[Impact of symptom onset to first medical contact time on the prognosis of patients with acute ST-segment elevation myocardial infarction].

Objective: To investigate the impact of symptom onset to first medical contact (SO-to-FMC)time on the prognosis of patients with acute ST-segment elevation myocardial infarction(STEMI). Methods: The clinical data of 341 consecutive STEMI patients, who were hospitalized to our hospital and received primary percutaneous coronary intervention(PCI) from August 2011 to April 2016, were retrospectively analyzed. The patients were divided into ≤90 min group (201 cases) and >90 min group (140 cases) according to the SO-to-FMC time. The treatment time, mortality and incidence of major adverse cardiac and cerebro-vascular events(MACCE) were analyzed. The risk factor of 1-year mortality after PCI and 1-year incidence of MACCE during the post-discharge follow-up period were analyzed by binary logistic regression analysis. The predictor of 4.5-year mortality after PCI was analyzed by multivariate Cox regression analysis. Methods The door to balloon time (104(88, 125) min vs. 111(92, 144)min, P=0.023), first medical contact to balloon time(146(119, 197) min vs. 177(125, 237)min, P=0.005), and symptom onset-to-balloon time(200(170, 257) min vs. 338(270, 474)min, P<0.001)were all significantly shorter in the ≤90 min group than in>90 min group. The 30-day mortality (2.99% (6/201) vs. 7.86%(11/140), P=0.042), 1-year mortality (2.89 (5/173) vs. 9.57(11/115), P=0.015), 1-year incidence of MACCE during the post-discharge follow-up period(1.16%(2/173) vs. 6.96%(8/115), P=0.021), and 4.5-year cumulative mortality(3.00% vs. 11.20%, P=0.007) after PCI were significantly lower in the ≤90 min group than in the >90 min group. Moreover, the 4.5-year incidence with free of MACCE (97.20% vs. 88.80%, P=0.025) during the post-discharge follow-up period was significantly higher in the ≤90 min group than in the >90 min group. In-hospital mortality was similar between the two groups (2.49%(5/201) vs. 6.43%(9/140), P=0.071). Results: The door to balloon time (104(88, 125) min vs. 111(92, 144)min, P=0.023) , first medical contact to balloon time(146(119, 197) min vs. 177(125, 237)min, P=0.005), and symptom onset-to-balloon time(200(170, 257) min vs. 338(270, 474)min, P<0.001) were all significantly shorter in the ≤90 min group than in >90 min group. The 30-day mortality(2.99% (6/201) vs. 7.86%(11/140), P=0.042), 1-year mortality (2.89(5/173) vs. 9.57(11/115), P=0.015), 1-year incidence of MACCE during the post-discharge follow-up period (1.16%(2/173) vs. 6.96%(8/115), P=0.021), and 4.5-year cumulative mortality (3.00% vs. 11.20%, P=0.007) after PCI were significantly lower in the ≤90 min group than in the >90 min group. Moreover, the 4.5-year incidence with free of MACCE (97.20% vs. 88.80%, P=0.025) during the post-discharge follow-up period was significantly higher in the ≤90 min group than in the >90 min group. In-hospital mortality was similar between the two groups (2.49%(5/201) vs. 6.43%(9/140), P=0.071). Results of binary logistic regression analysis showed that the SO-to-FMC time >90 min was the risk factor of 1-year mortality(OR=2.90, 95%CI 1.22-6.92, P=0.016) and 1-year incidence of MACCE (OR=5.19, 95%CI 1.21-22.20, P=0.026) during the post-discharge follow-up period. Multivariate Cox regression analysis demonstrated that the SO-to-FMC time >90 min was the risk factor of 4.5-year mortality after PCI in patients with STEMI (HR=2.88, 95%CI 1.10-7.53, P=0.031). Conclusion: Shorting the SO-to-FMC time can significantly reduce the treatment time of STEMI patients, short and long-term mortalities and the incidence of MACCE, and improve the prognosis of patients with STEMI.

Differential necrophoric behaviour of the ant Solenopsis invicta towards fungal-infected corpses of workers and pupae.

Necrophoric behaviour is critical sanitation behaviour in social insects. However, little is known about the necrophoric responses of workers towards different developmental stages in a colony as well as its underlying mechanism. Here, we show that Solenopsis invicta workers display distinct necrophoric responses to corpses of workers and pupae. Corpses of workers killed by freezing (dead for <1 h) were carried to a refuse pile, but pupal corpses would take at least 1 day to elicit workers' necrophoric response. Metarhizium anisopliae-infected pupal corpses accelerated the necrophoric behaviour of resident workers, with 47.5% of unaffected corpses and 73.8% infected corpses discarded by 1 day post-treatment). We found that fungus-infected pupal corpses had a higher concentration of fatty acids (palmitic acid, oleic acid and linoleic acid) on their surface. We experimentally confirmed that linoleic and oleic acids would elicit a necrophoric response in workers. The appearance of linoleic and oleic acids appeared to be chemical signals involved in recognition of pupal corpses, and M. anisopliae infection could promote the accumulation of fatty acids on surface of pupal corpses resulting in accelerated necrophoric responses of workers.

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters.

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20-29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30-39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer-assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age.

Cystic fibrosis transmembrane conductance regulator protein expression rate in healthy spermatozoa is not correlated with ovum fertilisation rate.

Our previous studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR) was important for capacitation and fertilisation in mouse, guinea pig and human spermatozoa. However, it is unclear whether CFTR is correlated with ovum fertilisation rate. The present study was to test the possible relationship between spermatozoa CFTR protein expression rate in healthy men and ovum fertilisation rate during in vitro fertilisation. Ninety-four couples for female factor infertility for IVF-ET treatments were retrospectively studied. All the patients were divided into three groups based on the fertilisation rate of ovum in vitro. It was performed to explore whether there were differences in sperm CFTR protein expression rate among the three groups and the relevance between CFTR protein expression rate and ovum fertilisation rate. Our study showed that there was no significant differences in sperm CFTR protein expression rate among the three groups (F = 0.614, P = 0.544), and the relevance between spermatozoa CFTR protein expression rate and ovum fertilisation rate was not significantly different (r = 0.013, P = 0.904). These results further suggest that CFTR protein expression rate in healthy men spermatozoa was not associated with ovum fertilisation rate and thus we cannot predict ovum fertilisation results by sperm CFTR protein expression rate.

Sperm phospholipases and acrosomal exocytosis.

At the time of fertilization, the sperm cell undergoes regulated exocytosis in response to the oocyte-associated agonists progesterone and zona pellucida. An early response generated by agonist-receptor interaction in spermatozoa is the activation of mechanisms leading to Ca2+ influx, this ion being essential for the activation of phospholipases and for the fusion of the plasma membrane with the outer acrosomal membrane. Both a phosphoinositide-specific, and a phosphatidylcholine-specific phospholipase C are involved in the generation of a variety of diacylglycerol molecular species. Phospholipase D, on the other hand, does not seem to play a significant role in the generation of diacylglycerol. Hydrolysis of phospholipids by phospholipase A2 generates free fatty acids and lysophospholipids; these are important either as substrates for the generation of other metabolites (e.g., eicosanoids) or having a direct, essential action in the final stages of membrane fusion. There is still much work to be done in the future in order to characterize phospholipase isozymes and their regulation during acrosomal exocytosis in spermatozoa.

Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa.

Phospholipase A(2) (PLA(2)) is activated in spermatozoa in response to progesterone and Ca(2+) ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA(2). We investigated whether PLA(2) is involved in ZP-stimulated acrosomal exocytosis, if Ca(2+) is required for activation of PLA(2), and signal transduction pathways modulating PLA(2) using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca(2+) medium with [(14)C]choline chloride or [(14)C]arachidonic acid and were then exposed to millimolar Ca(2+) and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca(2+) and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA(2) activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA(2) inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca(2+) or in medium with millimolar Ca(2+) and EGTA or La(3+) resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a G(i) protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA(2) plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA(2) activation requires Ca(2+) internalization, and that PLA(2) activation is regulated by signal transduction pathways involving G proteins and DAG.

Inhibition of hamster sperm acrosomal enzyme by gossypol is closely associated with the decrease in fertilization capacity.

To elucidate the mechanism of sterility induced by gossypol, we studied the relationship between the activities of acrosomal enzymes and their fertilizing capacity in the hamster. The results showed that the ability of spermatozoa to penetrate into bovine cervical mucus, hyperactivated motility (HAM) and fertility in vivo were significantly inhibited when spermatozoa were exposed to gossypol (2.5 microg - 60 microg/mL) for 15 min in vitro. Also, following administration of gossypol (12.5 mg/kg/day) for 6 weeks, sperm motility, HAM and rate of fertilization in vitro by the hamster cauda epididymal spermatozoa were significantly decreased and the extracts of testis delayed dispersion of the cumulus oophorus cells, suggesting that hyaluronidase and other acrosomal enzymes might be inhibited by gossypol. In addition, acrosin and arylsulfatase activities were also markedly inhibited. These data show that the inhibition of acrosin and arylsulfatase activities is the main cause of gossypol-induced infertility. The inhibition was dependent upon gossypol dose and the duration of administration. Thus, the assay of acrosin and arylsulfatase activities may provide a useful tool for monitoring sterility induced by gossypol.

[Capacitating action of GABA and progesterone in spermatozoa of human and guinea pig in vitro].

This study was designed to investigate whether GABA is involved in the capacitation effect and hyperactivated motility (HAM). Spematozoa from fertile men and retired guinea pigs were washed in modified BWW of 45% 90% Percoll gradient with 26 mg BSA/ml and in low Ca(2+)-MCM of 30%-55%-85% Percoll gradient (approximately 23 micromol/L Ca(2+)) respectively. The samples were preincubated for 2 h under 5% CO2 in air at 38.5 with or without GABA, progesterone (P(4)), GABA(A) receptor agonists or antagonists, and then exposed to 1 micromol/L (for human) or 5 micromol/L (for guinea pigs) calcium ionophore A 23187 for 15 min. The capacitation effect and HAM were assessed by using the chlortetracycline (CTC) staining method and phase-contrast microscopy. Motility was 80% 85% after all additions. The results showed that addition of GABA or P(4) at 5 micromol/L to the incubation medium resulted in a significant increase in the sum of B (characteristic of capacitated cell) and AR (acrosome recation) pattern (65.9% and 61.7% respectively), corresponding to capacitated spermatozoa in human, as compared to the control (37.3%). Likewise, the capacitating effect of GABA on spermatozoa in guinea pigs showed a concentration-dependent increase from 1 to 10 micromol/L(AR: 27.0 +/- 1.9% to 51.6 +/-2.8%). In addition, P(4) potentiated the capacitating effect of GABA when combined with GABA in the capacitation stage. The capacitating effect of GABA was mimicked by a GABAA receptor agonist muscimol. However, this effect was completely blocked by a GABA(A) receptor antagonist (-)-bicuculline and a GABA(A)/Cl(-) receptor antagonist picrotoxin. Furthermore, GABA markedly increased the HAM of P(4) on guinea pig spermatozoa, which was mediated obviously by the influx of extracellular Ca(2+) because the GABA-induced AR could be prevented by EGTA. These results indicate that GABA and P(4) are involved in the capacitation of spermatozoa in both human and guinea pigs through a GABA(A) receptor-mediated mechanism.

Effect of protein kinase C on guinea pig sperm acrosome reaction induced by progesterone.

AIM: To investigate the effect of protein kinase C (PKC) on the guinea pig sperm acrosome reaction (AR) induced by progesterone (P4). METHODS: Guinea pig spermatozoa were preincubated in MCM-Ca2+ DF medium at 38.5 degrees C under 5% CO2/air for 6-6.5 h for capacitation, and were then washed through a three-step gradient Percoll, resuspended in MCM-Ca2+ DF and adjusted to 5 x 10(9) cells/L. The AR was assessed by means of phase-contrast microscopy (expressed as percent of AR) 15 min after different reagents were added. RESULTS: In the presence of Ca2+ 2 mmol/L, phorbol 12,13-dibutyrate (PDB) or the permanent synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG) initiated the AR of spermatozoa in a concentration-related manner, whereas staurosporine (STA) exhibited an inhibition of the AR. PDB and OAG significantly enhanced P4-induced AR that was greater than that treated with either agonist separately. STA markedly inhibited the P4-induced AR. The AR induced by PDB was completely antagonized by inclusion of egtazic acid. Also, both egtazic acid and nifedipine antagonized the AR induced by P4 and PDB + P4. In addition, neomycin also prevented the P4-induced AR. This effect could be partly reversed by PDB. CONCLUSION: Activation or inhibition of PKC significantly plays an important role in the signal transduction pathway for the physiological AR.

[GABA initiates the acrosome reaction and fertilizing ability in human sperm].

The present study was undertaken to investigate whether GABA induced the acrosome reaction (AR) and fertilizing ability, as well as its possible mode of action in human spermatozoa. Spermatozoa from fifteen health fertile men isolated by the swim-up technique were preincubated in a modified BWW with 0.35% BSA for 1-11 h under 5% CO2 in 95% air at 37 degrees C. Aliquots of spermatozoa were collected at 0, 1, 3, 5, 7, 9 and 11 h of incubation for evaluation of the AR by chlortetracycline (CTC) staining. The sperm penetration assay (SPA) was carried out by using the zona-free hamster oocyte test. Intracellular calcium ([Ca2+]i) concentrations were determined by means of fluorescent probe Frua-2/AM. GABA at 1.25 mumol/L significantly induced the AR in human spermatozoa preincubated for 3 h, with a maximal response in preincubated for 9 h, and the effect changed in a concentration-dependent manner. The maximal stimulatory effect was observed with 1.25 mumol/L GABA, and the AR then decreased markedly with further increase of GABA concentration to 10 mumol/L. Exposure of preincubated spermatozoa to GABA in combination with progesterone resulted in a higher proportion of the AR as compared with that obtained with each agonist applied alone. In addition, GABA prompted a rapid increase in interacellular [Ca2+]i. Furthermore, the AR induced by GABA was prevented by inclusion of 1 mmol/L EGTA or 100 mumol/L La3+. Also, GABA enhanced significantly the ability of spermatozoa penetrating zona-free hamster oocytes and the index of fertilization. These results indicate that GABA may be involved in the modulation of the AR and the fertilization process in capacitated human spermatozoa through a calcium mediated mechanism, thus opening up possibilities for studies of signal transductions through activation of the GABAA receptor present on the sperm surface.

gamma-Aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa.

The sperm acrosome reaction takes place in response to progesterone and zona pellucida. Progesterone may act on more than one type of surface receptor, of which one is a gamma-aminobutyric acid (GABA) type A-like receptor. Although there is direct evidence of GABA initiation of mouse sperm acrosome reaction, there are conflicting results regarding GABA-induced exocytosis in human spermatozoa. We have examined whether GABA would initiate exocytosis in human spermatozoa using the chlortetracycline assay and a zona-free hamster oocyte test. Human spermatozoa preincubated for > or = 3 h in Biggers-Whitten-Whittingham medium with 0.35% bovine serum albumin underwent acrosome reactions in response to GABA, with maximal responses in spermatozoa preincubated for 9 h. The effect was concentration-dependent. Preincubated spermatozoa treated with GABA were able to fertilize a higher proportion of zona-free oocytes, with a higher number of spermatozoa penetrating each oocyte. Exposure of preincubated spermatozoa to GABA and progesterone together resulted in a higher proportion of acrosome reactions than when each agonist was used alone. The effect of GABA was mediated by the influx of extracellular Ca2+ because inclusion of EGTA or the Ca2+ channel antagonist La3+ prevented GABA-induced acrosome reactions. These results indicate that GABA can initiate exocytosis in capacitated human spermatozoa and open up possibilities for studies of signalling mechanisms activated upon occupancy of the GABAA receptor present on the sperm surface.

[The transducing pathway of Ca2+ influx during progesterone-initiated acrosome reaction of guinea pig sperm].

Progesterone and gamma-aminobutyric acid (GABA) could initiate the acrosome reaction (AR) in guinea pig spermatozoa, which was independent of extracellular Cl- in vitro, and a potentiation effect between progesterone and GABA was demonstrated. Antagonists of GABAA receptor/Cl- channel, picrotoxin and bicuculline, have no effect on the progesterone-induced AR, but they can significantly inhibit the GABA-induced AR in (Cl-)-containing medium. While in (Cl-)-deficient medium, picrotoxin has significant inhibitory effect on both progesterone- and GABA-induced AR, both progesterone- and GABA-induced AR are inhibited by the Ca2+ channel blocker, nifedipine. These results suggest that a GABAA/Cl- receptor complex and a Ca2+ channel might be involved in the influx of extracellular Ca2+ during the progesterone-induced AR.

Bicarbonate/CO2 is not required for zona pellucida- or progesterone-induced acrosomal exocytosis of mouse spermatozoa but is essential for capacitation.

We investigated whether bicarbonate/CO2 is required for capacitation or for acrosomal exocytosis triggered with zona pellucida or progesterone. Mouse spermatozoa, incubated for 90 min in a modified Tyrode's medium with bicarbonate and equilibrated with 5% CO2 in air, were washed in medium without bicarbonate but with Hepes, resuspended in media with or without bicarbonate, and then stimulated with 1 zona pellucida/microliter or 15 microM progesterone. Spermatozoa were able to undergo exocytosis, regardless of the medium in which they were resuspended, as ascertained via the chlortetracycline assay. If, however, spermatozoa were first incubated in a medium in which bicarbonate was replaced by Hepes and were then washed and resuspended in various media, they were unable to undergo exocytosis in response to zona pellucida or progesterone even when resuspended in bicarbonate-containing medium. This indicated that spermatozoa were not capacitated. Furthermore, the proportion of cells exhibiting the "B" pattern (characteristic of capacitated cells) after incubation in medium without bicarbonate was lower than that in cells incubated with the anion and the appropriate gas phase. Extended incubation in medium without bicarbonate (up to 3 h) did not increase the proportion of cells that exhibited the "B" pattern. These results demonstrate that bicarbonate is not required for acrosomal exocytosis but that it is essential for capacitation, exerting roles beyond its action as pH buffer.

Inhibition of rabbit sperm acrosomal enzymes by gossypol.

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12-76 microM gossypol. Hyaluronidase, beta-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 microM). Phospholipase C, alkaline phosphatase, and beta-N-Acetyl glucosaminidase were not inhibited even at 380 microM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 microM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurrence of gossypol-induced sterility.

Evidence that a GABAA-like receptor is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

We have investigated whether progesterone-triggered acrosomal exocytosis involves the activation of a gamma-aminobutyric acid (GABA) receptor, and whether activation of this receptor is linked to Ca2+ entry via Ca2+ channels. Mouse spermatozoa preincubated in a modified Tyrode's medium underwent exocytosis when stimulated with progesterone, as revealed by an increase in the number of cells exhibiting an "AR" pattern after staining with chlortetracycline; the effect was concentration dependent. Only capacitated spermatozoa underwent exocytosis in response to progesterone: cells preincubated for 15 min (uncapacitated) did not exocytose in response to this agonist, whereas cells preincubated for 120 min did. Stimulation of capacitated spermatozoa with GABA or muscimol, two GABAA receptor agonists, resulted in acrosomal exocytosis; this response was enhanced by half-maximal concentrations of progesterone. Bicuculline, a GABAA receptor antagonist, inhibited exocytosis stimulated by progesterone or GABA. Picrotoxin, another GABAA receptor antagonist, inhibited only GABA-stimulated exocytosis. These results suggest that progesterone effects are mediated by a GABAA receptor but that such receptor may not be identical to the neuronal GABAA receptor. The ability of progesterone, GABA, or muscimol to stimulate exocytosis was blocked by the Ca2+ channel antagonists verapamil or nifedipine. The Ca2+ channel agonist Bay K 8644, on the other hand, stimulated exocytosis in capacitated sperm cells. The stimulatory ability of progesterone and Bay K 8644 was additive. These results indicate that acrosomal exocytosis involves activation of a GABAA receptor apparently linked to Ca2+ channels.

[In vitro inhibition of celastrol on spermatozoa fertilization ability of guinea pig].

The effects of celastrol (Cel), isolated from Tripterygium wilfordii, on guinea pig sperm forward motility (FM), capacitation (Cap), the acrosome reaction (AR) and sperm penetration assay (SPA) were assessed in vitro. Cel (5 micrograms.ml-1) was found to inhibit these spermatozoal functions, and the inhibitions were proportional to the concentrations of Cel used. The potency of inhibition of Cel on the fertilizing ability in guinea pig spermatozoa in vitro seems to follow the order: Cap > FM > SPA > AR. The inhibitory effect appeared to be reversible after washing away Cel if the duration of exposure of spermatozoa to Cel was shorter than 3 h. In a comparative study, the inhibitory effects of Cel on guinea pig sperm FM and AR were significantly stronger than those of gossypol acetic acid.

Exocytosis in spermatozoa in response to progesterone and zona pellucida.

Exocytosis in mammalian spermatozoa (the acrosome reaction) is a process essential for fertilization. Both progesterone and zona pellucida induce exocytosis in spermatozoa, which may encounter both during penetration of the oocyte's vestments. When mouse spermatozoa were exposed first to progesterone and then to zona pellucida, exocytosis was enhanced to a greater degree than that seen when the agonists were presented together or in the inverse order, which suggests that the steroid exerts a priming effect. Progesterone similarly primed the generation of intracellular messengers evoked by zona pellucida. The effects triggered by progesterone were mimicked by gamma-aminobutyric acid (GABA) and were blocked by bicuculline, which indicates that the steroid acts on a GABAA receptor.

Inhibition of spermine on calcium influx during capacitation of guinea pig spermatozoa in vitro.

To investigate the mechanism of action of spermine, we measured the intracellular calcium ([Ca]i) of guinea pig spermatozoa using a probe of fluorescence, Quin 2. Results showed that spermine (0.25-2.0 mmol.L-1) suppressed the membrane permeability to Ca2+ during capacitation, which was similar to that of verapamil (a Ca2+ channel blocker). Furthermore, the rapid increase of [Ca]i induced by calcimycin (A-23187) was inhibited by spermine and verapamil, whereas trifluoperazine (an inhibitor of calmodulin) had no effect on it. The inhibition of the acrosome reaction caused by verapamil (5-100 mumol.L-1) or trifluoperazine (1-60 mumol.L-1) was reversed by calcimycin and cAMP, respectively. In addition, there was no effect on the initiation of the acrosome reaction when verapamil was added to capacitated spermatozoa. This result was in agreement with that of spermine. When addition of spermine (0.5 mmol.L-1) was combined with trifluoperazine (5 mumol.L-1), the acrosome reaction decline almost to zero, indicating that spermine might inhibit Ca2+ sensitive channel during sperm capacitation.

Effect of spermine on sperm capacitation of guinea pig in vitro.

Spermine (Sp) 10(-5) mM had vigorous activity of guinea pig spermatozoa, while it completely abolished sperm forward motility (SFM) at a concentration of 10(-3) mM. There appeared to be a dose relationship to inhibition to motility. 2-Difluoromethylornithine 10 mM antagonized the Sp-induced inhibition of SFM after 3 h of incubation. Capacitation of a guinea pig sperm was inhibited by Sp in a concentration-dependent manner. The majority of acrosome-reacted sperm did not display hyperactivated motility. Precapacitated sperm were able to undergo the acrosome reaction (AR) in the presence of Sp. Moreover, Sp-mediated inhibition of capacitation was a reversible process. Once sperm capacitation was completed, Sp no longer inhibited AR. Before capacitation, the content of Sp in spermatozoa was 4.5 +/- 0.5 micrograms/5 x 10(7) cells, whereas in case of capacitated spermatozoa it was significantly decreased (2.1 +/- 0.4 micrograms/5 x 10(7) cells). The penetration of spermatozoa into the zona-free hamster eggs in the presence of Sp was markedly decreased, but it did not affect the fertilizability of ova as compared to the control. These results suggest that Sp may be an inhibitory agent of sperm capacitation in guinea pig in vitro, and it may also be involved in the modulation of capacitation.