Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Single channel currents in adrenocortical cells

Single channel currents have been recorded from cell-attached patches of tumoral adrenocortical cells. Our experiments suggest the existence of three sets of potassium channels in the surface membrane of these cells. All channel types can be recorded in a given membrane patch but some patches have only one type of single channel currents. One channel type has a unitary conductance of about 103 pS. The other two channels have smaller conductances and opposite voltage dependence. In one case channels open on depolarization and have a single channel conductance of 31.6 pS. In the other case the probability of being in the open state increases on hyperpolarization and the single channel conductance is of 21 pS. These channels seem to be similar to the delayed and anomalous rectifying potassium channels seen in other preparations. The role of membrane ionic permeability in steroid release induced by ACTH is discussed.     

A voltage-sensitive cation channel present in clusters in lobster skeletal muscle membrane

The single channel properties of a voltage-sensitive cation channel are described in a study of ion channel activity in enzymatically induced blebs of lobster skeletal muscle membrane. This cation channel, one of several that are spontaneously active in excised patches from bleb membrane, can be distinguished from other channels on the basis of its large single channel conductance (293 pS), voltage-sensitive gating properties, the presence of a subconductance state of the fully open channel, and a weak selectivity for K>Na. At hyperpolarizing voltages, this channel displays flickering or bursting behavior, and a single state of the fully open channel is observed. At depolarizing voltages, the mean channel open time increases and a second longer-lived open state is observed. The voltage dependence of the mean channel open time and the linear i-V relation of this channel predict that the macroscopic current carried through this cation channel would be outwardly rectifying.Channels of this type are infrequently observed in this preparation, but when present in the patch are often present in multiple copies. We describe a statistical test for examining the clustering of ion channels in excised patches of membrane. The result of this test shows that the cation channels appear in clusters in the blebs.The authors gratefully acknowledge the technical assistance of Ms. Nicole Cherbuliez. M.K.W. was the recipient of an NRSA (5F32NS08605) and a Grass Foundation Fellowship (Marine Biological Laboratory, Woods Hole). This investigation was supported by a Program Project Grant (NS25915; E.A.K., Principal Investigator) and by grants from the US-Israel Binational Science Foundation and Muscular Dystrophy Association.     

Concanavalin A modulates a potassium channel in cultured Aplysia neurons

A novel 100 pS K(+)-selective ion channel is frequently observed in cell-attached membrane patches from cultured Aplysia neurons. The activity of this channel is moderately voltage-dependent, but channel openings are rare and brief even when the patch is strongly depolarized. However, the activity of the channel is increased dramatically by the addition of the lectin concanavalin A (Con A), to the patch pipette. The channel is also activated by Con A in the bathing medium, suggesting that the lectin's action is via an as yet unidentified intracellular second messenger. In the one single-channel patch studied, Con A had no effect on the channel mean open time; rather it decreased the average duration of the long closed times between bursts of openings. Thus Con A increases either the open probability of single channels, the number of functional channels in the patch, or both. The functional significance of the Con A-induced modulation of K+ channel activity remains to be determined.     

IS3 peptide-formed ion channels in rat skeletal muscle cell membranes

A 22-mer peptide, identical to the primary sequence of domain I segment 3 (IS3) of rat brain sodium channel I, was synthesized. With the patch clamp cell-attached technique, single channel currents could be recorded from the patches of cultured rat myotube membranes when the patches were held at hyperpolarized potentials and the electrode solution contained NaCl and 1 microM IS3, indicating that IS3 incorporated into the membranes and formed ion channels. The single channel conductances of IS3 channels were distributed heterogeneously, but mainly in the range of 10-25 pS. There was a tendency that the mean open time and open probability of IS3 channels increased and the mean close time decreased with the increasing of hyperpolarized membrane potentials. IS3 channels are highly selective for Na+ and Li+ but not for Cl- and K+, similar to the authentic Na+ channels.     

Sialyl glycoprotein distribution on the plasma membrane of ejaculated ram spermatozoa

Localization of sialyl residues on unfixed ejaculated ram sperm membrane using the direct covalent probes of either ferritin hydrazide or latex hydrazide revealed a unique regional distribution on the plasmalemma covering the sperm head only. Three different labelling zones were identified based on the intensity and the nature of the sialyl glycoconjugates: a patchy-like zone which included the plasma membrane overlaying the post-nuclear cap and the convex side of the apical body of the acrosome; highly ordered heavily labelled zones including the plasmalemma adjacent to the concave apical body of the acrosome and to the posterior part of the equatorial acrosomal segment; a paucity-labelling zone which included the plasma membrane underlying the principal acrosomal region and the anterior part of the equatorial acrosomal segment. The possible physiological role of the highly ordered labelled zones is discussed.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K~ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage-sensitive channels. Single ATP-sensitive K (K-ATP) channel currents were recorded in acutely dissociated rat neo-cortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intra-cellular ATP, but also by membrane potential ( Vm) , and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and di-versity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activa-tion-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary con-ductance of about 80 pS in detail ( n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane poten     

Methylation increases the open probability of the epithelial sodium channel in A6 epithelia

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.     

Sperm surface changes during the acrosome reaction as observed by freeze-fracture

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

ELECTRON MICROSCOPIC OBSERVATIONS OF GUINEA PIG SPERMATOZOA PENETRATING EGGS IN VITRO*

Guinea pig ovarian oocytes matured in vitro were inseminated in vitro with capacitated, acrosome-reacted spermatozoa and sperm penetration through the zona pellucida and into the egg cytoplasm were examined. Sperm heads passing through the zona pellucida had already lost all their acrosomal elements except for the inner acrosomal membrane and the equatorial segment. It was often observed that the texture of the zona material around the sperm head was distorted, giving the impression that the zona pellucida was parted, at least partially, by a shearing force produced by the sperm head advancing through the zona. When eggs were freed from their zonae pellucidae and inseminated, the acrosome-reacted spermatozoa immediately bound to the egg surfaces and began to fuse with the eggs; whereas the spermatozoa with intact acrosomes failed to do so. Fusion began between the egg plasma membrane and the sperm plasma membrane at the central region of the sperm head. The anterior half of the sperm head was engulfed by the egg in a phagocytic fashion, while its posterior half was incorporated into the egg by a fussion between egg and sperm plasma membranes. Incorporation of the sperm tail into the egg was achieved by fusion between the sperm and egg plasma membranes.     

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function. Project supported by the National Natural Science Foundation of China and Natural Science Foundation of Guangdong Province     

Assessing acrosomal status of bovine sperm using fluoresceinated lectins

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.     

Low-conductance states of K+ channels in adult mouse skeletal muscle

Single-channel currents were recorded from Ca2+-activated or ATP-sensitive K+ channels in inside-out membrane patches excised from isolated mouse toe muscles. In addition to the closed and fully open configurations, both types of channels may exhibit several intermediate low-conductance states which are clustered near multiples of elementary conductance units. The units are 1/8 or 1/6 of the channel conductance for Ca2+-activated channels and 1/4 or 1/3 for ATP-sensitive channels. Normally, low-conductance states are rare, but they occur more frequently directly after patch excision. An increased probability of low-conductance states of ATP-sensitive K+ channels was also observed in the presence and during washout of the internal channel blocker adenine. The results suggest that Ca2+-activated and ATP-sensitive K+ channels are composed of several membrane pores with strong positive cooperativity among the elementary conductance units.