Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods.

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.

Isolation and unique composition of purified head plasma membrane from rooster sperm.

The structure, composition, and function of membranes from organelles of mammalian spermatozoa differ from each other and from the sperm's plasma membrane. Avian sperm studies have suffered from the lack of a technique to isolate these various membranes, which the current study now provides. Nitrogen cavitation and differential centrifugation separated head plasma membranes (HPM) of rooster sperm from sperm debris, acrosomal membranes, and mitochondrial membranes and characterized these membranes enzymatically and microscopically. The HPM was enriched in acid phosphatase (marker enzyme for HPM; 1,814.81 +/- 470.43 micromol phosphate released/microg protein vs. 868.53 +/- 75.55 for whole semen; a 202.5 +/- 37.8% enrichment, mean +/- SE, P < 0.001), with less (P < 0.001) mitochondrial and acrosomal enzyme activity. The mitochondrial fraction had 515.1 +/- 167.6% more succinate dehydrogenase activity (marker for mitochondria, P < 0.001) and the acrosomal fraction had 315.4 +/- 61.2% more acetylglucosaminidase activity (marker for acrosome, P < 0.0001) than whole semen. Thin layer and gas chromatography showed that HPM lipids had more (P < 0.05) sphingomyelin and phosphatidylserine, and less phosphatidylcholine and phosphatidylethanolamine than did the sperm body membranes (SBM). Overall, HPM had less polyunsaturated fatty acids than SBM (36.8 +/- 3.4 vs. 44.5 +/- 1.7% of total phospholipids, P < 0.05). HPM had slightly more n3 (3.2 +/- 0.5 vs. 1.3 +/- 0.2%, P < 0.01) but much less n6 (33.6 +/- 3.3 vs. 43.3 +/- 1.9%, P < 0.01), specifically less C22:4n6. Future study of avian sperm will be able to reliably characterize the structure-function relationships of specific sperm membranes.

Response of human muscle spindle afferents to sinusoidal stretching with a wide range of amplitudes.

Impulses of human single muscle spindle afferents were recorded from the m. extensor carpi radialis, while 1 Hz sinusoidal movements for a wide range of amplitudes (0.05-10 deg, half of the peak-to-peak amplitude) were imposed at the wrist joint. The response was considered as linear when the discharge was approximately sinusoidally modulated. The linearity was further checked by a linear increase in the response with the amplitude and a constancy of the phase and mean level. Fifteen of 25 primary afferents were active at rest with a mean rate of 10.6 impulses s-1 (median). The linear response to sinusoidal stretching was limited to amplitudes lower than about 1.0 deg. The sensitivity was 5.6 impulses s-1 deg-1 (median) in the linear range and decreased at larger amplitudes. The other 10 primary afferents were silent at rest and lacked a linear response at low amplitudes. Nine secondary afferents were active at rest with a mean rate of 9.5 impulses s-1. The linear range extended up to about 4.0 deg with a sensitivity of 1.4 impulses s-1 deg-1. In the linear range, the phase advance of the response to sinusoidal stretching was about 50 deg and was similar between the two types of spindle afferents. In primary afferents, the phase advance increased to nearly 90 deg outside the linear range. The findings suggest that high sensitivity to small stretches is important in determining primary afferent firing during natural movements in intact humans.

Tactile directional sensibility: peripheral neural mechanisms in man.

Tactile directional sensibility, i.e. the ability to tell the direction of an object's motion across the skin, is an easily observed sensory function that is highly sensitive to disturbances of the somatosensory system. Based on previous psychophysical experiments on healthy subjects it was concluded that directional sensibility depends on two kinds of information from cutaneous mechanoreceptors; spatio-temporal information and information about friction-induced changes in skin stretch. In the present study responses to similar probe movements as in the psychophysical experiments were recorded from human single mechanoreceptors in the forearm skin. All slowly adapting type 2 (SA2) units were spontaneously active, and with increasing force of friction their discharge rates were modified by probe movements at increasing distances from the Ruffini end-organ, reflecting the high stretch-sensitivity of these units. Slowly adapting type 1 (SA1) and field units responded to the moving probe within well-defined skin areas directly overlying the individual receptor terminals, and compared to the SA2 units their response properties were less dependent on the force of friction. The results suggest that SA1 and field units have the capacity to signal spatio-temporal information, whereas a population of SA2 units have the capacity to signal direction-specific information about changes in lateral skin stretch.

Common modulation of motor unit pairs during slow wrist movement in man.

1. The activity of 36 pairs of single motor units were recorded with intramuscular wire electrodes from m. extensor carpi radialis while subjects performed slow wrist extension and flexion movements. Periods of steady position holding were interposed between movements. 2. The discharge trains from pairs of motor units were analysed statistically in the time and frequency domains. During extension movements, when the muscle recorded from was the agonist, coherence between motor units was significant below 12 Hz, with a peak at 6-12 Hz in 30 of 36 pairs (83 %). The magnitude of coherence decreased during position holding compared to movements in 26 pairs, while the difference in average firing rate was small. 3. During movements, but not during position holding, coherence estimates between single motor units and acceleration showed a significant peak at 6-12 Hz in 56 out of 62 motor units, suggesting that a modulation of motor unit discharge contributed to angular acceleration at these frequencies. Common motor unit modulation was present at 3 Hz as well, although the coupling between motor unit activity was weaker than at 6-12 Hz. 4. It is concluded that a 6-12 Hz common modulation of agonist motor units is a distinguishing feature of slow voluntary wrist movements, extending the previously established notion of an 8-10 Hz rhythmic organization of slow finger movements to more proximal limb segments. It is suggested that the 6-12 Hz input is specific for movements and is normally absent or much weaker during steady maintenance of position or force.

Single motor unit activity in relation to pulsatile motor output in human finger movements.

1. Forty-six single motor units in the common finger extensor, superficial finger flexor, and first dorsal interosseus muscles were recorded with intramuscular wire electrodes while subjects made voluntary flexion and extension finger movements at a single metacarpo-phalangeal joint. 2. Motor unit firing was analysed in relation to the 8-10 Hz discontinuities which previously have been shown to characterize these movements. Statistical time- and frequency-domain analyses of the activity of individual motor units in relation to the discontinuities showed that when the muscle was the agonist, all motor units in the common finger extensor muscle, and all units except one in the flexor muscles exhibited significant frequency modulation of their discharge in close temporal association with the joint acceleration. On the other hand, motor unit firing rate was not related to the frequency of the discontinuities. When the muscle recorded from was the antagonist, 21 of the 25 active units exhibited a similar frequency modulation. 3. When angular movement velocity was increased from 4 to 25 deg s-1, the strength of motor unit frequency modulation increased. Peak coherence between motor unit activity and acceleration increased by 74 %, on average, in the common finger extensor units. 4. The findings rule out a tentative mechanism attributing the discontinuities to newly recruited motor units firing at circa 8-10 Hz. Instead, a coherent 8-10 Hz input to the agonist and antagonist motoneurone pools is implied.

Dynamic response of human muscle spindle afferents to stretch during voluntary contraction.

1. The response of twenty-eight human muscle spindle afferents from m. extensor carpi radialis brevis to large amplitude ramp stretch and release at the wrist joint was recorded. The dynamic index was calculated as the difference in firing rate between that just before the end of stretch and that during the subsequent static phase of stretch. The value during steady voluntary contraction was compared with that during relaxation. 2. In twenty-three primary afferents, the dynamic index increased in eleven and decreased in twelve afferents with a range of -8 to +25 impulses s-1. In five secondary afferents the change was less than 2 impulses s-1. 3. The primary afferents abruptly stopped firing when the stretch was released in the relaxed muscle. This cessation was prevented during contraction in seventeen primary afferents. 4. The results suggest the presence of dynamic and static fusimotor actions on the human muscle spindles during voluntary contraction.

Coupling between single muscle spindle afferent and EMG in human wrist extensor muscles: physiological evidence of skeletofusimotor (beta) innervation.

OBJECTIVES: This study was conducted to find physiological evidence of skeletofusimotor innervation in man. METHODS: Discharges of 38 single muscle spindle afferents from m. extensor carpi radialis were recorded from 9 subjects during steady isometric contractions of wrist extension. Correlation between these afferents and rectified surface EMG was investigated by estimating cumulant density function. RESULTS: In the cumulant density estimate between spindle afferent and EMG, a positive EMG peak was obtained prior to afferent firing between -30 and -10 ms in 15 afferents (39%). CONCLUSIONS: The present finding indicates coupling between spindle afferents and extrafusal activity and suggests the widespread skeletofusimotor innervation in human muscle spindle.

Is human muscle spindle afference dependent on perceived size of error in visual tracking?

Impulses of 16 muscle spindle afferents from finger extensor muscles were recorded from the radial nerve along with electromyographic (EMG) activity and kinematics of joint movement. Twelve units were classified as Ia and 4 as II spindle afferents. Subjects were requested to perform precision movements at a single metacarpophalangeal joint in an indirect visual tracking task. Similar movements were executed under two different conditions, i.e. with high and low error gain. The purpose was to explore whether different precision demands were associated with different spindle firing rates. With high error gain, a small but significantly higher impulse rate was found in pooled data from Ia afferents during lengthening movements but not during shortening movements, nor with II afferents. EMG was also significantly higher with high error gain in recordings with Ia afferents. When the effect of EMG was factored out, using partial correlation analysis, the significant difference in Ia firing rate vanished. The findings suggest that fusimotor drive as well as skeletomotor activity were both marginally higher when the precision demand was higher, whereas no indication of independent fusimotor adjustments was found. These results are discussed with respect to data from behaving animals and the role of fusimotor independence in various limb muscles proposed.

Fusimotor and skeletomotor activities are increased with precision finger movement in man.

1. Impulses of eighteen muscle spindle afferents from finger extensor muscles were recorded from the radial nerve while subjects performed single joint finger movements of two kinds, i.e. routine and precision, which were nearly identical with regard to kinematics. 2. The firing rates of ten primary and two secondary spindle afferents were higher in the precision movements by more than 10%, although the difference reached statistical significance in only seven of them. In most cases when spindle firing was higher in precision movements the skeletomotor activity was higher as well. 3. The findings indicated that the fusimotor activity was often stronger with precision movements compared with routine movements. This result is in qualitative agreement with several studies on behaving cats, demonstrating higher fusimotor activity in more demanding motor tasks. On the other hand, the effects were much smaller in humans than in cats. Moreover, in contrast to findings from experiments in cats, no support was obtained for the hypothesis that fusimotor activity was adjusted independently of the skeletomotor activity in human finger muscles.

Receptive field characteristics of tactile units with myelinated afferents in hairy skin of human subjects.

1. Impulses in single nerve fibres from the lateral antebrachial cutaneous nerve were recorded using the microneurography technique in human subjects. 2. In a sample of fifty-five mechanoreceptive units with fast-conducting nerve fibres, five types were identified, i.e. SAI (slowly adapting type I, Merkel), SAII (slowly adapting type II, Ruffini), hair units, field units and Pacinian-type units. The latter three unit types were all rapidly adapting. 3. The detailed structure of thirty-five receptive fields of SAI, SAII, hair and field units was explored with a method which was objective and independent of the experimenter's skill and experience. A lightweight probe was used to scan the receptive field area in a series of tracks 0.23 mm apart while single-unit activity was recorded. 4. SAI fields were small and composed of two to four well-separated high-sensitivity spots and often, in addition, one minor spot of lower sensitivity. SAII units typically fired spontaneously at a low and regular rate. Most fields consisted of one single spot of high sensitivity with diffuse borders. The hair units innervated ten to thirty-three (or more) hairs, which were evenly distributed over a large area. The field units were characterized by a number of small and closely packed high-sensitivity spots with diffuse borders. A conservative estimate indicated eleven spots per unit. 5. The findings indicate that the sheet of mechanoreceptors on the skin of the forearm is distinctly different from that on the dorsum of the hand and in the face. It seems reasonable to assume that the former is more representative for the hairy skin covering the main parts of the body.

Composition and behavior of head membrane lipids of fresh and cryopreserved boar sperm.

Head plasma membranes were isolated from fresh or cryopreserved ejaculated boar spermatozoa and the lipids were extracted for determination of lipid fluidity (n = 6 for fresh and cryopreserved) and for compositional analysis (n = 5 for fresh, 6 for cryopreserved). Composition of the egg yolk extender was also determined. For fluidity determination, the mixed lipids were allowed to form natural liposomes. Bilayer fluidity of these liposomes was analyzed in the presence or the absence of 1 mM Ca2+ with the probes tPNA, which preferentially locates into gel-phase areas, and cPNA, which enters fluid and gel-phase areas equally and thus assesses bulk lipids. Fluidity of liposomes declined significantly during controlled-rate cooling for all samples. Compared to lipids from fresh membranes, gel lipids from cryopreserved cells lost fluidity at a significantly more rapid rate, as did bulk lipids in the presence of Ca2+ (P < 0.001). Fluidity increased during subsequent rewarming (5 to 50 degrees C), again at a slower rate for lipids from fresh cells, with the cryopreservation effect being significant for all probe/Ca2+ combinations (P < or = 0.05). Calcium altered the fluidity characteristics of membrane lipids from fresh but not cryopreserved sperm when analyzed during cooling with cPNA (P < 0.01) and during rewarming with cPNA (P < 0.0001) and tPNA (P < 0.05). Lipids from cryopreserved cells contained significantly less sphingomyelin (14.6 +/- 1.1 vs 22.4 +/- 1.6 mol%) and more phosphatidylcholine (51.5 +/- 2.0 vs 40.5 +/- 2.4%). The octadecanoate (18:0) content in both phosphatidylserine and phosphatidylethanolamine decreased after cryopreservation (P < 0.05). The polyunsaturated fatty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in sphingomyelin and phosphatidylinositol (P < 0.05). The alterations in the molecular interactions, composition, and Ca2+ sensitivity of membrane lipids may interfere with the normal membrane events of fertilization.

Discharge of muscle afferents during voluntary co-contraction of antagonistic ankle muscles in man.

The discharge of 38 tibialis anterior (TA) muscle spindle endings was recorded at rest and during isometric voluntary contraction of the ankle joint in eight healthy human subjects. With the ankle joint in 110 degrees plantarflexion, 24 endings (61%) were tonically active in the resting subject. During weak voluntary dorsiflexion, seven additional endings were activated, so that a total of 31 endings were active (82%). 24 of these were either newly recruited or discharged at a faster rate than at rest (average discharge rate 6.6 Hz at rest, 9.7 Hz during contraction). At matched levels of TA EMG, one ending was newly recruited and 10 were more active during co-contraction of dorsi and plantar flexors than during isolated dorsiflexion. 26 endings were equally active during the two tasks and one ending decreased its firing rate with co-contraction. Four of the 11 endings, which had a higher discharge rate during co-contraction than during dorsiflexion, discharged faster during plantarflexion than at rest although slower than during co-contraction. Plantarflexion had no effect on the discharge of three endings. The remaining four endings were not investigated during plantarflexion. It is suggested that the increased discharge rate of muscle endings during co-contraction is caused either by small changes in the length of the TA muscle or by a disproportionately high fusimotor drive during co-contraction.

Conduction velocity of low-threshold mechanoreceptive afferent fibers in the glabrous and hairy skin of human hands measured with microneurography and spike-triggered averaging.

The axonal conduction velocity (CV) of afferent fibers innervating low-threshold mechanoreceptors in the skin of the human hand was measured utilizing a spike-triggered averaging technique. Two tungsten microelectrodes were inserted into the median, or the ulnar, the superficial branch of the radial nerve, at two different points in the distal forearm. Unitary spike potentials were picked up with the proximal electrode. Unit-type SA (I and II), and FA (I and II), was determined from receptive field properties and response patterns to mechanical stimuli. Using these potentials as a trigger, the records from the distal electrode were averaged to reveal corresponding unitary potentials embedded in the background noise activities. CVs were calculated by dividing the interelectrode distance by the conduction time measured from the two neurograms. 122 mechanoreceptive afferent fibers in the glabrous and the hairy skin were recorded. The CVs of all sampled units were 36-73 m/s and the mean (SD) was 58.7 (7.4) m/s. The CVs did not differ between units in the three nerves, nor between units from the glabrous and the hairy skin. The mean CV of the FAI group was slower than the mean CV of the SA group by 4-5 m/s, but the overlap of the distributions was large.

Conduction velocities of alpha-motor fibers innervating human thenar muscles and order of recruitment upon voluntary contraction.

Conduction velocity (CV) of single alpha-motor fibers was measured using a combination of microneurographic and spike-triggered averaging techniques on 7 healthy subjects (aged 25 to 42). Two tungsten microelectrodes were inserted percutaneously into the median nerve at the wrist and 4 to 6 cm more proximally. Motor unit activities recorded from thenar muscles during voluntary isometric contraction with a third microelectrode, were isolated with a window discriminator, and used to trigger an averager. CVs were calculated by dividing the interelectrode distance by the conduction time between unitary activities in the proximally and distally averaged neurograms. The CVs of 100 units ranged between 34 and 66 m/s, with the peak at around 50 m/s. The relationship between CVs and recruitment orders was compared within sets of 2 or 3 units recorded simultaneously. Statistical analysis using a chi 2 test revealed that slower motor units tended to be recruited earlier than faster units (P less than 0.001).

Discrimination of different motor units by spike-triggered averaging of surface electromyograms.

An objective method was sought for the discrimination of individual motor units (MUs). Discrimination using the conventional needle electrode recording is difficult because the waveforms are easily changed by slight shifts of the electrode. A surface EMG recording was combined with the needle recording and MU activities were obtained by MU-triggered averaging of the surface EMGs. Waveforms of surface MU potentials were stable, irrelevant to changes in recording conditions of needle electrodes and thus were found useful for the purpose of discrimination. Efficacy of this method was tested in the MUs with known axonal conduction velocities.