A Sperm Cytoskeletal Protein That Signals Oocyte Meiotic Maturation and Ovulation M.A. Miller, V.Q. Nguyen, M.-H. Lee, M. Kosinski, T. Schedl, R.M. Capriloli, D. Greenstein

Supplementary Material

Supplemental Figure 1. Evolutionary relationship of the MSP and VAP protein families. (A) VAPs contain an NH2-terminal MSP-like domain (boxed), a variable region (black line), and a transmembrane domain (TM). Boxed values indicate percent amino acid identity compared to mouse VAP. (B) Results from maximum parsimony phylogenetic analysis raise the possibility that MSP is ancient. Bootstrap statistical support values are shown. A score of 80 or higher indicates very strong support for a particular relationship. Boxed values support the hypothesis that C. elegans VAP is more closely related to the other VAPs than MSPs. MSP sequences are from C. elegans (MSP Ce, GenBank accession number P53020) and Ascaris suum (MSP As, P27439). VAP sequences are from Arabidopsis thaliana (VAP At, BAA97151), Nicotiana plumbaginifolia (VAP Np, CAB65313), Drosophila melanogaster (VAP Dm, AAF45909), Mus musculus (VAP Mm, AAF23076), Aplysia californica (VAP Ac,Q16943), C. elegans (VAP Ce, AAB96705), and Saccharomyces cerevisiae (SCS Sc, P40075). PapD is from E. coli (PAPD Ec, 3DPA).

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Movies. Analysis of MSP signaling by time-lapse video microscopy (1/12 real time). For best viewing, set the monitor color depth on grays.

To view these movies, download a QuickTime viewer.

• Movie 1
  Wild type ovulation.mov (1.8 MB). Oocyte maturation and ovulation in a wild-type hermaphrodite, the first ovulation in the gonad arm is shown.

• Movie 2
  fog-2 mock injection.mov (1.1 MB). A buffer-injected fog-2(q71) female showing a low sheath cell contraction rate.

• Movie 3
  fog-2 MSP injection #1.mov (872 K). Oocyte maturation and ovulation in a fog-2(q71) female injected with sperm-purified MSP. Note that strong sheath contractions continue following ovulation, unlike the situation in the wild type.

• Movie 4
  fog-2 MSP injection #2.mov (1.4 MB). A fog-2(q71) female injected with sperm-purified MSP showing an increase in the rate and intensity of sheath cell contractions.

Methods

Phylogenetic analyses were performed using maximum parsimony and neighbor-joining methods. Amino acid sequences from MSP and MSP-like domains of several representative VAPs were used in the analyses. For parsimony, the heuristic search option of PAUP* 3.1 (1) was used for tree construction, with 200 random order taxon addition replicates and tree bisection and reconnection branch swapping. Bacterial PapD, which is structurally related to MSP, was used as the outgroup. The "protpars" matrix of PAUP* 3.1 was used to weigh amino acid substitutions. To obtain bootstrap values, 100 bootstrap replicates were performed using simple taxon addition with tree bisection and reconnection branch swapping.

References

1. D. Swofford, PAUP: Phylogenetic analysis using parsimony (Illinois National History Survey, Champaign, IL, 1993).