Mediators of the Jak/STAT signaling pathway in human spermatozoa

Abstract

In their journey to acquire the ability to fertilize the egg, numerous intracellular signaling systems are activated in spermatozoa, leading to an increase in protein tyrosine phosphorylation. Although the JAK/STAT signaling pathway is usually associated with the activation of transcription of specific genes, our laboratory previously demonstrated the presence of the IL6 receptor (IL6R) and the Janus kinase 1 (JAK1) in human spermatozoa, a cell that is mostly transcriptionally inactive. In order to determine the importance of the JAK/STAT signaling pathway, our objectives were to identify and characterize the mediators of this system in human sperm. Cell fractionation and surface biotinylation assays clearly demonstrated that IL6R is expressed at the sperm membrane surface. The kinase JAK1 is enriched in membrane fractions and is activated during human sperm capacitation as suggested by its increase in phosphotyrosine content. Many signal transducer and activator of transcription (STAT) proteins are expressed in human sperm, including STAT1, STAT3, STAT4, STAT5, and STAT6. Among them, only STAT1 and STAT5 were detected in the cytosolic fraction. All the detected STAT proteins were enriched in the cytoskeletal structures. STAT4 was present in the perinuclear theca, whereas JAK1, STAT1, and STAT5 were detected in the fibrous sheath. Indirect immunofluorescence studies showed that JAK1 and STAT1 colocalized in the neck region and that STAT4 is present at the equatorial segment and flagella. The presence of STAT proteins in sperm structural components suggests that their role is different from their well-known transcription factor activity in somatic cells, but further investigations are required to determine their role in sperm function.

FIG. 1.

IL6R in sperm subcellular fractions. Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were electrophoresed, and gels were silver stained (A) or transferred on PVDF membrane and subjected to immunoblotting using a polyclonal anti-IL6R antibody (B). Each line contains 1.5 and 3 μg of proteins in A and B, respectively. Data shown represent one of three experiments.

FIG. 2.

Surface localization of IL6R. Living sperm were incubated in the absence (Ctrl) or presence (Biot) of biotinylation reagents and, after washes, were subjected to solubilization and precipitation with Neutravidin-coated agarose-beads. Nonprecipitated proteins (sup, supernatant) and eluted proteins from Neutravidin beads (pellet) were electrophoresed and transferred onto PDVF membrane. Western blots were performed using an anti-IL6R antibody and an anti-HSPA5 antibody as an intracellular protein control. Data shown represent one of three experiments.

FIG. 3.

JAK1 in sperm subcellular fractions. A) Spermatozoa were fractionated using nitrogen cavitation and sonication and different centrifugation. B) Sperm were lysed in native IP buffer, and the soluble and insoluble fractions were separated by centrifugation. In both cases, proteins were submitted to electrophoresis and transferred onto PVDF membrane for immunoblotting with an anti-JAK1 antibody. The presence of tubulin α (TUBA) was also assessed in sperm subcellular fractions in A. Each line contains 3 μg of proteins in A and proteins extracted from 2 × 10⁶ sperm cells in B. Data shown represent one of three experiments.

FIG. 4.

Activation of JAK1 during capacitation. Human spermatozoa were incubated for 4 h under capacitation conditions in BWW medium. They were next lysed, and phosphotyrosine containing proteins were immunoprecipitated using PY20 antibody conjugated to agarose beads (IP PY20) following a first immunoprecipitation with commercial nonimmune mouse IgG as a negative control (mIgG). JAK1 was detected among immunoprecipitated proteins by Western blot using an anti-JAK1 antibody. Data shown represent one of three experiments.

FIG. 5.

STAT proteins in sperm subcellular fractions. A) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with anti-STAT1, STAT3, STAT4, STAT5, and STAT6 antibodies. Each line contains 3 μg of proteins. Data shown represent one of three experiments. B) The specificity of the STAT4 antibody was assessed on protein extracts from mouse and human testis and in human spermatozoa (spzs). PVDF membranes were incubated with anti-STAT4 antibody that has been preadsorbed or not with the antigenic peptide.

FIG. 6.

JAK/STAT mediators in sperm perinuclear theca and fibrous sheath. Proteins obtained during isolation of sperm perinuclear theca (A) and fibrous sheath (B) were submitted to electrophoresis and electrotransference on PVDF membrane for immunodetection with antibodies directed against JAK1, STAT1, STAT3, STAT4, or STAT5. The presence of IL6R was also assessed in these fractions; anti-ODF2 was used as a marker of outer dense fibers. Each line contains 2 μg of proteins. Data shown represent one of three experiments.

FIG. 7.

Localization of IL6R, IL6ST, JAK1, STAT1, and STAT4 on sperm. Fixed and permeabilized spermatozoa were submitted to indirect immunofluorescence using an anti-JAK1 or anti-STAT1 monoclonal antibody (D and E, respectively) or an anti-IL6R, anti-IL6ST, or anti-STAT4 polyclonal antibody (B, A, and C, respectively). Negative controls using total mouse or rabbit IgG (G or H, respectively) were shown. Corresponding phase images were shown for STAT1 (F) and rIgG (I). Equatorial segment is indicated by an arrow, flagella by an arrowhead, and neck region by an asterisk. Bar = 10 μm. Data shown represent one of three experiments.

FIG. 8.

Localization of IL6ST in human testis. Human testis sections were submitted to immunohistochemistry using an anti-IL6ST polyclonal antibody (A and C) or total rabbit IgG as a negative control (B and D). Positive staining is shown by a brownish precipitate. Spermatogonias (Sg), spermatocytes (Sc), and spermatids (Sd) are indicated. Bar = 50 μm (A and B) and 20 μm (C and D). Data shown represent one of three experiments.

FIG. 9.

Localization of STAT4 in human testis. Human (left panels) and mouse (right panels) testis sections were submitted to immunohistochemistry using anti-STAT4 polyclonal antibody (A–D) or preadsorbed STAT4 antibody (E and F) or total rabbit IgG (G and H) as a negative control. Positive staining is shown by a brownish precipitate. Spermatogonias (Sg), spermatocytes (Sc), and spermatids (Sd) are indicated. Bar = 50 μm (A, B, and E–H) and 20 μm (C and D).