Ethics Approval of Human Subjects and Animal Experiments

All protocols used in this study involving animals and human subjects were approved by the Ethics Review Committee of Shantou University Medical College according to the guidelines recommended by National Institute of Health involving human subjects and animal care and 1975 Declaration of Helsinki. Informed consent was obtained from all subjects. Human sperm samples of anonymous donors (22–45 years old healthy male, average 27 years old) and mature hamster egg-sperm penetration assay were performed according to established protocols25,34.

Preparations of human sperm specimens

Sperm samples (n = 105), which were obtained from undisclosed healthy donators by masturbation after 3 days of abstinence, were kept in a 5% CO 2 incubator at 37 °C for 30 min to allow liquefaction. The normal semen parameters of sperms were evaluated according to the WHO guidelines35. In order to prevent any contamination by other cells, such as premature sperm cells, epithelial cells, sertoli cells or leukocytes, the liquified semen samples were fractionated by the swim-up method as described following: Liquefied (0.2 ml in culture tube) semen sample were lay gently under 2 ml of Bigger-Whittem-Whittingham (BWW) medium containing 0.3% bovine serum albumin (BSA) and then incubated for 1 h at 37 °C in a CO 2 incubator. The supernatant in each tube was collected and pooled followed by centrifugation at 300 × g for 5 min at room temperature. The pellet of motile sperm was washed once and resuspended in BWW medium with 0.3% BSA to a final concentration of 1 × 106/ml for the subsequent assays.

Immunofluorescence stain

The prepared motile sperms were incubated at 37 °C in a CO 2 (5%) incubator for 4 h in a 100 μl drop of capacitated liquid, containing 1 μl of primary antibody such as rabbit anti-human IgG γ chain polyclonal antibody (1:100, Dako, Denmark), rabbit anti-human IgG λchain monoclonal antibody (1:100, ZSGB-BIO, China) or mouse anti-human IgG κ chain polyclonal antibody (1:100, Dako, Denmark) (See detail in Supplementary table S1). These sperm samples were fixed on slides with 4% paraformaldehyde (PFA) for 15 min at room temperature followed by washing with phosphate buffer solution (PBS) and then permeabilized with 0.1% Triton X-100 for 2 min. The Alexa Fluor 594-conjugated (Jackson, Immuno. Research Lab, USA) or Alexa Fluor 488-conjugated (Invitrogen, USA) secondary antibody (Supplementary table S1) was incubated with the sperms for 1h at RT. Nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Co. LLC, USA) and slides were mounted with Vectashield (Vector Laboratories, USA). Sperm samples incubated with PBS without the primary antibodies were used as negative controls. Protein localization was examined at a magnification of ×400 with a fluorescence microscope (Carl Zeiss Inc., USA).

Immuno-electron microscopy

Human sperm samples were fixed in 2% PFA with 0.2% glutaraldehyde in 0.1M PBS (pH 7.4) at 4 °C overnight. After dehydration in graded ethanol on ice, the samples were penetrated in LR White resin (Ted Pella Inc., USA). Then the samples were polymerized for 5–6 days under ultraviolet light at 4 °C. After that, a glass knife was used to make semi-thin sections (1–2 μm) under a dissection microscope (Motic, China) and the sections were stained with toluidine blue to locate areas of interest. Finally, ultra-thin (70–90 nm) sections, cut with a diamond knife, were collected on 200-mesh nickel grids. To stain the sections, the samples were first blocked in 5% BSA in phosphate buffer solution-tween (PBST) (0.1% Tween 20 in 0.1M PBS) for 1 h, followed by incubation with rabbit anti-human IgG γ chain specific polyclonal antibody (1:100, Dako, Denmark) at 4 °C overnight. After washing, the sections were blocked in PBST for 1 h and incubated with a secondary antibody for 1 h at room temperature. Then the sections were washed with PBS and distilled water. After the sections were air-dried and stained with 5% uranyl acetate, they were rinsed again in distilled water. Finally, the sections were viewed with a JEOL JEM-1400 transmission electron microscope (TEM) operating at 80 kV.

Western blot

Cell lysates were prepared from motile sperms using RIPA buffer according to the manufacturer’s instructions (Beyotime, China). Fifty-six micrograms of denatured human sperm total proteins were separated on a 10% SDS-PAGE gel with a Criterion® system (Bio-Rad, USA). Standard human IgG (0.2 μg, 0.5 μg and 0.1 μg respectively, Sigma-Aldrich Co. LLC, USA) was used as a positive control. The separated proteins were then transferred onto a nitrocellulose membrane. After blocked with 5% BSA, the nitrocellulose membrane were incubated with rabbit anti-human IgG γ chain specific polyclonal antibody (1:2500, Dako, Denmark), rabbit anti-human IgG λ chain specific monoclonal antibody (1:250, ZSGB, China) and mouse anti-human IgG κ chain specific polyclonal antibody (1:1000, Doka, Denmark). The appropriate fluorescein-labeled secondary antibodies were used. Finally, the membranes were analyzed in a two-color LI-COR Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer’s protocol.

RT-PCR assay

Total RNA from motile sperm and Raji cells ((human Burkitt’s lymphoma cell line, positive control, purchased from ATCC (June 12, 2008)) were extracted using a GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma-Aldrich Co. LLC, USA). Reverse transcription (RT) PCR of 5 μg total RNA was performed with the SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen, USA) according to the manufacturer’s instructions. The RT-PCR reaction was performed at 50 °C for 50 min.

PCR primers used for amplification of cDNA of the constant region of the IGHG1 including those of IgG λ chain and IgG κ chain were as previously described1,4 and were listed in Supplementary table S2. The sense and antisense primers spanned across introns were applied to facilitate the discrimination between transcripts and genomic DNA. The PCR products were analyzed by separation on 2% agarose gel in electrophoresis.

To analyze IgG V-D-J rearranged gene (RAG1, RAG2) expression, a nested RT-PCR assay was used (Supplementary table S2). Conserved active site of cytidine deaminase was selected as the target with primers as described previously (Supplementary table S2)1. For cDNA amplification of RAG1 and RAG2, DNase I (Amplification Grade) (Invitrogen, USA) was used to pretreat RNA samples to exclude any contamination by genomic DNA. The negative control was amplified with DNAase treated RNA as the template. Raji cells were used as the positive controls. Nested RT-PCR was also performed as described previously36.

The identification of PCR products was confirmed with DNA sequencing. The PCR primers used for amplification and the expected lengths of the resulting PCR products are presented in Supplementary table S2.

In Situ Hybridization

The cRNA probe against IGHG1 was prepared as described previously1. And the probe sequences were validated with DNA sequencing. Briefly, deparaffinized and dehydrated human spleen tissue sections (4 μm) were incubated in 0.1 M HCl for 10 min and heated to 95 °C in a microwave oven in 0.01 M citrate buffer (pH 6.0) for 20 min. The slides were cooled to room temperature, washed with PBS, fixed in 4% PFA for 10 min and hybridized overnight at 55 °C with human IGHG1 cRNA probe (sequence: acggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtg tggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc). After hybridization, sections were washed in 2 × SSC plus 50% formamide for 15 min and 2 × SSC twice for 15 min (50 °C) each. The samples were incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (AP) (dilution 1:500; Roche Diagnostics, Switzerland). 5-Bromo-4-chloro-3-in-dolyl phosphate (BCIP) and nitro-blue-tetrazolium (NBT) (Promega, USA) were used for the color reaction. For controls, slides were incubated with the corresponding sense probes. Finally, all sections were counterstained with methyl green.

Indirect ELISA to measure the concentration of asymmetric IgG in sperm

It was reported that a certain percentage of IgG in serum and placenta is glycosylated at one of its Fab arms to make the molecule asymmetric20. Such asymmetric antibodies are thought to protect instead of attack the molecules they bind to37. To detect such IgG, we added 50 μl of 2.6 μg/μl sperm total protein in 1200 μl ConA solution buffer. Then put 1 ml of the dilution into concanavalin A affinity column. Aberrant glycosylated IgG would specifically bind to concanavalin A. We collected unbound solution containing symmetric IgG in 15 ml centrifuge tubes. Then we used 1 ml solution buffer to wash the column and collected the solution in the 15 ml centrifuge tube again. Human IgG ELISA kit (Immunology Consultants Laboratory, USA) was used to measure the concentration of total IgG and that of symmetric IgG following the manufacturer’s instructions. The percentage of asymmetric IgG was then derived.

Zona-free hamster egg sperm penetration assay

To test the ability of fertilization of human sperm, the protocols for zona-free hamster egg-sperm penetration assay were performed according to previous report25. Briefly, gamete incubations were carried out in micro-drops under paraffin oil at 37 °C in a 5% CO 2 incubator. The swim-up sperm samples prepared as described were washed twice by centrifugation (5 min at 600 × g) in a 15 ml centrifuge tube. Hamster eggs were obtained from Golden Syrian hamsters by superovulation with PMSG (Pregnant Mare Serum Gonadotropin). Cumulus cells were removed by treating eggs with 0.1% hyaluronidase (Sigma-Aldrich Co. LLC, USA) for 3 min. The eggs were then pooled and washed with BWW medium containing 0.3% BSA with a pulled, heat-polished Pasteur pipette. Zona pellucida (ZP) was removed by treating the eggs with 0.1% trypsin (Sigma-Aldrich Co. LLC, USA) for 30 s followed by washing twice. The eggs were then distributed into the treatment group and the control group. One hour later, the zona-free hamster eggs were examined for penetration by sperms under a phase-contrast microscope after the denuded eggs were compressed between a slide and a coverslip. The zona-free hamster eggs were considered to be fertilized when swollen sperm head or pronucleus was observed inside the eggs with accompanying sperm tails.

Statistical analysis

Differences in fertilization rates of sperm bound eggs in the negative control groups and the experimental groups were analyzed with one way ANOVA test with Dunnet’s post-hoc test. Values are presented as means ± SE from four independent experiments and differences were considered to be statistically significant at p < 0.05.