Localization of FGFRs in human seminiferous epithelium.

<p>Immunohistochemical analysis of FGFRs in human testis using anti FGFR antibodies; rabbit IgG was included as control. The specimens were counterstained with hematoxylin. S: Sertoli cell, Sg: spermatogonia, Sc: spermatocyte, St: spermatid. Arrows indicate immunoreactivity for FGFRs in the flagellum of elongating/elongated spermatids and the arrow head indicates FGFR4 immunoreactivity in spermatid acrosome. Bar: 20 μm.</p

Activation of FGFR-related intracellular pathways in sperm exposed to FGF2.

<p>Sperm were incubated for a total 4-h period and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. (<b>A</b>) Immunolocalization of pERK and pAkt in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Sperm were processed for immunocytochemistry, stained with anti pERK or pAkt and FITC-conjugated secondary antibodies; nuclei were stained with propidium iodide. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pERK and anti pAkt after exposure to FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control. (<b>C</b>) Phosphorylation of ERK and Akt assessed by Western immunoblotting. Protein extracts from human sperm were subjected to SDS-PAGE and Western immunoblotting using anti pERK, ERK, pAkt and Akt antibodies. The estimated molecular weights of the protein bands are indicated on the right. (<b>D</b>) Densitometric analysis of Western immunoblotting results for pERK normalized to ERK and pAkt normalized to Akt. Results are expressed as mean ± SEM, n = 5 for ERK and n = 5 for Akt. * <i>P</i> < 0.05 and ** <i>P</i> < 0.01 compared with Control.</p

Localization of FGFRs in human sperm.

<p>Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; (<b>A</b>) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, (<b>B</b>) FITC-PSA, (<b>C</b>) merge.</p

Effect of sperm incubation with FGF2 on sperm motility.

<p>Sperm were incubated with 0, 10 and 100 ng/ml FGF2 in the absence or in the presence of 0.1 μM BGJ398 and subjected to computer-assisted sperm analysis. (<b>A</b>) Percentages of progressive (Grade a + b) and total motility (Grade a + b + c) for aliquots incubated in the absence (<b>left</b>) or in the presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 5. ** <i>P</i> < 0.01 compared with Control. (<b>B</b>) Individual recordings of the effect of sperm incubation with FGF2 (0, 10 and 100 ng/ml) on the percentage of total sperm motility in samples with low and high sperm motility. Each sample is identified with a different symbol (n = 12). (<b>C</b>) The percentages of sperm with Grade a, b, c and d motility in each condition (as defined in Materials and Methods) is depicted. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with the same Grade in Control (n = 12).</p

Expression of FGFRs in human testis and sperm.

<p>(<b>A</b>) Messenger RNA expression of testicular and sperm FGFRs assessed by RT-PCR. Messenger RNA extracted from MCF7 cells served as positive controls; negative controls without reverse transcriptase (RT Control) and without template (PCR Control) are shown. The amplicon sizes are indicated on the right. (<b>B</b>) Detection of testis and sperm FGFR protein forms using Western immunoblotting. Protein extracts from human testis and sperm were subjected to SDS-PAGE and Western immunoblotting using anti FGFR antibodies or rabbit IgG as control. The estimated molecular weights of the protein bands are indicated on the right. The experiments were performed at least 3 times obtaining similar results. Typical results are shown.</p

Activation of sperm FGFRs in response to FGF2.

<p>Localization of sperm pFGFRs by immunocytochemistry. Sperm were incubated for 4 h and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. Sperm were processed for immunocytochemistry, stained with anti pFGFR and FITC-conjugated secondary antibody; nuclei were stained with propidium iodide. (<b>A</b>) pFGFR immunolocalization in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pFGFR antibody after exposure to different concentrations of FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control.</p