Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

Ethics Statement

The study was approved by the Institutional Ethics Committee of the Hospital of Padova and written informed consent was provided by participants.

Semen sample collection and preparation

Human semen samples from volunteers were obtained by masturbation after 2–5 days of sexual abstinence in sterile containers. Samples were allowed to liquefy for 30 minutes at room temperature and semen volume, pH, sperm concentration, viability, motility, and morphology were determined following World Health Organization guidelines for semen analysis [35]. Sperm were separated by washing semen three times with Modified HTF medium with human serum albumin 5 mg/ml (Sperm washing medium, Irvine Scientific, Santa Ana, CA) and centrifuged at 300 g for 10 minutes. The pellet was used to collect the spermatozoa after swim-up technique (500 µl of sperm washing medium was layered gently up to the pellet, the tube was slanted to 45° and incubated for 1 h at 37°C in a 5% CO₂ incubator), the collected spermatozoa were used for subsequent analyses. Subjects have been described before [13], and sperm were investigated for HPV identification and localization (HPV DNA, HPV16-L1 capsid), by fluorescence in situ hybridization, immunofluorescence and PCR as described below.

Fluorescence in Situ Hybridization (FISH) for HPV in human sperm

Sperm samples containing at least 2×10⁶ sperm were fixed in a methanol-acetic acid solution for at least 1 hour at −20°C. To permeabilize sperm membrane, samples were treated with pepsin (1∶25,000, 0.01 mol/L) in prewarmed HCl 1 mol. for 10 minutes at 37°C. Permeabilization of the specimens was stopped with 4 min. washes in PBS 1×. Samples were then dehydrated in 70%, 80%, and absolute ethanol for 2 minutes and finally air-dried. Samples were then overlaid with 20 μL of hybridization solution containing biotin (BIO)-labeled HPV DNA probe (a mix of total genomes 7–8 Kb, containing the conserved HPV region) (Rembrandt in situ hybridization and detection kit HPV, Pan Path, Amsterdam, Netherlands). Each slide was covered with a glass coverslip and the edges were sealed with nail polish to prevent loss of the mixture during denaturation and hybridization. After denaturation of cellular target DNA and HPV DNA probe on a heating block for 5 min. at 95°C, hybridization was performed by incubating the samples at 37°C overnight in a humidified chamber. Thereafter, the coverslips were carefully removed and the slides were washed in PBS 1× for 10 minutes. After 15 minutes of incubation at 37°C with a wash solution to eliminate non-specific bound probe for use in situ hybridization (ISH) (R013R.0000 Pan Path, Amsterdam, Netherlands), the slides were washed three times in PBS 1× for 3 minutes. The negative control was processed in the same way, but omitting the viral probe. The biotin-labeled HPV probe was detected by incubation with 1∶200 streptavidin Texas red (Vector Laboratories, Burlingame, CA) for 40 minutes at room temperature. After detection, the slides were washed twice in PBS 1×/0.01% Triton and then twice in PBS 1× and mounted with a solution containing 4,6-diamino-2-phenylindole (DAPI 5 mg/mL) and antifade (BioBlue; BioView, Nes Ziona, Israel). Samples were analyzed using a fluorescence microscope (Nikon, Eclipse E600, Melville, NY, USA) equipped with a triple band-pass filter set (fluorescein isothiocyanate [FITC], tetrarhodamine isothiocyanate [TRITC], DAPI). For each slide 200 spermatozoa were analyzed. When nuclei were completely and homogeneously stained and when multiple small spots or single large signals were present, the sperm cells were classified as positive. The method was tested on control slides containing CaSki cells (R013R.0000 Pan Path, Amsterdam, Netherlands), human cervical carcinoma cell line with stably integrated and transcriptionally active HPV genomes that served as control for the specific probe.

Immunofluorescence for HPV16-L1 capsid protein in human sperm

Sperm were resuspended at a concentration of 20×10⁶ sperm/mL in sperm washing medium and incubated with 20 µg/ml HPV16-L1 (Gardasil, Sanofi Pasteur MSD, Lyon, France) for 1 h at 37°C. The reaction volume was 300 µL. After incubation, motile spermatozoa were harvested by the swim-up technique as described above. The swim-up sperm fraction was divided in two samples used for later HPV16-L1 capsid detection after or not induction of acrosome reaction by calcium Ionophore III (Sigma-Aldrich) at 10 µM. Sperm without incubation with HPV16-L1 were used as negative control.

For immunofluorescence, 10 µl of sperm sample was smeared on clean, grease-free slides, air-dried, and fixed in PBS/paraformaldehyde 4% for 15 minutes. The glasses were washed three times in PBS for 5 minutes at room temperature and then used for the HPV capsid (HPV16-L1) and acrosome (pisum sativum) detection.

For HPV16-L1 detection the sperm were incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1) (0.8 µg/mL, 1∶250, Santacruz, Santa Cruz, CA) for 120 minutes at room temperature, and then washed in 0.2% tween-PBS for 5 minutes. Immunoreaction was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody (1∶200, Vector Laboratories, Burlingame, CA) and streptavidin Texas red (1∶400, Vector Laboratories, Burlingame, CA) both for 60 minutes at room temperature. After that the slides were incubated with the acrosome specific Pisum Sativum (1 µg/ml) for 30 minutes at room temperature and then washed twice in PBS. Nuclei were counterstained with DAPI 5 mg/mL, slides were mounted with anti-fade buffer and 24×24 mm coverslip. Immunostaining was evaluated with Nikon ViCo Video Confocal Microscope.

HPV DNA (L1 gene) detection in human sperm

DNA extraction from swim-upped sperm was performed by QIAamp DNA mini kit (Qiagen, Valencia, CA). The presence of HPV DNA sequence (L1 gene) was investigated by nested polymerase chain reaction (PCR) using MY09/MY11 as outer primers and GP5þ/GP6þ as inner primers. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 2% agarose gel. PCR amplification was carried also with a positive control (sperm transfected with recombinant plasmid pIRES2-AcGFP1-E6E7, see below) and a negative control (no template).

Flow cytometry and immunofluorescence for CD138 (Syndecan-1) and HPV16-L1 capsid protein on human sperm

To evaluate the interaction between syndecan-1 and HPV16 capsid, fresh spermatozoa were washed twice in sperm washing medium and centrifuged (300 g for 10 minutes). The pellet was resuspended in PBS and divided into two aliquots with 40×10⁶ sperm cells with and without incubation with 3.5 U/ml Heparinase-III (Sigma Aldrich, St. Louis, MO) for 60 minutes at 37°C. Sperm were incubated with HPV16-L1 protein (20×10⁶ sperm/mL were resuspended in sperm washing medium and incubated with 20 µg/ml HPV16-L1 protein for 1 h at 37°C).

To detect CD138 (Syndecan-1) and HPV16-L1 in cytofluorimetry sperm were incubated with mouse anti-human CD138 monoclonal antibody, FITC conjugated, clone 1D4 (1∶5, Biolegend, San Diego, CA) or with mouse monoclonal antibody HPV16-L1 (CAMVIR-1) (1 µg/mL) (1∶200, Santacruz, Santa Cruz, CA) both on ice for 45 minutes in the dark. Sperm were washed twice in phosphate-buffered saline (PBS) and centrifuged at 300 g for 5 minutes. For HPV16-L1, the second layer phycoerythrin anti-mouse antibody (PE, 10 µM, Becton Dickinson, Franklin Lakes, NJ) was added to the cells and incubated for 45 minutes on ice. A multiple myeloma cell line U266 (ATCC, Manassas, VA, USA) were used as positive controls for syndecan-1, and the samples incubated with secondary antibody alone were used as negative controls for HPV16-L1. Samples were washed twice in PBS and centrifuged at 300 g for 5 minutes. The pellet was resuspended in 300 µl of PBS and then analyzed on a FACScalibur (Becton Dickinson, Oxford, UK) using Cellquest to acquire and analyze the data.

To detect CD138 (Syndecan-1) and HPV16-L1 colocalization in immunofluorescence, 10 µl of sperm samples were smeared on clean, grease-free slides, air-dried, and fixed in PBS/paraformaldehyde 4% for 15 minutes. The glasses were washed three times in PBS for 5 minutes, incubated with mouse anti-human CD138 monoclonal antibody, FITC conjugated, clone 1D4 (1∶10, Biolegend, San Diego, CA) overnight at 4°C, washed twice in PBS and incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1) (1∶250, 0.8 µg/mL, Santacruz, Santa Cruz, CA) for 120 minutes at room temperature. The slides were washed twice in 0.2% tween-PBS for 5 minutes. Immunoreaction for the HPV16-L1 was detected by sequential incubation with biotinylated goat anti-mouse immunoglobulin secondary antibody (1∶200 from Vector Laboratories, Burlingame, CA) and streptavidin Texas red (1∶400, Vector Laboratories, Burlingame, CA) both for 60 minutes at room temperature. The slides were washed twice in PBS, and nuclei were counterstained with (DAPI 5 mg/mL). Slides were mounted with anti-fade buffer and 24×24 mm coverslip. Immunostaining was evaluated with Nikon ViCo Video Confocal Microscope. Sperm without incubation with CD138 (Syndecan-1) and HPV16-L1 were used as negative control.

Construction of recombinant plasmid pIRES2-AcGFP1-E6E7

The expression vector for HPV E6/E7 genes and GFP was constructed as Figure 3a. The E6/E7 genes were amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 (Clontech, Mountain View, CA) by PCR and subcloned into plasmid pIRES2-AcGFP1 (Clontech, Mountain View, CA) to construct recombinant plasmid pIRES2-AcGFP1-E6E7. The PCR mixture consisted of 5 µL Expand high fidelity buffer with MgCl₂, 1 µL of PCR grade nucleotide mix (10×), 20 pmol of each primer with SalI and BamHI restriction sites (New England Biolabs, Ipswich, MA) including forward: 5′-GTGCGCGTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′-GTGCGCGGATCCGTGTGGTTTCTGAGAACAGATG-3′, 0,75 µL Expand high fidelity enzyme mix (Expand High Fidelity PCR System dNTPack, Roche Applied Science, Mannheim, Germany) and sterile H₂O in a final volume of 50 μL. The amplification program was as follows: denaturing at 94°C for 2 minutes, 10 cycles each at 94°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute, 20 cycles each at 94°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute plus 5 seconds cycle elongation for each successive cycle, followed by a final extension at 72°C for 7 minutes. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1% agarose gel and then purified using Microcon YM-100 (Millipore Corporation, Bedford, MA). Then the purified amplification products and plasmid pIRES2-AcGFP1 were digested by restriction enzyme SalI and BamHI (10 U at 37°C for 120 minutes). These digested PCR products were purified from a 0.8% agarose gel using the S.N.A.P. Gel Purification Kit (Invitrogen, Paisley, UK), according to the manufacturer's protocol. The purified PCR products and plasmid pIRES2-AcGFP1 were analyzed on a second 0.8% agarose gel to confirm recovery. Finally, these purified PCR products were recombined into plasmid pIRES2-AcGFP1 using T4 DNA ligase kit (New England Biolabs, Ipswich, MA) and subjected transformation into E. coli DH5α (Invitrogen, Paisley, UK) according to manufacturer instruction. The successful construction was confirmed by PCR, BamHI, and Sal I digestion and DNA sequencing.

Transfection of human sperm with plasmid pIRES2-AcGFP1-E6E7

Motile sperm were harvested by the swim-up technique as described above. The swim-up sperm fraction was divided in four samples: one used for the transfection with plasmid pIRES2-AcGFP1-E6E7, the second one used for the transfection with 2 µL Lipofectamine 2000 alone (Invitrogen, Paisley, UK), the third one used for the transfection with plasmid pIRES2-AcGFP1 and the last one as negative control.

Sperm were incubated in BWW supplemented with HEPES (10 mM, pH 7.4) and 3.5 mg/ml HSA (Human Serum Albumin) for capacitation at 37°C and 5% CO₂ for 4 hours. Three hours after the beginning of capacitation, sperm were exposed to plasmid pIRES2-AcGFP1-E6E7. Briefly, a total of 100 µL mixture containing 800 ng plasmid pIRES2-AcGFP1-E6E7 and 2 μL Lipofectamine 2000 (Invitrogen, Paisley, UK) in BWW was incubated at room temperature for 20 minutes, and then was added to sperm sample and kept in the incubator for another 1 hour. After that, the sperm sample was washed at least five times in 5 mL of fresh BWW.

The successful of human sperm transfection with plasmid pIRES2-AcGFP1-E6E7 was confirmed by PCR. The DNA was extracted from sperm by QIAamp DNA Mini Kit (Qiagen, Valencia, CA), according to the manufacturer's protocol. Plasmid pIRES2-AcGFP1-E6E7 and sterile ddH₂O were used as the positive and negative controls, respectively. The PCR mixture consisted of 5 µL 10× TaqGold buffer, 3 µL of MgCl₂ 25 mM, 5 µL of each deoxynucleosidetriphosphate 200 µmol/L, 20 pmol of each primer (forward CMV: 5′-CGCAAATGGGCGGTAGGCGTG-3′ and reverse IRES: 5′-CCTCACATTGCCAAAAGACG-3′), 2.5 U TaqGold polymerase and sterile H₂O in a final volume of 50 µL. The amplification program was as follows: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, followed by a final extension at 72°C for 10 minutes. The amplification products were analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1% agarose gel.

Hamster Egg Penetration Test (HEPT) with human sperm transfected with HPV E6/E7 plasmid

The HEPT was performed according to the World Health Organization [35] criteria with the use of commercially available frozen hamster eggs (Charles Rivers Laboratories, Wilmington, MA, USA) with human sperm transfected or not with plasmid pIRES2-AcGFP1-E6E7. After thawing, 120 eggs were washed three times in BWW and transferred to 0.1% trypsin to remove the zona pellucida. The zona-free hamster eggs were washed three times in BWW. Then, 15 eggs were placed in 100 µL containing 10⁶ motile spermatozoa under mineral oil. After 3 hours incubation at 37°C in a 5% CO₂ atmosphere oocytes were recovered from the droplets and washed to loose adherent spermatozoa. To evaluate the successful sperm penetration, 40 oocytes (10 for each condition) were analyzed with the nuclear acid staining SYBRGreen (Invitrogen, Paisley, UK) under phase contrast microscopy and under the fluorescence microscope at 400× magnifications (Leica DMLB, Wetzlar, Germany). Sperm egg penetration test was expressed as mean number of sperm penetrated for oocyte (HSA). Twenty-four hours after insemination, the remaining oocytes were investigated under the fluorescence microscope (Leica DMLB, Wetzlar, Germany) at 400× magnifications. The oocytes were collected and classified into two groups: oocytes with green fluorescence and oocytes without green fluorescence. The oocytes from two groups were washed three times in cold 1× PBS to remove serum from the medium and then each oocyte was transferred singly into a PCR tube for PCR and RT-PCR.

Single-Oocyte PCR for E6E7 genes

5 μL of cell lysis buffer were added to each oocyte, mixed then incubated at 65°C for 10 minutes. The cell lysate of oocyte was used as a DNA template. Plasmid pIRES2-AcGFP1-E6E7 and sterile ddH₂O as the positive and negative controls, respectively. The first round multiplex PCR was performed with mixture consisted of 5 µL 10× TaqGold buffer, 3 µL of MgCl₂ 25 mM, 5 µL of each deoxynucleosidetriphosphate 200 µmol/L, 20 pmol of each primer (forward CMV: 5′-CGCAAATGGGCGGTAGGCGTG-3′ and reverse IRES: 5′-CCTCACATTGCCAAAAGACG-3′), 2.5 U TaqGold polymerase and sterile H₂O in a final volume of 50 μL. The amplification program was as follows: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 2 minutes, followed by a final extension at 72°C for 10 minutes. This first round multiplex PCR is followed by a nested PCR reaction with one of the same outer primer (forward CMV) combined with a unique inner primer (reverse: 5′-GCATAAATCCCGAAAAGCAA-3′). The amplification products were routinely analyzed by staining with SYBR safe DNA gel stain, after electrophoresis on 1,5% agarose gel.

Single-Oocyte RT-PCR for E6/E7 expression

RNA extraction from each oocyte followed by RT-PCR was performed using the cells-to-cDNA II kit (Ambion, Austin, Texas). 10 µL of cold cell lysis buffer II were added to each oocyte, and incubated at 75°C for 10 minutes. 0.12 U DNase I was added to the mixture at 37°C for 30 minutes. Reverse transcription was preformed according to the kit protocol. PCR amplification was carried out with 10 µL cDNA of each sample from reverse transcription reaction as a template, E6/E7 specific primer pair (forward: 5′-GTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′- GCATAAATCCCGAAAAGCAA-3′) and two negative controls (no template and no reverse transcription). The amplification step was carried out using these conditions: denaturing at 94°C for 10 minutes, 37 cycles each at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. About 20 μL of each PCR product were made visible by staining with SYBR safe after electrophoresis on 1.5% agarose gel. This experiment was repeated three times under the same condition.

Immunofluorescence of oocytes after penetration of human sperm incubated with HPV16-L1

The sperm were incubated or not with HPV16-L1 20 µg/ml and then were selected by swim-up for 45 minutes at 37°C, capacitated for 3 hours, and washed in BWW. Both samples were resuspended in fresh medium and the concentration of motile sperm was adjusted to 5 mil/mL. Meanwhile, the oocytes were prepared as described before. Fifteen eggs were placed in 100 µL containing 10⁶ motile sperm under mineral oil. After 3 hours of incubation at 37°C in a 5% CO₂ atmosphere, oocytes were recovered from the droplets, washed free of loosely adherent spermatozoa. To perform immunofluorescence for HPV16-L1 in penetrated oocytes, samples were washed three times in BWW, allowed to air dry, fixed in PBS/paraformaldehyde 4% for 15 min and permeabilized with 3% PBS/Triton ×100 for 5 min (Sigma Aldrich, St. Louis, MO) at room temperature. All oocyte were then incubated with mouse monoclonal antibody HPV 16-L1 (CAMVIR-1) (0.8 µg/mL) (1∶250, Santacruz, Santa Cruz, CA) for 120 minutes at room temperature. Immunoreaction was then detected by sequential incubation with FITC-anti-IgG mouse secondary antibody (1∶200 from Bio-Rad, Berkeley, CA) for 60 minutes at room temperature. The slides were washed twice in PBS, and nuclei were counterstained with DAPI (5 mg/mL). Slides were mounted with anti-fade buffer and 24×24 mm coverslip. Finally, the oocytes were placed on to the slides and the immunostaining was evaluated with Nikon ViCo Video Confocal Microscope.