1 Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 355, 92140 Clamart, France and 2 Service d'Expérimentation Animale et de Transgénèse (SEAT), Centre National de la Recherche Scientifique (CNRS), 94800 Villejuif, France
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Abstract |
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Key words: genital organs/infected male/murine cytomegalovirus/virus transmission
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Introduction |
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CMV can be detected in the urine, saliva, milk, cervical secretions and semen (Terzian, 1998), and has been isolated from the spermatozoa of patients with mononucleosis caused by human cytomegalovirus (HCMV) (Lang and Kummer, 1972
), from CMV-seropositive patients (Rasmussen et al., 1995
; Mansat et al., 1997
; Witz et al., 1999
), and also from men consulting for infertility (Levy et al., 1997
). HCMV is more common in HIV-seropositive men (Detel et al., 1994
).
HCMV has been found in the semen of healthy donors (Mansat et al., 1997), and more recently, HCMV DNA has been reported in the semen of CMV-seronegative men (Witz et al., 1999
).
The presence of CMV in the semen of healthy men opens up the possibility that spermatozoa (Huang et al., 1986) or their surrounding medium could carry the virus to the oocyte at fertilization. CMV is known to be transmitted by sexual contact, but the route, the efficiency and frequency of this mode of transmission is unknown (Demler et al., 1986
; Liesnard, 1998
). Cytomegalovirus DNA has been found in some samples of cervical mucus of HCMV-seronegative women whose husbands had HCMV DNA in their ejaculate (Witz et al., 1999
). These questions are important because congenital HCMV is the most common infection leading to birth defects (Stagno et al., 1986
; Ranger-Rogez et al., 1999
).
Other viruses of the same group have transforming capacity in humans and in experimental systems (Huang et al., 1986; Smiley et al., 1988
). Species-specific murine CMV (MCMV) infection in mice is a valuable model, and has been the subject of intense investigation; indeed, it continues to yield intriguing information of potential value for understanding HCMV infection and disease. As in humans, the infection is not apparent in immunocompetent animals, but causes severe disorders in newborn mice and in immunodepressed adult mice (Dutko and Oldstone, 1979
).
One group (Baskar et al., 1986) infected the testes of mice in vivo and has detected MCMV DNA in the spermatids and spermatozoa; however, the effects on the offspring of such infection (the presence of infectious virus or MCMV DNA) have not been studied. In another study (Young et al., 1977
), infectious MCMV was isolated from one of 28 cultured mouse embryo fibroblasts (MEFs) after intrauterine insemination by spermatozoa containing MCMV. The Smith strain used in this study is very similar to the human CMV strain in terms of the disease it causes (Osborn, 1986
) and its genomic sequence (Rawlinson et al., 1996
). In the current study, adult male FVB/N mice were infected intratesticularly, and subsequently demonstrated the virus in their genital organs. These infected males were then mated with healthy females, and evidence sought of virus transmission in the uterine fluid, fertilized oocytes, blastocysts and newborn mice.
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Materials and methods |
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Cell cultures
Cultures of FVB/N MEF were prepared from minced 14- to 15-day-old embryos. The tissues were trypsinized, and the resulting cell suspensions diluted in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 UI/ml), streptomycin (100 µg/ml), 0.1% fungizone and 10% fetal calf serum (FCS). Monolayer cultures were obtained after 3 days of incubation at 37°C under 5% CO2, and used after two to eight passages.
Virus
The MCMV used in this study was the Smith strain, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA; VR-1399). The titre of this virus stock was 106.5 ID50/0.2 ml.
MCMV purification
Confluent MEF cells with viral suspension [second passage, 2x105 plaque-forming units (PFU) in 1 ml DMEM, 2% FCS] were cultured for 1 h at 37°C. The viral suspension was then replaced with fresh medium and the cells were incubated for 35 days, by which time 90% of the cells had characteristic MCMV cytopathic effects (CPE). The viral suspension containing released virus and infected cells were collected and stored at 80°C. Control cells were similarly treated in DMEM, except that the MCMV suspension was omitted.
The defrosted infected cells were dissociated, shaken, centrifuged at 6500 g (Beckman, rotor JS41) for 20 min at +4°C, after which the supernatant was removed and stored at +4°C. The pellet was suspended in 1 ml of medium and homogenized (Dounce homogenizer) by 20 pestle strokes on ice, and centrifuged at 12500 g for 10 min at +4°C to obtain a second supernatant. Both supernatants were pooled and centrifuged at 14000 g (Beckman, rotor SW28) for 3 h at 4°C to obtain a viral pellet which was stored on ice in a cold room overnight. The pellet was then re-sedimented by ultracentrifugation at 65000 g (Beckman, rotor SW 28) for 1 h at +4°C through a sucrose gradient (15% in phosphate-buffered saline; PBS) in polyallomer tubes. The viral pellet was finally suspended in 1 ml PBS and stored at 80°C (Figure 1A and B).
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Male infection
A total of 72 adult male mice was anaesthetized with avertin (tribromoethanol, amyl alcohol; Sigma-Aldrich, St Quentin Fallavier, France) (250 mg/kg, i.p.). Of these mice, 68 were mated (24 on D1, 17 on D3, 14 on D7 and 13 on D15), and 20 were killed and analysed. Each testis was injected with 2.5x105 PFU of MCMV in 25 µl DMEM (or 25 µl DMEM alone for controls) via a needle passed through the albuginea. The genital organs (testis, epididymis, pooled spermatozoa from the vasa deferentia) and target organs (spleen, salivary glands) were examined for virus (infectiousness, MCMV DNA).
Five adult males were injected intratesticularly with toluidine blue under the same experimental conditions to visualize fluid diffusion in testis compartments.
Mating and offspring
Uninfected female mice were superovulated with an i.p. injection of 10 IU pregnant mare's serum gonadotrophin (PMSG; Intervet, Angers, France), followed by 10 IU human chorionic gonadotrophin (HCG) 48 h later, just before mating (one or two females per male) with male mice infected 1, 3, 7 or 15 days previously. On the following morning the females were checked for the presence of a vaginal plug, and then sacrificed. The uterine fluid was flushed out and centrifuged at 20 800 g for 10 min. The resulting pellet was diluted in DMEM or in lysis buffer and examined for virus. Fertilized oocytes were recovered from the ampullae and either treated in the same way or cultured until the blastocyst stage.
Unsuperovulated females were placed with infected males; those with a vaginal plug were killed either during pregnancy or after birth. Fetuses (13 days) and newborn mice were analysed for virus. In some cases, the placenta and maternal salivary glands were also recovered for the detection of viral DNA.
Analysis of virus
Cytopathic effects
Tissue samples from infected and control mice were dissociated in DMEM with 2% FCS, and then centrifuged at 20 800 g for 10 min. The supernatants (101 and 102 dilutions for each sample, x100 µl/well) were added to confluent monolayers of MEFs and incubated at 37°C. The cultures were examined for CPE (rounded, swollen cells). Any cytopathic effect normally appeared 35 days after MCMV infection.
Detection of virus DNA by PCR
DNA was extracted from the tissues. Briefly, the organs were incubated overnight at 55°C in 500µl lysis buffer for only 2h [Tris:EDTA (10:1), pH 7.8, proteinase K); spermatozoa, oocytes and spermatozoa from uterine fluids were incubated for only 2 h. The DNA was extracted twice with 500µl phenol/chloroform (1:1) followed by precipitation with isopropanol, and then suspended in Tris:EDTA (10:1). Aliquots of DNA (200500 ng) were used with Ready-to-Go PCR beads (Amersham, Pharmacia Biotech, Orsay, France) and specific oligonucleotide primers. The oligonucleotide primers (GENSET, Paris, France) (sense: 5'-CGTCGCGAAGGAACACCTC-3'; antisense: 5'-GTCGCCATGCGGGAGGTTTC-3') were used to amplify a sequence of viral DNA (Klotman et al., 1990). The PCR product was a 258 bp fragment that covers a region within the BamH1-B fragment of the Smith strain MCMV genome.
PCR was performed under the following conditions: 94°C for 5 min; 65°C for 2 min and 70°C for 4 min for the first cycle; this was followed by 30 cycles at 91°C for 1 min, 65°C for 1 minute and 70°C for 4 min.
The PCR products from oocytes, blastocysts and spermatozoa from uterine fluid underwent a second PCR with the same primers. PCR products were electrophoresced on a 1.5% agarose gel (80 mA for 12 h) and stained with ethidium bromide.
Structural studies
Semi-thin sections (1 µm) were cut from Araldite-embedded blocks (see Ultrastructural procedure), mounted on glass slides, stained with 1% toluidine blue in 1% sodium borate, and examined under a light microscope to locate abnormal architecture, abnormal cells and cytomegalic inclusions.
Ultrastructural procedure
The cells were fixed in 2% glutaraldehyde in 0.1 mol/l caccodylate buffer (Sigma-Aldrich), washed in the same buffer containing 1 mol/l sucrose and postfixed in 1% OsO4 in caccodylate buffer. Cells were dehydrated (ethyl alcohol, 70° to 100°), incubated in Araldite plus ethanol, embedded in Araldite at room temperature and stored at 60°C for 3 days. Ultrathin sections were cut with an OmU3 ultramicrotome (Reichert, Vienna, Austria) and deposited on 600-mesh nickel grids. Sections were contrasted with 5% uranyl acetate in ethyl alcohol and lead citrate, and examined in a Phillips 301 electron microscope.
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Results |
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MCMV DNA detection
A representative gel of 258 bp of MCMV-specific PCR products amplified from the genital and target organs of infected male is shown in Figure 2. The specificity of amplification products was compared with two control samples consisting of stock virus (positive control) and uninfected organ (negative control).
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Infectious virus detection
Characteristic cytomegalic inclusions were observed in MEFs cultured with all the homogenates of organ samples of infected males on D1 and on D3 PI (four for testis, four for epididymis and pooled spermatozoa), whereas it was observed in the testes of only some animals (3/6) on D7 PI, and not in the epididymis or pooled spermatozoa. A viral CPE was rarely detected in testis tissue on D15 (1/3), and was always absent on D35. A CPE was never observed in the target organs, in peritoneal exudate, or in uninfected control samples.
Viral localization in the reproductive tract
Genital organs showing both virus DNA detected by PCR and cytopathic effect in cell culture were selected for examination by light (Figure 3AF) and by electronic microscopy (Figure 4AD
).
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Sertoli cells were maintained, spermatocytes were scarcely present, and only a few spermatids and spermatozoa were detected in the tubules.
With regard to the epididymis, few cells were infected on D3 PI in the connective tissue. No infected epithelia cells were observed on D3 PI or on D7 PI. However, lymphocyte infiltration occurred on D7 PI. Light microscopy did not permit the detection of virus on or in spermatozoa in the lumen of the tubes.
Electron microscopy showed testicular cells of the interstitium that appeared to be infected to different degrees (Figure 4A, B and D); some had capsids only in the nucleus. Isolated, dicapsidic or multicapsidic virions of the type typically recovered from mice, MCMV were present in the cytoplasm of the other cells (Figure 4D
), in the interstitial spaces, and close to erythrocytes in capillaries. The infected area in the testis always appeared to be limited to the interstitium; viruses similar to those present in high concentration in infected cells of the interstitium were never detected in seminiferous tubules. Virus was not detected in Sertoli cells or in germ cells (Figure 4B and C
).
The only infected cells in the epididymis were those of the connective tissue. No infected epithelial cells or virions were found inside the epididymal lumen or in spermatozoa extracted from the vasa deferentia.
Mating and early development
Sixty-eight infected males were mated with uninfected females at each stage (D1, D3, D7 and D15 PI). The consistency of the vaginal plug of the mated females prevented PCR from being performed.
Spermatozoa from uterine fluids
Spermatozoa recovered from uterine fluids were pooled at each stage (D1, D3, D7 and D15 PI; 38 females in total) and analysed for virus. No cytopathic effect was observed in MEFs cultured with these spermatozoa samples, whatever the day of mating. Moreover, the band characteristic of MCMV DNA was not detected by PCR, or even by double PCR analysis (D1, nine females tested; D3, 11 females; D7, 10 females; D15, eight females).
Oocytes and blastocysts
The fertilization rate and the in-vitro development capacity of fertilized oocytes from eight females mated with infected males on D3 and D7 were normal until the blastocyst stage (147 blastocysts/232 fertilized oocytes), as compared with four control females (31 blastocysts/45 fertilized oocytes). Neither the oocytes immediately recovered in the ampullae (n = 63) nor the blastocysts (n = 147) obtained from culture contained any MCMV DNA.
Embryos and newborns
Six mated females maintained for gestation had 31 embryos and 22 new offspring. These neonates were tested and showed no anatomical abnormalities or virus. Neither the salivary glands nor the placenta of the corresponding mothers contained viral DNA.
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Discussion |
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MCMV in the testis of male mice caused inflammatory infiltrate, infection of interstitial cells and degeneration of the seminiferous tubules. The swelling and lysis of cells in the seminiferous tubules led to complete disorganization and a reduction in spermatozoa formation with maintenance of Sertoli cells. Such seminiferous tubule disorders have been reported in cases of interstitium infection with other viruses (Tearle et al., 1996; Fountain et al., 1997
).
Leydig cells have been found to be infected with MCMV, supporting the hypothesis that such cells are preferential targets for viruses of the herpes viridae family (Baskar et al., 1983; Blunden et al., 1998
). The fact that Leydig cells were infected indicates altered testosterone synthesis, most likely affecting the well-known role of these cells in regulating spermatogenesis (Dadoune and Demoulin, 1991
; Jegou et al., 1995
; Saez et al., 1995
).
Among the several cell types of the interstitium forming the bloodtestis barrier (Dadoune, 2000), endothelial cells were found with typical cytomegalic inclusions and cell junction damage leading to the presence of haemorrhagic areas between cells. The presence of virions inside vascular vessels suggests that the virus spreads throughout the organism via the blood. Similar pathological features of endothelial cells infected with CMV and other viruses of the same family have been reported (Price et al., 1990
; Grefte et al., 1993
; Tearle et al., 1996
; Blunden et al., 1998
). In these infected males, peritubular cells were degenerative or, more rarely, infected. In contrast, the Sertoli cells, by the production of antiviral factors such as interferons (Dejucq et al., 1998
; Dejucq and Jegou, 1999
) or by their specific junction system, may represent potent means to prevent MCMV virion occurrence in the seminiferous tubules.
Viral particles were not detected inside the tubules (i.e. in the germinal cells, from spermatogonia to spermatozoa, or in the Sertoli cells). However, there may have been a germinal compartment infection, closely linked to the testicular infiltrates. Some stained seminiferous tubules were obtained in the control injection system (toluidine blue), demonstrating the aleatoric means of inoculation.
The sperm pool from the vasa deferentia was infectious in MEFs until 3 days post-inoculation, but no virions were found in the spermatozoa using electron microscopy. This suggests that virionspermatozoon interactions are rare or absent, and that the virus is carried by spermatozoa, by other cells, or remains free in the semen. CMV-like particles have been found in sperm cells, but the spermatozoa were always abnormal (Baskar et al., 1986; Huang et al., 1986
). The key issue of whether immature germ cells and/or spermatozoa can bind virus should therefore be investigated using complementary techniques.
MCMV DNA was also detected by PCR in spermatozoa isolated from the testis and vas deferens duct up to 15 days after inoculation, in parallel with the virus infectivity. The relationship between free viral DNA and spermatozoa should be taken into account because CMV can exist in both complete and incomplete forms, as well as free viral DNA. The first evidence for the uptake of a heterologous virus genome by mammalian spermatozoa was reported in 1971 (Brackett et al., 1971), and this was followed by investigations of sperm-mediated virus genes (Baskar et al., 1986
; Pao et al., 1996
; Kotronias and Kapranos 1998
; Brossfield et al., 1999
). In IVF, the transport of a virus gene into oocytes by incubation of DNA in a sperm suspension has been well documented (Lavitrano et al., 1989
; Camaioni et al., 1992
; Auvray et al., 2000
). Transgenic offspring produced with sperm cells as vectors to introduce foreign DNA have been recently obtained by intracytoplasmic sperm injection (ICSI) (Perry et al., 1999
). It was found that virus DNA persists, as a latent form, for a long period of time in the genital tract of male mice. The term `latency' is defined as failure to detect infectious virus despite the presence of viral genome (Koffron et al., 1998
). Persistent virus and molecular latency appear to be characteristic of CMV, and they become reactivated principally in immunosuppressed hosts (Jordan, 1983
).
No virus and/or MCMV DNA were found in spermatozoa recovered from the female genital tract on the day after mating. Similarly, no virus was found during the first stages of embryogenesis, or in the offspring, whilst the placenta and the maternal organs studied also contained no virus. Within the limit of the delay respected for mating after male infection (i.e. 15 days after infection), it could be concluded that the protective barriers (of male and/or female origin) are active in in-vivo fertilization in these FVB/N immunocompetent animals. The differences in fertilization between mice (intrauterine insemination, vaginal plug formation) and humans (intravaginal insemination and cervical mucus crossing) might explain why in some cases HCMV DNA has been found in the cervical mucus of an uninfected woman (Witz et al., 1999). However, no infected babies have been reported, despite the presence of virus in the cervical mucus of the mother. These reports, together with the current results, indicate that there is very little chance that motile, fertilizing spermatozoa carry the virus to the oocyte during natural fertilization, even if the spermatozoa are obtained from an acutely infected partner.
However, in in-vitro systems (particularly ICSI) the selected spermatozoon is picked at random from the testis, epididymis or from the ejaculate and injected into the ooplasm. It is possible that, under these conditions, the defective spermatozoon might carry the virus, or the CMV might be carried to the ooplasm by the surrounding medium. This question is important, as abnormal lineages have been obtained by injecting purified MCMV DNA into the pronuclei of male mice (Baskar et al., 1993).
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Acknowledgements |
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Notes |
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References |
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Submitted on December 29, 2000; accepted on June 14, 2001.