Murine Cytomegalovirus Infects Spermatogenic Cells

Murine Cytomegalovirus Infects Spermatogenic CellsAuthor(s): Francis J. Dutko and Michael B. A. OldstoneSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 76, No. 6 (Jun., 1979), pp. 2988-2991Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/69900 .

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Proc. Natl. Acad. Sci. USA Vol. 76, No. 6, pp. 2988-2991, June 1979 Medical Sciences

Murine cytomegalovirus infects spermatogenic cells (sperm/solution and in situ hybridization/nude mice)

FRANCIS J. DUTKO AND MICHAEL B. A. OLDSTONE

Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Communicated by Frank J. Dixon, April 2, 1979

ABSTRACT Murine cytomegalovirus replicated in reproductive tissue of male mice infected with the virus. We examined three strains of mice latently infected by injection at birth with 100 plaque-forming units of the virus. As adults, these mice contained within their testes 4-6 viral genomic equivalents per 100 cells, as tested by hybridization between mouse DNA and cytomegalovirus DNA. Acutely infected male adult CBA mice homozygous for the nude gene (athymic: nude/nude) produced infectious virus in thir testes, the amounts of which varied ac- cording to the animal's age at the time of infection. Heterozy- gous (nude/+) litter mates contained significantly less virus than nude/nude mice. At the peak of virus replication hybridization between virus DNA and mouse DNA indicated the presence of 3.3 viral genome equivalents per testicular cell. Both in situ hybridization studies and phenol emulsion reassociation of virus DNA to DNA from purified spermatozoa localized this viral DNA to immature and mature sperm cells. Hence, murine cytomegalovirus can be harbored in testes during both acute and latent infections and can replicate in male germ-line cells.

Because of its destructive impact on human health, cytomegalovirus (CMV) infection is among the most challenging problems in biomedical research. CMV infection is a leading cause of birth defects (1, 2) and a suspected cause of subtle visual and auditory deficiencies in children (3). CMV commonly infects patients given organ transplants (4, 5), receiving blood transfusions (6), or undergoing immunosuppression (7). Epi- demiological surveys indicate that few humans escape infection from this virus during their life (reviewed in ref. 1). Germane biological questions focus around identifying the cells that harbor CMV, learning how it exists in a latent state and is later activated, and determining the mechanisms(s) by which CMV persists in the face of a vigorous host immune response.

We have used CMV infection in mice (MCMV) as a model system of human CMV disease because both disorders are similar and because virologic and immunologic manipulations are relatively easy in mice. In studying the cells that harbor MCMV in vivo and the role that these cells play in the patho- genesis of disease, we detected MCMV in the reproductive tissues of both latently and acutely infected male mice. Further, the MCMV was localized to spermatozoa by in situ hybridization of MCMV RNA to murine DNA and by phenol emulsion hybridization analysis of MCMV DNA to DNA isolated from spermatozoa purified by centrifugal elutriation (8).

MATERIALS AND METHODS

DNA-DNA Hybridization. To isolate DNA, we homoge- nized tissues, treated them with proteinase K and 1% sodium dodecyl sulfate for 2 hr at 37?C, and extracted the DNA with phenol and chloroform (9). Urea (8 M) and phosphate buffer (0.24 M) were added, and the mixture was placed on a 2-cm diameter hydroxylapatite (Bio-Gel HTP, Bio-Rad) column.

The publication costs of this article were defrayed in part by page charge payment. This article niust therefore be hereby marked "ad- vertisement " in accordance with 18 U. S. C. ? 1734 solely to indicate this fact.

After washing with 1 vol of 8 M urea, and 7 vol of 0.12 M phosphate buffer, DNA was eluted with 0.48 M phosphate buffer, concentrated to 5 mg/ml by lyophilization, jnd sheared by sonication to a fragment size of 6 *8 S, as analyzed by sedimentation in alkaline sucrose gradients. Hybridization reactions were carried out at 65?C in 0.48 M phosphate buffer. The MCMV probe had a specific activity of 5 X 106 cpm/,g of DNA. The radioactivity in single-stranded DNA and double- stranded DNA was determined by hydroxylapatite chromatography as described by Britten and Kohne (10).

In Situ Hybridization. 3H-Labeled RNA complementary to MCMV DNA (cRNA) was prepared by incubating purified MCMV DNA with Escherichia coli DNA-dependent RNA polymerase (Miles) and [5-3H]uridine triphosphate (NET-287, New England Nuclear) (11). Cytohybridization was performed on 6-,um sections of mouse testes. Tissues were fixed by treatment at 40C with ethanol/acetic acid, 3:1 (vol/vol) for 15 min. After ethanol washes and air drying, the DNA was denatured in situ by treatment with 75 mM NaOH for 3 min at 37?C. The slides were washed three times in 95% ethanol and dried in air. The cytohybridization was performed by incubating the section with 0.1 ml of [3H]cRNA (5 X 106 Cpm/ml in 6 X SSC where 1 X SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7) at 65?C for 22 hr. The sections were washed with 2 X SSC, treated with ribonuclease (40 ,ug/ml, Worthington), washed with 2 X SSC, and dehydrated by washes with 95% ethanol. Autoradiography was performed with NTB-2 emulsion (Eastman Kodak), ex- posure at -70?C for 6 days, and development in Kodak D-19 developer. The sections were stained with hematoxylin and eosin.

Purification of Mouse Spermatozoa. To purify spermatozoa from other cell types found in testes, we modified the method of Grabske et al. (8). Briefly, eight testes were pooled, cut to 1- to 2-mm sections, and incubated with 0.05% trypsin and 0.02% EDTA (Flow Laboratories, Rockville, MD). After 20 min at 370C, fetal calf serum (final concentration 8% vol/vol) was added to inactive trypsin and the cell suspension was first fil- tered through nylon mesh and then injected into the JE-6 rotor (Elutriator rotor, Beckman Instruments) driven by a Beckman J21C preparative centrifuge. We used a rotor speed of 3000 rpm, with a flow rate of 11 ml/min of phosphate-buffered sa- line/0.5% bovine serum albumin. Under these conditions, cells with a maximum sedimentation velocity of 2.5 mm hr-lg-1 or less flowed through the rotor's separation chamber. These cells were collected in two 150-ml centrifuge bottles and found by morphologic criteria to be greater than 99.9% spermatozoa. Purified spermatozoa were pelleted by centrifugation at 1500 rpm for 20 min in a CRU-5000 centrifuge (International Equipment Centrifuge) and resuspended in 0.24 M phosphate buffer/5 mM EDTA, at pH 7.

Phenol Emulsion Reassociation Technique. DNA isolated from purified spermatozoa was analyzed for MCMV genome

Abbreviatioiis: CMV, cytomegalovirus; MCMV, murine CMV; PFU, plaque-forming unit; i.p., intraperitoneally.

2988



Medical Sciences: Dutko and Oldstone Proc. Natl. Acad. Sci. USA 76 (1979) 2989

equivalents per cell by hybridization by using a phenol emulsion system (12). Briefly, 0.1-ml samples containing 50 ,ug of DNA per ml (absorbance at 260 nm of 1), 0.48 M phosphate buffer (pH 7), MCMV [3H]DNA, and 9% (vol/vol) phenol were treated at 110?C for 1 min. Samples were immediately cooled in an ice bath, and then mixed at room temperature on a Vortex mixer. Ten-microliter samples were removed at various times, and the radioactivity in single- and double-stranded DNA was measured by hyroxylapatite chromatography.

RESULTS

MCMV Detected in Testes during Latent Virus Infection. C3H/St, CBA/WEHI, and SWR/J newborn mice were infected intraperitoneally (i.p.) with 100 plaque-forming units (PFU) of tissue-culture-passed MCMV. Complete descriptions of the virus stock, inoculations, and fates of such animals have been published (13, 14). For this report we examined adult mice that survived MCMV infection and were, by reported criteria (13, 14), carriers of latent virus. Mice were killed when 2-6 months old, and their tissues were examined by hybridization between MCMV DNA and mouse DNA (10) for evidence of MCMV. No virus was detected in brain, thymus, liver, or kid- neys. In contrast, by solution hybridization assay, MCMV DNA was present in splenic and testicular tissues at the level of 4-6 viral genomes per 100 cells. These results were obtained in three separate experiments using tissue pooled from seven mice in each experiment, as well as in assays using tissues from four individual mice.

13ecause the sensitivity of detection of our in situ hybridization technique is 20 gene copies per cell, the small number of viral genome equivalents in cells from the testes precluded using this technique to determine which cell types(s) in the testes harbored the virus. Therefore, to determine whether MCMV replicated in male germ-line cells, we turned to a model of acute MCMV infection.

MCMV Replicated in Testes during Acute Virus Infection. Aduilt athymic CBA/WEHI mice (homozygous nude/nude) and heterozygous litter mates (nude/+) mice born with normal thivniuses were inoculated i.p. with 105 PFU of MCMV. At various intervals after infection, testicular DNA was analyzed bv hybridization of MCMV DNA. The results shown in Fig. 1 indicate a peak of 3.3 viral genome equivalents per cell at 15 days after the initiation of infection. Values were lower at all other times tested: 1.1, 1.1, and 1.0 viral genome equivalents per cell at 5, 10, and 20 days after infection, respectively. A corresponding difference in PFU/g of testes was noted over this interval with 1.3 X 105 PFU at day 6-7, 4.6 X 105 PFU at day 13-14, and 1.2 X 105 PFU at day 20-21. Consistently, adult liomiozvgous nude mice contained larger amounts of MCMV than their heterozygous litter mates of the same age. Two weeks after infection, significantly more MCMV was detected in testes of the homozygous nude mice than in those of heterozygous litter mates (Table 1). Table 1 also shows that testes from young adult nude/nude mice had higher titers of virus than testes from older nude/nude mice (4 weeks > 6 weeks > 11 weeks).

MCMV Replicated in Male Germ-Line Cells. Immature arl(l ndature sperm cells from the testes of acutely infected tiuide/nude mice contained MCMV DNA. As shown in Fig. 2, s)erniatozoa in the seminiferous tubules contained MCMV 1i3H IIRNA.MCMV DNA hybrids, as indicated by the presence of grains after in situ hybridization. Overall, at least 0.01% of .sI)rnmatazoa contained 20 or more MCMV DNA gene copies. T he.se resullts wTere obtained in at least three random sections froml te.ste s of five separate mice (nude/nude). In contrast, no gralins wsere found in spermatozoa from the same number of

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5,000 10,000

Cot, mol sec liter-'

FIG. 1. MCMV DNA-murine DNA hybridization analysis of testes. At 4 weeks of age, CBA/WEHI nude/nude mice were inoculated i.p. with 105 PFU of MCMV passaged in mouse embryo cell cultures. At various times after infection, testes were removed. DNA was isolated from the testes and analyzed by DNA-DNA hybridization. The fraction of single-stranded DNA was calculated from cpm in single-stranded DNA divided by the sum of the cpm in single- stranded DNA and the cpm in double-stranded DNA. Then 1/(fraction of single-stranded DNA) was plotted against the Cot (the product of the initial DNA concentration in mol/liter and the time of incu- bation in seconds). Cot1/2 was interpolated from the line obtained by linear regression analysis. The number of viral genome equivalents per cell was calculated by comparison of the Coti/2 with an assumed Cot1/2 for unique mouse DNA sequences of 3000. 0, 5 days after infection; A, 10 days after infection; 0, 15 days after infection; 0, 20 days after infection; *, uninfected mouse DNA.

sections from uninfected nude/nude mice or in other cell types in the testes of MCMV infected mice.

MCMV DNA Was Present in Purified Spermatozoa. The purity of the centrifugally elutriated spermatozoa from MCMV-infected mice was greater than 99.9% (Fig. 3), based

Table 1. Age-dependent resistance of CBA/WEHI mice to infection with MCMV

Age at infection, PFU/g testes weeks nude/nude mice nude! + mice

4 3.0 X 104 <1.0 X O' 6 1.9 X 103 <1.0 X 10

11 4.0 X 1O' <1.0 X 101

CBA/WEHI nude/nude and nude/ + mice of various ages were inoculated i.p. with 105 PFU of MCMV. Two weeks after infection, testes were removed and a 10% (wt/vol) homogenate was made in a Dounce tissue grinder. After centrifugation at 1000 x g for 10 mi, the supernatants were analyzed for infectivity by plaque assay on confluent monolayers of secondary mouse embryo cell cultures. Plaques were fixed with formaldehyde and stained with crystal violet at 5 days after infection. Tissue infectivities are expressed as PFU per g of testes.



2990 Medical Sciences: Dutko and Oldstone Proc. Natl. Acad. Sci. USA 76 (1979)

FIG. 2. In situ hybridization of testes from nude mice. CBA/ WEHI nude/nude mice were infected at 4 weeks of age by inoculation i.p. of 105 PFU of MCMV. At 3 weeks after infection, testes were removed, placed into test tubes, and snap frozen in liquid nitrogen. Six-micrometer sections of testes were analyzed by in situ hybridization. Specific MCMV [3H]RNA-MCMV DNA hybrids are indicated by silver grains. These grains were observed over spermatozoa in the testes of two separate (Left, Right) nude mice. (X650.)

on microscopic counting of 1000 or more cells. DNA isolated from both purified spermatozoa and unfractionated testes was analyzed independently for MCMV genome equivalents by the phenol emulsion reassociation technique. The results of three experiments are shown in Table 2. Spermatozoa obtained from nude mice contained a higher concentration of MCMV DNA (4.7 genome equivalents per diploid genome) than did unfractionated testes (3.2 genome equivalents per diploid genome). Further, the hybridization results were specific for MCMV-infected spermatozoa because DNA from MCMV- infected mouse spermatozoa did not hybridize to herpes simplex virus [3H]DNA and DNA from uninfected spermatozoa did not hybridize to MCMV [3H]DNA.

DISCUSSION This report makes two main points. First, MCMV is harbored in reproductive tissues of adult male mice several months after the initiation of a neonatal infection. Second, after acute infection of adult athymic mice, MCMV replicated to high titers in testes and localized predominantly in immature and mature sperm cells. MCMV did not replicate to any significant degree in testes of acutely infected thymic-competent adult mice.

Our observations of MCMV DNA in male germ-line cells raise the possibility that MCMV infection can be harbored in

Table 2. Phenol emulsion reassociation of viral [3H]DNA to DNA from purified spermatozoa

No. of viral DNA genome equivalents per diploid

cell genome using DNA MCMV HSV type 2

isolated from [3H]DNA [3H]DNA

Infected spermatozoa 4.7 <0.2 Infected testes 3.2 <0.2 Uninfected spermatozoa <0.2 <0.2 Uninfected testes <0.2 <0.2

Four-week-old CBA/WEHI nude/nude mice were inoculated i.p. with 105 PFU of MCMV. Two weeks after infection, testes were removed and the DNA from either testes or purified spermatozoa was analyzed for MCMV DNA by the phenol emulsion reassociation technique. The number of viral genome equivalents per diploid cell genome was calculated in the same manner as in the legend for Fig. 1 except that the assumed Cot112 for unique mouse DNA sequences was 3 mol-sec per liter. HSV, herpes simplex virus.

FIG. 3. Cytocentrifuge preparations of purified spermatozoa and unfractionated testes. Spermatozoa were purified from testes by centrifugal elutriation. An aliquot of the cells was pelleted onto glass slides by cytocentrifugation, air dried, fixed in methanol for 10 min at room temperature, and stained with Giemsa. (X275.) (Left) Puri- fied spermatozoa fraction; (Right) unfractionated starting mate- rial.

and transmitted by sperm. In preliminary studies, Brautigam and Oldstone* found MCMV DNA within ovarian perifolli- cular cells of acutely infected female mice by in situ hybridization and in ovaries of latently infected mice by hybridization of MCMV DNA and mouse DNA. Coupled with a recent report that fetuses from mice persistently infected with MCMV contained MCMV DNA when tested by DNA-DNA hybridization and viral antigen as seen by immunofluorescence (15), our data are consistent with the hypothesis that CMV may be transmitted vertically as well as horizontally. However, in our system we do not yet know whether sperm cells infected with MCMV remain viable. Using electron microscopy, Lang and coworkers (16, 17) initially reported the association of sperm with CMV by detecting human CMV in the semen. In these studies (16, 17), CMV virions were found only as extracellular aggregates and not within sperm heads. Other results suggest a relationship between CMV and spermatozoa (11), and MCMV has been recovered from prostate gland cells explanted from latently infected mice (18).

Replication of a virus within germ-line cells leads to several interesting speculations. First, if germ-line cells harbor virus, they can maintain the continuity of infection through vertical transmission. Such transmission could perpetuate the infectious agent in a population, even in the absence of susceptible subjects needed for horizontal transmission. Second, viruses may alter chromosomes (19). Could such a mechanism occur during CMV infection and partially explain associated birth defects? Third, virus may integrate into chromosomes. Although there is no evidence at present that CMV DNA does this, the generalized principle that viruses can integrate into chromosomes has been shown with Moloney leukemia virus in relation to murine germ-line cells (20) and with simian virus 40 in the 7th chro- mosome of a transformed line of human cultured cells (21).

Note Added in Proof. Neighbour and Fraser have reported on the recovery of MCMV from epididymal sperm and seminal vesicles and from uterine sperm collected from mated female mice (22).

We thank David T. Kingsbury and David Kohne for their help and interest as well as Alan Brautigam and Clark Huang. The technical assistance of Muriel Caruana is gratefully appreciated. This is publication no. 1522 from the Department of Immunopathology, Scripps

* Brautigam, A. & Oldstone, M. B. A. (1978) in 4th International Congress on Virology, The Hague, The Netherlands (abstr.).



Medical Sciences: Dutko and Oldstone Proc. Natl. Acad. Sci. USA 76(1979) 2991

Clinic and Research Foundation, La Jolla, CA. This research was supported by U.S. Public Health Service Grant AI-07007, National Foundation March of Dimes Grant 1-364, and Biomedical Research Support Program Grant RRO-5514. F.J.D. is supported by National Institutes of Health Fellowship 1-F32-AI05596.

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Murine Cytomegalovirus Infects Spermatogenic Cells