Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 °C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 μm and a radius of 0.4 μm with an osmotically inactive cell volume, V_b, of 0.61V_o, where V_o is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L_pg and activation energy, E_Lp). The predicted best-fit permeability parameters ranged from, L_pg = 0.021–0.038 μm/min-atm and E_Lp = 27.8–41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.     

Stimulation of forward motility of goat cauda epididymal spermatozoa by a serum glycoprotein factor

Blood sera of humans, rats, goats, and buffalo have been shown to possess a forward motility-stimulating factor (FMSF) that markedly stimulated goat cauda epididymal sperm forward motility, as assayed by a microscopic method in the presence of epididymal plasma (1.2 mg protein/ml) that had sufficient anti-sticking activity to eliminate the possibility of cell-sticking artifacts in motility assays. The specific activity of FMSF was greatest in buffalo blood serum compared to the sera of the other species. Buffalo serum at a concentration as low as 8.5 mg protein/ml induced forward motility in nearly 45% of the cells. The buffalo serum FMSF was heat-stable, nondialyzable, and sensitive to the action of trypsin. Purified proteins--casein, serum albumin, ovalbumin, myoglobin, and beta-lactoglobulin--showed little or relatively low FMSF activity. FMSF is a glycoprotein, as it binds with high affinity to concanavalin A-agarose. A major portion of the serum protein (approx. 70%) did not bind to the affinity matrix, and this unretained serum protein fraction showed little FMSF activity. The FMSF activity of buffalo serum was confirmed by estimating sperm forward motility spectrophotometrically: an objective method of assessing sperm motility.     

Ecto-cyclic AMP-receptor in goat epididymal intact spermatozoa and its change in activity during forward motility

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

Inhibition of bovine spermatozoa by caudal epididymal fluid: I. Studies of a sperm motility quiescence factor

Spermatozoa from all portions of the bovine epididymis are essentially quiescent when examined in vitro without dilution. However, sperm from the caudal epididymis, particularly the distal portion, develop full motility when they are diluted into seminal plasma or simple isotonic buffers. Dilution of the sperm into neat cauda epididymal fluid (CE fluid) does not result in the initiation of motility. The initiation of motility upon dilution into buffers is complete within 10-20 min, while the inhibition induced by CE fluid is nearly instantaneous. CE fluid concentration, but not sperm concentration, controls sperm motility. Therefore, an inhibitory component of this fluid, but not sperm-sperm interactions, is responsible for the inhibition. CE sperm, which have been diluted into isotonic buffers and are consequently motile, become quiescent when resuspended in CE fluid; thus, this process is fully reversible. No elevations in sperm cyclic AMP levels can be detected concomitant with the induction of motility but high concentrations of cyclic AMP phosphodiesterase inhibitors can overcome the quiescence induced by CE fluid. The inhibitors of CE sperm motility reported for other species, e.g., the high-viscosity mucin, immobilin ; carnitine; calcium; or glycerylphosphorylcholine , do not appear to be of importance in the bovine caudal epididymis. The quiescence produced by bovine CE fluid is strongly dependent upon the extracellular pH; i.e., motility is inhibited at pH 5.5 but not at pH 7.6.     

Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa

This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen from beef bulls was collected by electroejaculation and extended 1:3 in TL-Hepes containing 100 micro g/ml pyruvate and 6 mg/ml BSA. In solutions of 150-1200 mOsmolal (mOsm), bull spermatozoa behaved as linear osmometers (r(2) = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 micro m(3). Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270-360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92-103% of their isosmotic cell volume. Motility following a one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively, compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol as a CPA.     

Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.     

Changes in calmodulin level and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa

Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.     

Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa

Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.     

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca²⁺-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca²⁺-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca²⁺-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.     

Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。