Patients
Semen samples were obtained from 138 men, including 91 infertile men (Group I) and 47 healthy donors enrolled in the sperm donor program (Group II). The informed consent was obtained from all patients.
Clinical material
Semen samples were fractionated by gradient centrifugation with SupraSperm reagent (Origio, Jyllinge, Denmark) according to the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (2010). The fraction of motile spermatozoa (MS) was washed two times in 2 ml of Dulbecco modified Eagle Medium (DMEM; Paneko, Moscow, Russia) by centrifugation and used as described below.
Virus and cell culture
HCMV AD 169 strain was provided by the Russian Federation State Collection of Viruses. The virus was propagated and titrated in human embryo lung fibroblasts (HEF).
Rapid culture method (RCM)
RCM was used for detection of HCMV infectious activity in the samples. The material (0.2 ml) was injected into each well of 24-well culture plate (Costar, Washington DC, USA) with HEF confluent monolayer, incubated for 1 h at 37°C in an atmosphere of 95% air/5% CO2. The cells were washed 2 times in serum-free culture medium, incubated for 48 h in 1 ml of DMEM with 2% fetal calf serum (FCS; Gibco, Carlsbad, CA, USA), washed 2 times in PBS and fixed in cold methanol. HCMV was identified by immunoperoxidase staining with monoclonal antibody (Mab) against HCMV pp65 protein (DAKO, Glostrup, Denmark). Immunolabeled cells were calculated in an inverse light microscope LABOVEWRT FS (Leitz, Oberkochen, Germany).
HCMV DNA detection and quantification by real-time PCR
HCMV DNA extraction was performed from 200 μl of samples using QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. In brief, 200 μl of sample and 10 μl of the STI-87 positive internal control (PIC; Interlabservice, Moscow, Russia) were added to 200 μl of AL-buffer and heated at 56°C for 15 min. 200 μl of 96% ethanol was added, applied to columns and washed as per the manufacturer's instruction with the final elution in 200 μl of the kit AE-buffer preheated to 50°C. Real-time PCR was performed using the Amplisense CMV-screen/monitor-FL kit (Interlabservice) according to the manufacturer's protocol. Real-time amplification was carried out using 10 μl DNA eluate combined with 10 μl PCR-mix-1-FL and 5 μl PCR-mix-2-FL using Rotor-Gene 6000 Instrument (Corbett Research, Doncaster, Australia) with the following cycling parameters: pre-denaturation at 95°C for 15 min, 95°C for 5 s, 60°C for 20 s and 72°C for 15 s for 45 cycles. Data acquisition was performed in both JOE/Yellow (for HCMV DNA) and ROX/Orange (for the STI-87 PIC) channels during the annealing (60°C) stage. For quantification of HCMV DNA two standard positive sample KSG1 (104 copies per reaction mixture) and KGS2 (102 copies per reaction mixture) (Interlabservice) were included in the run. Calculations of Ct, preparation of standard curve and quantification of DNA in each sample were performed by Rotor-Gene Operating Software, version 1.8 (Corbett Research).
PCR in situ
Washed-off MS were transferred to glass slides, centrifuged for 5 min at 1500 rpm in Cytospin 4 (Thermo Electron, Waltham, USA), air dried, fixed in 10% formaldehyde for 4 h and washed twice in 0.05 M Tris-HCl. The preparations were then incubated with proteinase K (DAKO) for 30 min at 37°C. Amplification with biotinilated primers (Gentech, Moscow, Russia) was carried out using T1 cycler (Biometra, Goettingen, Germany). Viral DNA was detected with the biotin-streptavidin-peroxidase complex (DAKO) and diaminobenzidene (Sigma-Aldrich, St Louis, MO, USA). The proportion of sperm cells containing HCMV DNA was calculated after analysis of at least 2000 cells.
Spermiological and quantitative karyological analysis
Spermiological analysis was performed according to the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (2010). The immature germ cells (IGC) in sperm samples were identified by morphological criterions under a light microscope BX51 (Olympus, Tokyo, Japan). At least 200-300 IGC were calculated in each slide. The proportions of spermatids and primary spermatocytes at early stages (leptotene, zygotene, pachytene and diplotene) and cells that could not be identified (classified as unidentified and/or degenerated) were calculated as described previously [14].
Organotypic culture of human testis explants
The procedures followed were in accordance with ethical standards of the Helsinki Declaration and were approved by the local Ethics Committee of the D.I. Ivanovsky Institute of Virology of Ministry of Health and Social Development of Russian Federation; informed consent was obtained from all patients. Testis samples from 3 patients with prostate cancer (62, 65 and 67 years old) were transported in fresh medium on ice immediately following orchidectomy. Testicular tissues were carefully dissected with scissors into 3 mm3 fragments. In each well of a six-well plate, two fragments were placed onto a permeable membrane insert (Falcon Labware, Mt Pritchard, NSW, Australia) and incubated at the interface between air and 2 ml of DMEM with 10% FCS (Gibco), 1 mmol/l sodium piruvate, 100 ng/ml vitamin A, 50 ng/ml vitamin C and 200 ng/ml vitamin E (all from Sigma-Aldrich), 4 mmol/l glutamine, 10 μg/ml insulin, 5 μg/ml transferrin and 50 μg/ml gentamycin (all from Paneko) at 37°C in an atmosphere of 95% air/5% CO2. Culture medium was replaced every other day.
HCMV-infection of testicular explants
Fragments were incubated with 0.025 ml of HCMV inoculate for 1 h at 37°C. Multiplicity of infection (MOI) was 0.0001-0.001 plaque forming units (PFU) per cell. Control uninfected cultures were incubated in DMEM under the same conditions. Then explants were washed three times in 1 ml DMEM and the culture was established for up to 14 days as described above. Viral load was estimated every other day in culture medium by PCR and RCM starting from Day 2. Three explants were analyzed in each point.
Light microscopy
For histological analysis, testicular explants (3 mm3) were fixed in neutral buffered 10% formaldehyde for 24 h at 4°C, dehydrated in a series of graduated ethanol concentrations (70%, 96% and 100%), embedded in paraffin, sectioned at 4.0 μm and stained with Caracci's hematoxylin (BisVitrum, S.-Peterburg, Russia) for examination. The following morphological criteria were used for analysis of germ cells viability and explants architecture: cell number, cell size and location, signs of apoptosis, and histological characteristics of seminiferous tubules, including the basal membrane structure.
Immunostaining
To reveal HCMV proteins in the testis explants immunostaining with Mab to HCMV pp65 was performed on formaldehyde-fixed, paraffin-embedded tissues. Antigen retrieval was performed as follows: deparaffinized and dehydrated sections were treated for 20 min at 750 Wt in microwave oven (Sanyo, Moriguchi, Osaka, Japan) in 10 mM citrate buffer (pH = 6.0, DAKO) and then washed in 0.05 mol/l phosphate-buffer saline (PBS, pH 7.6; Gibco). Endogenous peroxidase was inactivated in deparaffinized sections by 5-min treatment in PBS with 3% H2O2. Slides were processed by PBS supplemented with 2% bovine serum albumin (BSA, Sigma-Aldrich) to block the non-specific sites, before overnight incubation at 4°C with the Mab to HCMV pp65 (0.02 ug/ml) (DAKO) diluted in PBS with 1% BSA. Next steps were performed at room temperature. Reagents from UltraVision LP Large Volume Detection System kit (Thermo Scientific, Fremont, USA) were added according to the manufacturer's protocol after washing 4 times in PBS. The following substrate was used: 0.5 mg/ml diaminobenzidine (Sigma-Aldrich) in 0.05M TRIS-HCL, pH 8.0 with 3% H2O2. Sections were prepared immediately after tissue dissection and on days 2, 4, 7 and 14 after introducing in culture. Stained cells were identified and photographed with a BX51 microscope coupled to a digital macro camera U-CMAD3 (Olympus).
Transmission electron microscopy (TEM)
TEM was performed immediately after tissue dissection and on days 2, 4, 7 and 14 of culturing. Two infected and two uninfected explants were analyzed in each point. The explants were fixed in 2.5% glutaraldehyde in 0.1 M buffered sodium cacodylate (pH 7.4) for 24 h at 4°C, postfixed in 1% OsO4 in 0.1 M sodium cacodylate buffer at room temperature for 40 min, then dehydrated in gradual ethanol concentrations (70%, 96% and 100%) and, subsequently, submitted to progressive impregnations in epon resin (Sigma-Aldrich). Polymerization was carried out at 60°C for 48 h. Ultrathin sections were cut in an ultratome III (LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate (Sigma-Aldrich) and examined in a JEM-100 S electron microscope (JEOL, Tokyo, Japan) at 80 kV.
Statistics and data analysis
The analysis was performed in StatXact 8 (Cytel, Cambridge, MA, USA) using Student's paired t-test, χ2 test and Mann-Whitney test. P < 0.05 was considered statistically significant.