In this study, we have used SIV and SHIV infected macaques to determine the characteristics of post-acute SIV and SHIV infection in the testis and epididymis. Our data demonstrate (i) significant SIV RNA viral load in both testis and epididymis upon establishment of viremia, (ii) productive infection of immune cells of the testis and epididymis, (iii) SIV and SHIV infection of spermatogonia, but not the later germ cells, (iv) the presence of a DC population in the monkey testis and (v) the presence of immature testicular germ cells (spermatocytes and round spermatids) in the epididymal lumen, which may be related to the infection. Although the animal cohort was relatively small in size and the animal maturation status differed between animals of both groups, this study clearly demonstrates that SIV and SHIV infection of the MGT is an important site of viral replication during SIV and SHIV infection. Based in the VLs observed in the testis and epididymis we conclude that these two organs do not significantly contribute to the overall viremia. However, because free virions and infected cells are shed from these organs into semen, the observed VLs are potentially very significant in terms of male to female viral transmission.
With disease progression, HIV-1 infection disrupts the testis morphology and spermatogenesis, reviewed in . Our data show potentially significant levels of viral RNA within the testis tissue even within 11–13 weeks and 23 weeks of infection for SHIV-infected versus SIV infected macaques, well prior to the development of simian AIDS. At week 23, despite high SIV viral loads, the percentage of CD4 T-cells was relatively preserved for SIV infected macaques but was substantially reduced in SHIV infected macaques. Moreover, infected immune cells were observed in the testis interstitium and epididymal stroma. Thus, infection of both testicular and epididymal cells could potentially act as a reservoir for semen, throughout the duration of infection , reviewed in .
In acute infection, plasma viral loads correlate with viral loads in semen, suggesting that the MGT (prostate, seminal vesicles, urethra, testis, epididymis) may be targeted by the virus in the very early stage of infection . While seminal plasma and seminal leukocytes originate mainly from the infected prostate and seminal vesicles, most of the cells present in semen are germ cells originating from the testis and epididymis. Macrophages and T cells in the testis of the infected macaques were infected. It is possible that these infected cells are passed onto the epididymis and contribute to viral shedding in semen early in infection. This might occur at the rete testis where translocation of T cells and macrophages into the lumen of the duct can occur [20–22]. There is also evidence that macrophages and T cells found in the epididymal epithelium may also cross into the epididymal duct [23, 24]. It is however important to note that we have shown no evidence that infected T cells or macrophages move from the testis or epididymal tissue into the duct. The more rapid progression of SHIVmn229 compared to SIV infection of macaques was consistent with previous reports [25, 26]. As expected, plasma and lymph node viral load was significantly higher in SHIV infection when compared with SIV infection, but there was no significant difference in viral load in the testis and epidydimis from SHIV infected animals compared with SIV-infected animals. This possibly suggests that infection of the MGT occurs relatively early following infection and is not dependent on disease progression, the degree of immunosuppression of the host or co-receptor usage.
Infection of macrophages in the MGT with SIV and SHIV was infrequent. Most DC-SIGN+ cells were negative for p24 staining in the SHIV challenged monkeys. Similarly, a low number of infected macrophages has been demonstrated in the female genital tract via in situ hybridization . Using the more specific method of double IF, our data reinforce these findings. We were unable to comment on MGT cell loss (i.e. T cell loss due to infection versus other maturation related cell numbers) due to SIV and SHIV infection as we did not have access to serial biopsies or maturation matched tissues from naive animals. Although DCs have been observed previously in the rodent testis and very recently in the human testis , the identification of a dendritic cell population in macaque testis is a novel finding.
There have been very few studies of the non-human primate reproductive tract during the pre-pubertal period . In the course of this study, in at least two animals infected with SHIV and one SIV infected animal, we have interestingly observed the presence of spermatocytes and round and elongated spermatids in the epididymal lumen. Autofluorescence of spermatocytes, spermatids and mature sperm made it difficult to determine whether these cells are infected in the epididymis of SIV and SHIV macaques. These round and elongated spermatids did not stain with the antibody to p24, suggesting that they are uninfected. We are in the process of establishing a technique that will specifically determine the presence of SIV and SHIV RNA and/or DNA in these cells. Whether their presence in the epididymal lumen is a consequence of the viral infection of this organ or simply a normal maturation process in non-human primate sperm maturation biology remains to be determined. The spermatogonia of these animals were positive for p24 antigen, suggesting ongoing productive infection within the seminiferous tubules. This would lead to impaired spermatogenesis even in early HIV infection.
Infection of spermatogonia has only been previously reported by one study , though not via IF. Meiotic and post-meiotic germ cells could potentially get infected by HIV-1 due to clonal infection (i.e. from one infected germ cell to the next) . Thus, HIV-1 DNA has been detected within different categories of more mature germ cells, possibly through clonal infection. We saw no evidence of later germ cells being productively infected suggesting that either infected spermatogonia die rather than develop further, a conclusion that supports the previously described arrest of spermatogenesis in men that have died from AIDS , or later germ cells are latently infected and do not support productive infection. Spermatogonia would represent the initial target for HIV-1 infection. The spermatogonia are located outside the Sertoli cell tight junctions, making them readily accessible to virions present in the interstitium. While the presence of the galactosylceramide receptor has been reported on their surface , the presence of other HIV receptors on spermatogonia remains unclear. Two studies have reported the presence of viral RNA or proteins within germ cells [31, 8]. We detected productive infection in spermatogonia located in seminiferous tubules using IF. A productive infection would lead to the release of free HIV-1 particles into the seminiferous tubules lumen and subsequently in the seminal fluid. It is not clear how infection of these cells is initiated, although one possibility would be virus transcytosis through the blood testis barrier, a phenomenon that is yet to be observed.