Table 1.
Primer information.
Figure 1.
Presence of UPEC inside testes of infected rats 7 days after inoculation in the vas deferens.
(A) Genomic DNA was extracted from testicular tissue and 200 ng DNA from each sample were amplified with PCR using UPEC pili primers. DNA isolated from one explanted testis immediately after direct UPEC injection served as a positive control. PCR products were separated on 1.5% agarose gel and stained with ethidium bromide. The same amount of DNA from each sample without PCR amplification was subjected to agarose gel electrophoresis and served as a loading control. Lane 1∶100 bp DNA marker; lane 2–4: samples from saline injected animals; Lane 5∼7: samples from UPEC infected rat testes; Lane 8: UPEC positive control; Lane 9: negative control. (B) Testicular homogenates from saline injected (left panel) and UPEC infected rats (right panel) were streaked on agar plates without antibiotics and kept at 37°C overnight. Colonies were counted under translucent light. (C) Cryosections of testis from control (left panel) and UPEC infected rats (right panel) were probed with anti-E. coli antibody and decorated with secondary anti-rabbit IgG antibody conjugated to Cy-3 (orange). DAPI (blue) was used for nuclear counterstain (x20 objective). (D) Semithin cross-section of a seminiferous tubule (asterisk) with adjacent interstitial space. Microbial presence in interstitial space is visible (arrow in the black frame, x20 objective). Inset: Electron microscopical examination on the same area (primary magnification x3,000).
Figure 2.
Electron microscopical analysis reveals intact blood-testis barrier (BTB) and blood-epididymis barrier (BEB).
(A) Ultrastructural analysis shows that intercellular tracer penetration does not extend beyond the junctional complex of the BTB (arrow in inset) within the seminiferous epithelium of UPEC infected rats (x3,000 magnification, inset x20,000 magnification). SC = Sertoli cells, orientation of the luminal and basal compartment are highlighted (B) Ultrastructural analysis of a UPEC infected epididymis demonstrates intercellular tracer penetration (x3,000 magnification). Inset is a magnification of the area represented in the black frame showing the tight junctions. (x20,000 magnification).
Figure 3.
Morphological changes and histological evaluation of the testis and epididymis.
(A) Testicular weight of control (n = 8) and infected rats (n = 10) are presented as mean ± standard deviation (SD). Student’s t-test was employed for statistical analysis and the level of significance is indicated as **p<0.001. (B) Sperm concentration was assessed in seven animals of each group and the results are presented as mean ± SD. Statistical analysis was performed with Student’s t-test and statistical significance is denoted as *p<0.05. (C) Tissue sections of paraffin embedded testes were stained with hematoxylin and eosin. Histopathological assessment was performed on control (n = 5) and UPEC infected (n = 9) testes using light microscopy. The images were captured using Axioplan 2 Imaging system at magnification x20 and representative figures are shown. Various forms of impairment of spermatogenesis are visible exemplified by a Sertoli cell only tubule (star) and a hypospermatogenic tubule (triangle). (D) Histopathological images of caput (D top panels) and cauda epididymis (D bottom panels, x20 objective). Representative results from control (n = 5) and infected (n = 9) rats are depicted.
Figure 4.
Increase of TUNEL positive cells in UPEC infected testis.
(A) DNA strand breakage in testicular cells from control (upper panel) and UPEC infected (lower panel) rats were analyzed using TUNEL assay. Nuclei were counterstained with DAPI (blue). TUNEL (+) cells (green) with ring-like nuclear stain are indicated with arrows. (B) Numbers of TUNEL (+) cells are presented as mean ± SD/seminiferous tubule. Student’s t-test was used for statistical analysis and the level of significance is indicated as **p<0.01. (x20 objective).
Figure 5.
RNA expression pattern of the bcl-2 family genes in the testis.
The expression of the anti-apoptotic gene bcl-2 (A), pro-apoptotic genes bax (B), bid (C), bim (D) and bak (E) in the testis were determined with quantitative real time PCR. Target gene expression levels were normalized with the endogenous control ß-2-microglobulin (ß2M). Data are present as 2ΔCt, ΔCt = Cttarget gene-Ctß2M. The Mann-Whitney U test was employed for statistical analysis (* p<0.05). Each single symbol (circle and triangle) represents one individual testis sample.
Figure 6.
Caspase-1, -3, -6 and-8 are not activated in UPEC infected testis.
Total testis protein (20 µg) from four different animals in each group were separated on 15% SDS-PAGE. Immunoblots were probed with anti-caspase-8 (A), anti-caspase-3 (B, upper panel), anti-caspase-6 (B, lower panel) and anti-caspase-1 (C) antibodies and detected using chemiluminescence. RAW 264.7 cells treated with sodium nitroprusside (SNP) served as a positive control. (D) The intensity of target bands on the films was measured with the ImageJ software (http://rsbweb.nih.gov/ij/). Semi-quantitative results are presented as mean ± SD and Student’s t-test was used for data analysis (Caspase-8, p = 0.875; Caspase-3, p = 0.686; Caspase-6, p = 0.486; Caspase-1, p = 0.343).
Figure 7.
Oligonucleosomal DNA fragmentation measurement and ultrastructural examination of UPEC infected testis.
(A) Genomic DNA was extracted from testes after seven days of infection. For gel electrophoretic analysis, 5 µg of DNA from each sample were separated on 1.5% agarose gels and stained with ethidium bromide. Lane 1∶1 kbp DNA ladder. Lane 2–4: DNA extracted from testes of control rats (n = 3). Lane 5–7: DNA samples extracted from testes of infected rats (n = 3). Lane 8: untreated RAW 264.7 cells. Lane 9: RAW 264.7 cells were treated with 0.5 mM H2O2 for 24 h as a positive control for apoptotic DNA laddering. Lane 10: RAW 264.7 cells were frozen and thawed repeatedly as a positive control for necrotic DNA fragmentation. (B) Electron microscopical examination of control rat testis shows normal morphology of the seminiferous epithelium (x1,100). (C) A representative ultrastructural image (x1,100) of infected testes demonstrates a hypospermatogenic seminiferous epithelium with germ cells displaying necrotic nuclei (arrows) and SC with strong cytoplasmic vacuolization (right panel, asterisk) and various large lipid droplets (right panel, arrowheads).
Figure 8.
HMGB1 expression and localization in the testis.
(A) For Western blot analysis, 20 µg of protein extracted from total testis of control (n = 4) and UPEC infected (n = 4) rats were separated on a 12.5% SDS-PAGE. HMGB1 was detected by immunoblot using anti-HMGB1 polyclonal antibody and chemiluminescence. ß-actin served as a loading control. (B) Intensity of target bands was measured with the ImageJ software (http://rsbweb.nih.gov/ij/) and data are presented as the relative intensity = intensity of HMGB1/intensity of ß-actin. (C) Testis cryosections were probed with anti-HMGB1 antibody decorated with Cy3-labeled secondary antibody (orange) and nuclei were counterstained with DAPI (blue, images taken with x40 objective). In control samples (left column) some Sertoli and peritubular cells are indicated by arrows and arrowheads, respectively. Representative results from two independent experiments are shown.
Figure 9.
NF-κB pathway is not activated in UPEC infected testis.
(A) Total testis proteins were separated on 10% SDS-PAGE. Immunoblots were labeled with mouse anti-IkBα antibody. ß-actin served as a loading control. (B) The intensity of target bands was measured with the ImageJ software (http://rsbweb.nih.gov/ij/) and results are presented as the relative intensity = intensity of p65/intensity of ß-actin. (C) Testis cryosections were probed with anti-p65 antibody labeled with Cy3-linked secondary antibody (orange) and the nuclei were counterstained with DAPI (blue, images taken with x40 objective). Representative results from at least two independent experiments are shown.