Nitric oxide-targeted protein phosphorylation during human sperm capacitation

Figure 1. Effect of GSNO, l-NAME and AG on PKA substrates (PKAs-P) and tyrosine phosphorylation (Tyr-P). Sperm were incubated for 4 h under capacitating conditions in the absence of any treatments (CONTROL) or in the presence of GSNO, a NO donor, and l-NAME and AG (both NOS inhibitors). (a,b) Sperm protein extracts (n = 7) were analyzed for phosphorylation by Western blot using anti-PKAs-P or anti-Tyr-P as first antibodies, respectively. (c) β-tubulin (β-TUB) was used as a protein loading control. For signal quantification, each lane was normalized to its β-TUB optical density value. (d–g) Relative amount of signal quantified in each membrane using ImageQuant TL v8.1 software for PKAs-P and Tyr-P, respectively. Different letters (a, b, c) indicate statistically significant differences (P < 0.05) between groups. Images (a–c) were cropped from the corresponding blot showed in Supplementary Fig. S1 (lanes 3–6).

Figure 2. Effect of L-Arginine (R), GSNO, l-NAME and AG on PKA substrates (PKAs-P) and tyrosine phosphorylation (Tyr-P). Sperm were incubated for 4 h under capacitating conditions in the absence of any treatments (CONTROL) or in the presence of L-Arginine, the substrate for NO synthesis, GSNO, a NO donor, l-NAME and AG (both NOS inhibitors). (a,b) Sperm protein extracts (n = 7) were analyzed for phosphorylation by Western blot using anti-PKAs-P or anti-Tyr-P as first antibodies, respectively. (c) β-tubulin (β-TUB) was used as a protein loading control. For signal quantification, each lane was normalized to its β-TUB optical density value. (d–g) Relative amount of signal quantified in each membrane using ImageQuant TL v8.1 software for PKAs-P and Tyr-P, respectively. SM: Sperm Medium. Different letters (a, b, c) indicate statistically significant differences (P < 0.05) between groups. Images (a–c) were cropped from the corresponding blot showed in Supplementary Fig. S2 (lanes 3–7).

Figure 3. Effect of follicular fluid (FF), GSNO, l-NAME, and AG on PKA substrates (PKAs-P) and tyrosine phosphorylation (Tyr-P). Sperm were incubated for 4 h under capacitating conditions in the absence of any treatments (CONTROL) or in the presence of follicular fluid, GSNO, a NO donor, l-NAME and AG (both NOS inhibitors). (a,b) Sperm protein extracts (n = 7) were analyzed for phosphorylation by Western blot using anti-PKAs-P or anti-Tyr-P as first antibodies, respectively. (c) β-tubulin (β-TUB) was used as a protein loading control. For signal quantification, each lane was normalized to its β-TUB optical density value. (d–g) Relative amount of signal quantified in each membrane using ImageQuant TL v8.1 software for PKAs-P and Tyr-P, respectively. SM: Sperm Medium. Different letters (a, b, c) indicate statistically significant differences (P < 0.05) between groups. Images (a–c) were cropped from the corresponding blot showed in Supplementary Fig. S3 (lanes 3–7).

Figure 4. Effect of L-Arginine (R), follicular fluid (FF), GSNO, l-NAME, and AG on PKA substrates (PKAs-P) and tyrosine phosphorylation (Tyr-P). Sperm were incubated for 4 h under capacitating conditions in the absence of any treatments (CONTROL) or in the presence of L-Arginine, the substrate for NO synthesis, follicular fluid, GSNO, a NO donor, l-NAME and AG (both NOS inhibitors). (a,b) Sperm protein extracts (n = 7) were analyzed for phosphorylation by Western blot using anti-PKAs-P or anti-Tyr-P as first antibodies, respectively. (c) β-tubulin (β-TUB) was used as a protein loading control. For signal quantification, each lane was normalized to its β-TUB optical density value. (d–g) Relative amount of signal quantified in each membrane using ImageQuant TL v8.1 software for PKAs-P and Tyr-P, respectively. SM: Sperm Medium. Different letters (a, b, c) indicate statistically significant differences (P < 0.05) between groups. Images (a–c) were cropped from the corresponding blot showed in Supplementary Fig. S4 (lanes 3–8).

Figure 5. Venn diagram illustrating different functional groups for the proteins identified via HPLC-ESI-Q-TOF–MS/MS. The figure was created using the web-based tool InteractiVenn88.

Figure 6. Protein–protein interactions network produced by NetworkAnalyst 3.0. Significant factors (seeds) are highlighted with a blue circle. The size of the nodes is based on their degree (connectivity) values, with a big size for large degree values. The color of the nodes is proportional to their betweenness centrality values, starting from red for higher values.

Figure 7. Experimental design. Human spermatozoa were capacitated for 4 h in the presence/absence of a NO donor (GSNO) and two NOS inhibitors (l-NAME and AG). The experimental groups were supplemented or not with L-Arginine and/or follicular fluid (FF). Sperm proteins were subjected to electrophoresis followed by Western Blot to analyze the phosphorylation levels of phospho-PKA substrates (PKAs-P) and tyrosine residues (Tyr-P). The amount of signal in each membrane was determined by chemiluminescence and, subsequently, quantified. Specific protein bands, that showed significant differences amongst the treatments mentioned above, were subjected to in-gel trypsin digestion, followed by mass spectrometry analysis (HPLC-ESI-Q-TOF–MS/MS). Testis-specific protein–protein interaction and fertility-related knockout phenotypes were also analyzed.