Kosinski M et al. (2005) Development "C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes."

Greenstein D, Schwartz J, Kosinski M, Yamamoto I, McDonald K

[Development, 2005]

The major sperm protein (MSP) is the central cytoskeletal element required for actin-independent motility of nematode spermatozoa. MSP has a dual role in Caenorhabditis elegans reproduction, functioning as a hormone for both oocyte meiotic maturation and ovarian muscle contraction. The identification of the signaling function of MSP raised the question, how do spermatozoa, which are devoid of ribosomes, ER and Golgi, release a cytoplasmic protein lacking a signal sequence? Here, we provide evidence that MSP export occurs by the budding of novel vesicles that have both inner and outer membranes with MSP sandwiched in between. MSP vesicles are apparently labile structures that generate long-range MSP gradients for signaling at the oocyte cell surface. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly. We propose that MSP generates the protrusive force for its own vesicular export.

Smith HE et al. (1997) International C. elegans Meeting "SPERM MOTILITY IN C. ELEGANS: STRUCTURE-FUNCTION ANALYSIS OF THE MAJOR SPERM PROTEIN"

Ward S, Smith HE

[International C. elegans Meeting, 1997]

In nematodes, sperm are amoeboid cells which crawl via an extended pseudopod. Unlike other crawling cells, this pseudopod contains no actin or myosin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility is derived from the regulated assembly and disassembly of MSP. Filaments composed of MSP dimers are thought to provide the motive force. Mutants defective in MSP-MSP interactions would provide insights into the process of filament formation. Because there are 28 MSP genes expressed in C. elegans, which precludes traditional mutational analysis, the yeast two-hybrid system was used to investigate MSP-MSP interactions. Fusions of MSP to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) exhibit interaction and drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones which fail to interact with LEXA-MSP. The mutations were mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted by the crystal structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. In addition, multiple mutations were obtained in the carboxy-terminal beta strand, a region not predicted to be involved in folding or dimer formation. Compensating mutations which restore the interaction also map to this region. The data suggests that the carboxy-terminal beta strand is directly involved in interactions required for MSP filament assembly.

Sung Min Han et al. (2007) International Worm Meeting "Identifying receptors that interact with major sperm protein in Caenorhabditis elegans."

Michael A. Miller, Sung Min Han

[International Worm Meeting, 2007]

An evolutionary conserved feature of oogenesis is that oocytes arrest in meiotic prophase. Oocytes resume meiosis in response to extracellular signals by a process called oocyte meiotic maturation, which prepares the oocyte for fertilization and embryogenesis. The Caenorhabditis elegans major sperm protein (MSP) is the most abundant protein in sperm. It is exported by a budding mechanism from the sperm cytoplasm to the extracellular environment, where it induces oocyte meiotic maturation and ovulation. During evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions. Proteins with MSP-related domains have been found in diverse metazoans, raising the possibility that MSP has a conserved signaling function. Previous work using an in situ MSP-FITC binding assay identified the VAB-1 Eph receptor as an MSP receptor. However, vab-1 null gonads still bind to and respond to MSP. These results suggest that MSP binds to multiple receptors that act redundantly to induce oocyte maturation and sheath contraction. To identify additional MSP receptors, I conducted an RNAi-based screened of 58 receptor candidates whose mRNAs are expressed during oogenesis, but not spermatogenesis. Five receptor candidates have been identified that are required for MSP binding. These encode members of evolutionarily conserved receptor classes. Currently, I am testing whether any of five candidates directly binds to MSP and whether mutations in the candidates cause defects in MSP signaling.

Mary E Kosinski et al. (2003) International Worm Meeting "Release of the major sperm protein from spermatids and spermatozoa in vivo"

Jonathan Holder, Ginger Winfrey, David Greenstein, Mary E Kosinski, Gary Olson

[International Worm Meeting, 2003]

Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. During meiotic maturation, C. elegans oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in a spatially restricted manner such that the most proximal (-1) oocyte matures, is ovulated, and fertilized. Sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using the major sperm protein (MSP) as a signaling molecule. The discovery of MSPs signaling role raised the intriguing question of how sperm release MSP to signal oocytes and sheath cells at a distance. The release of MSP from sperm likely occurs through a non-canonical mechanism since spermatozoa do not possess cellular components required in standard models of protein secretion, such as ribosomes, ER, or Golgi. Moreover, MSP does not have a N-terminal leader sequence, and in vitro release does not involve protein processing. Non-canonical or leaderless secretory pathways are widespread, and in general, poorly understood, despite figuring prominently in cell signaling, disease, and host defense. Using specific antibodies, we detect MSP both inside and outside of spermatids and spermatozoa in vitro. MSP release does not require motility or a pseudopod, and does not involve sperm lysis. We observe extracellular MSP up to approximately 90 m away from spermatozoa in the spermatheca, frequently reaching the -1 oocyte. Examination of extracellular MSP reveals it is localized in a graded distribution with the highest levels of MSP adjacent to spermatozoa and lower levels distally. MSP receptors on the -1 oocyte appear to bind and concentrate MSP resulting in a sharp boundary of staining, which is eliminated when trafficking of oocyte membrane proteins is blocked. Confocal microscopy shows extracellular MSP to be punctate. We observe large (~100-200 nM) MSP-staining puncta near spermatids or spermatozoa in the uterus. We speculate that these puncta are MSP-containing vesicles. Using immuno-EM we examined MSP localization in spermatozoa to pinpoint its localization in the cell. In addition to localization of MSP within the pseudopod, we also found MSP located in the cell body. Interestingly, we found a fraction of MSP associated with the plasma membrane of the spermatozoa. Based on these results we propose a model in which spermatids and spermatozoa shed vesicles containing MSP. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical tests of this model are underway.

Smith HE et al. (1998) J Mol Biol "Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans."

Ward S, Smith HE

[J Mol Biol, 1998]

In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal beta strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal beta strands are directly involved in interactions required for MSP filament assembly.

Burke DJ et al. (1983) J Mol Biol "Identification of a large multigene family encoding the major sperm protein of Caenorhabditis elegans."

Ward S, Burke DJ

[J Mol Biol, 1983]

DNA fragments corresponding to genes encoding the MSP of Caenorhabditis elegans sperm have been isolated by recombinant DNA techniques. Analyses of individual genomic clones suggest that there are multiple MSP genes that are dispersed in the genome. From restriction enzyme digests of genomic DNA fractionated and hybridized with an MSP complementary DNA probe, there appear to be more than 30 MSP genes in the genome. Despite the occurrence of this large dispersed multigene family, the MSP messenger RNA from both males and hermaphrodites is homogene in size. There are at least three different proteins of identical molecular weight but different isoelectric point that cross-react with anti-MSP antisera. Each protein is a primary translation product with no detectable post- translational modifications, suggesting that at least three of the MSP genes are expressed.

Mary E Kosinski et al. (2005) International Worm Meeting "C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes"

David Greenstein, Mary E Kosinski, Joel Schwartz, Ikuko Yamamoto, Kent McDonald

[International Worm Meeting, 2005]

Intercellular signaling facilitates multiple steps of the fertilization process. In many species, sperm prepare the oocyte for fertilization by providing signals for meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, nuclear envelope breakdown, and meiotic spindle assembly. C. elegans sperm signal oocyte meiotic maturation using the major sperm protein (MSP) as a hormone. Interestingly, the MSP also functions as the central cytoskeletal protein required for the amoeboid motility of nematode sperm. The discovery of MSPs signaling role raised the question of how sperm export MSP to signal oocytes at a distance. MSP lacks a hydrophobic leader sequence and C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. We used correlative light and electron microscopy to analyze the mechanism of MSP release from sperm. We will present evidence showing that sperm bud novel MSP vesicles to signal distant oocytes. These 150-300 nm MSP vesicles contain both an inner and an outer membrane, with MSP sandwiched in between. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly, suggesting that MSP may generate the protrusive force for its own vesicular export. MSP vesicles are labile structures that generate long-range MSP gradients for signaling at oocyte and sheath cell surfaces. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Spermatozoa generate a long-range, short-acting signal, whereas spermatids generate a long-acting signal. EM results suggest that differential vesicle stability affects the physical and temporal range of signaling.

Scott AL et al. (1989) Mol Biochem Parasitol "Major sperm protein genes from Onchocerca volvulus."

Scott AL, Ward S, Sussman DJ, Yenbutr P, Dinman J

[Mol Biochem Parasitol, 1989]

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 lambda gt11 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 genes 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

Mary Kosinski et al. (2001) International C. elegans Meeting "Control of Oocyte Meiotic Maturation and Gonadal Sheath Cell Contraction by MSP Signaling"

Mary Kosinski, David Greenstein, Michael A Miller

[International C. elegans Meeting, 2001]

Oocyte meiotic maturation and ovulation are essential biological processes required for reproduction. During meiotic maturation, C. elegan s oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in an assembly-line-like fashion. Recently we found that the major sperm cytoskeletal protein (MSP) is the sperm derived signal that induces both oocyte meiotic maturation and gonadal sheath cell contraction (1). Many questions remain about this newly discovered signaling pathway. For example, how does a cytoskeletal protein function as a signaling molecule? How does MSP signal two distinct responses? To examine MSP signaling in vivo , we analyzed extracellular MSP localization. Previous studies reported the intracellular localization of MSP during spermatogenesis (2). We stained mated fog-2 females with anti-peptide antibodies generated to the N- and C- terminal regions of MSP, as well as previously described antibodies (2). In addition to intracellular staining within spermatozoa, we observed an extracellular MSP gradient, with the highest levels of MSP within the spermatheca and lower levels in the proximal gonad arm. The average extent of the observable MSP gradient is approximately 30 m m. Detection of this extracellular MSP gradient is dependent on the number of sperm present and their position in the spermatheca. By mating females with Mr. Vigorous, we have been able to detect the MSP gradient extending as far as 90 m m. These data suggest that a fraction of MSP is released from sperm in vivo, and the most proximal oocyte receives the highest concentration of released MSP. MSP is a bipartite signal: MSP (106-126) is sufficient to promote gonadal sheath cell contraction, and MSP (1-106) is sufficient to promote oocyte maturation (1). In solution MSP exists as a dimer (3). To determine if dimerization is necessary for signaling, we injected dimerization mutants (4) into females and measured oocyte maturation and sheath cell contraction rates. The dimerization mutants tested signal both oocyte maturation and gonadal sheath cell contraction at rates similar to wild-type MSP. To further identify regions responsible for promoting oocyte maturation and gonadal sheath cell contraction, we tested whether Ascaris suum MSP could signal in C. elegans . Ascaris MSP shares about 80% identity with C. elegans MSP at the amino acid level. Our results indicate that Ascaris MSP can signal maturation and sheath cell contraction in C. elegans at rates comparable to C. elegans MSP. We are currently targeting regions for deletion experiments that are conserved between both C. elegans and Ascaris . 1. Miller et al. (2001). Science 291:2144-2147 2. Ward & Klass. (1982). Dev. Bio. 92:203-208 3. Haaf et al.(1996). J. Mol. Biol. 260:251-260 4. Smith & Ward. (1998). J. Mol. Biol. 279:605-619 We would like to thank T. Roberts and P. Griffin for Ascaris clones and S. Ward and H. Smith for dimerization mutants and antibodies.

Scott AL et al. (1989) Parasitology "Major sperm protein and actin genes in free-living and parasitic nematodes."

Sussman DJ, Scott AL, Dinman J, Ward S

[Parasitology, 1989]

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.