Tail Membrane Research Articles

Silane-coated silica particle colloid processing of human sperm.

The purpose of this study was to determine differences in the quality of human sperm processed through different lots of silane-coated silica particle colloid solutions. The objectives were to compare (a) sperm kinematic parameters, (b) the sperm acrosome status, (c) the membrane integrity of the head and tail regions, (d) the DNA normality, and (e) the heat-inducible hyperactivation motility after processing sperm through either a Silane-coated silica particle colloid solution, a Percoll solution, or a simple centrifuge sperm wash (control). Sperm cells were derived from pooled cryopreserved-thawed specimens of several donors (n = 10). The pooled sperm were divided and processed through either the centrifuge wash, the 90:47% two-layer Percoll, or one of three lots of silane-coated silica particle colloidal solutions from three vendors. Aliquots of sperm cells were analyzed using the Hamilton-Thorn HTM-C motility analyzer for differences in kinematics and hyperactivation. Sperm were also analyzed for membrane integrity at both head and tail regions, normal morphology, acrosome status, and viability. Sperm undergoing apoptosis were determined using the acridine orange stain. Processed sperm were also incubated at 40 degrees C for 4 hr and the quality of the sperm was assessed using the heat-induced hyperactivation and motility parameter. The data showed that after sperm processing, the number of sperm recovered was higher for the three lots of colloids (silane-coated silica particle colloid solutions) compared with Percoll processing. Total sperm motility was higher in the colloidal washes compared with the control. There were no differences in motility between Percoll- and colloid-processed sperm. In contrast, the percentages of sperm exhibiting progressive motility or hyperactivation varied among the different lots of colloid solutions. The Percoll wash solution yielded the highest percentage of sperm with intact tail membranes, whereas some lots of colloid solutions disrupted sperm head membranes. The percentages of sperm undergoing apoptosis varied for the different lots of colloid solutions. There was a marked increase in hyperactivation associated with one colloid solution after heat induction. The results demonstrated variability in the different lots of silane-coated silica particle colloid solutions for processing sperm. Each lot of colloid solution excelled at improving different sperm parameters. The silane-coated silica particle colloid solutions were shown to be effective in recovering motile sperm compared with Percoll but the types of motility and sperm quality varied for the different lots of colloid solutions. Due to the variability in lots of silane-coated silica colloid solutions, reported studies based on only one lot or one source of colloid solution may be difficult to interpret. Furthermore, it may be advantageous to select the best lot of silane-coated silica particle colloid solution to produce the highest number of sperm exhibiting the ideal parameters for use in assisted reproduction technologies.

Mechanical properties of bat wing membrane skin

The skin of the bat wing in functionally unique among mammals: it serves as a major locomotor organ in addition to its protective and regulatory functions. We used tensile testing to investigate the mechanical capabilities of wing membrane skin, and compared stiffness, strength, load at failure, and energy absorption among specific wing regions and among a variety of bat taxa. We related these characteristics to the highly architectural fibrous supporting network of the wing membrane. We found that all material properties showed a strong anisotropy. In particular, wing membrane skin shows maximum stiffness and stregth parallel to the wing skeleton, and greatest extensibility parallel to the wing's trailing edge. We also found significant variation among wing regions. The uropatagium (tail membrane) supported the greatest load at failure, and the plagiopatagium (proximal wing membrane between laterl body wall and hand skeleton) is the weakest and most extensible part of the wing. We believe that the increased load bearing ability of the uropatagium relats to its key role in capture of insect prey, and that the great extensibility of the plagiopatagium promotes development of camber near the wing's centre of lift. In interspecific comparisons, energy absorpion and load to failure were greatest in Artibeus jamaicensis, the largest bat in our sample and the species with the highest wing loading, suggesting that wing loading may play a role in dictating the fuctional design of wing membranes.

Evidence for Ca2+-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm

Membrane preparations from mouse sperm heads and tails were used in a gamma 32P-ATP hydrolysis assay to investigate Ca(2+)-dependent ATPase activity. In membranes from sperm heads, but not tails, a Ca2(+)-dependent ATPase that was further stimulated by calmodulin was detected. The addition of partially purified mouse sperm decapacitation factor (DF) to head membrane preparations significantly stimulated Ca(2+)-ATPase activity, this effect being further increased in the presence of DF plus calmodulin; in contrast, no response was observed when the same treatment was applied to tail membranes. Sperm preincubated in the presence of trifluoperazine (TFP), a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing 45Ca2+ +/- DF, their ability to accumulate 45Ca2+ was significantly lower in the early stages after resuspension in the presence of DF than in its absence. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in medium only, there was a noticeable shift from the F pattern (characteristic of uncapacitated cells) to the B pattern (characteristic of capacitated cells), but the reintroduction of DF caused a significant reversion to the F pattern. Finally, using a monoclonal antibody to somatic cell Ca2(+)-ATPase, we have obtained evidence that the enzyme is particularly localized to the postacrosomal region of the mouse sperm head; specific binding was observed only in permeabilized cells, indicating that the epitope involved in the binding has an intracellular location. Based on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca(2+)-ATPase activity and thus ensures maintenance of a low intracellular Ca2+ concentration. As capacitation proceeds, DF is lost and Ca2(+)-ATPase activity declines, allowing intracellular Ca2+ to rise and promoting capacitation-related changes. The fact that inhibitors of Ca(2+)-ATPase and calmodulin appear to accelerate capacitation in several mammalian species, as determined by chlortetracycline analysis, suggests that Ca(2+)-ATPase activity may play an important role in modulating capacitation in many or even all mammals.

Identification of heterotrimeric G proteins in human sperm tail membranes.

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein alpha-subunits, and A 86, which detects all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein alpha-subunits failed to detect any specific antigens in enriched tail membranes. AS 36, recognizing the beta 2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive alpha-subunits. However, membrane preparations of nonpurified human spermatozoa contained alpha i2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein alpha-subunits; the putative beta-subunit was identified as a beta 2-subunit.

Insect pursuit, prey capture and echolocation in pipestirelle bats (Microchiroptera)

The foraging and echolocation behaviour of three European pipistrelles ( Pipistrellus pipistrellus, P. nithusii and P. kuhlii) was studied under natural conitions. The pipistrelles were photographed with two 35 mm cameras under stroboscopic illumination, and their echolocation signals were recorded simultaneously. This permits a three-dimensional reconstruction of the flight paths of bat and prey, and allows the details of echolocation behaviour to be studied in the context of natureal foraging behaviour. The general relationships between foraging and echolocation behaviour were consistent among the three species. Foraging behaviour consisted of four stages: search flight (before detection of prey), approach flight (pursuit after detection of prey), capture and retrieval of prey. These stages correlated with phases in echolocation behaviour: search, approach, and terminal phase followed by a pause. Detection of prey occurred at distances of 1·14−2·20 m. The search cone extending from the bat's mouth was up to 150° wide. The pipistrelles caught prey in mid-air, either with the tail membrane alone or by funnelling it with a wing onto the tail membrane. Except for some intra- and interspecific differences in sound duration, pulse interval, bandwidth and terminal frequency in search phase, the structure and pattern of the echolocation signals were similar in the three pipistrelles. In the approach and terminal phases, pulse duration and pulse interval decreased with the approach to the target, while bandwidth and sweeprate increased. While pursuing insects, the pipistrelles precisely avoided an overlap between outgoing signal and the echo returning from the prey. Furthermore, the bats stopped emitting signals several centimeters before they reached the insect.

Improvement in quality of cryopreserved human spermatozoa by swim‐up before freezing

Selecting a population of spermatozoa by the swim-up technique yields, after freezing and thawing, a population of cells that contains proportionally more spermatozoa which are morphologically normal, fewer spermatozoa with damaged tail membranes, and a greater percentage of progressively motile spermatozoa with greater velocities and amplitudes of head displacement than those obtained after freezing and thawing the same semen samples in the normal way. This pattern was found for the semen from 10 patients and five volunteers. However, the cells selected by swim-up were as susceptible to the stresses caused by freezing and thawing as unselected spermatozoa in the original semen sample, and the improvement came from the better quality of the initial sample.

White blood cells in semen affect hyperactivation but not sperm membrane integrity in the head and tail regions

The presence of high numbers of peroxidase-positive PML in ejaculated semen significantly reduced sperm HA, an important step leading to sperm capacitation. Sperm membranes at both the head and tail regions, as assessed by the hypo-osmotic viability parameter and the hypo-osmotic sperm swelling test, respectively, were not affected by peroxidase-containing leukocytes. Sperm motility was not affected, but sperm curvilinear and straight line velocity parameters were reduced in the presence of high concentrations of leukocytes in the ejaculate. The results suggested that the effect of leukocytes on sperm was through a reduction in sperm hyperactive motility but not through alterations in the sperm head and tail membranes.

Assessment of sperm for cryopreservation using the hypoosmotic viability test

In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated that the integrity of the sperm membranes at the head were more important than the tail membrane.

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.

Membrane association of flagellar creatine kinase in the sperm phosphocreatine shuttle.

The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.

Combined supravital staining and hypoosmotic swelling test

The parameter of sperm viability in hypoosmotic solution (VHOS) provides information concerning the membrane integrity of the sperm head. The objective of this study was to determine the association between the VHOS parameter and the sperm penetration assay. The VHOS parameter correctly predicted 70.0-71.4% of the failed sperm penetration assay samples in the short duration preincubation groups. A combination of both the VHOS parameter and the hypoosomotic sperm swelling (HOS) test significantly reduced the number of false negative results. In general, a sperm sample with an abnormal VHOS result and an abnormal HOS test result would be associated with a negative sperm penetration assay. Washing by centrifugation appeared to weaken the sperm head membranes while the swim-up method selected for sperm with strong head and tail membranes. After the various processing methods unique changes in the integrity of the sperm head and tail membranes for each sperm sample may help to identify the optimal method of preparation for individual patients undergoing the newer assisted reproductive technologies such as sperm microinjection.

Intracellular pH regulates bovine sperm motility and protein phosphorylation.

Bovine sperm in neat caudal epididymal fluid become motile in response to either pH elevation or dilution of the fluid. Buffers containing permeant weak acids at physiologic concentrations are able to mimic these effects of caudal fluid. These observations lead to the hypothesis that a pH-dependent epididymal fluid quiescence factor regulates bovine sperm motility by modulating sperm intracellular pH (pHi). Here we report that sperm pHi, measured with the fluorescent pH probe carboxyfluorescein, increases by approximately 0.4 units in response to either of these motility-initiating manipulations. At least 26 discrete phosphoprotein bands are distinguishable by sodium dodecylsulfate-polyacrylamide gel electrophoresis after incubation of intact caudal sperm with 32PO4. A prominent phosphoprotein, with Mr approximately 255,000 (pp255) and a relatively high specific radioactivity, is reversibly dephosphorylated in response to elevations in pHi that initiate sperm motility. Unlike most of the sperm phosphoproteins, the extraction of pp255 requires reducing agents. This phosphoprotein cosediments with the sperm heads but not the tail, midpiece, soluble, or plasma membrane fractions. No other pHi-dependent phosphorylation changes are apparent in gels of whole sperm extracts. However, subcellular fractionation allows the detection of increased phosphorylation of two plasma membrane phosphoproteins (Mr approximately 105,000 and 97,000) and decreased phosphorylation of another plasma membrane phosphoprotein (Mr approximately 120,000) in response to increasing pHi. This is the first report describing changes in endogenous phosphoproteins from intact motile and nonmotile bovine sperm that are regulated by pHi.

A study of the electrical characteristics of sodium currents in single ventricular cells of the frog.

1. The generation of action potentials elicited from enzymatically dispersed ventricular cells from the frog, Rana catesbeiana, has been shown to be due to the influx of both Na+ and Ca2+. The maximum rate of rise, the amplitude and the duration at 50% repolarization of the action potential were estimated to be 26.4 +/- 5.1 V/s (n = 8), 110 +/- 2.7 mV (n = 8) and 601 +/- 180 ms (n = 8) at 15 degrees C, respectively. 2. Inward Na+ current (INa) was studied in these ventricular cells by the whole-cell patch clamp technique in a medium where Ca2+ current was eliminated by substituting extracellular Mg2+ for Ca2+ and K+ current was suppressed by applying Cs+ intracellularly. All the voltage clamp experiments were carried out at 4 degrees C. 3. INa elicited by single depolarizing steps from a holding potential (VH) of -80 mV had a threshold of -50 mV and was maximal at -20 mV. Peak currents in normal Ringer solution containing 113.5 mM-Na+ were of the order of 0.01-0.02 mA/cm2. Maximum Na+ conductance (gNa) was calculated to be 5.9 mS/cm2. 4. Under normal conditions the reversal potential for INa was determined to be 50 mV, which is close to the value predicted from the Nernst equation. The reversal potential changed by 59 mV per tenfold change in the activity of extracellular Na+ (aNa). 5. The instantaneous relation between INa tail currents and membrane potential is linear, crossing the abscissa at the reversal potential for INa. 6. Reconstructions of INa were made in terms of the parameters of the Hodgkin-Huxley model for the squid axon, using constants obtained from the frog ventricular cells. 7. The falling phase of INa and the development of inactivation measured by the double-pulse method could be well fitted by a single-exponential function. 8. The time course for recovery of INa from inactivation exhibited a single time constant.

Regional differentiation of the sea urchin sperm plasma membrane.

In order to study the molecular basis for the functional localization and behavioral control of sperm, we have partially characterized plasma membranes prepared from isolated head and tail fractions. These membranes have similar amounts of the Na+ pump (as reflected by (Na+,K+)-ATPase activity), whereas they differ in protein composition, binding sites for Ca2+ channel antagonists, and in the localization of enzymes of cyclic nucleotide metabolism. The Ca2+ channel antagonist D600 (and related phenylalkylamines) binds to plasma membrane preparations from sperm heads and tails with much higher affinity than do the dihydropyridine antagonists. This binding is inhibited greatly by certain monovalent (but not divalent) ions, especially Na+, Tris+, glycine ethyl ester+, and methylamine+.K+,Li+, and choline+ are less effective. In media of ionic composition resembling seawater, sperm tail membranes exhibit 6.5-fold more binding sites for D600 than do membranes from sperm head. cGMP phosphodiesterase and adenylate cyclase are also enriched in plasma membranes from the tail. Thus, the highly polarized sperm cell exhibits a regional differentiation of plasma membrane proteins implicated in behavioral control.

Acetylcholinesterase in sea urchin spermatozoa

AbstractFlagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris‐buffered (pH 8.0), Ca, Mg‐free artificial sea water (F‐ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para‐aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500‐fold increase in specific activity.Twenty‐four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5‐75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X‐100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A8 and A4.Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10−4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.

Zipper lines in the flagellar plasma membrane of the squid ( Loligo) spermatozoon

Spermatozoa of the common squid, Loligo vulgaris, have been examined in a combined thin-sectioning and freeze-fracture study. Zipper-lines were seen in freeze-fracture replicas as rows of particles located on the proximal end of the axoneme. In thin section granules were visible on both sides of the tail membrane surrounding the axoneme at the proximal level, but another structure that could be identified as a zipper-line was also seen in thin section; this structure links accessory fibres to the tail membrane of the sperm. The role(s) of zipper-lines is discussed.

Regional differentiation in bull sperm plasma membranes

Bull sperm heads and tails have been separated by proteolytic digestion (trypsin) and plasma membranes have been isolated, using discontinuous sucrose density gradient centrifugation. Plasma membrane bound Ca2+-ATPase is shown to be associated mostly with the tail membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization experiments indicate a more fluid lipid phase in the tail region. Differences in surface charge distribution have been found, using 1-anilinonaphthalene-8-sulfonate and Tb3+ as fluorescent probes.

Scanning Electron Microscopy of Some Salamander Spermatozoa

Spermatozoa of 11 species of salamanders, representing nine genera and three families, were studied with the scanning electron microscope. The barbed acrosomes were found to be distinctive at the family level but intrafamilial differences were not evident. Familial differences were also noted in the presence, position and texture of the spermatozoan cytoplasmic droplet. Ambystomatid spermatozoan tails were unique among the families studied in the presence of a tail membrane and rough surface topography. The scanning electron microscope was useful in elucidating sperm characteristics not readily visible with the light microscope nor easily obtainable with the transmission electron microscope.

Phénologie et variations du dermecos chez quelques espèces deSpinturnicidae (Acarina, Mesostigmata)

Original cards allow to make the first study exclusively on phenology and dermecos of Spinturnicidae. Phenology. Species studied of the genera Spinturnix and Eyndhovenia have reproduction and maximum population during summer: In winter some males and females are resistant stages. On the contrary Paraperiglischrus rhinolophinus is a winter species. Dermecos. Species of the genus Spinturnix are found on the wing membranes of bats all the year. E. euryalis is located on the wing membrane in summer and in the coat bordering on this one during winter. Males of P. rhinolophinus are on the wing membranes females and nymphs on back tail membrane.

Bat Ticks of the Genus Argas (Ixodoidea: Argasidae). A. (Carios) macrodermae, New Species, from Queensland, Australia1,2

Argas (Carios) macrodermae , n. sp., is described from a female, a male, 63 nymphs, and 36 larvae from a large cave complex inhabited by the ghost bat, or false vampire, Macroderma gigas Dobson, near Rockhampton, Queensland, Australia. Some of the larvae were removed from wing and tail membranes of these bats. The other specimens were taken climbing on cave walls, from guano or boulders on the cave floor, or in a specially devised tick trap at least 14 m below the high ceilings where the bats roosted. This tick, which probably shelters in crevices near the host's ceiling roosts, is exceptionally difficult to find in the multichambered cave complex. Specimens taken on or near the apparently unfavorable environment of the cave floor, mostly partially fed nymphs, were probably dislodged during the sudden, unexpected flight of the excessively wary ghost bat when disturbed by humans entering the caves. A. (C.) macrodermae is a distinctive, very large tick with long, spiderlike legs and numerous setae on the body and legs. It is the 6th species described in the Argas subgenus Carios and the 4th member of Carios known from Australia.