Major Sperm Protein Research Articles

Actin and major sperm protein in spermatids and spermatozoa of the parasitic nematode Heligmosomoides polygyrus.

Nematode spermatozoa are amoeboid cells. In Caenorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, namely major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton, which is supposed to act in the absence of actin.

The Motile Major Sperm Protein (MSP) from Ascaris suumis a Symmetric Dimer in Solution

The major sperm protein (MSP) of Ascaris suummediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exists in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.

Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction.

Progesterone initiation of te human sperm acrosome reaction (AR) includes stimulation of a transient Ca2+ influx and a transient Cl- efflux. A role for one or more plasma membrane receptors has been suggested, but, except for evidence supporting the involvement of a sperm GABAA-like receptor/Cl- channel, there is little information about possible receptor identity. Here, we attempt to identify plasma membrane progesterone receptors in human sperm using a mouse monoclonal antibody (mAb-C262) raised against the C-terminal steroid-binding domain of the human intracellular progesterone receptor. C-262 inhibited the progesterone-initiated AR in a dose-dependent manner. Maximum inhibition was 77% as detected by fluorescein isothiocyanate (FITC)-concanavalin A (conA). Motility was unaffected. A control mouse mAb (h-151) raised against the human estrogen receptor did not inhibit the progesterone-initiated AR. Western blotting with C-262, but not with h-151, detected a major sperm protein band of 50-52 kDa. In indirect immunofluorescence localization studies, live and ethanol-fixed uncapacitated sperm and fixed capacitated sperm incubated with C-262, but not with h-151, displayed fluorescence at the equatorial segment region of the sperm head plasma membrane. In spectrofluorometric studies using capacitated sperm loaded with the Ca2+ probe Fura-2 or the Cl- probe MEQ, C-262 but not h-151 inhibited both Ca2+ influx and Cl- efflux. These ion fluxes could be due to the binding of progesterone to two different receptor/channels or to its binding to one and cross talk with the other. Our results strongly support the involvement of sperm plasma membrane receptors in the progesterone-initiated AR and provide a candidate for one such receptor.

Reconstitution In Vitro of the Motile Apparatus from the Amoeboid Sperm of Ascaris Shows That Filament Assembly and Bundling Move Membranes

We have developed an in vitro motility system from Ascaris sperm, unique amoeboid cells that use filament arrays composed of major sperm protein (MSP) instead of an actin-based apparatus for locomotion. Addition of ATP to sperm extracts induces formation of fibers ∼2 μm in diameter. These fibers display the key features of the MSP cytoskeleton in vivo. Each fiber consists of a meshwork of MSP filaments and has at one end a vesicle derived from the plasma membrane at the leading edge of the cell. Fiber growth is due to filament assembly at the vesicle; thus, fiber elongation results in vesicle translocation. This in vitro system demonstrates directly that localized polymerization and bundling of filaments can move membranes and provides a powerful assay for evaluating the molecular mechanism of amoeboid cell motility.

Nematode sperm locomotion

The simplicity and specialization of the amoeboid motility of nematode sperm can give intriguing insights into the molecular mechanisms underlying movement in more conventional actin-based systems. Amoeboid motility in nematode sperm is based on their major sperm protein. Advances over the past year in understanding the assembly of this protein in vivo and in vitro have underlined the importance of vectorial assembly and filament bundling. In this system, it is possible that these two properties may be sufficient to generate motility that closely resembles that seen in conventional actin-based systems.

Chapter 12 Cell Biology of Nematode Sperm

Publisher Summary This chapter reviews methods that have been developed for working with the amoeboid sperm of nematodes. The chapter focuses on the free-living Caenorhabditis elegans and the pig parasite Ascaris suum. The superb genetics and ease in obtaining DNA clones containing desired gene sequences make C. elegans the system of choice for genetic and molecular biological studies. Greater than 50% of the cells in a mature C. elegans male are sperm. When C. elegans males are squashed under hydraulic force, they burst open and release their sperm into the surrounding medium under conditions where other tissues remain relatively intact. This permits straightforward purification of near-homogeneous sperm, the vast majority of which are in the spermatid stage. Ascaris offers three principal advantages over C. elegans for the study of sperm cell biology. A robust Ascaris male contains 5 X l07 spermatids in its seminal vesicle, compared with 3 X l03 in a virgin C. elegans male. The volume of each spermatid from Ascaris is about 10-fold greater than its counterpart from C. elegans. Activation in Ascaris produces motile spermatozoa with a highly ordered cytoskeleton composed of major sperm protein (MSP) filaments grouped into long, branched fiber complexes that can be viewed in real time in live cells by light microscopy.

Supramolecular assemblies of the Ascaris suum major sperm protein (MSP) associated with amoeboid cell motility

Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of locomotion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.

The Motile Major Sperm Protein (MSP) of Ascaris suum Forms Filaments Constructed from Two Helical Subfilaments

The amoeboid motility nematode sperm is mediated by cytoskeletal filaments composed of major sperm protein (MSP). We have used electron microscopy and image processing to show that MSP filaments are constructed from two subfilament strands which are themselves formed from a helical arrangement of subunits. The subfilaments are based on left-handed helices of pitch 9 nm that then coil along right-handed helical tricks of pitch 22.5 nm to form filaments. The subfilaments appear to be indistinguishable from the helices present in orthorhombic crystals of MSP. Because in filaments the subfilaments are themselves helical, not all subunits are able to participate in protein-protein interactions between different strands. One consequence of this interaction geometry is that the same molecular interactions that function to assemble subfilaments into filaments can also be used to assemble filaments into larger supramolecular assemblies such as the macrofibres formed in vitro and the fibre bundles found in vivo in sperm pseudopods. These results indicate the importance of filament bundling in addition to vectorial filament assembly in amoeboid cell motility.

Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH.

The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction.

Crystallization of the Motile Major Sperm Protein (MSP) of the Nematode Ascaris suum

We have obtained orthorhombic crystals (space group P 2 12 12 1, a = 72 Å, b = 78 Å, c = 452 Å) of the major cytoskeletal protein associated with the amoeboid motility of Ascaris sperm. These crystals diffract past 3·5 Å and appear to be constructed from arrays of helical fibres of the major sperm protein. The fibres within the crystals appear to be closely analogous to those seen in vitro and in vivo.

Evolution of pro‐protamine P2 genes in primates

Protamines P1 and P2 form a family of small basic peptides that represent the major sperm proteins in placental mammals. In human and mouse protamine P2 is one of the most abundant sperm proteins. The protamine P2 gene codes for a P2 precursor, pro-P2 which is later processed by proteolytic cleavages in its N-terminal region to form the mature P2 protamines. We have used polymerase chain amplification to directly sequence the pro-P2 genes of the five major primate families: red howler (Alouatta seniculus) is a New World monkey (Cebidae); the two macaque species, Macaca mulatta and M. nemistrina are Old World monkeys (Cercopithecidae), the gibbon, Hylobates lar, represents one branch of the apes (Hylobatidae); the orangutan, Pongo pygmaeus, gorilla, Gorilla gorilla and two species of chimpanzee Pan paniscus and Pan troglodytes represent a second ape family (Pongidae). These pro-P2 genes are compared with that of human [Domenjoud, L., Nussbaum, G., Adham, I. M., Greeske, G. & Engel, W. (1990) Genomics 8, 127-133]. The overall size and organization of the genes are conserved within the group. The mean length of pro-P2 is 101 residues, with an increase to 102 in M. nemistrina and a decrease to 99 residues in red howler (A. seniculus). In gorilla and red howler one of two 79-bp tandem repeats that occurs 3' of the gene is deleted. Of the 101 deduced amino acids examined, an amino acid change occurs in one or more primates at 45 positions. Considering only the most recently diverged group, the human/gorilla/chimpanzee clade, this represents a very high mutation rate of 0.99 changes/100 sites in 10(6) years. This rapid mutation rate is characteristic of both members of the protamine gene family, P1 and P2. Consideration of the variable nature of the sequences at the multiple sites of proteolysis during the processing of the pro-P2 indicates either that there are several processing enzymes of differing specificities, or more likely that the folded structure of the pro-P2 limits accessibility of a non-specific protease to certain exposed sites.

The diagnostic antigen encoded by gene fragment Dv3-14: A major sperm protein of Dictyocaulus viviparus

The Dv3-14 gene fragment of Dictyocaulus viviparus is 471 bp, of which about 90% is translated. The calculated molecular mass of the translation product is 15.5 kDa. DNA sequence homologies were found with major sperm proteins (MSPs) from Ascaris suum, Caenorhabditis elegans and Onchocerca volvulus. Within a segment of 62 amino acids the predicted amino acid sequence of Dv3-14 shows 84% homology with an A. suum MSP and 84% homology with a C. elegans MSP. Computer analysis of the protein sequence identified three segments of immunodominant antigenic sites as putative candidates for synthetic peptide antigens. Immunoblotting analysis of the translated product showed that it is adult stage specific, water soluble and not membrane bound and can be demonstrated in the supernatant fluids of cultures from adult worms kept in cell culture medium. The results suggest that the protein encoded by Dv3-14 is a MSP of D. viviparus.

The Caenorhabditis elegans spe-6 gene is required for major sperm protein assembly and shows second site non-complementation with an unlinked deficiency.

Caenorhabditis elegans spermatozoa move by crawling. Their motility requires thin cytoskeletal filaments assembled from a unique cytoskeletal protein, the major sperm protein (MSP). During normal sperm development the MSP is segregated to developing sperm by assembly into filaments that form a paracrystalline array in a transient organelle, the fibrous body-membranous organelle. Mutations in the spe-6 gene cause sterility because they lead to defective primary spermatocytes that do not form spermatids. In these mutant spermatocytes the MSP fails to assemble into fibrous body filaments. Instead, the unassembled MSP distributes throughout the cytoplasm and nucleus. Thus, the spe-6 gene product is necessary for normal MSP localization and assembly during sperm development. In addition to their MSP assembly defect, spe-6 mutant spermatocytes arrest meiosis at diakinesis although their spindle pole bodies still replicate and separate. This results in spermatocytes with four half-spindles surrounding condensed, but unsegregated, chromosomes. All four spe-6 alleles, as well as a chromosome III deficiency that deletes the spe-6 gene, fail to complement two small overlapping chromosome IV deficiencies, eDf18 and eDf19. This non-allele-specific second site non-complementation suggests a concentration-dependent interaction between the spe-6 gene product and products of the gene(s) under eDf18 and eDf19, which include a cluster of sperm-specific genes. Since MSP filament assembly is highly concentration-dependent in vitro, the non-complementation might be expected if the sperm-specific gene products under eDf18 and eDf19 were needed together with the spe-6 gene product to promote MSP assembly.

The development and evolution of actin‐containing organelles during spermiogenesis of a primitive nematode

Spermatogenesis in the primitive marine nematode Sphaerolaimus hirsutus (Chromadoria, Sphaerolaimidae) was investigated by examining the ultrastructure and cytochemistry. Spermatozoa are lenticular cells of about 15 microns in diameter and are devoid of flagellum and acrosome as in other nematodes. In spermatocytes, dictyosomes produced transient structures, the fibrous body-membranous organelle complexes (FB-MO). In spermatids, the FBs were arranged as cartwheel spokes, with the FBs in the centre and the MOs at the periphery. The FBs were first made up of parallel fibres and surrounded by a membrane, then, in a later stage, showed a dense central structure with a surrounding vermiculate region and were devoid of membrane. The FBs contain actin as shown by immunofluorescence using a monoclonal anti-actin antibody and by affinity cytochemistry using fluorescent phalloidin. MOs contained mainly F-actin as shown by their labelling by phalloidin. In spermatozoa, the MOs were no longer peripheral but arranged on a ring in the central region of the cell and the FBs disappeared to form the cytoskeleton of the cell outer region. It was assumed, by analogy with the ultrastructure of other nematodes, that this cytoskeleton was made up of major-sperm-protein (MSP). Labelling of spermatids of Caenorhabditis elegans also revealed the presence of actin, but cells and actin spots were very small. This demonstrates the interest of Sphaerolaimus as a giant sperm model in which fibrous bodies and membranous organelles can be distinguished. In the few species in which it has been studied (C elegans and Ascaris suum), MSP is thought to constitute in spermatozoa a motile cytoskeleton excluding the presence of actin. However, the present study of Sphaerolaimus shows that the actin cytoskeleton is present during nematode spermiogenesis.

Sperm-activating peptide type-V (SAP-V), a fifth member of the sperm-activating peptide family, purified from the egg-conditioned media of the heart urchin Brissus agassizii

1. 1. A novel type of sperm-activating peptide named sperm-activating peptide type-V (SAP-V) was isolated from the egg-conditioned media (egg jelly) of the heart urchin ( Brissus agassizii and the primary structure of the peptide was determined by fast atom bombardment mass spectrometry as follows: ▪. 2. 2. SAP-V and [Met(O) 9]SAP-V stimulated the respiration of B. agassizii spermatozoa with half-maximal concentrations of 0.5 and 0.3 nM, respectively. However, half-maximal stimulation of the sperm respiration required 40 nM of S-carboxymethylated SAP-V. 3. 3. SAP-V induced significant increases in the cyclic AMP and cyclic GMP levels in B. agassizii spermatozoa in a concentration-dependent manner. 4. 4. The addition of SAP-V to B. agassizii spermatozoa resulted in a mobility shift of a major sperm protein (mol. wt from 133,000 to 129,000) on sodium dodecyl sulfate-polyacrylamide gels.

Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55-66, (1989)). Examination by video-enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled rearward at the same rate (10-50 microns/min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as weak acids and protonophores, caused the fiber complexes to disassemble completely in 4-5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30-60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3-. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.

Derivatives of sperm-activating peptide III (SAP-III) associated with eggs of sand dollars, Clypeasterjaponicus and Astriclypeusmanni

1. 1. NIne sperm-activating peptides were isolated from the egg jelly of sand dollars Clypeaster japonicus and Astriclypeus manni, and their amino acid sequences were determined. 2. 2. The peptides elevated cyclic GMP and cyclic AMP concentrations in spermatozoa of respective species and caused a shift in the apparent molecular weight of a major sperm protein of the respective species of sand dollars. 3. 3. The peptides stimulated their own sperm respiration rate one half-maximally at about 0.1–1.9 nM under slightly acidic pH. 4. 4. Egg jelly extract from Peronella japonica contained a substance(s) which stimulates C. japonicus sperm respiration.

Major sperm protein genes from Onchocerca volvulus

Nematode spermatozoa, unlike their mammalian counterparts, are nonflagellated crawling cells. The pseudopod of these cells contains the major sperm protein (MSP) which comprises more than 15% of the protein in the sperm. MSP is presumed to function as a cytoskeletal element involved in motility. An Ascaris MSP cDNA sequence was used as a probe to identify and isolate Onchocerca volvulus MSP clones from a λgt11 genomic library. Two clones, OVGS-1 (765 bp) and OVGS-2 (1765 bp), were characterized by restriction endonuclease mapping and sequence analysis. Both genomic clones contain MSP protein coding regions of 99 and 282 bp separated by an intervening sequence of 153 bp. The génes OVGS-1 and OVGS-2 are 95% similar in nucleotide sequence in the protein coding regions, but only 79% similar in their intron sequences. A number of potential regulatory sequences in the flanking regions and at the exon/intron junctions of the O. volvulus MSP genes are in good agreement with consensus sequences in other eukaryotic cells. The nucleotide sequence of the O. volvulus MSP genes were over 80% similar to the Ascaris MSP cDNA sequence and 79% similar to the Caenorhabditis MSP-3 cDNA. The predicted amino acid sequence of the O. volvulus MSPs were 96% similar to each other, 90–91% similar to Ascaris MSP and 81–82% similar to Caenorhabditis MSP-3. These results offer evidence that the MSP sequences have been highly conserved throughout nematode evolution but are variable in their genomic organization and the presence of introns.

Major sperm protein and actin genes in free-living and parasitic nematodes

The DNA from a number of free-living and parasitic nematode species was examined to determine the genomic number and distribution of DNA sequences encoding two evolutionarily conserved proteins; the major sperm protein (MSP) and nematode actin. Ascaris and Caenorhabditis MSP cDNA sequences and Ascaris genomic actin sequences were used to probe Southern blots of Eco RI and Hin d III digested nematode DNA. The number of MSP genes varied widely between the 1 MSP gene in Ascaris and the 60 MSP genes in Caenorhabditis. Filarial nematodes appeared to have 1-4 MSP genes while the plant and insect parasitic species showed from 5-12 MSP-hybridizing restriction fragments. Mammalian intestinal parasites showed between 1 and 13 bands hybridizing with the MSP probes. Blots probed to estimate the number of actin genes showed that, with the exception of Ascaris which contains more than 20 germ line sequences that encode actin, all of the nematodes tested had between 3 and 9 bands that hybridized to the Ascaris genomic actin probe. The possible use of highly conserved sequences such as MSP and actin to differentiate between nematode species in diagnostic and taxonomic studies is discussed.

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum.

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.