Requirement of an extracellular energy substrate for the guinea pig sperm acrosome reaction induced by calcium ionophore

It is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore-induced AcR of guinea pig spermatozoa. It was found that 0.3 microM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate-free modified Tyrode's medium containing 0.3 microM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore-induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore-induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore-induced AcR of guinea pig spermatozoa.     

Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig

The presence of caltrin-like proteins in reproductive tract fluid (RTF) and seminal vesicle content from male guinea pigs has been determined. Two fractions with electrophoretic mobility corresponding to Mr = 6200 (main band) and 5100 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Isoelectric focusing in thin-layer agarose gels revealed three bands with acidic pIs of 5.3, 6.0, and 6.2, respectively. RTF prevented the enhancement of calcium permeability induced by incubating guinea pig epididymal spermatozoa in medium for capacitation. Spermatozoa incubated for 2 h in minimal culture medium plus pyruvate and lactate containing RTF accumulated less than 30% of the 45Ca2+ accumulated by cells maintained in absence of this fluid. Calcium uptake by preincubated spermatozoa was also inhibited by RTF. Inhibition of calcium transport activity by RTF and seminal vesicle proteins was not decreased by heating the dialyzed preparations at 60 degrees C for 5 min. After this treatment, the inhibitory activity and the protein pattern were stable for 3 wk when stored at 4 degrees C. Unheated extracts lost calcium transport inhibitory activity after 2 or 3 days at 4 degrees C. In spite of the differences in pIs among the proteins from the guinea pig reproductive tract and bovine caltrin, several features indicate they may play a similar role in both species by controlling Ca2+ movement across the plasma membrane. By this mechanism, these proteins could regulate physiologic events essential for the fertilization process.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Glucose effect on respiration: possible mechanism for capacitation in guinea pig spermatozoa.

Guinea pig sperm respiration was determined in minimal capacitation medium (MCM) with different energy sources. The ZO2 observed for spermatozoa suspended in media containing pyruvate and lactate was 35.7 +/- 5.9, pyruvate alone, 27.9 +/- 3.8 and D-glucose alone 3.4 +/- 1.1. When D-glucose was added to spermatozoa rapidly respiring in media containing pyruvate as the only exogenous energy source, an immediate suppression in respiration was observed. Further reduction was caused by continued addition of D-glucose. Fructose and mannose also produced a suppression in respiratory rate. However, lactose, fucose, sucrose, L-glucose, and galactose did not alter the respiratory rate. The suppression of respiration by metabolizable sugars is paralleled by a suppression of acrosome reaction in guinea pig spermatozoa. The possibility that suppression of respiration is the mechanism for retardation of capacitation and the subsequent acrosome reaction by D-glucose and other metabolizable sugars is suggested.     

On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre-incubated in Tyrode's pyruvate lactate glucose medium (T-PLG) or Tyrode's glucose solution (T-G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM-PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome-reacted spermatozoa were observed within 5 min when cells were transferred to MCM-PL solution, after preincubating them for 60–120 min either in T-PLG or T-G media. By 15 min in MCM-PL the percentage of acrosome-reacted spermatozoa reached values similar to those obtained in cells pre-incubated from the beginning in MCM-PL medium (P > 0.05 in both) but significantly different from T-PLG and T-G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T-PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose-6-phosphate (as 2-deoxyglucose-6-phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

A requirement of bicarbonate for Ca2+-induced elevations of cyclic AMP in guinea pig spermatozoa

Ca2+ causes less than 2-fold elevations of guinea pig sperm cyclic AMP concentrations when cells are incubated in a minimal culture medium in the absence of bicarbonate (HCO3-). However, in the presence of HCO3-, Ca2+ increases cyclic AMP by as much as 25-fold within 1 min. The (Ca2+, HCO3-)-induced elevations occur in either the presence or absence of the permeant anions, pyruvate and lactate. In the absence of extracellular Ca2+, HCO3- elevates cyclic AMP only slightly. The effect of HCO3- is concentration-dependent, with maximal responses obtained at concentrations of greater than 25 mM. Ca2+ (25 mM HCO3-) at concentrations of less than 100 microM causes one-half-maximal elevations of cyclic AMP. The (Ca2+, HCO3-)-induced elevations of cyclic AMP are observed at various extracellular pH values (7.5-8.5) and in the presence or absence of extracellular Na+ or K+. NH4Cl does not elevate sperm cyclic AMP concentrations and does not greatly alter the (Ca2+, HCO3-)-induced elevations. the putative Ca2+ transport antagonist, D-600 (100 microM), completely blocks the (Ca2+, HCO3-)-induced elevations of cyclic AMP. A23187, in the presence but not in the absence of extracellular Ca2+, increases sperm cyclic AMP but does not further elevate cyclic AMP in HCO3(-)-treated cells. These studies establish that Ca2+-dependent elevations of cyclic AMp in guinea pig spermatozoa are dependent on the presence of HCO3- and suggest that HCO3- is required for the uptake (exchange) or membrane sequestration of small amounts of physiologically active Ca2+.     

Ca2+ uptake during capacitation of mouse spermatozoa and the effect of an anion transport inhibitor on Ca2+ uptake

With a specially constructed chamber, Ca2+ uptake by mouse spermatozoa was monitored continuously during capacitation and the acrosome reaction. It was shown, using calcium ion-selective microelectrodes, that there was an initial uptake of Ca2+ by spermatozoa undergoing capacitation. Such net transport was also promoted by the divalent cation ionophores A23187 or ionomycin. An anion inhibitor, SITS, produced dose-dependent inhibition of Ca2+ uptake. This inhibitor reduced the incidence of capacitation as revealed by a reduction in the B pattern by chlortetracycline (CTC) assay and thus inhibited fertilization, suggesting that anions are involved in calcium uptake in mouse spermatozoa.     

In vitro capacitation of goat spermatozoa

Goat epididymal and ejaculated spermatozoa were incubated in Krebs-Ringer bicarbonate buffer containing pyruvate and lactate as energy source. A 3 hr incubation for epididymal and 4 hr for ejaculated spermatozoa was required for the capacitation and acrosome reaction to take place. Calcium is an essential requirement which was needed for motility maintenance/activation and for the initiation of acrosome reaction. A 2-fold increase in cAMP content was measured over 3 hr period of incubation of goat epididymal spermatozoa which was not seen when calcium ions were either omitted or chelated with EGTA. There is thus a definite involvement of Ca2+ ions and cAMP in capacitation and acrosome reaction of goat spermatozoa.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Induction of the acrosome reaction in guinea pig spermatozoa by cGMP analogues

The effect of cyclic nucleotide analgoues upon the immediate induction of the guinea pig acrosome reaction (AR) was studied. Dibutyryl (dB) CGMP and 8-bromo-cGMP, when added to sperm suspensions after varying periods of preincubation in glucose-free BWW medium (NaCl 94.59 mM, KCl 4.7 mM, CaCl2 1.71 mM, KH2PO4 1.19 mM, MgSO4 1.19 mM, NaHCO3 25.07 mM, pyruvate 0.25 mM, lactate 21.58 mM, and bovine seru albumen 1 g/liter), induced the AR in a large proportion of spermatozoa relative to controls. The proportion of ARs induced upon the addition of dB cGMP or 8-bromo-cGMP (10mM) at 1 h was equivalent to that obtained after a 5-h incubation in glucose-free BWW alone. The effect of cGMP analogues was concentration dependent over the tested range of 2-12 mM (less than 1-20%). The simultaneous addition if imidazole (10 mM), a cAMP phosphodiesterase stimulator, potentiated the effect (imidazole + 12 mM 8-bromo-cGMP: 73%). cAMP analogues were without effect. The presence of extracellular Ca++ was required, and it is suggested that a rise in the cGMP/CAMP ratio triggers Ca++ influx and the AR.     

Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa

Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45 min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20 μM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5 μM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.     

Mechanism for procaine-mediated hyperactivated motility in guinea pig spermatozoa

Hyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine-treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca²⁺ did not inhibit procaine-induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca²⁺ for sperm to express hyperactivated motility. © 1994 Wiley-Liss, Inc.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate.

Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Calcium dependence of human sperm fertilizing ability

The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.     

Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane in mezaton and histamine regulation of electrical and contractile activity in smooth muscle cells from the guinea pig ureter

Influence of Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane on electrically-induced action potential and contraction of smooth muscle cells from guinea pig ureter was examined with the methods of the double sucrose gap junction. Mesatone (10 microM) and histamine (10 microM) induced prolongation of the action potential and elevation of smooth muscle cell contraction, whereas hyperosmic medium (+150 mM sucrose), and recovery of solution osmolality in hyposmic condition (70 mM NaCl) after a single contraction. Inhibitor Na+,K+,2Cl(-)-cotransport bumetanide (10 microM) and chloride permeability blockers niflumic acid (10-100 microM) and SITS (10-500 microM) attenuated stimulating effects of mesatone, histamine and hyperosmic medium. In opposite to adenylate cyclase activation with forskolin (1 microM), guanylate cyclase activation with sodium nitroprusside (SN, 100 microM) decreased both inhibitory action of bumetanide, niflumic acid and activating effects of mesatone, histamine on action potential and elevation contraction of smooth muscle cells. Influence of forskolin rather and not SN on AP and SMC C was inhibited with tetraethylammonium (5 mM). These results suggest that influence of Na+,K+,2Cl(-)-cotransport on electrical and contractil properties of ureter smooth muscle cells is mediated by stimulation of Ca(2+)-activated chloride permeability of the cell membrane and modulated by intracellular cGMP, but not triggered by Ca2+ release from sarcoplasmic reticulum.