14 research outputs found
Seminal CD38 Enhances Human Sperm Capacitation through Its Interaction with CD31
<div><p>Human sperm have to undergo a maturational process called capacitation in the female reproductive tract. Capacitation confers upon the sperm an ability to gain hypermotility and undergo acrosome reaction. Previous studies have suggested that seminal plasma proteins induce the capacitation of sperm in the female reproductive tract for the successful fertilization of the oocyte. However, the function of seminal plasma proteins in capacitation remains largely unclear. To the end, we found that soluble CD38 (sCD38) in seminal plasma increases the capacitation of sperm via specific interactions between sCD38 and the CD31 on the sperm. Upon the association of sCD38 with CD31, tyrosine kinase Src phosphorylates CD31, a process blocked by Src inhibitors. Shc, SHP-2, Grb2, and SOS, as well as Src kinase were found to associate with the phosphorylated CD31. The sCD38-induced phosphorylation of CD31 initiates a cascade reaction through the phosphorylation of Erk1/2, which results in the acrosome reaction, and sperm hypermotility. These processes were prevented by Src, Ras and MEK inhibitors. Taken together, these data indicate that the sCD38 present in seminal plasma plays a critical role in the capacitation of sperm.</p></div
Interaction of sCD38 and CD31 and sCD38-induced tyrosine phosphorylation of proteins in sperm.
<p>(A) Interaction of sCD38 and CD31 as demonstrated by PLA on sperm cross-linking with DSS. Human sperm were incubated with or without 2 μg/ml sCD38 for 1 hour at 4°C and cross-linked with 3 mM DSS prepared in DMSO for 30 minutes at room temperature, then fixed with formalin. Sperm were subjected to immunofluorescence staining with antibodies against CD38, CD31 or CatSper, and then visualized with confocal laser-scanning microscopy. Isolated human sperm by the immediate washout were incubated in BWW medium supplemented with different concentration of sCD38 (B) and were stimulated with 600 ng/ml sCD38 for the indicated times (C). Western blot analyses were performed with anti-pY antibodies or anti-actin as controls.</p
Acrosome reaction and motility of human sperm are increased by sCD38.
<p>(A) Human sperm were preincubated in either the presence or absence of sCD38 for 90 minutes. The specific inhibitors were added for the last 10 minutes of the preincubation, and A23187 was added for 30 minutes. The percentage of acrosome reacted cells were determined using FITC-conjugated PSA. *P < 0.05 versus control (PBS treatment); #P < 0.05 versus group treated with sCD38. (B) sCD38-induced sperm motility or hyperactivation was analyzed with the CASA system. Sperm were preincubated in either the presence or absence of the specific inhibitors for 10 minutes and sCD38 (600 ng/ml) was added. Sperm motility was measured using CASA. Results are mean ± S.D. from four experiments. *P < 0.05 versus control (no treatment); #P < 0.05 versus group treated with sCD38.</p
The heterophilic activation of CD31 by sCD38 transduces outside-in signal.
<p>(A) Human sperm were stimulated with sCD38 for 10 minutes. The sperm lysates were immunoprecipitated with anti-CD31 antibodies and then immunoblotted with anti-pY antibodies. (B) Isolated sperm were stimulated with 600 ng/ml sCD38 for 0 minute, 15 minutes and 30 minutes, and CD31-associated proteins were examined by co-immunoprecipitation. Heterophilic interaction of CD31 by sCD38 enhanced its association with the intracellular protein Src, SHP2 and Grb2, which were detected by anti-Src, GRB2, Shc and SOS antibodies. (C) Src is required for CD31 tyrosine phosphorylation in sperm. Sperm were pre-incubated with 50 μM Su6656. After incubating for 30 minutes at 37°C, CD31 activation was induced by addition of 600 ng/mL sCD38. After a further incubation for 30 minutes, cells were lysed in Triton X-100, and CD31 immunoprecipitates were prepared. Immunoblot analysis was performed to determine the extent of CD31 tyrosine phosphorylation, using an anti-pY antibodies. Input represents ten percent of the content in the cell lysate of the molecules that were immunoprecipitated; in the bottom panel, analyses of band intensity are presented as the percentage of immunoprecipitated proteins per total input.</p
The sCD38-induced tyrosine phosphorylation of sperm protein is mediated by ERK pathway.
<p>(A) CD31 activation of human sperm by sCD38 stimulated tyrosine phosphorylation of sperm protein mediated by ERK1/2. Human sperm were incubated in capacitating medium supplemented with sCD38 as time-dependent manner. Following incubation, sperm were solubilized in lysis buffer and prepared for immunoblotting with anti-phospho-ERK1/2 and anti-phospho-p38. Total ERK and p38 was detected as a control for sample loading. (B) Sperm were preincubated with inhibitors (50 μM SU6656, 3 μM FTI-277 and 50 μM PD98059) for 30 min, then treated with (+) or without (-) 600 ng/ml sCD38 for 0 or 60 minutes, sperm proteins were immunoblotted with the anti-phospho-ERK1/2 antibody and total ERK1/2.</p
A schematic model showing signaling pathways underlying β-AR-mediated NAADP-synthesizing enzyme and CD38 activation.
<p>Binding of ISO to β-AR stimulates AC, thus activating PKA. PKA induces Ca<sup>2+</sup> influx that leads to activation of an NAADP-synthesizing enzyme (NSE) to produce NAADP. NAADP-mediated Ca<sup>2+</sup> mobilization from the acidic Ca<sup>2+</sup> stores results in activation of CD38. cADPR produced by CD38 in the endocytic vesicles induces Ca<sup>2+</sup> release from SR Ca<sup>2+</sup> stores. cADPR-mediated Ca<sup>2+</sup> release induces SOCE, resulting in a sustained Ca<sup>2+</sup> signal. NE: norepinephrine; SR: sarcoplasmic reticulum; NSE: NAADP-synthesizing enzyme; AC, adenylyl cyclase; PKA, protein kinase A; SOCE, store-operated Ca<sup>2+</sup> entry.</p
ISO-stimulated Ca<sup>2+</sup> increase in cardiomyocytes.
<p><b>(A)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to 2 μM ISO in the presence of an adenylate cyclase inhibitor and a cAMP antagonist. <b>(B, C)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to 2 μM ISO in the presence of thapsigargin (Thap), 8-Br-cADPR, xestospongine C (XesC), or bafilomycin A1 (Baf). <b>(D)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to 2 μM ISO or 50 nM NAADP-AM in the presence or absence of extracellular Ca<sup>2+</sup>. <b>(E)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to NAADP-AM after pretreatment with or without 8-Br-cADPR. <b>(F)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to 100 nM deaza-cADPR in the absence and presence of extracellular Ca<sup>2+</sup>. <b>(G)</b> Representative tracings of the [Ca<sup>2+</sup>]<sub>i</sub> response to 2 μM ISO in cardiomyocytes after transfection with scrambled or Stim1 siRNA. Values are the mean ± SEM of three independent experiments.</p
ISO-stimulated Ca<sup>2+</sup> increase and cADPR and NAADP production in wild-type (WT) and CD38 knock-out (KO) mice.
<p><b>(A)</b> Representative tracings of the Ca<sup>2+</sup> response to ISO in cardiomyocytes obtained from WT and CD38 KO mice. (right panel) A direct comparison of the mean [Ca<sup>2+</sup>]<sub>i</sub> during sustained increases in [Ca<sup>2+</sup>]<sub>i.</sub> The data shown are analyzed at 150 s. <b>(B)</b> ISO-stimulated cADPR production in WT and CD38 KO mice. <b>(C)</b> ISO-stimulated NAADP production in WT and CD38 KO mice. *, <i>P</i>< 0.05 versus WT controls. #, <i>P</i> < 0.05 versus CD38 KO controls. §, P<0.05 versus WT + ISO. Values are the mean ± SEM of three independent experiments.</p
Echocardiographic assessment of CD38 WT and CD38KO mice at baseline.
<p>Echocardiographic assessment of CD38 WT and CD38KO mice at baseline.</p
ISO-stimulated cADPR and NAADP production in cardiomyocytes.
<p><b>(A)</b> Time course of cADPR and NAADP production following ISO treatment. <b>(B and C)</b> Differential effects of Ca<sup>2+</sup> second messenger inhibitors on ISO-induced cADPR and NAADP formation. *, P<0.05 versus control (Con) cADPR level. #, P<0.05 versus control (Con) NAADP level. §, P<0.05 versus ISO-induced cADPR level. ¶, P<0.05 versus ISO-induced NAADP level. Values are the mean ± SEM of three independent experiments.</p