Calcium regulation of flagellar curvature and swimming pattern in triton X-100--extracted rat sperm

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10(-7) M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 x 10(-6) M Ca2+ to assume a tight, hook-like bend at concentrations of 10(-5) to 10(-4) M free Ca2+. Sodium vanadate at 2 x 10(-6) M blocked flagellar motility, but did not inhibit the Ca2+-mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 microM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+-dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+-treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1-5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+-induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.     

Protocadherin alpha3 acts at sites distinct from classic cadherins in rat testis and sperm

The testis expresses a variety of cadherin superfamily members including classic cadherins and protocadherins. This report describes the first localization of a protocadherin protein in testis and sperm. After cloning rat cDNAs for protocadherin alpha3 and alpha4, isoform-specific polyclonal antibodies were generated against protocadherin alpha3. Western blotting of rat testis showed that protocadherin alpha3 was solubilized completely by Triton X-100, in contrast to the adhesion junction components N-cadherin, beta-catenin, and p120 catenin. Corroborating this data, protocadherin alpha3 was immunolocalized to the spermatid acrosomal area, intercellular bridge, and flagellum, but not classic cadherin-based adhesion junctions. Acrosome-associated protocadherin alpha3 was first detected at step 8 of spermiogenesis, and this association remained on cauda epididymal sperm. Acrosome immunostaining was reduced, but present, in acrosome-reacted sperm. Spermatid intercellular bridges became positive for protocadherin alpha3 coincident with the appearance of plectin, occurring at spermiogenic steps 8 to 9, and elongate spermatid bridges remained positive throughout spermatogenesis. The developing flagellum was uniformly immunostained for protocadherin alpha3 up to approximately spermiogenic step 17. Subsequently, flagellar immunostaining was confined to the principal piece, and this pattern continued in cauda epididymal sperm. These data show that protocadherin alpha3 performs functions unique from classic cadherins in spermatogenesis and suggest a role for protocadherin alpha3 in organizing germ cell-specific structures including the intercellular bridge, flagellum, and acrosome.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

Synthesis and assembly of flagellar surface antigens during zoosporogenesis inPhytophthora cinnamomi

Summary Cryomicrotomy and immunofluorescence microscopy employing three different categories of monoclonal antibody (MAb) that label antigens on the surface of one or both flagella ofPhytophthora dnnamomi have been used to follow the synthesis and assembly of flagellar surface components. MAb Zf 1 binds to the surface of both the anterior tinsel and posterior whiplash flagella, as well as to a nuclear component. The labeling of the flagella is punctate in nature, is brighter at the flagellar base, and does not always extend to the distal tip of the flagella. MAbs in the Zt group recognise an antigen that is located along the sides of the tinsel flagellum and may be associated with the base of the mastigonemes. Immunodot-blot analysis has shown that binding of Zt MAbs is abolished by pretreatment with either pronase or periodate oxidation indicating that the antigen is a glycoprotein. MAbs in the Zg group bind to the mastigonemes on the tinsel flagellum and to packets of mastigonemes in the cytoplasm of zoospores. Zt and Zg antigens increase in abundance during zoosporogenesis and are present throughout the life cycle of the fungus, whereas the non-nuclear localisation of the Zf antigen appears only during sporulation. Prior to association with the flagellar surface, all three components become clustered in the groove region of zoospores. They do not become associated with the flagellar surface until at least 15 min after the flagellar axoneme has formed.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMF dimethylformamide - lgG₁ immunoglobulin G₁ - MAbs monoclonal antibodies - NIM non-immune mouse antibodies - PBS phosphate-buffered saline - PBST phosphate-buffered saline with 0.5% Tween 20 - PIPES 1,4-piperazinediethanesulfonic acid - PPD paraphenylenediamine dihydrochloride - RT room temperature - TBS tris-buffered saline - TEST tris-buffered saline with 0.05% Tween 20     

Germinal angiotensin I-converting enzyme is totally shed from the rodent sperm membrane during epididymal maturation

Acquisition of sperm fertilizing ability is due, in part, to the reorganization of plasma membrane proteins that occurs during epididymal sperm transit. Using polyclonal antibodies against angiotensin I-converting enzyme (ACE), we showed that this enzyme is immunolocalized mainly on the middle piece of rat and mouse testicular sperm and with less intensity along the initial part of the principal piece of the flagellum. In both species, only some sperm from the caput epididymis were still reactive, whereas no labeling was observed on cauda epididymal sperm. The 105- to 110-kDa germinal ACE was absent from the rat testicular fluid but appeared in the fluid of the anterior epididymis. Thereafter, its molecular weight shifted to 94 kDa in the corpus epididymal fluid and remained at this weight in the caudal region. The 105- to 110-kDa immunoreactive protein was present in testicular rat sperm extract but was completely absent from epididymal sperm extracts. Western blot analysis of testicular and epididymal tissue extracts from the rat and mouse also confirmed that the germinal enzyme was absent from the epididymal sperm cell. Our results demonstrated that the rodent germinal ACE is released from the testicular sperm membrane when sperm enter the epididymis, a process similar to that observed in domestic mammals. This result is discussed in view of the suggested role for this enzyme in sperm fertility.     

Compartmentalization of a unique ADP/ATP carrier protein SFEC (Sperm Flagellar Energy Carrier, AAC4) with glycolytic enzymes in the fibrous sheath of the human sperm flagellar principal piece

The longest part of the sperm flagellum, the principal piece, contains the fibrous sheath, a cytoskeletal element unique to spermiogenesis. We performed mass spectrometry proteomics on isolated human fibrous sheaths identifying a unique ADP/ATP carrier protein, SFEC [AAC4], seven glycolytic enzymes previously unreported in the human sperm fibrous sheath, and sorbitol dehydrogenase. SFEC, pyruvate kinase and aldolase were co-localized by immunofluorescence to the principal piece. A homology model constructed for SFEC predicted unique residues at the entrance to the nucleotide binding pocket of SFEC that are absent in other human ADP/ATP carriers, suggesting opportunities for selective drug targeting. This study provides the first evidence of a role for an ADP/ATP carrier family member in glycolysis. The co-localization of SFEC and glycolytic enzymes in the fibrous sheath supports a growing literature that the principal piece of the flagellum is capable of generating and regulating ATP independently from mitochondrial oxidation in the mid-piece. A model is proposed that the fibrous sheath represents a highly ordered complex, analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products with SFEC serving to mediate energy generating and energy consuming processes in the distal flagellum, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse. An immunohistochemical study

Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuraminidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation

A cAMP-induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA-mediated signaling and negatively by the PI3K/PDK1-mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)-treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30-min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr-197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180-min incubation. A potentially active form of PDK1 (54/55-kDa phospho-PDK1) was detected in the principal piece of the spermatozoa during the 90-min incubation. Another potentially active form (59-kDa phospho-PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180-min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of PI3K-dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti-PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa.     

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.     

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: Unique distribution of glutamylated tubulin

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti α and β-tubulin, 6-11B-1 anti-acetylated α-tubulin, and GT335 anti-glutamylated α and β-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated α-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated α and β-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum. © 1996 Wiley-Liss, Inc.     

Reversible intracellular ATP changes in intact rat spermatozoa and effects on flagellar sperm movement.

The initiation of motility and modification of energy metabolism of rat caudal epididymal spermatozoa can be induced by dilution in a saline medium. We have investigated in these cells the relationships between the energy reserve (sperm ATP content measured by bioluminescence) and flagellar movement (high speed videomicrography, 200 frames/sec). A steady state was observed in sperm ATP content, progressive velocity (Vp) and flagellar beat frequency (F) with sperm dilution in a medium with glucose, lactate, pyruvate and acetate substrates after 30 minutes of incubation. Without these substrates, changes in metabolic pathways occurred immediately and initially disturbed the relationship between ATP levels and F, suggesting differences in motility initiation when energy is from an endogenous origin via mitochondrial oxidative phosphorylation. This "energy crisis" was reversed by the addition of substrates to the medium. The three-dimensional flagellar movement observed in the presence of substrates quickly became two-dimensional in their absence. The flagellar beat envelope became more splayed, the mean amplitude of lateral head displacement increased and F decreased. The resulting high flagellar beat efficiency can be compared to that observed during hyperactivation which is a physiological event related to a fall in intracellular ATP level. In both media, the displacement of the flagellum in relation to the wave axis varied sinusoidally. The sine period increased with time when the spermatozoa were incubated in the medium without substrates. These results suggest a gradual slowing-down of the velocity of wave formation in the proximal part of the flagellum.     

Dynamic distribution of Spatial during mouse spermatogenesis and its interaction with the kinesin KIF17b

The Spatial gene is expressed in highly polarized cell types, such as epithelial cells in the thymus, neurons in the brain and germ cells in the testis. In this study, we report the characterization and distribution of Spatial proteins during mouse spermatogenesis. Besides Spatial-epsilon and -delta, we show that the newly described short isoform Spatial-beta is expressed specifically in round spermatids. Using indirect immunofluorescence, we detected Spatial in the cytosol of the early round spermatid. By the end stages of round spermatids, Spatial is concentrated at the opposite face of the acrosome near the nascent flagellum and in the manchette during the elongation process. Finally in mature sperm, Spatial persists in the principal piece of the tail. Moreover, we found that Spatial colocalizes with KIF17b, a testis-specific isoform of the brain kinesin-2 motor KIF17. This colocalization is restricted to the manchette and the principal piece of the sperm tail. Further, coimmunoprecipitation experiments of native proteins from testis lysates confirmed Spatial-KIF17b association through the long Spatial-epsilon isoform. Together, these findings imply a function of Spatial in spermatid differentiation as a new cargo of kinesin KIF17b, in a microtubule-dependent mechanism specific to the manchette and the principal piece of the sperm tail.     

Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus

A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein-a component of the flagella central apparatus-was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse

Summary Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuramainidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.     

Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum

Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3' UTR and a different 5' UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.     

Two distinct Ca(2+) signaling pathways modulate sperm flagellar beating patterns in mice

Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca(2+). We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca(2+) signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca(2+) channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca(2+) release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca(2+) and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca(2+) increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca(2+) signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca(2+) from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm.     

Analysis of the flagellar bending waves of ejaculated ram sperm

The variability of flagellar movement, illustrated by the highly heterogeneous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 X 36 camera system. The multiple-moving-exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.     

Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation

We have previously shown that sperm-egg recognition in the mouse is mediated by the binding of galactosyltransferase (GalTase) on the sperm surface to its appropriate glycoside substrate in the egg zona pellucida [L. C. Lopez, E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur (1985) J. Cell Biol. 101, 1501-1510]. In the present study, we have defined the spatial and temporal expression of surface GalTase during spermatogenesis and epididymal maturation. Purified populations of spermatogenic cells were isolated by unit gravity sedimentation, and surface GalTase expression was determined by indirect immunofluorescence and by direct enzymatic assay. GalTase is present on the surface of all spermatogenic cells assayed. During differentiation, there is a progressive redistribution of GalTase from an initially diffuse and uniform localization on the surface of primary spermatocytes to a restricted plasma membrane domain overlying the dorsal aspect of the mature acrosome. This apparent redistribution of surface GalTase was confirmed by direct enzymatic assays, which show that surface GalTase activity, normalized per cell, remains relatively constant throughout spermatogenesis, despite a drastic reduction in cell surface area. When normalized to the relevant cell surface area, the GalTase concentration per square micrometer increases 77-fold from pachytene spermatocytes to cauda epididymal sperm. Cell surface GalTase is thought to be a cytoskeletally associated transmembrane protein [N. L. Shaper, P. L. Mann, and J. H. Shaper (1985) J. Cell Biochem. 28, 229-239]; consequently we examined whether cytoskeletal components may be involved in the redistribution of GalTase during spermatogenesis. beta-Tubulin, monomeric actin, and filamentous actin were found to be present during spermatogenesis, as assayed by indirect immunofluorescence and by Western immunoblotting. alpha-Actinin and vinculin were not detectable under these conditions and served as negative controls. During spermatogenesis, the distribution of tubulin coincides with the appearance of the mitotic spindle, flagellum, and manchette. On the other hand, the distribution of filamentous actin coincides with surface GalTase, suggesting that actin-containing microfilaments may participate in the redistribution of surface GalTase during spermatogenesis.