Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are present and active in human sperm  Osmond J D’Cruz, Ph.D., Alexei O Vassilev, Ph.D., Fatih M Uckun, M.D., Ph.D.  Fertility and Sterility  Volume 76, Issue 2, Pages 258-266 (August 2001) DOI: 10.1016/S0015-0282(01)01896-9

Figure 1 Identification of the TYK 2, STAT 1, and STAT 4 proteins in human sperm. Whole-cell lysates prepared from motile fraction of sperm and Jurkat cells, derived from acute T-cell leukemia, were fractionated by SDS/PAGE (8%) and transferred to polyvinylidene difluoride membranes and subjected to Western blot analysis. Blots were probed with highly specific polyclonal anti-JAK 1, JAK 2, JAK 3, TYK 2, STAT 1, STAT 2, STAT 3, STAT 4, STAT 5, and STAT 6 antibodies followed by immunostaining with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using the enhanced chemiluminescence system. The positions of specific proteins are indicated with arrowheads. D’Cruz. Sperm JAK/STAT proteins. Fertil Steril 2001. Fertility and Sterility 2001 76, 258-266DOI: (10.1016/S0015-0282(01)01896-9)

Figure 2 Laser scanning confocal fluorescence images of sperm TYK 2, STAT 1, and STAT 4 immunoreactivity. Two-color labeling of fixed and permeabilized sperm by indirect immunofluorescence with anti-TYK 2 (A), anti-STAT 1 (B) and anti-STAT-4 (C), and ALEXA-488-conjugated secondary antibody (green) and counterstaining of nuclear DNA with TOTO-3 (blue). With anti-TYK 2 antibodies (A), specific immunoreactivity was localized to sperm tail as well as the apical region of sperm head; reactivity of anti-STAT 1 (B) and anti-STAT 4 (C) was localized to the apical region of the sperm head (arrows). (Original magnification, ×600). Insets show anti-STAT 1 and anti-STAT 4 antibodies binding to the apical region of sperm head at higher magnification. D’Cruz. Sperm JAK/STAT proteins. Fertil Steril 2001. Fertility and Sterility 2001 76, 258-266DOI: (10.1016/S0015-0282(01)01896-9)

Figure 3 IFN-α and IFN-γ enhance tyrosine phosphorylation of STAT 1 in human sperm. Capacitated human sperm were incubated for 1 hour in medium alone (BWW-3% BSA) or with indicated concentrations of IFN-α and IFN-γ. One half of the whole sperm lysates were resolved by SDS-PAGE (8%), transferred to polyvinylidene difluoride membranes and blotted with phospho-STAT 1 antibody, and the other half was blotted with an antibody to STAT 1. Western blot was revealed using enhanced chemiluminescence. The arrowheads indicate the position of STAT 1 doublet (91 kD). The insets below the panels show the signal intensities of protein bands of the representative blot expressed as densitometry scanning units. Phosphorylation index represent the ratio between the density of the band corresponding to STAT 1 (91 kD) revealed by phospho-STAT 1 (Tyr 701) antibodies, to the density of the band correspondent to STAT 1 (91 kD) revealed by anti-STAT 1 antibodies, respectively, in the same samples. D’Cruz. Sperm JAK/STAT proteins. Fertil Steril 2001. Fertility and Sterility 2001 76, 258-266DOI: (10.1016/S0015-0282(01)01896-9)

Figure 4 IL-12 enhances tyrosine phosphorylation of STAT 4 in human sperm. Capacitated human sperm were incubated for 3 hours in medium alone (BWW-3% BSA) or with indicated concentrations of IL-12. One half of the whole sperm lysates were resolved by SDS-PAGE (8%), transferred to polyvinylidene difluoride membranes and blotted with antibody to phosphotyrosine, and the other half was blotted with an antibody to STAT 4. Western blot was revealed using enhanced chemiluminescence. The arrowhead indicates the position of STAT 4. The insets below the panels show the signal intensities of protein bands of the representative blot expressed as densitometry scanning units. Phosphorylation index represent the ratio between the densities of the band corresponding to STAT 4 revealed by anti-phosphotyrosine antibodies, to the density of the band correspondent to STAT 4 revealed by anti-STAT 4 antibodies in the same samples. D’Cruz. Sperm JAK/STAT proteins. Fertil Steril 2001. Fertility and Sterility 2001 76, 258-266DOI: (10.1016/S0015-0282(01)01896-9)

Figure 5 Ca2+ ionophore A23187 and progesterone inhibit tyrosine phosphorylation of STAT 4 in human sperm. Capacitated human sperm were incubated for 6 hours in BWW medium-3% BSA with 10 μM A23187 or 10 μM progesterone. One half of the whole sperm lysates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with antibody to phosphotyrosine, and the other half was blotted with an antibody to STAT 4. Immunoreactive bands were visualized using enhanced chemiluminescence. The arrowhead indicates the position of STAT 4. The insets below the panels show the signal intensities of protein bands expressed as densitometry scanning units. Phosphorylation index represent the ratio between the densities of the band corresponding to STAT 4 revealed by anti-phosphotyrosine antibodies, to the density of the band correspondent to STAT 4 revealed by anti-STAT 4 antibodies in the same samples. D’Cruz. Sperm JAK/STAT proteins. Fertil Steril 2001. Fertility and Sterility 2001 76, 258-266DOI: (10.1016/S0015-0282(01)01896-9)