Experimental inoculation of the adult rat testis with Sendai virus: effect on testicular morphology and leukocyte population

N. Melaine1, A. Ruffault2, N. Dejucq-Rainsford1 and B. Jégou1,3

1 GERM-INSERM U435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes and 2 Laboratoire de Virologie, Centre Hospitalier Régional de Ponchaillou, Rennes, Bretagne, France

3 To whom correspondence should be addressed. e-mail: bernard.jegou{at}rennes.inserm.fr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Surprisingly little is known about the interactions between viruses and the male uro-genital tract. These are important, as viral testicular orchitis, induced by mumps or human immunodeficiency virus (HIV) infection for example, can lead to sterility. Moreover, semen is an essential vector in the propagation of sexually transmissible viral diseases. Here, we studied the effects of testicular infection with Sendai virus, a virus related to mumps virus, on the cellular distribution of viral particles and on testicular morphology, with particular attention to the testicular leukocyte population. METHODS: At 5, 9, 11 or 24 h post-injection of Sendai virus through the scrotum, the testes were fixed for morphological and immunohistological studies. Localization of virus particles and numeration of leukocytes were performed using specific antibodies and morphological criteria. RESULTS: As early as 5 h post-injection, a rapid and massive infiltration of leukocytes was observed in the interstitial tissue. The peritubular cell layer and the most external part of the basal portion of the seminiferous tubules were altered. The virus was diffusely located within the interstitial tissue 9 h following the injection whereas, after 24 h, viral proteins were restricted to the cytoplasm of infiltrated leukocytes. The number of leukocytes increased with time post-injection. Thus, 24 h post-injection, CD3+ T-cell number was 3-fold higher, ED1+ monocyte number was 4-fold higher and polynuclear cell number was 600-fold higher than in the control testes (P < 0.001 all observations). In contrast, the population of resident macrophages was unaffected by Sendai virus. CONCLUSIONS: Testicular viral infection causes inflammation including rapid recruitment of leukocytes. The experiments presented here provide a model for further studies on the etiopathology of viral orchitis, in particular that caused by mumps virus.

Key words: leukocytes/orchitis/Sendai virus/testis/viral infection


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Virus-induced inflammation of the testis (viral orchitis) is a major health problem as it can cause sterility. Semen is also a major vector of virus transmissible disease (Dejucq and Jégou, 2001Go). Viruses can enter the testis either via the blood and lymphatic vessels present in the interstitial cells or via the rete testis. A number of viruses including mumps virus (in Leydig cells; Aiman et al., 1980Go), human immunodeficiency virus (HIV; in germ cells; Muciaccia et al., 1998Go), hepatitis B and C viruses and herpes simplex virus (in sperm; Kotronias and Kapranos, 1998Go) have been found in the interstitial tissue and/or seminiferous tubules of men (Mikuz and Damjanov, 1982Go; Csata and Kulcsar, 1991Go; Lang, 1993Go; Liu et al., 1994Go; Dejucq and Jégou, 2001Go) and found to be associated with an intratesticular influx of leukocytes (Gall, 1947Go; Charny and Meranze, 1948Go; Hedger, 1997Go). However, despite the deleterious effects of these viruses on testicular function and although semen is an essential vector for the transmission of diseases, the etiopathology of testicular infection is unknown.

The most well-known virus involved in human testicular disorders is the mumps virus (Dejucq and Jégou, 2001Go). Sendai virus, which belongs to the same family as mumps virus (the paramyxovirus family) and infects the rat, was used here as a model virus. It previously has been shown to induce in isolated testicular cells the production of type I and II interferons (Dejucq et al., 1995Go, 1998) and of the antiviral proteins 2'5' oligoadenylate synthetase (2'5'AS), double-stranded RNA-activated protein kinase (PKR) and Mx proteins (Dejucq et al., 1997Go; Melaine et al., 2003Go).

This work aimed at studying the effect of testicular injection of Sendai virus on testicular morphology in the rat. We also studied the distribution of viral particles as well as the kinetics and the nature of the cellular infiltrate that occurs within the testis post-infection.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Adult, male Sprague–Dawley rats were purchased from Elevage Janvier (Le Genest Saint Isle, France).

Antibodies
The mouse monoclonal ED2 antibody, raised against resident rat macrophages, and ED1 antibody, which labels a lysosomal antigen specific to monocytes, dendritic cells and some macrophages, were purchased from Biosource International (CA, USA). The goat polyclonal antibody, which recognizes the {epsilon} subunit of the CD3 complex associated with the T-cell antigen receptor, was purchased from Santa-Cruz Biotechnology Inc. (Santa Cruz, CA, USA) (sc-1127). Two mouse monoclonal antibodies directed against two Sendai virus proteins [the nucleocapsid protein (anti-NP-m52) and the haemagglutinin (HA)-neuramidase protein (anti-HN-m57)] were kindly provided by Dr K.Kaul (Department of Virology and Molecular Biology, St Jude Children’s Research Hospital, Lauderdale, Memphis, TN, USA; Lyn et al., 1991Go; Coronel et al., 2001Go).

Preparation of Sendai virus
Sendai virus (Pasteur Institute, Paris, France) was injected into the allantoic sac of quail eggs that had been fertilized 9 days previously. The eggs were incubated for 3 days in a humidified incubator at 36–37°C and then overnight at 4°C. They were cleaned with an iodophor detergent (Gifrer & Barbezat, Decines, France) and the shell above the air sac removed. The allantoic fluid was harvested, centrifuged at 3000 g and tested for sterility. To determine the viral titre in the fluid, the fluid was diluted 1:10 with phosphate-buffered saline (PBS) and then serially 1:2 in 0.2 ml of PBS in the wells of a plastic microtitre plate. A 200 µl aliquot of a 0.5% chick red blood cell suspension subsequently was added to each well and the plates were incubated for 1 h at room temperature. After this time, the HA titres were read.

In vivo exposure to Sendai virus
Adult Sprague–Dawley male rats were anaesthetized (i.p., 50 mg/kg pentobarbital), and 200 µl of 1000 HA units (HAU)/ml of Sendai virus in allantoic fluid was slowly injected through the scrotum skin into the testis. Controls were injected with the same volume of allantoic fluid. The rats were killed 5, 9, 11 or 24 h post-injection (virus-induced lethality prevented analysis of a longer time course, n = 3 for each time point). Just after collection, as classically done, the albuginea of testis was carefully cut on a few millimetres at each pole of the testis and the organ dropped for 12 h in a vial containing Bouin’s fixative. After this first bath, the testis was cut in two halves for a further fixation period of 24 h. It was then dehydrated by immersion in a series of alcohol concentrations, and embedded in paraffin wax. To verify that the effects induced by the virus were homogeneous throughout the testis, half a testis of each animal was cut into 5 µm sections and stained with haematoxylin (100 mg/ml AlKO8S2, 2 mg/ml hamatein, 2% acetic acid).

Immunohistochemistry
Endogenous peroxidase was quenched with 3% H2O2 for 5 min. After 10 min in the presence of 1% PBS–bovine serum albumin (BSA), testes sections (5 µm) were incubated with mouse monoclonal anti-HN and anti-NP antibodies (1:10), mouse monoclonal antibodies ED1 or ED2 (1:100) or goat polyclonal anti-CD3 (1:50) antibody. Normal serum, control IgG and 1% Tris-buffered saline (TBS)–BSA were used as negative controls. Bound antibodies were visualized by avidin–biotin–peroxidase complex amplification (DAKO, Trappes, France; Stephan et al., 2000Go). The colour reaction was developed using a diaminobenzidine (DAB) substrate-chromogen (Sigma-Aldrich, Saint-Quentin Fallavier, France). The cells were counterstained with haematoxylin, dehydrated and mounted in Eukitt solution (Labonord, Villeneuve d’Ascq, France).

Quantification of CD3+, ED1+, ED2+ and polynuclear cells
Cells stained by anti-CD3, ED1 or ED2 antibodies in the interstitial space of rats treated with Sendai virus or with allantoic fluid were counted under a light microscope (BH2-RFCA, Olympus France SA, Rungis, France). All of the immunopositive cells and polynuclear cells in 60 randomly chosen fields (magnification x100) were counted. The results are expressed as means ± SEM. Duncan’s Multiple Range test was used to analyse variance. Differences were considered to be significant if P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to the literature, in the rat, ED2-positive macrophages can represent up to 25% of the total number of the interstitial cells (Wang et al., 1994Go; Dijkstra et al., 1985Go). This contrasts with the lower numbers found in the present study. As very careful countings were performed here by several independent examiners, one tentative explanation for this is that, for reasons relying on the nature of the antibodies used and of the immunohistochemistry procedure adopted (ED2 and CD3 antibodies), we only detected a fraction of the total population of lymphocytes normally present within the rat interstitial tissue. However, there was no reason to suspect differences in the proportion of macrophages evidenced between virus-exposed and control testes as the numbers counted were relatively equivalent between individual animals in all cases.

Morphological effects of in vivo injection of Sendai virus
Apart from the hole created by the injection needle, no particular damage that could be related to the injection itself was observed in the testes of rats injected with allantoic fluid alone (Figure 1A). At 24 h after injection with Sendai virus, a massive infiltration by leukocytes was observed in the interstitial tissue (Figure 1B). The seminiferous epithelium often became detached from the tunica propria of the tubules (Figure 1B). The virus-induced morphological changes were homogenous throughout the testis parenchyma.



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Figure 1. Effects of Sendai virus on testicular morphology. Adult rats were injected intratesticularly with 200 µl of allantoic fluid (A) or with the same volume of allantoic fluid containing 1000 haemagglutinin units (HAU) of Sendai virus (B). At 24 h post-injection, a massive infiltration of cells was observed in the interstitial tissue (IT) of the virus-injected testes (B) but not of the allantoic fluid-injected testes (A). The tunica propria surrounding the seminiferous tubules frequently became detached in the virus-injected testes (B; arrows). (A and B, bar = 50 µm).

 
Localization of viral particles after injection of Sendai virus
At 9 h post-injection, viral nucleocapsid proteins were scattered throughout the interstitial space (Figure 2A and B). The diffuse nature of this signal did not allow us to determine precisely which interstitial cell type was involved. However, after 24 h, the viral protein was, beyond doubt, located in the cytoplasm of the leukocytes that had invaded the interstitial spaces, whereas the leukocytes that remained in the blood vessels were never labelled (Figure 2C and inset, and D). Viral particles were never observed in Sertoli cells or germ cells.



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Figure 2. Immunolocalization of Sendai virus within the testis. Adult rats were injected intratesticularly with 200 µl of allantoic fluid containing 1000 HAU of Sendai virus (A–D) and killed at 9 (A and B) or 24 h (C, inset and D) post-injection. The localization of the Sendai virus was determined using NP-m552 mouse monoclonal antibodies (A–D). The anti-HN-m57 antibody gave the same result (data not shown). After 9 h (A and B), the Sendai virus was localized in the interstitial tissue (IT) in only a few fields, in a strong but diffuse manner that did not allow us to identify any particular host cell (A and B, arrows). In contrast, 24 h post-injection (C and D), the Sendai virus was homogeneously localized in the interstitial tissue (IT), within leukocytes (arrows), whereas the leukocytes in the neighbouring blood vessels were not labelled (C inset, arrow). (A and C, bar = 50 µm; C inset, B and D, bar = 10 µm).

 
Quantification of T cells, monocytes, polynuclear cells and macrophages
At 5 h post-injection (Figure 3A, C, E and G), immunostaining with anti-CD3 and ED1 antibodies and morphological observations revealed that the interstitial cells had been infiltrated by T cells (Figure 3A), polynuclear cells (Figure 3C) and monocytes (Figure 3E). The numbers of these three cell types increased throughout with time (Figure 3B, D and F). The CD3-positive cell number was 3-fold higher 24 h after the administration of the virus than in control testes that had been injected with control allantoic fluid (P < 0.001, Figure 4). After 11 h, the polynuclear cell number had reached a plateau, corresponding to ~600 times as many cells as in the controls (P < 0.001, Figure 4). The ED1-positive cell number was ~4 times higher in the injection group than in the control group after 5 h, ~5 times higher after 9 h, ~8 times higher after ~11 h and ~17 times higher after 24 h (P < 0.001 for all time points, Figure 4). In contrast, the ED2-positive cell number, corresponding to resident macrophages, did not change (Figures 3G and H, and 4). While no specific antibodies against rat CD45 cells were available at the time we started this study, we tested an anti-human CD45 antibody (anti-human CD45, Santa-Cruz; which recognizes all leukocytes) that did not allow us to reveal any specific labelling in the rat testis (not shown). Therefore, we were unable to characterize these cells after virus injection.



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Figure 3. Effects of testicular exposure to Sendai virus on cell infiltration in vivo. Adult rats were injected intratesticularly with 1000 HAU of Sendai virus (A–H). The testes of the injected rats were stained with haematoxylin (C and D). The polyclonal CD3-{epsilon} antibody (A and B), the monoclonal ED1 antibody (E and F) and the monoclonal ED2 antibody (G and H) were used to determine the locations of T cells, monocytes and resident macrophages, respectively. Bound antibodies were visualized by adding an avidin–biotin–peroxidase complex. At 5 h post-injection (A, C, E and G), CD3-positive cells (A; arrows) and ED1-positive monocytes (E; arrows) were seen in the interstitial tissue. The numbers of positive cells increased with time (24 h, B and F, respectively). The number of ED2-positive cells did not change with time (5 and 24 h, G and H; arrows). Polynuclear cells could be seen in the interstitial tissue 5 h post-injection (C, arrows), and the number of these cells increased with time (24 h, D; arrows). (A, B and E–H, bar = 50 µm; C and D, bar = 10 µm).

 


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Figure 4. Number of cells infiltrating the testes of rats exposed or not exposed to Sendai virus. Adult rats were injected intratesticularly with 200 µl of allantoic fluid (allantoic fluid-injected testis, filled circles) or with the same volume of allantoic fluid containing 1000 HAU of Sendai virus (virus-injected testes, filled triangles). The values at time 0 h represent the numbers of cells found in the non-injected testes. The rats were killed at 5, 9, 11 and 24 h post-injection. The polyclonal anti-CD3-{epsilon} antibody (CD3+) was used to detect T cells, the monoclonal ED1 antibody (ED1+) to detect monocytes and the monoclonal ED2 antibody (ED2+) to detect resident macrophages. Polynuclear cells were easily identified morphologically. The number of cells in 60 different fields was counted under a x100 lens using a BH2-RFCA Olympus microscope. The data are expressed as the means ± SEM for three different animals. Duncan’s Multiple Range test was used to analyse variance (*P < 0.01, ***P < 0.001).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In their early and preliminary work, Gall (1947Go) and Charny and Meranze (1948Go) showed that in the case of orchitis caused by mumps, the interstitial tissue becomes oedematous and the testicular blood vessels become congested and surrounded by lymphocytes. Degeneration of germ cells also occurred, whereas Sertoli cells and Leydig cells were apparently unaffected. Lymphocyte infiltration (Rogers and Klatt, 1988Go; Tebourbi et al., 2001Go), loss of germ cells and interstitial fibrosis were also observed in AIDS patients (Chabon et al., 1987Go; Rogers and Klatt, 1988Go; De Paepe and Waxman, 1989Go; Yoshikawa et al., 1989Go).

In the present study, we chose a route of injection of the testis—intratesticular injection—which does not mimic the ‘natural’ routes, which are generally thought to be the vascular route or through the rete testis. However, systemic injection would have led to lethality for the animals and, experimentally, ‘clean’ intra-rete testis injection of viruses is not possible to perform.

Our present in-vivo findings using the mumps virus-related Sendai virus reveal that leukocytes are rapidly mobilized within the interstitial tissue of the testis. Just 5 h post-injection, the interstitial tissue had been invaded by considerable numbers of CD3+ cells, ED1+ cells and polynuclear cells. After 24 h, substantial numbers of these cells had accumulated within the interstitial space, associated with damage to the basal part of a number of seminiferous tubules. The kinetics of leukocyte recruitment observed here were similar to those of the induction of several testicular chemoattractant molecules within the seminiferous tubules secreted by testicular macrophages, Leydig cells, peritubular cells and Sertoli cells, namely MCP-1, RANTES, IP-10 and GRO/KC, previously observed by us following Sendai virus stimulation (Le Goffic et al., 2002Go). These chemokines are known to be involved in leukocyte mobilization at sites of inflammation. The close concordance between these phenomena strongly suggests that the activation of chemokine production is responsible for the recruitment of leukocytes.

The interstitial tissue contains large numbers of ‘resident’ leukocytes, chiefly macrophages (el-Demiry et al., 1987Go; Pöllänen and Niemi, 1987Go; Hedger, 1997Go). Regardless of whether resident macrophages are involved, inflammation results in an influx of circulating monocytes, lymphocytes and neutrophils. The production of inflammatory cytokines by these cells may activate the testis-specific lymphocytes, resulting in autoimmune orchitis or antibody development (for a review see Hedger, 1997Go).

We found viral proteins in the leukocytes that had invaded the interstitial tissue, but not in those located within the lumen of the blood vessels. This clearly indicates that the virus only infected these cells within the interstitial tissue. Sendai virus can infect monocytes and macrophages (Pirhonen et al., 1999Go), and parainfluenza virus 1, another paramyxovirus, is able to infect and to replicate in leukocytes (Bogen et al., 1977Go). Interestingly, 24 h following injection of Sendai virus, Leydig cells, unlike leukocytes, did not appear to be infected in situ. This is compatible with the apparent absence of damage of the Leydig cells in human testes of patients suffering from mumps-induced orchitis (Gall, 1947Go; Charny and Meranze, 1948Go). This may be due to the ability of these cells to protect themselves and to induce antiviral proteins. We recently showed that testicular cells produce large amounts of antiviral proteins (2'5'AS, PKR and Mx) after exposure to interferons or Sendai virus in vitro (Dejucq et al., 1997Go; Melaine et al., 2003), thus creating an antiviral system that may protect germ cells against virus infection. This is of prime importance as contamination of the latter cells would not only threaten the fertility of the individual, but may potentially lead to the propagation of the viral genome to the haploid germ cells and therefore to the offspring (Dejucq and Jegou, 2001Go).

It is noteworthy that the testicular damage induced by Sendai virus is restricted to the interstitium and to the seminiferous tubule basal lamina. Due to the virus-induced lethality observed at 24 h post-injection, it was not possible to know whether the virus-induced damage would have reached the seminiferous epithelium itself after a longer time exposure. However, the apparent absence of virus-induced damage to the seminiferous epithelium 24 h post-injection could also result from the ability of Sertoli cells to protect themselves and germ cells against a viral attack, as shown previously by our group in vitro (Dejucq et al., 1997Go). This is consistent with previous anatomopathological observations made in testes of patients affected with mumps virus which show no apparent damage to Sertoli cells (Gall, 1947Go; Charny and Meranze, 1948Go).

In conclusion, our results show that a testicular viral infection causes inflammation and a rapid mobilization of leukocytes. As this mimics testicular orchitis, the in vivo model presented here supports the need for further studies on the characterization of proteins induced by viral inflammation such as antiviral proteins or chemokines.


    Acknowledgements
 
We would like to thank Dr K.Kaul (Department of Virology and Molecular Biology, St Jude Children’s Research Hospital, Lauderdale, Memphis TN) for providing us with anti-Sendai antibodies, A.Le Nedic for his technical assistance with the in vivo work, and Brigitte Lemarchand (Hopital Ponchaillou, CHR Rennes, France) for preparing the Sendai virus. This study was funded by INSERM, the Ministère de l’Education Nationale, de la Recherche et de la Technologie and the Fondation Langlois.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 26, 2002; resubmitted on January 8, 2003; accepted on May 1, 2003.





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