Divalent cation activation of flagellar ATP-phosphohydrolase from bull sperm

The apparently inconsistent reports on flagellar ATPase properties may be resolved by elimination of adenylate kinase from the system. Removal of the adenylate kinase from alkaline M/2 KCl extracts of bull sperm flagella yields a spermosin-ATPase which liberates only the terminal phosphate of ATP. In any case spermosin is preferentially activated by calcium. However, combination of spermosin with flactin (e.g., by addition of digitonin and MgCl₂ to the extraction medium) produces an ATPase much more highly activated by magnesium. But flactospermosin has so far resisted purification from its adenylate kinase contaminant. In divalent cation activation, pH optima and nature of ATP hydrolysis, the flagellar contractile protein system closely parallels the muscle system.     

Mechanisms of flagellar motility deduced from backward-swimming bull sperm

Under certain conditions of cryopreservation, bull spermatozoa undergo an interesting structural alteration. The sperm tail becomes bent back on itself to form a hairpin shape. The bend in the tail occurs at a very precise point, 11 microns behind the neck, and it causes the tail to become kinked. Flagellar microtubules and dense fibers become broken and the ninefold symmetry of the flagellum is greatly distored. Although the portion of the flagellum between the kink and the sperm head does not propagate a wave, the distal portion of the flagellum propagates a base-to-tip wave, causing the spermatozoan to progress backward. These observations suggest that the mammalian spermatozoon does not need basal structures to propagate a flagellar wave.     

Effects of mechanical and chemical disruption on the ATP-phosphohydrolase activity and ultrastructure of sperm flagella.

The Ca²⁺- and Mg²⁺-ATP-phosphohydrolase (ATPase) activities measurable in suspensions of and extracts from bull epididymal spermatozoa flagella, sonicated for 2 min at 8 W, were compared with those of control flagella after 0, 3, and 18 h dialysis against an alkaline 0.5 M KCl extracting solution. Activity was measured in the absence of and in the presence of oligomycin. The effects of sonication and extraction on structural components within the flagella were visualized electron microscopically. Sonication caused fragmentation of flagella, extensive disruption of mitochondria and an immediate (0 h) increase in both Ca²⁺- and Mg²⁺-ATP-ase activity. Prolonged dialysis resulted in solubilization of specific flagellar structures, partial disruption of mitochondrial integrity, and increases in ATPase activity. Mg²⁺-ATPases of flagellar suspensions and extracts were greater than Ca²⁺-ATPases, and a part of this Mg²⁺-ATPase activity was inhibited by oligomycin. Therefore, Mg²⁺-ATPases from disrupted mitochondria contribute to the Mg²⁺-ATPase activities measurable in suspensions of and extracts from bull sperm flagella. This study emphasizes the necessity of evaluating the effects of the mechanical and chemical treatments used in fractionating cells before interpreting the biochemical information derived from their isolated components.     

Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts.

The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.     

Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.     

A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit.     

Analytical subcellular distribution of cAMP-dependent protein kinase activity in bull sperm

The distribution of the cAMP-dependent protein kinase activity in bull ejaculated sperm has been investigated. This activity proved to be mainly present in a soluble form inside the cell. Sperm fractionation into heads and flagellar fragments, using differential centrifugation or centrifugal elutriation, has shown that the particulate cAMP-dependent protein kinase activity was mainly associated with the flagellar structures. A much activity was shown to be associated with the head fraction. Some activity could also be detected in the purified plasma membrane fraction.     

Isolation of the outer acrosomal membrane from bull sperm

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction.Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both α-fucosidase and β-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.     

Biochemical characterization of tektins from sperm flagellar doublet microtubules

Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

Cryopreservation of epididymal sperm obtained at necropsy from goats

In the field of transgenic production, the ability to carry a male's genetic contribution beyond its natural life span is remarkably important. The ability to successfully collect and cryopreserve sperm from the epididymis at necropsy may prove to be a useful technique for preserving valuable genes. Thirty-two bucks ranging in age from 13 days to 7 years were examined in this study and 25 had epididymal sperm extracted at necropsy. Seven bucks yielded clear fluid with no spermatozoa; all were under four months of age. Testes were removed from the scrotal sac, small lateral incisions made across the convoluted tubules, pressure applied to the tail of the epididymis and small droplets of sperm pipetted into equilibrated extender. The average initial analysis of wave motion (0 to 5, 5 being rapid wave motion), live/dead sperm percentage and acrosomal integrity of 25 fresh epididymal samples were 5.0, 92%, and 100%, respectively. By comparison, the same parameters obtained from 206 fresh ejaculated samples were 3.0, 86%, and 95%, respectively. After being cryopreserved in liquid nitrogen, one straw from each sample was thawed after 3 to 60 days of cryostorage. Results of post-thaw analysis of 25 cryopreserved epididymal sperm samples for live/dead percentage and acrosomal integrity were 82% and 84%, respectively. By comparison, results of post-thaw analysis of 206 cryopreserved ejaculated sperm samples for live/dead percentage and acrosomal integrity were 60% and 89%, respectively. To assess the competence of the frozen epididymal sperm, IVF and AI were performed. In parallel IVF experiments, 40% of the oocytes showed cleavage patterns, with 6% developing to the blastocyst stage using frozen epididymal sperm, while 37% of the oocytes showed cleavage patterns and 4% developed into blastocysts using frozen ejaculated sperm. One artificial insemination out of 20 resulted in a pregnancy using frozen epididymal sperm, while 7 of 18 artificial inseminations resulted in a pregnancy using frozen ejaculated sperm. This data documents the successful collection and cryopreservation of epididymal sperm from the goat and its use for in vitro fertilization and artificial insemination.     

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

Isolation of the outer acrosomal membrane from bull sperm.

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Selected proteins of "prostasome-like particles" from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull

During epididymal transit, spermatozoa acquire selected proteins secreted by epithelial cells. We recently showed that P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. To further characterize the interactions between PLPCd and epididymal spermatozoa, PLPCd were prepared by ultracentrifugation of bull epididymal fluid, then surface-exposed proteins were biotinylated and coincubated in different conditions with caput epididymal spermatozoa. Western blot analysis revealed that only selected proteins are transferred from PLPCd to spermatozoa. MALDI-TOF analysis revealed that these transferred proteins are closely related. The pattern of distribution of the PLPCd transferred varied from one sperm cell to the other, with a bias toward the acrosomal cap. This transfer appeared to be temperature sensitive, being more efficient at 32-37 degrees C than at 22 degrees C. Transfer of PLPCd proteins to spermatozoa was also pH dependant, the optimal pH for transfer being 6.0-6.5. The effect of divalent cations on PLPCd protein transfer to caput spermatozoa was investigated. Whereas Mg(2+) and Ca(2+) have no effect on the amount of proteins remaining associated with spermatozoa following coincubation, Zn(2+) had a beneficial effect. These results are discussed with regard to the function of PLPCd in epididymal sperm maturation.     

Motility of undiluted bull epididymal spermatozoa collected by micropuncture

Motility of whole undiluted semen collected from different regions of the bull epididymis by micropuncture was determined by examining a droplet under paraffin oil. Bull caudal spermatozoa showed vigorous motility in undiluted semen. This motility was less in samples collected from nearer the testis: samples from the distal caput showed weak but detectable motility while those from the proximal and mid-caput were completely quiescent. Motility of spermatozoa from the distal caput and the proximal corpus was markedly increased after incubation at 34 or 37 degrees C for 1 h, but was depressed by incubation at 25 degrees C. Similar but smaller effects were observed with spermatozoa collected from the mid-corpus and the mid-cauda, except that motility of spermatozoa from the mid-corpus was reduced after incubation for 1 h at 37 degrees C. The inhibitory effect of low temperature was completely reversible. Incubation of caudal spermatozoa under anaerobic conditions produced partial and reversible inhibition of sperm motility. The results suggested that bull epididymal spermatozoa may not be completely quiescent in their native environment as previously assumed.