenesis within the testis.
In summary, the present study demonstrates that MuVinduced TNF-α upregulates CXCL10 expression in Sertoli cells in an autocrine manner, and that CXCL10 induces male germ cell apoptosis though the activation of caspase-3. The results provide novel insights into the mechanism underlying MuV-impaired spermatogenesis. The CXCL10/CXCR3 system in testicular cells might be considered as a therapeutic target for male infertility caused by MuV infection. Cell isolation. Testicular cells were isolated from 4-week-old mice based on previously described procedures. 45 In brief, mice were anesthetized with CO 2 and then killed by cervical dislocation. The testes were decapsulated and incubated with 0.5 mg/ml collagenase type I (Sigma) in PBS at room temperature for 15 min with gentle oscillation. The suspensions were filtered using 80 μm copper meshes to separate interstitial cells and seminiferous tubules. Interstitial cells were cultured in F12/DMEM (Life Technologies, Grand Island, NY, USA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum (FCS, Life Technologies). After 24 h, Leydig cells were detached by treatment with 0.125% trypsin for 5 min. Testicular macrophages were not detached in this treatment. The purity of Leydig cells were more than 95% based on staining for LHR (a maker of Leydig cells). 46 Macrophages in Leydig cell preparations were less than 3% based on the immunostaining for F40/80 (a marker of macrophages). 47 The seminiferous tubules were recovered and suspended in collagenase type 1 at room temperature for an additional 15 min to remove the peritubular myoid cells. The tubules were cut into small pieces (~1 mm) and incubated with 0.5 mg/ml hyaluronidase (Sigma) at room temperature for 10 min with gentle pipetting to dissociate Sertoli and germ cells. Cell suspensions were cultured in F12/DMEM medium supplemented with 10% FCS at 32°C for 6 h. Germ cells were recovered by collecting non-adherent cells. The purity of the germ cells was 495% based on immunostaining for MVH, a marker of germ cells. 48 Sertoli cells were cultured at 37°C for another 24 h and treated with a hypotonic solution (20 mM Tris, pH 7.4) for 1 min to remove the germ cells that adhered to Sertoli cells. Sertoli cells purity was495% based on immunostaining for WT1, a marker of Sertoli cells. 49 Co-cultures of Sertoli and germ cells. Sertoli cells were seeded in sixwell plates at a density of 1 × 10 5 cells per well. After 24 h, 1 × 10 6 germ cells were added to Sertoli cells in each well. Twenty four hours later, non-adherent germ cells were removed by washing twice with culture media and the co-cultures were infected with MuV.
MuV infection. MuV (SP-A strain) was isolated from a mumps patient 50 and obtained from the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). MuV was amplified and titrated in Vero cells. MuV preparations were diluted in 1 × PBS at a density of 1 × 10 9 PFU/ml and stored at − 80°C. MuV was added to cell cultures at a multiplicity of infection of 5 for in vitro infection. For in vivo infection, 5-week-old mice were anesthetized with pentobarbital sodium (50 mg/kg) and the testis were surgically exposed. The testis was locally injected with 1 × 10 7 PFU MuV in 10 μl of PBS using 30-gauge needles. The testis of control mice was injected with an equal volume of PBS.
Western blot analysis. Cells or tissues were lysed using RIPA lysis buffer containing protease inhibitor cocktail (Sigma). The protein concentration was determined using the bicinchonic acid protein assay kit (Applygen Technologies Inc., Beijing, China). The proteins (20 μg/lane) were separated on 10% SDS-PAGE gel and electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked on Tris-buffered saline (pH 7.4) containing 5% non-fat milk at room temperature for 1 h and incubated with the primary antibodies overnight at 4°C. The membranes were washed twice with appropriate HRPconjugated secondary antibodies (Zhongshan Biotechnology Co.) at room temperature for 1 h. Antigen/antibody complexes were visualized using an enhanced chemiluminescence detection kit (Zhongshan Biotechnology Co.).
Histological analysis and IF staining. For histological analysis, the testis of 5-week-old mice was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The paraffin sections (5 μm in thickness) were cut with a rotary microtome Reichert 820 HistoSTAT (Reichert Technologies, Depew, NY, USA). The sections were stained with hematoxylin and eosin for histological analysis on spermatogenesis.
For IF staining, the slides were soaked in citrate buffer and then heated in a microwave at 100°C for 10 min to retrieve the antigens or the cells were fixed with pre-cooled methanol for 3 min and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking with 5% preimmune goat sera in PBS for 1 h at room temperature, the sections were incubated with primary antibodies overnight at 4°C. After washing twice with PBS, the sections were incubated with FITC-or TRITCconjugated secondary antibodies at room temperature for 1 h. The slides were mounted by antifade mounting medium with DAPI (Zhongshan Biotechnology Co.). Negative controls were incubated with preimmune animal serum instead of the primary antibodies. The sections were counterstained with hematoxylin and mounted with neutral balsam (Zhongshan Biotechnology Co.).
Enzyme-linked immunosorbent assay. Cells were cultured in six-well plates. Culture media were collected at 24 h after MuV infection. The testis was lysed by grinding in 1 × PBS and the supernatants of the lysates were collected after centrifugation at 1000 × g for 5 min. TNF-α and CXCL10 levels were measured using ELISA kits in accordance with the manufacturer's instructions.
Flow cytometry. Male germ cells were washed twice in 1 × PBS and labeled with Annexin V-FITC using apoptosis detection kit (FXP018, Beijing 4A Biotech Company) following the manufacturer's instructions. The cells were analyzed with a BD Accuri C6 flow cytometer (BD Biosciences, Franklin lakes, NJ, USA).
AO/EB staining. Testicular cells were cultured in six-well plates with 2 ml media. At 24 h after MuV infection, 2 μl fluorescent staining solution containing 100 μg/ml AO and 100 μg/ml EB (Sigma) was added to each well and incubated for 1 min at room temperature. Cells were observed under a fluorescent microscope (BX51, Olympus, Tokyo, Japan). Apoptotic cells were shown as 'orange' and living cells as 'green'.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Paraffin sections of the testis were prepared for analyzing germ cell apoptosis. Apoptotic germ cells in situs were detected using a TUNEL kit (Maibio Biotechnology Co., Shanghai, China) according to the manufacturer's instruction. The sections were stained by IF staining with antibodies to MVH, a germ cell marker.
Statistical analysis. All data are presented as the mean ± S.E.M. of at least three independent experiments. Statistical significance between individual comparisons was determined by Student's t-test. One-way ANOVA with Bonferroni's (selected pairs) post hoc test was used for multiple comparisons. The calculations were performed using SPSS Version 13.0 (SPSS Inc., Chicago, IL, USA) and Po0.05 was considered statistically significant.