Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa

Herrero, Marı́a Belén; Martı́nez, Silvina Pérez; Viggiano, J.M.; Polak, J.M.; Gimeno, M.F.

doi:10.1071/rd9960931

Cited by 90 publications

(68 citation statements)

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“…In epididymal mouse sperm loaded with 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate (DAF-FM DA) the distribution of NO changed from the head and midpiece to the midpiece upon capacitation (Fig. 4A, upper panels a and c), consistent with previously reported NOS relocalization (Herrero et al 1996).…”

Section: Gst-spink3 Treatment Correlates With Reduced No Production Isupporting

confidence: 79%

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SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

Zalazar

Lancellotti²,

Clementi

et al. 2012

Reproduction

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Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca 2C )-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activity in vitro while the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca 2C concentration, using either intracellular (BAPTA-AM) or extracellular Ca 2C (EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), and S-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.

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Section: Gst-spink3 Treatment Correlates With Reduced No Production Isupporting

confidence: 79%

“…SPINK3 involves NO signalling in sperm immunofluorescence using antibodies that cannot distinguish among NOS forms (Herrero et al 1996). However, de Lamirande & Lamothe (2009) observed, using the same probe, an increase of NO signal on the head in human sperm during capacitation.…”

mentioning

confidence: 99%

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

Zalazar

Lancellotti²,

Clementi

et al. 2012

Reproduction

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“…In the present study, the pre-and post-bleaching images for the FRET data revealed the close interaction of PMCA4 and the NOSs on the sperm head, neck (Figures 2 and 3), and on the proximal principal piece of the flagellum, reflecting their co-localization. This is the first report of the localization for eNOS in murine sperm (Figures 1a and 3) and our results provide confirmation of that reported for nNOS by Herrero et al (1996). These localizations in murine sperm are similar to what has been reported for nNOS and eNOS in human sperm (Andrews et al, 2015;Herrero et al, 1996; O'Bryan, Zini, Cheng, & Schlegel, 1998), where they were also shown to co-localize (Andrews et al, 2015).…”

Section: Resultssupporting

confidence: 79%

“…Thus, when WT and nulls were compared, UNCAP and CAP, the median fluorescence units for each increased ∼2-fold for nulls versus WT-sperm (Figure 5a,b). 4 | DISCUSSION 4.1 | PMCA4 and the NOSs co-localize and intimately interact in murine sperm in a Ca 2+ -dependent manner While PMCA4 (Aravindan et al, 2012;Wennemuth et al, 2003) and nNOS (Herrero, Perez Martinez, Viggiano, Polak, & Gimeno, 1996) have been localized in murine sperm on the head, over the acrosome, Figure 1a, we show that nNOS is more abundant and more widely distributed on the sperm, being found in the distal principal piece where eNOS and PMCA4 do not reside. In the present study, the pre-and post-bleaching images for the FRET data revealed the close interaction of PMCA4 and the NOSs on the sperm head, neck (Figures 2 and 3), and on the proximal principal piece of the flagellum, reflecting their co-localization.…”

Section: Resultsmentioning

confidence: 81%

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Olli

Galileo

et al. 2017

Journal Cellular Physiology

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Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca 2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca 2+ efflux pump, PMCA4. This is the major Ca 2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca 2+ ] in capacitated and Ca 2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca 2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca 2+ -dependent manner. In Pmca4 −/− sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca 2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4 −/− males.They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.asthenospermia, calcium efflux pump, infertility, oxidative stress, sperm damageCapacitation is the series of changes that occur in the final maturation of mammalian sperm in the female in order for them to gain fertilizing competence, and subsequently undergo the acrosome reaction. Both of these processes are dependent on elevated levels of cytosolic Ca 2+ concentration ([Ca 2+ ] c ) (de Lamirande, Leclerc, & Gagnon, 1997;Fraser, 1987). After the calcium influx required to meet this high calcium demand, there needs to be a return to resting cytosolic calcium concentration ([Ca 2+ ] c ) levels (50-100 nM [Herrick et al., 2005]). In mice, in the absence of the major calcium efflux pump, Plasma Membrane Calcium ATPase 4 (PMCA4) (Wennemuth, Babcock, & Hille, 2003), calcium levels remain elevated with loss of sperm motility (Asthenospermia, AS) and resulting male infertility (Okunade et al., 2004;Schuh et al., 2004 (de Lamirande et al., 1997;Fraser, 1987) which rapidly activates them (Knowles & Moncada, 1994). Thus, as PMCA4 extrudes Ca 2+ , the NOSs are held in a locale or microdomain with a relatively low [Ca 2+ ] c, compared to the surrounding environment where it is globally high. In this Ca 2+ -extruding locale, NO...

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“…NO has been demonstrated to play a role in a variety of functions in the human reproductive tract, including sperm motility (Lewis et al 1996), chemotaxis (Miraglia et al 2007), and sperm-zona pellucida binding ability (Sengoku et al 1998). NOS isoforms have been localized in the acrosome and tail of human, mouse, and bovine spermatozoa (Herrero et al 1996, Meiser & Schulz 2003, and low motility spermatozoa have been shown to exhibit aberrant patterns of NOS3 immunostaining (O'Bryan et al 1998). It has been reported that low concentrations of NO (25-100 nM sodium nitroprusside) enhance the motility of human spermatozoa (Hellstrom et al 1994, Zhang & Zheng 1996.…”

Section: Introductionmentioning

confidence: 99%

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway

Miraglia¹,

Angelis²,

Gazzano³

et al. 2011

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Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90 min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 mm/s vs 34, 35, and 35.5 mm/s), curvilinear velocity (81, 83, and 84 mm/s vs 68 mm/s), and average path velocity (52, 57, and 54 mm/s vs 40, 42, and 42 mm/s) at 5 mM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60 min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO-and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

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Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa

Cited by 90 publications

References 0 publications

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway

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