Immunoglobulin G Expression in Human Sperm and Possible Functional Significance

Abstract

Immunoglobulin G (IgG), the major molecule of the immune system, which was traditionally thought to be produced by differentiated B-lymphocytes, had recently been found in non-immune cells including spermatozoa of rabbit testis. To study if human sperms could produce IgG that might play a role in fertilization, we employed immunofluorescent staining, Western blot, in situ hybridization, RT-PCR (reverse transcription polymerase chain reaction) and immunoelectron microscope and found that human sperms were capable of synthesizing IgG. IgG protein and mRNA were detected in the cytoplasm, mainly the neck region of the sperm and IgG immunoreactivity was found to cover the entire sperm cell. The essential enzymes necessary for IgG synthesis and class switching, RAG1 (recombination activating gene 1), RAG2 (recombination activating gene 2) and AID (activation-induced cytidine deaminase), were also detected in the sperm cells. Furthermore, we found that anti-IgG antibody could inhibit sperm from penetrating Zona-free hamster egg with statistical significance. These discoveries suggested that immunoglobulin G could be produced by human sperms and it might play a role during fertilization.

Figure 1. Immunofluorescence stains show that human sperms were positive for IgG.

(a) The primary antibody is rabbit anti-human IgG γ chain, the secondary antibody is donkey anti-rabbit IgG (H + L) labeled with Alexa Fluor® 594 and the positive stain is red in color. (b) The primary antibody is mouse anti-human IgG κ chain antibody, the secondary antibody is goat anti-mouse labeled with Alexa Fluor® 488 and the positive stain is green in color. (c) The primary antibody is rabbit anti-human IgG λ chain, and the secondary antibody is donkey anti-rabbit Alexa Fluor® 594 IgG (H + L) and the positive stain is red in color. (d) Negative control. DAPI was used to stain the nucleus of sperm (blue). The scale bar is 30 μm for all photos.

Figure 2. IgG could be detected in the neck and the tail of human sperm with immuno-electron microscopy.

Normal human sperm was processed and stained with rabbit anti-human IgG antibody (γ chain specific) and the secondary antibody to rabbit IgG was labeled with 10 nm colloidal gold. The positive stain was shown by highly electron-dense gold particles. (a) The neck region of sperm contains abundant gold particles (white arrow) showing the subcellular distribution of IgG. Nu is nucleus and Ce is centriole. (b) Lower magnification of (a). (c) The middle piece of a sperm tail. The gold particles (white arrow) depict IgG distribution. Mi is mitochondria and Ax is axoneme. (d) Lower magnification of (c). The results demonstrate that both the neck region and the middle piece of the tail of human sperm contain synthesis IgG γ chain molecule.

Figure 3. Western blot assay indicated the expression of IgG. RT-PCR amplification detected the expression of IgG mRNA in sperm.

(a) Results of Western blot. The first lane is total protein extracted from human sperm (56 μg), the second lane to the forth lane is 0.2 μg /0.5 μg /2 μg human standard IgG molecule respectively (positive control). Anti-human IgG γ chain antibody, anti-IgG λ chain antibody and anti-IgG κ chain antibody were used as primary antibodies respectively. Molecular weight of protein was labeled on the right of each panel. (b) Expression of RAG-1 (327 bp) and RAG-2 (193 bp) genes were detected in human spermatozoa. Lane 1 is PCR product of RAG1 and RAG2 gene extracted from Raji cells (positive control). Lane 2 is DNase treated cDNA of Raji cells was used as a template (negative control). Lane 3 is cDNA of normal human sperm extract was used as a template. RAG1 and RAG2 genes were amplified. Lane 4 is DNase treated template of normal human sperm. No RAG1 or RAG2 gene was detected. (c) RT-PCR amplification of mRNA transcripts of IgG λ chain, AID, IGHG1, CD19 and GAPDH in human sperm. There was no contamination from B cells in human sperm since CD19 was negative. Human sperm could express transcripts of IgG λ chain, AID, IGHG1 and GAPDH. M is DNA ladder, W is water as a negative control, S represents human sperm, Raji cell was used as the positive control. CD19 is a marker for Raji cell. The results show that human sperm can express the essential genes for immunoglobulin G V-D-J rearrangement. In addition, human sperm can synthesis IgG in both the protein level and the mRNA levels.

Figure 4

(a) In situ hybridization assay detected the expression and location of IgG mRNA. (A,B) were human sperms incubated with anti-sense probe and sense probe against IgG γ chain respectively. The positive stain (purple) in (A) demonstrated mRNA expression of IgG which was mainly located in the neck of human sperm. There was no positive stain with sense probe as shown in (B). (C,D) were human normal spleen tissue served as additional controls. (C) was incubated with anti-sense probe against IgG γ chain (positive control) and (D) was incubated with sense probe against IgG γ chain (negative control). Bar was 20 μm in (A,B). Bar was 25 μm in (C,D). (b) Anti-IgG antibody could inhibit sperm-egg penetration. After being pre-incubated with anti-human IgG antibody (γ chain specific) for 4 h at 37 °C, zona-free hamster egg-sperm penetration assay were performed and the penetration ratio was calculated. The sperms without anti-IgG antibody treatment were used as a control for comparison. Data show means ± SE of four independent experiments. The mean penetration ratio in the treatment group is 27%, while the mean ratio in the control group is 15%. The difference was statistically significant between the two groups (*p < 0.05, One way ANOVA, Dunnet’s post-hoc test). n is the number of zona-free hamster eggs used in the penetration assay for each group. The result shows that IgG produced by sperm may play a role in sperm-egg fusion.