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
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Abstract |
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Key words: leukocytes/orchitis/Sendai virus/testis/viral infection
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Introduction |
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The most well-known virus involved in human testicular disorders is the mumps virus (Dejucq and Jégou, 2001). 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., 1995
, 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., 1997
; Melaine et al., 2003
).
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.
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Materials and methods |
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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 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 Childrens Research Hospital, Lauderdale, Memphis, TN, USA; Lyn et al., 1991
; Coronel et al., 2001
).
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 3637°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 SpragueDawley 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 Bouins 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% PBSbovine 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 avidinbiotinperoxidase complex amplification (DAKO, Trappes, France; Stephan et al., 2000). 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 dAscq, 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. Duncans Multiple Range test was used to analyse variance. Differences were considered to be significant if P < 0.05.
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Results |
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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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Discussion |
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In the present study, we chose a route of injection of the testisintratesticular injectionwhich 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., 2002). 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., 1987; Pöllänen and Niemi, 1987
; Hedger, 1997
). 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, 1997
).
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., 1999), and parainfluenza virus 1, another paramyxovirus, is able to infect and to replicate in leukocytes (Bogen et al., 1977
). 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, 1947
; Charny and Meranze, 1948
). 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., 1997
; 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, 2001
).
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., 1997). 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, 1947
; Charny and Meranze, 1948
).
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.
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Acknowledgements |
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References |
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Submitted on June 26, 2002; resubmitted on January 8, 2003; accepted on May 1, 2003.