Herpes simplex virus inoculation in murine rete testis results in irreversible testicular damage

Abstract

This study aimed to establish the influence of herpes simplex virus (HSV) on testis morphology and germ cell development using a model of ascending urogenital HSV infection in mice. Adult C57BL/6J mice were inoculated with 100 plaque-forming units of HSV1 in rete testis. Viral proteins and HSV DNA were detected from 3 days postinoculation (DPI), while capsids and virions could be visualized at 6 DPI. Infectious activity of HSV was revealed by rapid culture method in testes from 3 to 14 DPI, and virus DNA by PCR - from 3 to 100 DPI. Germ and Sertoli cells were infected during the early stages of the infection, whereas interstitial cells only occasionally contained the virus at 21 and 45 DPI. Microscopic analysis revealed severe degeneration of the germinal epithelium in the infected testes. By 21 DPI, testes became atrophic and most Sertoli cells were destroyed. No testicular regeneration and no spermatozoa in the epididymis were observed at 45 and 100 DPI. From 3 DPI, inflammatory cells accumulated in the interstitium between damaged tubules; a significant increase in the number of CD4(+), CD8(+) T lymphocytes and F4/80(+) cells was observed in the infected testes. This study shows that in the case of HSV retrograde ascent into seminiferous tubules, the acute viral infection results in irreversible atrophy of the germinal epithelium, orchitis and infertility. These results may be used to further study viral orchitis and the influence of HSV on spermatogenesis and male fertility.

Keywords: Sertoli cells; herpes simplex virus; orchitis; spermatogenesis; testicular damage; testis.

Figure 1

In situ hybridization of HSV DNA on testis sections at 3 DPI (a–c), 6 DPI (d, e) and 45 DPI (f). Signals of viral DNA are clearly seen in spermatogonia and Sertoli cells located near the basement membrane of seminiferous tubules, spermatocytes (arrowheads), round spermatids (arrows) and elongated spermatids (asterisks). Sections are counterstained with haematoxylin. Scale bars = 20 μm.

Figure 2

Dynamics of testicular HSV infection. Vertical axis indicates the percentage of gB⁺ seminiferous tubules in the testes of infected mice; horizontal axis indicates days postinfection (DPI). The number of gB⁺ tubules was counted in three cryosections of the testis, and the percentage of gB⁺ tubules relative to the total number of tubules examined was calculated. Data are mean ± SEM; n = 3 testes per time point. The largest number of gB⁺ tubules was detected at 6 DPI (*P < 0.05 in relation to other time points).

Figure 3

Immunofluorescent localization of HSV proteins in the testes of infected mice. Viral proteins (red) are seen in the interstitium (a) and in the seminiferous tubule (b) at 21 DPI (a) and 45 DPI (b). Nuclei are counterstained with DAPI (blue). (c–f) Representative confocal microscopy images of the mouse testis at 6 DPI. Viral proteins (green) are seen in the cytoplasm of individual Sertoli cells (c, d); in the group of Sertoli cells and germ cells near the basement membrane of the seminiferous tubule (e); in all layers of the germinal epithelium (f). Nuclei are counterstained with PI (red). The primary antibody used was rabbit polyclonal anti-HSV1 antibody. Scale bars = 100 μm for (a, b, f); 20 μm for (c); 30 μm for (d); 40 μm for (e).

Figure 4

EM and IEM detection of HSV in infected testis. (a–e) EM of HSV-infected testis. (a) Basal part of the seminiferous tubule from the infected mouse at 6 DPI. (b, c) Magnified fragments of (a), demonstrating HSV capsids (arrows) in Sertoli cell nucleus (b) and in spermatocyte nucleus (c). (d) Luminal compartment of the seminiferous tubule from the infected animal. (e) Magnified fragment of (d) – the cytoplasmic droplet of the elongating spermatid, containing viral capsids (arrows). (f–h) IEM of the HSV-infected testis using rabbit polyclonal anti-HSV1 antibody. (f) Immunolabelled viral capsids (arrows) in Sertoli cell nucleus and dense bodies (arrowheads) in cytoplasm. (g, h) Magnified fragments of (f). 1 – basement membrane, 2 – interstitium, 3 – Sertoli cell, 4 – spermatocyte, 5 – elongating spermatid. Scale bars = 5 μm for (a, d), 1 μm for (b, c, e); 2 μm for (f); 250 nm for (g, h).

Figure 5

Testicular histopathology. (a) Degenerating germ cells in the lumina of seminiferous tubules (asterisks) and the arrest of spermatogenesis (arrow) at 3 DPI. (b) Disorganization of the germinal epithelium at 3 DPI. (c) Reduction in the thickness of the germinal epithelium (below dashed line), seminiferous tubules occupied by degenerating cells (asterisks) at 6 DPI. (d) Damaged Sertoli cells (arrowheads), multinucleated germ cells (arrow) and pyknotic germ cells (P) in abnormal tubules at 6 DPI. (e) Seminiferous tubules containing either only Sertoli cells or Sertoli cells and a few germ cells at 14 DPI. (f) Cellular debris (D) in tubules at 14 DPI. (g) Decrease in the number of Sertoli cells in seminiferous tubules at 21 DPI, arrows indicate margins of disintegrated tubules. (h) Testicular atrophy at 100 DPI, the normal tubule is indicated (asterisk). (i) Normal testicular morphology in mock-infected mice. Sections are stained with haematoxylin and eosin. Scale bars = 100 μm for (a, c, e, f, h, i); 40 μm for (b, g); 20 μm for (d).

Figure 6

Quantitative analysis of testicular morphology during HSV infection. Vertical axis indicates the percentage of normal seminiferous tubules in the testes of infected mice; horizontal axis indicates days postinfection (DPI). The number of normal tubules (without degenerative changes) was counted in three histological sections of the testis, and the percentage of normal tubules relative to the total number of tubules examined was calculated. Data are mean ± SEM; n = 3 testes per time point. *P < 0.05 in relation to the previous time point.

Figure 7

Representative images of immunofluorescent staining with the antibody against Sertoli cell marker Wt1 in sections of control mouse testes (a, b) and infected testes at 14 DPI (c, d). Nuclei are stained with DAPI. It is seen that only about half of the seminiferous tubules contain Sertoli cells at 14 DPI. Scale bars = 200 μm.

Figure 8

Leukocytosis in the pathogenesis of HSV-induced orchitis. (a) Leucocyte infiltration of HSV-infected testis at 6 DPI, light microscopy. (b) Leucocyte infiltration of testicular interstitium at 6 DPI, EM. (c) Several sperm heads inside the macrophage from the epididymis of the infected mouse at 6 DPI, EM. 1 – basement membrane, 2 – interstitium, 3 – macrophage, 4 – spermatozoon. Scale bars = 100 μm for (a), 5 μm for (b, c).

Figure 9

Flow cytometric analysis of testicular interstitial cells at 10 and 21 DPI. (a) Representative flow cytometry histograms of CD4, CD8 and F4/80 expression in the testis of mock-infected (control) mice and HSV-infected mice at 10 and 21 DPI. The percentages of stained cells are indicated. (b) Absolute number of T lymphocytes and F4/80⁺ cells in the testes. (c) Absolute number of CD4⁺ and CD8⁺ T lymphocyte subsets in the testes.*P < 0.05 in relation to control, ^#P < 0.05 in relation to 10 DPI.