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From the * Departamento de Farmacología,
Facultad de Medicina, Universidad Nacional Autónoma de México,
Ciudad Universitaria, Mexico; and the
Departamento de Bioquímica, Centro de
Investigación y de Estudios Avanzados del Instituto Politécnico
Naciona, Mexico.
| Correspondence to: Dr Marco T. González-Martínez, Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México. Ciudad Universitaria, CP 04510. Apartado Postal 70-297 México, D.F., México (e-mail: tuliog{at}servidor.unam.mx). |
| Received for publication November 28, 2007; accepted for publication May 14, 2008. |
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Abstract |
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Key words: cAMP, intracellular calcium, progesterone
The calcium transport mechanism involved in the calcium influx induced by
progesterone has not been identified yet; however, there is evidence of
biochemical modulation. In this regard, the calcium influx induced by
progesterone is markedly stimulated during capacitation in human sperm
suspensions (Baldi et al, 1998;
González-Martínez et al,
2002; Bedu-Addo et al,
2005). During this process that confers sperm the ability to
undergo the acrosome reaction induced by progesterone or by the egg zona
pellucida glycoprotein ZP3, different signal transduction systems are
triggered. Initially, there is an increase in cAMP produced by a bicarbonate
and calcium-dependent, soluble adenylyl cyclase (sAC) that in turns activates
the protein kinase A (PKA; Uhler et al,
1992; Lefievre et al,
2002). At a late step, a tyrosine kinase is activated,
phosphorylating 
105 and 85 kd proteins, which are presumably kinase A
anchor proteins (Carrera et al,
1996). Additionally, capacitation is accompanied by small
increases in resting intracellular pH (pHi;
Cross NL and Razy-Faulkner,
1997). As for this effect, it has been suggested that bicarbonate
stimulates the progesterone-induced calcium influx via a mechanism involving
sperm alkalization rather than cAMP production
(Aitken et al, 1998).
In this work, we report that a brief incubation with papaverine, a phosphodiesterase (PDE) inhibitor, produces a remarkable increase in the calcium influx induced by progesterone. Interestingly, part of the stimulated signal was reversed by the PKA inhibitor H89 and an additional part by the tyrosine kinase inhibitor genistein. The results presented here suggest that the calcium transport mechanism activated by progesterone is markedly stimulated by PKA activation.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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100
x 106) were separated from seminal plasma using isotonic
Percoll gradients (75/50% Percoll in 150 mM NaCl + 10 mM HEPES, pH 7.4) as
reported in Linares-Hernandez et al
(1998), and washed in
HEPES-buffered human sperm medium (HHSM), in 117.5 mM NaCl, 8.6 mM KCl, 2.5 mM
CaCl2, 0.5 mM MgCl2, 0.3 mM NaHPO4, 0.25 mM
Na pyruvate, 19 mM Na lactate, 25 mM HEPES, adjusted with NaOH to pH 7.6. The
cells were loaded with either 2 µM fura ff-AM for 40 minutes or with 0.5
µM BCECF-AM for 30 minutes as described in Neri-Vidaurri et al
(2006). In some cases, fura
ff-loaded sperm (3 x 107 cells) were incubated for 4
hours at 36°C in 15 mL capacitating medium, that is, HHSM medium
containing 15 mM HEPES, supplemented with 3 mg/mL BSA and 25 mM
NaHCO3. Reagents used to prepare HHSM medium were obtained from
Sigma Chemical Co (St Louis, Missouri), Mallinckrodt Baker (Phillipsburg, New
Jersey), or Merck (Whitehouse Station, New Jersey). Papaverine,
pentoxifylline, genistein, and H89 were obtained from Sigma. Fura ff-AM and
BCECF-AM were from Molecular Probes (Invitrogen, Carlsbad, California).
Induction of cAMP Increase
The increase in intracellular cAMP in noncapacitated sperm (1 x
107) was achieved by incubation with 0.5 mM papaverine (a PDE
inhibitor), in 0.25 mL HHSM, at 36°C for 5 minutes. After 5 minutes
incubation with papaverine or with papaverine + genistein and/or H89 (both
added 35 minutes before papaverine addition), the sample was diluted from 0.25
mL to 1.25 mL with fresh HHSM medium, centrifuged, and the pellet was
resuspended in 2.5 mL HSM and used for cell viability assayed by eosine (0.5%
in PBS, 1:1 dilution with sperm suspensions) exclusion test,
[Ca2+]i determination (in fura ff-loaded cells), PKA activity
measurement, determination of cAMP content or, in some cases, for pHi
determination (in BCECF-loaded cells) or for identification of
tyrosine-phosphorylated proteins on Western blots, as described below. Washed
pellets were used because papaverine severely affected fura ff fluorescence
(not shown). Using washed cells barely altered the Rmax and
Rmin values used to calibrate fura ff signals. In some
intracellular calcium measurements, pentoxifylline was used under identical
conditions.
Measurement of cAMP
cAMP was measured with the Cyclic AMP Competitive EIA kits from Zymed
Laboratories (San Francisco, California) and Cayman Chemical (Ann Harbor,
Michigan). Sperm pellets (1 x 107 cells) obtained from
different experimental conditions (see above) were treated with 1 mL 0.05 M
HCl. The sample was boiled for 3 minutes and then cooled in ice. The sample
was microcentrifuged for 10 minutes and the supernatant used for cAMP
determination. The sample and the appropriate cAMP standards were alkalized
with 200 µl of 4 M KOH and acetylated with 50 µl acetic anhydride. The
acetylated samples (50 µl) were added to the well plate, and the
competition assay proceeded by adding 50 µl of either acetyl
cholinesterase-cAMP or alkaline phosphatase-cAMP complexes and the cAMP
antiserum. Once washed, the plate was reconstituted with 200 µl buffer
containing either p-nitrophenyl phosphate or the Ellman's reagent for 90
minutes in the dark, under constant stirring, and read at 405 nm.
Measurement of PKA Activity
The activity of PKA was measured in sperm extracts as the incorporation of
32P from ATP-32P to a kemptide substrate, provided by an
Up-State kit (Lake Placid, New York). The sperm pellets (1 x
107) were resuspended with 0.25 mL of nondenaturating lysis buffer
containing 1 mM Na-ortovanadate, 20 mM MOPS, 25 mM β-glycerophosphate, 5
mM EGTA, 1 mM dithiothreitol, and 1% triton X-100, pH 7.2 at 4°C. After 10
minutes of bath sonication, the sample was microcentrifuged at 10 000 g for 10
min and the supernatant used for determination of PKA activity. The extract
was supplemented with 12.5 mM MgCl2, 83 µM ATP, 0.33 µM PKC
inhibitor peptide, 3.3 µM CaMK (calmoduline dependent kinase) inhibitor
R24571, and ATP-32P (6 µCi). The reaction started upon 1 mM
kemptide addition and proceeded for 15 minutes at 30°C. A negative control
containing 1 µM PKA inhibitor peptide was included in the measurements. A
sample (25 µl) of the reaction mixture was blotted on P81 nitrocellulose
paper and washed 3 times in phosphoric acid. After a last single wash in
acetone, the paper was dried and counted for 32P radioactivity in a
scintillation counter. The negative control produced 10% of the counts found
in control nontreated sperm and was rested as unspecific binding.
Detection of Tyrosine Phosphorylated Proteins by Western Blot
Sperm (1 x 107 cells) pellets were solubilized with
0.05 mL lysis buffer (62.5 mM Tris-HCl, 25% glycerol pH 7.6, 2% SDS, pH 6.8),
sonicated for 10 minutes (bath sonicator) and microcentrifuged for 10 minutes.
Sperm proteins were separated by SDS-PAGE electrophoreses (10%) and
transferred electrophoretically onto nitrocellulose membrane. The
nitrocellulose membrane was then incubated in 3% defatted milk-PBS overnight
at 4°C and then incubated with antibody anti-phosphotyrosine (1:2000)
coupled with peroxidase (Sigma) for 6 hours. The membrane was washed with
PBS-Tween (0.05%) and then with PBS. The phosphotyrosine proteins were
detected with enhanced chemoluminiscence (ECL, Amersham, Buckinghamshire,
United Kingdom) and revealed with Kodak Biomax light film (Rochester, New
York).
Measurement of Intracellular Calcium and Intracellular pH
Fura ff-loaded sperm pellets (1 x 107) were added to
the fluorescence cuvette containing 2.5 mL HHSM medium. The cuvette was kept
at 36°C and continuously stirred with a magnetic bar. Once the signal was
stabilized (1 minute later), 4 µM progesterone was added, and the effect
was followed for one more minute. The fluorescence was detected at 488 nm with
an optical filter (Andover Corp, Salem, New Hampshire), alternately exciting
at 340 nm and 380 nm at 0.83 Hz. The 340/380 fluorescence ratios were
converted to intracellular calcium values using the Grynckievicz equation and
a Kd = 5.5 µM as described in Neri-Vidaurri et al
(2006). On the other hand,
fluorescence of BCECF-loaded sperm was detected at 550 nm with an optical
filter (Andover Corp), alternately exciting at 500 and 439 nm. The 500/439
ratios were converted to pH as previously described
(Fraire-Zamora and
Gonzalez-Martínez, 2004).
Statistical Analysis
Data are reported as means ± standard error of the mean (SEM), with
n meaning number of individuals tested. Nontreated and treated groups were
compared using analysis of variance (ANOVA; Newman-Keuls test) or with paired
t-test. Results with P < .05 were considered
statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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5-fold stimulation. Even though the stimulation was remarkable, it did
not reach the stimulation values (ie, close to 7.5-fold) found in sperm
incubated 4 hours in capacitating medium. In noncapacitated sperm, papaverine
treatment also produced an increase in resting [Ca2+]i, as
indicated by the dashed line on the traces. The resting [Ca2+]i
detected in fura ff-loaded sperm increased from a normal 147 ± 23 nM to
523 ± 40 nM (n = 6, SEM) in papaverine-treated sperm. The resting value
obtained in fura ff-loadedsperm incubated in capacitating medium (127 ±
34 nM, n = 6, SEM) was statistically indistinct (P = .4) from the one
obtained in noncapacitated cells.
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Since papaverine is a phosphodiesterase inhibitor, it was pertinent to compare its effect with pentoxifylline, another PDE inhibitor that is widely used to stimulate sperm motility. As shown in Figure 2, pentoxifylline also stimulated the progesterone-induced calcium influx in noncapacitated human sperm, but to a lower extent as compared with papaverine. This was particularly evident at low PDE inhibitor concentrations, as shown in the dose-response curve (Figure 2B). At 2 mM, pentoxifylline produced maximum stimulating effect in the range tested, whereas papaverine tended to decrease it. Interestingly, preincubation with pentoxifylline did not increase resting [Ca2+]i as compared with papaverine (Figure 2A). Given the higher effect of papaverine on the progesterone-induced calcium influx, its effect at 0.5 mM, which produced maximum effects, was further explored.
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41% and 48% respectively
(Figure 3). Under these
conditions, sperm maintained 81 ± 0.9% and 86 ± 0.5% viability
(n = 7, SEM) respectively, according to the eosine exclusion test, with values
close to control (96 ± 0.2%). When both inhibitors were used together,
a condition that maintained sperm viability of 83 ± 0.85%, the
transient calcium influx induced by progesterone was further inhibited, to
89%. The increase in resting [Ca2+]i induced by papaverine
exposure was not reversed by H89 and/or genistein, and these inhibitors did
not affect the resting [Ca2+]i of intact (nontreated with
papaverine) noncapacitated cells (traces not shown).
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14% average)
stimulation in cAMP production induced by H89 was observed in 5 out of 7
experiments, which was consistent with observations indicating that PKA
inhibition results in stimulation of soluble adenylyl cyclase
(Nolan el al, 2004); the lack
of significant stimulation was possibly related to the high activity, perhaps
close to maximum, of adenylyl cyclase in papaverine-treated sperm, so that in
this condition H89 had a small margin for further stimulation. In contrast,
genistein and genistein + H89 decreased the cAMP content, reducing the effect
of papaverine to 5-fold with respect to untreated cells. The PKA activity
determined in these conditions also showed a 9-fold increase induced by
papaverine preincubation (Figure
4B). In the presence of H89, the PKA activity was significantly
(P < .05) inhibited by 80%; hence, a still significant 2.5-fold
stimulation with respect to untreated cells was still observed. Genistein also
reduced (P < .05) the papaverine-induced PKA activation by 42%, a
result that can be explained by the reduced amount of cAMP produced in this
condition. Preincubation with genistein + H89 produced a summed inhibition of
the papaverine-stimulated PKA activity that brought it to values close to
control (no papaverine added). The positive relationship between PKA activity
and progesterone-induced calcium influx, obtained from the above described
conditions (Figure 5),
supported the hypothesis that PKA activation stimulated progesterone
action.
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Figure 4C shows the 107 and 95 kd proteins that were intensively tyrosine phosphorylated after 4 hours incubation in capacitating medium (not shown) and should correspond to the 105 and 85 kd sperm bands previously reported in Carrera et al (1996). Tyrosine phosphorylation of these proteins was slightly increased in papaverine-treated noncapacitated sperm as compared with nontreated cells. As expected, preincubation with genistein reduced tyrosine phosphorylation protein bands in papaverine-treated sperm to levels below untreated cells.
Since it has been reported that bicarbonate, an activator of soluble
adenylyl cyclase, stimulates progesterone-induced calcium influx via a
mechanism involving sperm alkalization rather than cAMP production, the effect
of papaverine on pHi and on progesterone-induced calcium influx was explored.
Papaverine (0.5 mM) preincubation for 5 minutes slightly alkalized the cells,
from a resting 6.64 ± 0.03 to 6.79 ± 0.04 (n = 5, SEM).
Figure 6 shows a series of
experiments that compare the calcium influx induced by progesterone in
papaverine-treated sperm and the calcium influx induced by progesterone in
cells treated 15 seconds before with 5 mM NH4Cl, which alkalizes
the pHi to 6.87 ± 0.02 (n = 5, SEM), a value even more alkaline that
the one produced by papaverine preincubation. Even though in some cases there
was stimulation, the average increase remained nonsignificant (n = 10,
P = .12, paired t-test) as previously reported
(Fraire-Zamora and
González-Martínez, 2004). In papaverine-treated
sperm, the addition of 5 mM NH4Cl produced a higher alkalization
(7.07 ± 0.03, n = 5, SEM) and significantly stimulated (increase of
27%) the calcium influx induced by progesterone (n = 10, P =
.004, paired t-test).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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5-fold. This conclusion is
supported by the fact that an important fraction of the papaverine-stimulated
calcium influx induced by progesterone is inhibited by H89, a PKA inhibitor.
The partial inhibition was expected since H89 did not produce a total
inhibition of the PKA activity, a result that might be explained because all
determinations were performed with washed cells in fresh media. Consequently,
the 2.5-fold increase in PKA activity still observed in the presence of H89
should be reflected on the calcium influx induced by progesterone.
Unexpectedly, genistein was also able to reduce the papaverine-induced
increase of both cAMP content and the PKA activity, an effect that was also
consistent with a diminished papaverine-stimulated [Ca2+]i increase
induced by progesterone. Other effects of genistein (see below) or H89 could
also affect other pathways. In this regard, H89 inhibits PKB/AKT (Davies et
al, 2001) a serine-threonine kinase present in human sperm
(Aquila et al, 2000);
consequently, a contribution of this kinase to the stimulated signals observed
here should not be discarded. Nevertheless, the fact that a combined effect of
both inhibitors produced a summed decrease on both PKA activity and
[Ca2+]i increase induced by progesterone supports the hypothesis
that the calcium transport mechanism activated by progesterone is up-regulated
by PKA activation. Consistently, PKA inhibitors block the acrosome reaction
induced by progesterone in human capacitated sperm
(Harrison et al, 2000). Thus,
it is conceivable that the activation of the soluble adenylyl cyclase/cAMP/PKA
system observed during capacitation stimulates the calcium entry mechanism
triggered by progesterone and contributes to its capability to induce the
acrosome reaction. Interestingly, induction of cAMP production mediated by
particulate adenylyl cyclase, and PKA activation, has been involved in calcium
influx induced by bourgeonal, an odorant receptor agonist that, like
progesterone (Teves et al,
2006), induces chemotaxis in human sperm
(Spehr et al, 2004).
Unlike the results presented here, Aitken et al (1998) have reported that exposure to 3 mM pentoxifylline and 5 mM dibutiryl cAMP in bicarbonate-free medium does not stimulate the calcium influx induced by progesterone in human sperm. Here we show that under our experimental condition, basically related to 5 minutes of exposure to the PDE inhibitor in sperm suspensions, pentoxifylline alone stimulates the progesterone-induced calcium influx, although to a lower extent as compared with papaverine (in the range of PDE inhibitor tested). Additionally, Aitken et al have reported that the progesterone-induced calcium influx is stimulated by pHi alkalization induced in pH 8.6 medium and suggested that bicarbonate, the sAC activator, stimulates progesterone-induced calcium influx via pHi alkalization. In this work, we observed that papaverine induced a slight alkalization that could consequently contribute to the signal. However, this effect is negligible since pHi alkalization induced with ammonium, at a value even more alkaline than the one induced by papaverine, produced no significant effects on progesterone action as previously reported (Fraire-Zamora and González-Martínez, 2004); however, in sperm exposed to papaverine, the calcium influx induced by progesterone was significantly stimulated by pHi alkalization, suggesting that pHi alkalization may have stimulatory effects on progesterone action unless the cAMP-PKA system is sufficiently activated.
Exposure to papaverine produced a slight increase in tyrosine phosphorylation activity on the 2 major tyrosine phosphorylated proteins that appear during capacitation in human sperm (Carrera et al, 1996), and preincubation with genistein decreased this activity to levels even below the control. As mentioned above, the inhibitory effect of genistein on the calcium influx induced by progesterone may be mediated by its decreasing effect on cAMP content and consequently PKA activity. Consistently, it has been observed that the induction of hyperactivated motility by progesterone, a process mediated by cAMP production, is inhibited by genistein (Parinaud and Milhet, 1996). Thus, it is possible that the adenylyl cyclase requires a sustained, basal tyrosine kinase activity to have full activation. In this regard, it has been suggested that the adenylyl cyclase can be up-regulated by tyrosine kinases in glial and vascular smooth muscle cells (El-Mowafy and White, 1998; Roymans et al, 2001). On the other hand, tyrosine kinases could directly stimulate PKA activity; as for this hypothesis, it has been proposed that human sperm PKA might be activated during sperm capacitation independently of cAMP (Lefievre et al, 2002). In addition, the effect of genistein could be related to its stimulating effect on a cystic fibrosis transmembrane conductance that produces hyperpolarization in mouse sperm (Hernández-González et al, 2007). This phenomenon might be significant given that the calcium influx induced by progesterone diminishes in hyperpolarized human sperm (Guzmán-Grenfell et al, 2004). It is important to note that a lack of genistein effect on the [Ca2+]i increase induced by progesterone has been observed (Martínez et al, 1999; Kirkman-Brown et al, 2002). The higher amount of genistein used in this study or the use of washed cells for [Ca2+]i measurements might explain this discrepancy. On the other hand, others have documented inhibitory effects of genistein only on the sustained calcium influx induced by progesterone in capacitated human sperm (Bonaccorsi et al, 1995).
The mechanism by which PKA activation stimulates calcium entry pathway(s) triggered by progesterone remains to be established. The increased sustained calcium influx induced by progesterone in capacitated sperm has been attributed to the slight increase in resting [Ca2+]i (detected with the high-affinity detector fura 2) that occurs during sperm capacitation (Baldi et al, 1991), which would increase the number of functional progesterone receptors (Mendoza and Tesarik, 1993). We must mention that we could not detect this increase in fura ff-loaded sperm, perhaps because of its low affinity for calcium (Kd = 5.5 µM; Neri-Vidaurri et al, 2006). A high resting [Ca2+]i could be related to the stimulating effect of papaverine on the calcium influx induced by progesterone because papaverine increases resting [Ca2+]i, However, this seems not to be the case since the stimulation was inhibited by genistein or H89 or the sum of both, without reversing the enhanced [Ca2+]i resting values caused by papaverine. In this regard, the lack of effect of H89 and genistein on the papaverine-induced increase on basal [Ca2+]i suggests direct participation of either cAMP and/or cGMP or cAMP targets different from PKA, such as the exchange proteins activated directly by cyclic AMP (EPAC), on the calcium transport mechanisms that set the resting [Ca2+]i in human sperm. Interestingly, pentoxifylline incubation did not increase the resting [Ca2+]i, an effect that has been observed by Nassar et al (1998). It is possible that this difference reflects a preferential effect of papaverine on a PDE involved in cAMP present near the calcium transport system that sets the resting [Ca2+]i in human sperm and on that cAMP regulate them.
In summary, we provide evidence suggesting that the calcium transport systems triggered by progesterone in human sperm are highly stimulated when protein kinase A is activated. This regulation may contribute to the enhanced [Ca2+]i increase induced by progesterone in capacitated sperm, a phenomenon that might be required for a successful egg fertilization.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aquila S, Middea E, Catalano S, Marsico S, Lanzino M, Casaburi I, Barone I, Bruno R, Zupo S, Andò S. Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway. Hum Reprod. 2000;22: 2594 –2605.[CrossRef]
Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G.
Intracellular calcium accumulation and responsiveness to progesterone in
capacitating human spermatozoa. J Androl. 1991; 12: 323
–330.
Bedu-Addo K, Lefievre L, Moseley FL, Barratt CL, Publicover SJ.
Bicarbonate and bovine serum albumin reversibly `switch' capacitation-induced
events in human spermatozoa. Mol Hum Reprod. 2005; 11: 683
–691.
Blackmore PF, Neulen J, Lattanzio F, Beebe SJ. Cell surface-binding
sites for progesterone mediate calcium uptake in human sperm. J
Biol Chem. 1991;266: 18655
–18659.
Bonaccorsi L, Luconi M, Forti G, Baldi E. Tyrosine kinase inhibition reduces the plateau phase of the calcium increase in response to progesterone in human sperm. FEBS Lett. 1995; 364: 83 –86.[CrossRef][Medline]
Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, Moss SB. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol. 1996;180: 284 –296.[CrossRef][Medline]
Cross NL, Razy-Faulkner P. Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone. Biol Reprod. 1997; 56: 1169 –1174.[Abstract]
Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351: 95 –105.[CrossRef][Medline]
El-Mowafy AM, White RE. Evidence for a tyrosine kinase-dependent activation of the adenylyl cyclase/PKA cascade downstream from the G-protein-linked endothelin ETA receptor in vascular smooth muscle. Biochem Biophys Res Commun. 1998; 251: 494 –500.[CrossRef][Medline]
Francavilla F, Romano R, Santucci R, Macerola B, Ruvolo G, Francavilla S. Effect of human sperm exposure to progesterone on sperm-oocyte fusion and sperm-zona pellucida binding under various experimental conditions. Int J Androl. 2002; 25: 106 –112.[CrossRef][Medline]
Fraire-Zamora JJ, González-Martínez MT. Effect of
intracellular pH on depolarization-evoked calcium influx in human sperm.
Am J Physiol (Cell Physiol). 2004; 287: C1688
–1696.
González-Martínez MT, Bonilla-Hernández MA, Guzmán-Grenfell AM. Stimulation of voltage dependent calcium channels during capacitation and by progesterone in human sperm. Arch Biochem Biophys. 2002;408: 205 –210.[CrossRef][Medline]
Guzman-Grenfell AM, Gonzalez-Martinez MT. Lack of voltage-dependent
calcium channel opening during the calcium influx induced by progesterone in
human sperm. Effect of calcium channel deactivation and inactivation.
J Androl. 2004;25: 117
–122.
Harrison DA, Carr DW, Meizel S. Involvement of protein kinase A and
A kinase anchoring protein in the progesterone-initiated human sperm acrosome
reaction. Biol Reprod. 2000; 62: 811
–820.
Hernández-González EO, Treviño CL, Castellano
LE, De la Vega-Beltrán JL, Ocampo AY, Wertheimer E, Visconti PE,
Darszon A. Involvement of cystic fibrosis transmembrane conductance regulator
in mouse sperm capacitation. J Biol Chem. 2007; 282: 24397
–24406.
Kirkman-Brown JC, Lefievre L, Bray C, Stewart PM, Barratt CL,
Publicover SJ. Inhibitors of receptor tyrosine kinases do not suppress
progesterone-induced [Ca2+]i signalling in human spermatozoa. Mol
Hum Reprod. 2002;8: 326
–332.
Lefievre L, Jha KN, De Lamirande E, Visconti PE, Gagnon C.
Activation of protein kinase A during human sperm capacitation and acrosome
reaction. J Androl. 2002; 23: 709
–716.
Linares-Hernández L, Guzman-Grenfell AM, Hicks-Gomez JJ, González-Martínez MT. Voltage dependent calcium influx in human sperm assessed by simultaneous detection of intracellular calcium and membrane potential. Biochim Biophys Acta. 1998; 1372: 1 –12.[Medline]
Martinez F, Tesarik J, Martin CM, Soler A. Mendoza C. Stimulation of tyrosine phosphorylation by progesterone and its 11-OH derivatives: dissection of a Ca(2+)-dependent and a Ca(2+)-independent mechanism. Biochem Biophys Res Com. 1999; 255: 23 –27.[CrossRef][Medline]
Meizel S, Turner KO, Nuccitelli R. Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction. Dev Biol. 1997; 182: 67 –75.[CrossRef][Medline]
Mendoza C, Tesarik J. A plasma-membrane progesterone receptor in human sperm is switched on by increasing intracellular free calcium. FEBS Lett. 1993; 330: 57 –60.[CrossRef][Medline]
Nassar A, Mahony M, Blackmore P, Morshedi M, Ozgur K, Oehninger S. Increase of intracellular calcium is not a cause of pentoxifylline-induced hyperactivated motility or acrosome reaction in human sperm. Fertil Steril. 1998;69: 748 –754.[CrossRef][Medline]
Neri-Vidaurri PV, Torres-Flores V, González-Martínez MT. A remarkable increase in the pHi sensitivity of voltage-dependent calcium channels occurs in human sperm incubated in capacitating conditions. Biochem Biophys Res Commun. 2006; 343: 105 –109.[CrossRef][Medline]
Nolan MA, Babcock DF, Wennemuth G, Brown W, Burton KA, McKnight GS.
Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP
signaling for male fertility. Proc Natl Acad Sci U S
A. 2004;101: 13483
–13488.
Parinaud J, Milhet P. Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm. J Clin Endocrinol Metab. 1996; 81: 1357 –1360.[Abstract]
Roymans D, Grobben B, Claes P, Slegers H. Protein tyrosine kinase-dependent regulation of adenylate cyclase and phosphatidylinositol 3-kinase activates the expression of glial fibrillary acidic protein upon induction of differentiation in rat c6 glioma. Cell Bio Int. 2001;25: 467 –474.[CrossRef]
Senuma M, Yamano S, Nakagawa K, Irahara M, Kamada M, Aono T. Progesterone accelerates the onset of capacitation in mouse sperm via T-type calcium channels. Arch Androl. 2000; 47: 127 –134.
Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Neuhaus EM,
Hatt H. Particulate adenylate cyclase plays a key role in human sperm
olfactory receptor-mediated chemotaxis. J Biol Chem. 2004; 279: 40194
–40203.
Teves ME, Barbano F, Guidobaldi HA, Sanchez R, Miska W, Giojalas LC. Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa. Fertil Steril. 2006; 86: 745 –749.[CrossRef][Medline]
Uhler ML, Leung A, Chan SY, Wang C. Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril. 1992; 58: 1191 –1198.[Medline]
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[Ca2+]i), corresponds to the
representative traces depicted in the lower panel (progesterone addition is
indicated by the arrows). The bars show mean values ± SEM (n = 10).
*P < .05 (Newman-Keuls test).
