FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muciaccia, B.
Right arrow Articles by Stefanini, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muciaccia, B.
Right arrow Articles by Stefanini, M.
(The FASEB Journal. 1998;12:151-163.)
© 1998 FASEB

Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization

Barbara Muciacciaa, Stefania Uccinib, Antonio Filippinia,c, Elio Ziparoa,c, François Paraired, Carlo D. Baronib and Mario Stefaninia,1

a Department of Histology and Medical Embryology
b II Chair of Pathology and Immunopathology Section of the Department of Experimental Medicine and Pathology
c Istituto Pasteur Fondazione Cenci Bolognetti, University of Rome ‘La Sapienza’, Rome, Italy
d Laboratory of Pathology and Forensic Medicine, Hôpital Poincaré, Garches, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular distribution of HIV-1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV-1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell-free virus is suggested. In the testes of AIDS-deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV-seropositive men produce infected spermatozoa that are released in the genital tract.—Muciaccia, B., Uccini, S., Filippini, A., Ziparo, E., Paraire, F., Baroni, C. D., Stefanini, M. Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization. FASEB J. 12, 151–163 (1998)


Key Words: testis • stem cell infection • AIDS • germ cell


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ARREST OF GERM CELL MATURATION, thickening of seminiferous tubular wall, interstitial fibrosis, and irreversible testicular atrophy are the major pathologic features observed in the testes of AIDS-deceased men (1–3). However, the pathogenetic mechanisms responsible for testis impairment during HIV infection have not been clarified. Consistent evidence in favor of testicular inflammatory processes and vascular pathologic changes has never been reported, and a low incidence of opportunistic testicular infections has been observed (1, 3, 4). The hypothesis of an autoimmune orchitis is considered highly unlikely (5), and the possibility that the observed testicular atrophy may be secondary to impairment of gonadotropin regulation of testicular function does not correlate with the normal number and appearance of Leydig cells (3, 4).

HIV particles have been described in the testis by electron microscopy, without a defined cellular localization (6). By immunocytochemistry, HIV-related p17 protein was observed in germ cells of AIDS-deceased patients, suggesting that the virus may infect germ cells, which may contribute to semen infection (7). By in situ hybridization and immunocytochemistry, HIV-infected macrophages and lymphocytes were observed within the interstitial compartment and also migrating into the seminiferous epithelium (8). More recently, HIV infection of the testis of AIDS-deceased men has been demonstrated by in situ polymerase chain reaction (PCR) 2 (9). In particular, infected spermatogonia, spermatocytes, and, less frequently, round spermatids have been observed (9). On the basis of these observations, it has been suggested that degeneration of infected germ cells may contribute to germ cell maturative arrest, as well as to the diffusion of the virus into the male genital tract (9).

These observations, mostly performed at the final stages of infection, do not permit us to define the timing of HIV testicular localization with respect to the natural history of the disease. This limits understanding of the pathogenetic mechanisms responsible for testicular damage and hampers establishing whether the testis represents an early site of HIV infection and a source of viral spreading into the male genital tract.

As for the detection of HIV-1 in the semen, the virus has been observed in the mononuclear cells present in the pre-ejaculatory (10, 11) and ejaculatory fluids (12–18) as well as in mononuclear cells infiltrating the genital tract (19). Diffusion of the virus to the seminal fluid through infected cells infiltrating the wall of the genital tract is also suggested by the presence of HIV in the semen of azoospermic vasectomized HIV-positive patients (20). In addition, seminal fluid of both seropositive and AIDS men contains measurable amounts of cell-free virus particles (14, 18, 21); evidence has demonstrated the presence of the virus in the seminal fluid as early as a few weeks after the onset of primary HIV-1 infection (22), as well as intermittent shedding of the virus in the semen during the disease (23).

The presence of HIV on ejaculated spermatozoa has been controversial for a long time. However, evidence has consistently demonstrated the presence of the virus on the surface and/or in the cytoplasm of ejaculated spermatozoa (24–28). In addition, HIV-1 sequences have been detected in seminal spermatozoa from HIV-infected men by standard (15–17) and in situ PCR (26, 27). Since the virus may penetrate the membranes of human sperm (26–28), it is possible that spermatozoa become infected in the genital tract (29). However, the possibility that the testis may contribute, from the early stages of the disease, to the spreading of the virus into the genital tract by releasing infected spermatozoa and/or cell-free virus remains to be evaluated.

It must be considered that the testis is an immunologically privileged site of the body (30–36). Germ cell-linked autoantigens, expressed from puberty onward, are physiologically tolerated in situ, but elicit strong autoimmune reactions if injected elsewhere in the body (32). The testis could therefore represent an early and tolerated site of HIV localization and replication and a precocious route for virus spreading into the genital ducts.

To explore this hypothesis, we have studied, by in situ PCR hybridization, the presence and cellular distribution of HIV-1 proviral DNA in the testes of infected men who died during either early or advanced stages of the disease. Our observations demonstrate the presence of HIV-1 proviral DNA in autoptic testes from HIV-seropositive asymptomatic subjects. In particular, infected germ cells were observed at all stages of differentiation, from spermatogonium to spermatozoon, in seminiferous tubules characterized by normal spermatogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues
Testicular tissues from 14 subjects were available for this study. Eight testes were from HIV-seropositive asymptomatic subjects; of these: 1) seven (obtained from the Service d'Anatomie Pathologique et de Médecine Légale, Hôpital R. Poincaré, Garches, France) were collected at autopsy of young men who died of unnatural causes, mostly heroin overdose. Serology for HIV and a complete postmortem were carried out in each case: all were HIV positive, none had lesions suggestive of AIDS or AIDS related complex; there was no lymphadenopathy, no significant visceral inflammation, and no opportunistic infections or tumors (37); 2) one was a testicular surgical biopsy (courtesy of Dr. G. Bertalot, Ospedale di Leno, Brescia, Italy). Five testes were autoptic from AIDS-deceased men (courtesy of Dr. L. Vago, V Anatomia Patologica, University of Milano, Italy). One was a control testicular surgical biopsy from an HIV-uninfected patient affected by leydigioma, (courtesy of Dr. L. Vago, V Anatomia Patologica, Università di Milano, Italy).

In addition, epididymal biopsies from two seronegative men who had undergone surgery for testis neoplasia (courtesy of Professor A. Maver, Ospedale Malpighi, Bologna, Italy), and a lymph node biopsy from an HIV-infected man, were studied. Tissues were fixed in neutral-buffered 10% formalin and embedded in paraffin by routine procedures. Sections of 5 µm thickness were cut, mounted, and routinely stained. Fragments obtained from autoptic testes from AIDS-deceased men were also processed for immunohistology.

Immunohistology
The material available for immunohistologic analysis from HIV-seropositive asymptomatic subjects was represented only by paraffin-embedded blocks. Therefore, only antibodies reacting with this material were used. In particular, a monoclonal antibody anti-HLA-DR (Chemicon International, El Segundo, Calif.) was used to identify activated lymphocytes, macrophages, and endothelial cells espressing MHC II class antigen. Immunostaining was revealed by an avidin-biotin technique in which a biotinilated secondary antibody reacts with several peroxidase-conjugated streptavidin molecules (Dako LSAB kit), followed by incubation with 0.03% H2O2 and 0.06% 3,3'-diaminobenzidine (Sigma, St. Louis, Mo.). Counterstaining with hematoxylin was then performed.

Fragments of autoptic testes from AIDS-deceased men were embedded in optimal cryopreserving tissue compound (Miles, Elkhart, Ind.), snap frozen in liquid nitrogen, and stored at -80°C until sectioning in a Leitz cryostat (Leitz Wetzlar GMBH, Wetzlar, Germany). Sections (6 µm) were fixed for 10 min in ice-cold aceton and immunostained with the following monoclonal antibodies: anti CD3, CD4, CD8, HLA-DR (Ortho, Rartian, N.J.), and CD68 (Dakopatts, Golstrup, Denmark), as previously described (Dako LSAB kit). The slides were counterstained with hematoxylin and mounted in Canadian balsam. In control slides, primary antibody was omitted.

DNA isolation from paraffin-embedded tissues
Total DNA was isolated from paraffin blocks as described (38). Briefly, five sections of 10 µm each were cut from each block, placed into sterile microfuge tubes, dewaxed twice with xylene, and washed with 100% ethanol. Dried pellets were resuspended in lysis buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween 20, 400 µg/ml proteinase K) and incubated overnight at 55°C. After phenol/chloroform extraction, DNA was precipitated, dried, and dissolved in 50 µl of sterile water.

Phase-solution PCR
The presence of HIV-1 sequences in extracted DNA was monitored by standard PCR using specific primers for gag and env genes. Primer pairs used were SK38/SK39, which amplify a 115 bp region of gag gene (39); SK68/SK69, which amplify a 141 bp region of env gene (39); and SK40 (5'-ACCCTTCAGGAACAAATAGGATGGATGA-3') and SK41 (5'GAACCGGTCTACATAGTCTCTAAAAGGT-3') for the gag gene. The SK40 and SK 41 primers were designed as outer primers to SK38 and SK39 in order to perform nested PCR on HIV-gag gene.

PCR was performed on total DNA extracted from paraffin-embedded tissues in a mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 200 µM dNTPs, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase (Boehringer Mannheim, Germany), and 0.5 µM of each primer. After initial denaturation for 5 min at 94°C, PCR reactions were performed for 40 s at 94°C, 1 min at 60°C, and 1 min at 72°C, for 30 cycles.

For nested PCR, genomic DNA was first amplified using primers SK 40/SK 41. An aliquot of the reaction was then amplified with primers SK38/SK39.

For each amplification reaction, DNA extracted from paraffin-embedded sections of HIV-negative testes, HIV-positive lymph nodes, and the HIV chronically infected cell line 8E5/LAV (40) was used for comparison.

PCR in situ hybridization
In situ PCR
In situ detection of HIV-1 proviral DNA PCR products was performed as previously described (9, 41–43), with minor modifications. Briefly, histological sections mounted on silanized slide (Perkin Elmer in situ PCR glass slide) were deparaffinized in xylene and hydrated through graded alcohols. Slides were then washed in phosphate-buffered saline (PBS), postfixed in 4% para-formaldehyde for 20 min, and incubated with 20µg/ml proteinase K at 37°C for 15 min.

After digestion, slides were washed in PBS, dehydrated through graded alcohols, and air dried. Sections were then subjected to Hot-Start PCR amplification by using a GeneAmp In situ PCR System 1000 (Perkin-Elmer, Norwalk, Conn.). Slides were assembled at 70°C in the Assembly Tool, using Amplicover discs and clips (Perkin Elmer). PCR reaction mixture consisted of 200 µM dNTPs, 0.5 µM of each primer, 3 mM MgCl2, 1x PCR buffer (20 mM (NH4)2SO4, 75 mM Tris-HCl pH 9, 0.01% Tween), and 2U Thermoprime plus DNA polymerase (Advanced Biotechnologies, Leatherhead, U.K.). After initial denaturation, 30 cycles of amplification, each at 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min were performed. Primers used to detect HIV-1 proviral DNA were as described (see above). For the ß-globin gene in situ amplification, a commercial primer pair (PC04 and GH20; Perkin Elmer) was used (44). For each experiment of in situ amplification, the following controls were executed: 1) specificity controls with heterologous primers; 2) amplification control in the absence of either Taq polymerase or primers; 3) sensitivity control using primers for an endogenous gene (ß-globin).

In addition, sections of HIV-negative testis and HIV-positive lymph node (see above) were processed for in situ PCR, together with each testis sample studied, for comparison.

Probes for in situ hybridization
Digoxigenin probes to detect the PCR products by in situ hybridization were produced by standard molecular cloning techniques (45). In particular, genomic DNA extracted from the 8E5/LAV cell line (40) and amplified by PCR was blunt-ligated into pUC18 vector (Pharmacia, Uppsala, Sweden). Probes obtained were: a 115 bp genomic region of HIV-gag produced using SK38/39 primers; a 141 bp genomic region for HIV-env amplified by using SK68/69 primers; and a 268 bp genomic region of ß-globin amplified using PCO4 and GH20 primers. Probes were labeled with digoxigenin by direct incorporation of DIG-dUTP (DIG-dUTP: dTTP 1:2 labeling ratio) during amplification reaction. The yield of each labeled probe was estimated in a dot-blot spot test, according to DIG system user's protocols guide (Boehringer Mannheim, Germany).

In situ hybridization
PCR products were detected on tissue sections by in situ hybridization with the corresponding digoxigenin-labeled DNA probes as described (41, 46). Slides were washed in 1x standard sodium citrate (SSC)/0.05% Triton-X and prehybridized in 50% formamide, 2X SSC, 2X Denhardt's, 10 mM EDTA, and 200 µg/ml salmon sperm for 1–3 h at 37°C. After washing, slides were hybridized in 50% formamide, 2X SSC, 10% dextran sulfate, 2X Denhardt's, 10 mM EDTA, 200 µg/ml salmon sperm, and 300 ng/ml of the specific probe overnight at 42°C in a humidified chamber. After hybridization, slides were washed as follows: 2x SSC at room temperature (RT), 1x SSC at RT, 0.2% SSC at 42°C, and finally, 0.2% SSC at RT. Sections were then incubated for 15 min in blocking solution (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1% BSA). To detect the amplified products, sections were incubated for 2 h at RT with alkaline phosphatase-conjugated anti-digoxigenin antibodies (Boehringer Mannheim, Germany) diluted 1:300 in blocking solution and then with chromogen substrate (NBT, BCIP) at RT in the dark. Sections were not counterstained.

Sperm chromatin decondensation
To verify whether the absence of in situ amplification of HIV-1 proviral DNA, as well as of the ß-globin gene observed on maturing spermatids (see Results), could be the consequence of the inability of proteinase digestion to get access to the highly condensed chromatin of these cells, we performed in situ amplification after putting decondensed sperm chromatin through a specific procedure (47). Decondensation was performed on histological sections from two human bioptic epididymides from HIV serum-negative subjects and from all testes of the HIV-seropositive cases studied. In particular, dewaxed tissue sections were incubated at 37°C for 40 min in detergent solution (10 mM Tris, pH 8, 5 mM DTT, 1% Triton-X), washed in 2X SSC, dehydrated, and used for in situ PCR amplification.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphological observations
HIV-seropositive asymptomatic subjects
In all HIV-seropositive subjects studied (eight in number), well-preserved spermatogenesis was observed. Germ cells at all phases of differentiation were regularly arranged in the typical cell associations of the human seminiferous epithelium ( Fig. 1). The peritubular wall was not increased in thickness, and Leydig cells appeared to be normal in quantity and morphology. Few lymphomononuclear cells were observed isolated in the interstitial compartment; in two of the eight cases studied, they were arranged in small clusters surrounding postcapillary venules.



View larger version (178K):
[in this window]
[in a new window]
  		Figure 1. Autoptic testis from an HIV-seropositive asymptomatic subject. A well-preserved spermatogenesis and germ cells regularly arranged in the cell associations, typical of the human testis, are observable; Leydig cells are regularly arranged in small clusters, and lymphomononuclear cells, scattered in the interstitium or clustered around postcapillary veins (asterisks), are present. Staining with hematoxylin and eosin, x200.

AIDS subjects
Extensive testicular hypoplasia was observed in AIDS-deceased subjects, with reduction of tubular diameter and mild to severe impairment of spermatogenesis characterized mostly by the absence of postmeiotic cells ( Fig. 2 a, b). Conspicuous interstitial fibrosis, thickening of the peritubular wall, and the presence of lymphomonocytes scattered in the interstitial compartment were also noticed ( Fig. 2a, b).



View larger version (174K):
[in this window]
[in a new window]
  		Figure 2. Autoptic testes from AIDS-deceased men. Spermatogenesis is impaired, and only spermatogonia and few spermatocytes are present. Peritubular wall is increased in thickness and a conspicuous interstitial fibrosis is evident, with mild infiltration of lymphomonocytes (arrows). Staining with hematoxylin and eosin: x200 (a); x400 (b)

Immunohistology
In testes from HIV-positive subjects, HLA-DR+ cells were observed around the vessels as well as isolated in the interstitial compartment ( Fig. 3a, b) HLA-DR+ cells were also present within the peritubular wall ( Fig. 3b), but never in the seminiferous epithelium.



View larger version (156K):
[in this window]
[in a new window]
  		Figure 3. Immunostaining for HLA-DR antigen on testes from HIV-seropositive men. HLA-DR + endothelial cells are observed in the vast majority of capillaries. Some of the lymphomononuclear cells scattered in the interstitial compartment and clustered around the blood vessels are HLA-DR + (a). HLA-DR + cells are present within the peritubular wall (b, arrows), but never in the seminiferous epithelium. Counterstaining with hematoxylin: X 200 (a); X 400 (b).

In cryostatic sections of testis from AIDS-deceased men, the lymphomononuclear infiltrates were mainly scattered. They consisted of low numbers of CD3+ lymphocytes, predominantly represented by CD8+ cells. CD68+ macrophages were numerous; endothelial cells of small blood vessel were constantly HLA-DR+ (data not shown).

Detection of HIV-1 proviral DNA by PCR analysis
The presence of HIV sequences by means of PCR and nested PCR analysis on DNA extracted from paraffin-embedded testicular sections was demonstrated in all autoptic testes from HIV-seropositive asymptomatic and AIDS subjects studied ( Fig. 4). A specific band of 115 bp was obtained in all cases and confirmed by nested PCR analysis. Control experiments, performed on DNA extracted from the testes of HIV-negative subjects, from lymph nodes of HIV-positive subjects, and the 8E5/LAV cell line confirmed these results ( Fig. 4).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. HIV-gag gene detection by PCR and nested-PCR analysis from paraffin-embedded testes. Samples, lanes 1–5: AIDS cases; lane 6: uninfected testis; lane 7: 8E5 cell line; lane 8: HIV-positive lymph node; lanes 9–16: HIV-seropositive asymptomatic cases; M: 100 bp ladder. Primers, lanes 1–4 and 7: gag SK38/39 (inner primer pair); lanes 5, 6, and 8–16: first amplification gag SK40/41 (outer primer pair), second amplification gag SK38/39 (inner primer pair). The PCR products were analyzed by electrophoresis on a 2% agarose gel and ethidium bromide stained.

Cellular localization of HIV-1 proviral DNA in the testis by in situ PCR hybridization
HIV-seropositive asymptomatic subjects
Cellular localization of the virus was detected in the testes of all HIV-seropositive asymptomatic subjects studied. A strong positive signal was observed in the nuclei of germ cells at all stages of differentiation from spermatogonium to round spermatid ( Fig. 5a–d) but Sertoli cells were always negative ( Fig. 5c, d). HIV-infected lymphomononuclear cells were observed in some of the interstitial infiltrates ( Fig. 5e), but were absent in the peritubular wall ( Fig. 5a–d).



View larger version (133K):
[in this window]
[in a new window]
  		Figure 5. In situ PCR hybridization for HIV-1 proviral DNA (primers SK40/SK41 for HIV-gag gene, digoxigenin DNA gag probe) on sections from autoptic testes of HIV-seropositive asymptomatic men (a), and on a bioptic testis from an HIV-seronegative man (f). In several tubules, a limited number of infected germ cells at various stages of differentiation are present (a, b). Positive cells are spermatogonia (a, arrows), spermatocytes (a, arrowheads), and round spermatids (d, asterisks). They are often arranged in clusters of cells at the same stage of differentiation. Maturing spermatids (c, empty arrows) and Sertoli cells (c, d, double arrows) are never labeled. Lymphomononuclear positive cells are observed only in the interstitial compartment (e). Stained cells are never observed in the control testis (f). Magnification: X 200 (a, b); X 400 (c, d X 250 (e); X 150 (f).

HIV-infected germ cells had a patchy distribution, with cohorts of positive cells all at the same differentiative stage. This hybridization pattern was observed at all stages of germ cell differentiation. Cohorts of positive spermatogonia, spermatocytes, and spermatids were observed alone or in association ( Fig. 4a–d). Tubules with infected germ cells were observed adjacent to completely negative tubules. When HIV-1 proviral DNA was amplified in situ after proteinase K digestion, maturing spermatids, characterized by their highly condensed chromatin, were never stained ( Fig. 5c). On the other hand, the same negative result was obtained when the same experiment was performed to detect the ß-globin gene ( Fig. 6a, b).



View larger version (141K):
[in this window]
[in a new window]
  		Figure 6. In situ PCR hybridization for the ;hb-globin gene on sections from autoptic testes of HIV-seropositive men after proteinase digestion (a, b) and from epididymides of HIV-seronegative subjects after sperm chromatin decondensation (c, d). Proteinase digestion does not allow access to the condensed chromatin of maturing spermatids; these cellular types are in fact always negative (empty arrows), whereas all other germ and somatic cells are always positive. Chromatin decondensation performed on epididymis sections allows in situ amplification of the ;hb-globin gene on sperm nuclei (c, d, arrows); stained with a digoxigenin-labeled DNA ;hb-globin probe. Magnification: X 150 (a) X 300 (b) X 700 (c); X 1000 (d).

The inability to detect HIV-1 proviral sequences, as well as the ß-globin gene, on maturing spermatids could be related to the high degree of chromatin condensation of these cells. To verify this hypothesis, we performed in situ PCR hybridization after sperm chromatin decondensation. The method was set up using epididymides from HIV-negative subjects and after amplification for ß-globin gene.

The results obtained demonstrated that the technique used for sperm chromatin decondensation was adequate to amplify DNA sequences. Approximately 40–50% of the sperm observed in the epididymal lumen were positive for ß-globin ( Fig. 6c, d). Head volume of positive sperm was increased by up to fourfold. The vast majority of negative sperm heads had a normal morphology, suggesting either an absence of or incomplete decondensation.

When in situ PCR hybridization was performed on the testis after treating the sections for sperm chromatin decondensation, ß-globin-positive maturing spermatids were observed ( Fig. 7a, b) Equally positive results on maturing spermatids were obtained in the testes of HIV-seropositive asymptomatic subjects by in situ amplification for gag and env HIV genes ( Fig. 7c, d). Staining was localized in the nuclei, which appeared larger than those of unstained spermatids, thus confirming that only decondensed cells had undergone in situ DNA amplification. HIV-positive maturing spermatids were infrequent with respect to infected round spermatids; however, a low number of stained testicular spermatozoa was observed when in situ amplification after decondensation was performed for the ß-globin gene. This latter observation suggests that incomplete decondensation as well as cellular damages induced by the technical procedures may result in underscoring of HIV-1 sequences in maturing spermatids.



View larger version (77K):
[in this window]
[in a new window]
  		Figure 7. In situ PCR hybridization for ;hb-globin gene (a, b) and HIV proviral DNA (primers SK 40/SK 41 for gag gene) (c, d) after decondensation on sections from autoptic testes of HIV-seropositive asymptomatic men. Maturing spermatids positive for ;hb-globin (a, b) and HIV (c, d) are present. Stained with a digoxigenin-labeled DNA specific probe. Magnification: X 700 (a, c); X 1100 (b, d).

AIDS subjects
In the testes of AIDS-deceased men, the few spermatogonia and primary spermatocytes observed were mostly positive for HIV-1 proviral sequences, as demonstrated by in situ PCR hybridization ( Fig. 8c, d) In addition, at variance with the HIV-seropositive samples, Sertoli cells with a positive hybridization signal were occasionally observed ( Fig. 8a). In the interstitial lymphomononuclear infiltrates, HIV-infected cells were present ( Fig. 8a. b).



View larger version (154K):
[in this window]
[in a new window]
  		Figure 8. a-d) In situ PCR hybridization for HIV proviral DNA (primers SK40/SK41 for HIV-gag gene) on sections from autoptic testes of AIDS-deceased men. Positive spermatogonia (arrows) and primary spermatocytes (arrowheads) are present in several tubules. Positive Sertoli cells are also observable (aterisks). Infected lymphomonocytes are present in the interstitial compartment (empty arrows), but never in the seminiferous epithelium or within the peritubular wall. Stained with a digoxigenin-labeled DNA gag probe. Magnification: X 200 (a, b); X 300 (c, d).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our observations demonstrate that HIV-1 is present in the testes of seropositive asymptomatic men. Proviral HIV-1 DNA sequences have been detected by in situ PCR hybridization in the nuclei of germ cells at all stages of differentiation, from spermatogonium to spermatozoon. The presence of HIV-1 proviral DNA in the nuclei of germ cells was not accompanied by morphological signs of cell damage and was associated with normal spermatogenesis.

It has previously been suggested that degeneration of infected germ cells observed in the testis of AIDS-deceased men could contribute to germ cell maturative arrest, as well as to the diffusion of the virus into the male genital tract (9). Our data demonstrate instead that, at least in the early stages of the disease, HIV virus is not cytopathic for germ cells, does not interfere with the progression of spermatogenis, and seropositive asymptomatic men are able to produce HIV-infected spermatozoa.

The mild lymphomononuclear infiltration observed in the cases studied indicates, on immunohistological grounds, that the presence of the virus in the testis is not accompanied by a local immune response.

Although no clinical information was available to correlate each subject studied to defined stages of the natural history of the disease on the basis of postmortem serology and epicrisis (37), these results (obtained in asymptomatic subjects) demonstrate that the testis represents an early site of viral localization and a potential HIV-1 reservoir that fails to elicit an immunological response, as demonstrated for other districts (48, 49).

Immunotolerance for the virus could be related to the immunological privilege of the testis (30–36). Several mechanisms responsible for this physiologic function have been identified, such as the blood–testis barrier, which segregates the interstitial compartment from autoantigens expressed on the meiotic and postmeiotic cell surface (33), and the production of immunosuppressive molecules by Sertoli cells (34). More recently, production of the CD95 (Fas or Apo 1) ligand by mouse Sertoli cells has been demonstrated and interpreted as the molecular counterpart for the testis immune privilege.

HIV-1 testis infection is potentially of great interest, considering that proviral DNA was observed in the nuclei of spermatogonia, a cell population directly exposed to the blood vessel compartment that includes stem cells of the germ cell line. Infection of stem cells could become chronic. Experiments are under way to verify whether HIV germ cell infection is productive in asymptomatic seropositive subjects, as has been reported to occur in AIDS-deceased men (9). We also intend to evaluate whether HIV-1 proviral DNA is integrated in the germ cell genome. Since spermatogonia are proliferating cells, the conditions required for DNA proviral integration into host chromosomes (50, 51) could be satisfied.

The HIV hybridization pattern observed in the seminiferous epithelium of seropositive men—i.e., cohorts of infected germ cells all at the same stage of differentiation—is compatible with clonal infection of germ cells; therefore, the virus could infect single spermatogonia and be transmitted to all their progeny up to spermatozoa. Spermatogonia, located in the basal compartment of the seminiferous epithelium, are directly exposed to the vascular compartment (33). They could therefore represent the first epithelial target for HIV present outside the tubules.

The mechanisms responsible for viral entry in the testicular germ cells remain to be clarified. HIV-infected lymphomononuclear cells have been observed in the interstitial compartment, but never in the peritubular wall or within the seminiferous epithelium. HLA-DR+ cells observed within the tubular wall, which possibly are tissue monocytes normally present in the peritubular wall of the human testis (52, 53), were always HIV negative. On the other hand, Sertoli cells, known to have some phenotypic characteristics similar to those of macrophages (54, 55), were infected only in the advanced stages of the disease. In autoptic testes from AIDS-deceased subjects, in addition to the already reported presence of the virus in the residual germ cells (9), we detected HIV-1 proviral DNA in some Sertoli cells.

These observations suggest the possibility of direct infection of the germ cells by cell-free virus. HIV infection in CD4-negative cells has been described in several tissues (56–59). The presence of a galacto-glycero lipid on the cell membrane of spermatozoa and spermatogonia able to bind the viral gp120 envelope glycoprotein has been recently claimed (60, 61).

Inability to detect the presence of HIV-1 proviral sequences in maturing spermatids, by in situ PCR hybridization after proteinase K digestion, appears to be due to technical reasons, since the ß-globin gene was also undetectable in these cellular types with the same procedure. In addition, no signs of degeneration were observed in infected round spermatids that could justify their disappearance. We considered, therefore, the possibility that proteinase K treatment could be inadequate to gain access to the highly condensed chromatin of maturing spermatids. In the nucleus of maturing spermatids and spermatozoa, histones have been replaced by protamines (62), and DNA is tightly packaged with a high degree of disulfide bonding. The problem was overcome by chromatin decondensation with reducing agents that disrupt disulfide bonds (15, 47) prior to in situ PCR hybridization. By this means, we were able to detect maturing spermatids positive for the ß-globin gene as well as for HIV-1 proviral sequences in the testis of HIV-seropositive subjects.

Localization of the HIV virus in germ cells of the testis of an asymptomatic HIV-infected man was previously reported by Nuovo et al. (9). In that single case studied, the virus was reported to be present only in spermatogonia and spermatocytes. In light of our data, absence of the virus in spermatids seems questionable, since its detection requires sperm chromatin decondensation.

The fate of testicular-infected spermatozoa is a subject for speculation. They are certainly released from the seminiferous epithelium, since no morphological evidence of sperm damage and/or delayed spermiation was observed. These cells may either degenerate in the male genital tract or reach the semen and be ejaculated. In both cases, they may contribute to the spreading of the virus in the recipient sexual partner. Horizontal transmission of retroviruses via sperm has been demonstrated in the mouse (63, 64). Infected germ cells released from the testis could be involved in sexual transmission of the infection, beginning with the early stages of the disease. The presence of spermatozoa harboring HIV-1 proviral DNA in their nuclei must be considered in relation to the problem of HIV-1 transmission by donor semen (65–68).

Finally, the functional study of spermatozoa from HIV-infected men indicates that infected spermatozoa may be motile (27). The possibility that infected spermatozoa fertilize human oocytes has recently been demonstrated in vitro (26). It remains to be verified whether fertilization by infected spermatozoa is followed by abortion.


   ACKNOWLEDGMENTS
 
We are indebted to Professors Michel Durigon and Françoise Gray, Laboratory of Pathology and Forensic Medicine, Hôpital Poincaré, Garches, France, for providing us with autoptic testes from HIV-seropositive asymptomatic subjects, which were instrumental in realizing this study. We also thank Dr. Luca Vago for providing autoptic testes from AIDS subjects; Dr. Giovanni Bertalot for a testicular biopsy from an HIV-seropositive subject; Professor Armando Maver for bioptic epididymal tissues; Professor Fioretta Palombi and Dr. Antonio Volpi and for critical reading of the manuscript; and Dr. Fabrizio Colelli and Miss Stefania De Grossi, who provided excellent technical support. This work was supported by grants from Istituto Superiore di Sanita and by AIDS projects #9306-23 and 9405-08 to M.S. and #9403-13 to C.D.B.


   FOOTNOTES
 
1 Correspondence: Department of Histology and Medical Embryology, University of Rome 'La Sapienza', Via Antonio Scarpa 14, 00161 Rome, Italy. E-mail: mstefanini{at}axrma.uniroma1.it

2 Abbreviations: RT, room temperature; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; SSC, standard sodium citrate.

Received for publication October 10, 1997. Accepted for publication October 20, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chabon, A. B., Stenger, R. J., and Grabstald, H. (1987) Histopathology of the testis in acquired immune deficiency syndrome. Urology 29, 658–663
  2. De Paepe, M. E., and Waxman, M. (1989) Testicular atrophy in AIDS: a study of 57 autopsy cases. Hum. Pathol. 20, 210–214[Medline]
  3. Dalton, A. D. A., and Harcourt-Webster, J. N. (1991) The histopathology of the testis and epididymis in AIDS: a post-mortem study. J. Pathol. 163, 47–52[Medline]
  4. Rogers, C., and Klatt, E. C. (1988) Pathology of the testis in acquired immunodeficiency syndrome. Histopathology 12, 659–665[Medline]
  5. De Paepe, M. E., Vuletin, J. C., Lee, M. H., Rojas-Corona, R. R., and Waxman, M. (1989) Testicular atrophy in homosexual patients: an immune-mediated phenomenon? Hum. Pathol. 20, 572–578[Medline]
  6. Lecatsas, G., Houff, S., Macher, A., Gelman, E., Steis, R., Reichert, C., Masur, H., and Sever, J. L. (1985) Retrovirus-like particles in salivary glands, prostate and testes of AIDS patients. Proc. Soc. Exp. Biol. Med. 178, 653–655[Abstract]
  7. Da Silva, M., Shevchuk, M. M., Cronin, W. J., Armenakas, N. A., Tannenbaum, M., Fracchia, J. A., and Ioachim, H. L. (1990) Detection of HIV-related protein in testes and prostates of patients with AIDS. Am. J. Clin. Pathol. 93, 196–201[Medline]
  8. Pudney, J., and Anderson, D. (1991) Orchitis and human immunodeficiency virus type 1 infected cells in reproductive tissues from men with the acquired immune deficiency syndrome. Am. J. Pathol. 139, 149–160[Abstract]
  9. Nuovo, G. J., Becker, J., Simsir, A., Margiotta, M., Khalife, G., and Shevchuk, M. (1994) HIV-1 nucleic acid localize to the spermatogonia and their progeny. Am. J. Pathol. 144, 1142–1148[Abstract]
  10. Ilaria, G., Jacobs, J. L., Polsky, B., Koll, B., Baron, P., MacLow, C., and Armstrong, D. (1992) Detection of HIV-1 DNA sequences in pre-ejaculatory fluid (letter). Lancet 340, 1469
  11. Pudney, J., Oneta, M., Mayer, K., Seage, G., and Anderson, D. (1992) Pre-ejaculatory fluid as potential vector for sexual transmission of HIV-1 (letter). Lancet 340, 1470
  12. Zagury, D., Bernard, J., Leibowitch, J., Groopman, J. E., Feldman, M., and Gallo, R. C. (1984) HTLV-III in cells cultured from semen of two patiets with AIDS. Science 226, 449–451[Abstract/Free Full Text]
  13. Ho, D. D., Schooley, R. T., Rota, T. R., Kaplan, J. C., and Flynn, T. (1984) HTLV-III in the semen and blood of a healthy homosexual man. Science 226, 451–453[Abstract/Free Full Text]
  14. Krieger, J. N., Coombs, R. W., Collier, A. C., Ross, S. O., Chaloupka, K., Cummings, D. K., Murphy, V. L., and Corey, L. (1991) Recovery of human immunodeficiency virus type 1 from semen: minimal impact of stage of infection and current antiviral chemotherapy. J. Infect. Dis. 163, 386–388[Medline]
  15. Van Voorhis, B. J., Martinez, A., Mayer, K., and Anderson, D. J. (1991) Detection of HIV-1 in semen from seropositive men using culture and PCR DNA amplification techniques. Fertil. Steril. 55, 588–594[Medline]
  16. Mermin, J. H., Holodniy, M., Katzenstein, D. A., and Merigan, T. C. (1991) Detection of human immunodeficiency virus DNA and RNA in semen by polymerase chain reaction. J. Infect. Dis. 164, 769–772[Medline]
  17. Hamed, K. A., Winters, M. A., Holodniy, M., Katzenstein, D. A., and Merigan, T. C. (1993) Detection of human immunodeficiency virus type-1 in semen: effects of disease stage and nucleoside therapy. J. Infect. Dis. 167, 798–802[Medline]
  18. Vernazza, P. L., Eron, J. J., Cohen, M. S., van der Horst, C. M., Troiani, L., and Fiscus, S. A. (1994) Detection and biologic characterization of infectious HIV-1 in semen of seropositive men. AIDS 8, 1325–1329[Medline]
  19. Mostad, S. B., and Kreiss, J. K. (1996) Shedding of HIV-1 in the genital tract. AIDS 10, 1305–1315[Medline]
  20. Anderson, D. J., Politch, J. A., Martinez, A., Van Voorhis, B. J., Padian, N. S., and O'Brien, T. R. (1991) White blood cells and HIV-1 in semen from vasoectomized seropositive men (letter). Lancet 338, 573–574
  21. Borzy, M. S., Connell, R. S., and Kiessling, A. A. (1988) Detection of human immunodeficiency virus in cell-free seminal fluid. J. Acquir. Immune Defic. Syndr. 1, 419–424
  22. Tindall, B., Evans, L., Cunningham, P., McQueen, P., Hurren, L., Vasak, E., Mooney, J., and Cooper, D. A. (1992) Identification of HIV-1 in semen following primary HIV-1 infection. AIDS 6, 949–952[Medline]
  23. Krieger, J. N., Coombs, R. W., Collier, A. C., Ho, D. D., Ross, S. O., Zeh, J. E., and Corey, L. (1995) Intermittent shedding of human immunodeficiency virus in semen: implications for sexual transmission. J. Urol. 154, 1035–1040[Medline]
  24. Baccetti, B., Benedetto, A., Burrini, A. G., Collodel, G., Elia, C., Piomboni, P., Renieri, T., Sensini, C., and Zaccarelli, M. (1991) HIV particles detected in spermatozoa of patients with AIDS. J. Submicrosc. Cytol. Pathol. 23, 339–345[Medline]
  25. Dussaix, E., Guetard, D., Dauguet, C., D'Almeida, M., Auer, J., Ellrodt, A., Montaignier, L., and Auroux, M. (1993) Spermatozoa as potential carriers of HIV. Res. Virol. 144, 487–495[Medline]
  26. Baccetti, B., Benedetto, A., Burrini, A. G., Collodel, G., Costantino-Ceccarini, E., Crisà, N., Di Caro, A., Estenoz, M., Garbuglia, A. R., Massacesi, A., Piomboni, P., Renieri, T., and Solazzo, D. (1994) HIV-particles in spermatozoa of patients with AIDS and their tranfer into the oocyte J. Cell Biol. 127, 903–914
  27. Bagasra, O., Farzadegan, H., Seshamma, T., Oakes, J. W., Saah, A., and Pomerantz, R. J. (1994) Detection of HIV-1 proviral DNA in sperm from HIV-1-infected men. AIDS 8, 1669–1674[Medline]
  28. Scofield, V. L., Rao, B., Broder, S., Kennedy, C., Wallace, M., Graham, B., and Poiesz, B. J. (1994) HIV interaction with sperm. AIDS 8, 1733–1736[Medline]
  29. Marx, J. L. (1989) Do sperm spread the AIDS virus? (letter). Science 245, 30
  30. Head, J. R., Neaves, W. B., and Billingham, R. E. (1983) Immune privilege in the testis. Basic parameters of allograft survival. Transplantation 36, 423–431[Medline]
  31. Head, J. R., and Billingham, R. E. (1985) Immune privilege in the testis. Evaluation of potential local factors. Transplantation 40, 269–275[Medline]
  32. Tung, K. S. K. (1980) Autoimmunity in the testis. In Immunological Aspects of Infertility and Fertility Regulation (Dhindsa, D. H., and Shumacher, G. F. B., eds) 33–91, Elsevier/North-Holland, New York
  33. Dym, M., and Fawcett, D. W. (1970) The blood-testis barrier of the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308–326[Abstract]
  34. De Cesaris, P., Filippini, A., Cervelli, C., Riccioli, A., Muci, S., Starace, G., Stefanini, M., and Ziparo, E. (1992) Immunosuppressive molecules produced by Sertoli cells cultured in vitro: biological effects on lymphocytes. Biochem. Biophys. Res. Commun. 186, 1639–1646[Medline]
  35. Bellgrau, D., Gold, D., Selawry, H., Moore, J., Franzusoff, A., and Duke, R. (1995) A role for CD95 ligand in preventing graf rejection. Nature (London) 377, 630–632[Medline]
  36. Sanberg, P. R., Borlongan, C. V., Saporta, S., and Cameron, D. F. (1996) Testis-derived Sertoli cells survive and provide localized immunoprotection for xenografts in rat brain. Nature Biotechnol. 14, 1692–1695[Medline]
  37. Gray, F., Lescs, M. C., Keohane, C., Paraire, F., Marc, B., Durigon, M., and Gherardi, R. (1992) Early brain changes in HIV infection: neuropathological study of 11 HIV seropositive, non-AIDS cases. J. Neuropathol. Exp. Neurol. 51, 177–185[Medline]
  38. Jackson, D. P., Hayden, J. D., and Quirke, P. (1991) Extraction of nucleic acid from fresh and archival material. In PCR: A Practical Approach (McPherson, M. J., Quirke, P., and Taylor, G. R., eds) 29–50, Oxford University Press, Oxford
  39. Ou, C. Y., Kwok, S., Mitchell, S. W., Mack, D. H., Sninsky, J. J., Krebs, J. W., Feorino, P., Warfield, D., and Schochetman, G. (1988) DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239, 295–297[Abstract/Free Full Text]
  40. Folks, T. M., Powell, D., Lightfoote, M., Koenig, S., Fauci, A. S., Benn, S., Rabson, A., Daugherty, D., Gendelman, H. E., Hoggan, M. D., Venkatesan, S., and Martin, M. A. (1986) Biological and biochemical characterization of a cloned Leu-3 cell surviving infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164, 280–290[Abstract/Free Full Text]
  41. Haase, A. T., Retzel, E. F., and Staskus, K.A. (1990) Amplification and detection of lentiviral DNA inside cells. Proc. Natl. Acad. Sci. USA 87, 4971–4975[Abstract/Free Full Text]
  42. Bagasra, O., Hauptman, S. P., Lischer, H. W., Sachs, M., and Pomerantz, R. J. (1992) Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. N. Engl. J. Med. 362, 1385–1391
  43. O'Leavy, J. J., Chetty, R., Graham, A. K., and McGee, J. O'D. (1996) Review article: In situ PCR: pathologist's dream or nightmare? J. Pathol. 178, 11–20[Medline]
  44. Bauer, H., Ting, Y., Greer, C., Chambers, J., Tashiro, C., Chimera, J., and Reingold, A. (1991) Genital human papilloma virus infection in female university students as determinated by a PCR-based method. J. Am. Med. Assoc. 265, 472–477[Abstract]
  45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) In Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Plainville, New York
  46. Nuovo, G. J. (1992) PCR in situ Hybridization: Protocols and Application, Raven Press, New York
  47. Vidal, F., Morages, M., Català, V., Torello, M. J., Santalò, J., Calderòn, G., Gimenez, C., Barri, P. N., Egozcue. J., and Veiga, A. (1993) Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study. Human Reprod. 8, 1740–1743[Abstract/Free Full Text]
  48. Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., Hermankova, M., Chadwick, K., Margolick, J., Quinn, T.C., Kuo, Y. H., Brookmeyer, R., Zeiger, M. A., Barditch-Crovo, P., and Siliciano, R. F. (1997) Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature (London) 387, 183–188[Medline]
  49. Perelson, A. S., Essunger, P., Cao, Y., Vesanem, M., Hurley, A., Saksela, K., Markowitz, M., and Ho, D. D. (1997) Decay characteristics of HIV-1-infected compartments during combination therapy. Nature (London) 387, 188–191[Medline]
  50. Zack, J. A., Arrigo, S. J., Weistsman, S. R., Go, A. S., Haislip, A., and Chen, I. S. Y. (1990) HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61, 213–222[Medline]
  51. Bukrinsky, M. I., Sharova, N., Dempsey, M. P., Stanwick, T. L., Bukrinskaya, A. G., Haggerty, S., and Stevenson, M. (1992) Active nuclear import of human immunodeficiency virus type 1 preintegretion complexes. Proc. Natl. Acad. Sci. USA 89, 6580–6584[Abstract/Free Full Text]
  52. Hermo, L., and Clermont, Y. (1976) Light cells within the limiting membrane of rat seminiferous tubules. Am. J. Anat. 145, 467–484[Medline]
  53. Hermo, L., and Lalli, M. (1978) Monocytes and mast cells in the limiting membrane of human seminiferous tubules. Biol. Reprod. 19, 92–100[Abstract]
  54. Filippini, A., Russo, M. A., Palombi, F., Bertalot, G., De Cesaris, P., Stefanini, M., and Ziparo, E. (1989) Modulation of phagocytic activity in cultured Sertoli cells. Gamete Res. 23, 367–375[Medline]
  55. Riccioli, A., Filippini, A., De Cesaris, P., Barbacci, E., Stefanini, M., Starace, G., and Ziparo, E. (1995) Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells. Proc. Natl. Acad. Sci. USA 92, 5808–5812[Abstract/Free Full Text]
  56. Cheng-Mayer, C., Rutka, J. T., Rosenblum, M. L., McHugh, T., Stites, D. P., and Levy, J. A. (1987) Human immunodeficiency virus can productively infect cultured human glial cells. Proc. Natl. Acad. Sci. USA 84, 3526–3530[Abstract/Free Full Text]
  57. Tateno, M., Gonzalez-Scarano, F., and Levy, J. A. (1989) Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells. Proc. Natl. Acad. Sci. USA 86, 4287–4290[Abstract/Free Full Text]
  58. Harouse, J. M., Bhat, S., Spitalnik, S. L., Laughlin, M., Stefano, K., Silberberg, D. H., and Gonzalez-Scarano, F. (1991) Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide. Science 253, 320–323[Abstract/Free Full Text]
  59. Fantini, J., Cook, D. G., Nathanson, N., Spitalnik, S. L., and Gonzalez-Scarano, F. (1993) Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc. Natl. Acad. Sci. USA 90, 2700–2704[Abstract/Free Full Text]
  60. Brogi, A., Presentini, R., Piomboni, P., Collodel, G., Strazza, M., Solazzo, D., and Costantino-Ceccarini, E. (1995) Human sperm and spermatogonia express a galactoglycerolipid which interacts with gp120. J Submicrosc. Cytol. Pathol. 27, 565–571[Medline]
  61. Brogi, A., Presentini, R., Solazzo, D., Piomboni, P., and Costantino-Ceccarini, E. (1996) Interaction of human immunodeficiency virus type-1 envelope glycoprotein gp120 with galactoglycerolipid associated with human sperm. AIDS Res. Hum. Retrov. 12, 483–489[Medline]
  62. Meistrich, M. (1989) Histone and basic nuclear protein transections in mammalian spermatogenesis. In Histones and Other Basic Nuclear Proteins (Hnilica, L. S., Stein, G. S., and Stein, J. L. eds) 165–182, CRC Press, Boca Raton, Florida
  63. Kiessling, A. A., Crowell, R. C., and Connell, R. S. (1987) Sperm-associated retroviruses in the mouse epididymis. Proc. Natl. Acad. Sci. USA 84, 8667–8671[Abstract/Free Full Text]
  64. Portis, J. L., McAtee, F. J., and Hayes, S. F. (1987) Horizontal transmission of murine retroviruses. J. Virol. 61, 1037–1044[Abstract/Free Full Text]
  65. Stewart, G., Cunningham, A., Driscoll, G., Tyler, J., Barr, J., and Gold, J. (1985) Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet 2, 581–584[Medline]
  66. Guinan, M. (1995) Artificial insemination by donor: safety and secrecy. J. Am. Med. Assoc. 273, 890–891[Medline]
  67. Araneta, M. R. G., Mascola, L., Eller, A., O'Neil, L., Ginsberg, M. M., Bursaw, M., Marik, J., Friedman, S., Sims, C. A., Rekart, M. L., and Collie, F. (1995) HIV transmission through donor artificial insemination. J. Am. Med. Assoc. 273, 854–858[Abstract]
  68. Stewart, G., and Downie, J. (1996) Transmission of HIV-1 by donor semen (Letter). Lancet 347, 970



This article has been cited by other articles:


Home page
 Hum ReprodHome page
B. Muciaccia, S. Corallini, E. Vicini, F. Padula, L. Gandini, G. Liuzzi, A. Lenzi, and M. Stefanini
HIV-1 viral DNA is present in ejaculated abnormal spermatozoa of seropositive subjects
Hum. Reprod., November 1, 2007; 22(11): 2868 - 2878.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
L. Bujan, M. Daudin, N. Moinard, P. Plante, J. Parinaud, and C. Pasquier
Azoospermic HIV-1 infected patients wishing to have children: proposed strategy to reduce HIV-1 transmission risk during sperm retrieval and intracytoplasmic sperm injection: Case Report
Hum. Reprod., September 1, 2007; 22(9): 2377 - 2381.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
T. Persico, V. Savasi, E. Ferrazzi, M. Oneta, A.E. Semprini, and G. Simoni
Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure
Hum. Reprod., June 1, 2006; 21(6): 1525 - 1530.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
L. Mencaglia, P. Falcone, G. M. Lentini, S. Consigli, M. Pisoni, V. Lofiego, R. Guidetti, P. Piomboni, and V. De Leo
ICSI for treatment of human immunodeficiency virus and hepatitis C virus-serodiscordant couples with infected male partner
Hum. Reprod., August 1, 2005; 20(8): 2242 - 2246.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
L. Barzon, M. Boscaro, and G. Palu
Endocrine Aspects of Cancer Gene Therapy
Endocr. Rev., February 1, 2004; 25(1): 1 - 44.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
C. Habasque, F. Aubry, B. Jegou, and M. Samson
Study of the HIV-1 receptors CD4, CXCR4, CCR5 and CCR3 in the human and rat testis
Mol. Hum. Reprod., May 1, 2002; 8(5): 419 - 425.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
N. Dejucq and B. Jegou
Viruses in the Mammalian Male Genital Tract and Their Effects on the Reproductive System
Microbiol. Mol. Biol. Rev., June 1, 2001; 65(2): 208 - 231.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muciaccia, B.
Right arrow Articles by Stefanini, M.
Right arrow Search for Related Content