A Sperm Cytoskeletal Protein That Signals Oocyte Meiotic Maturation and Ovulation By Michael Miller

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A Sperm Cytoskeletal Protein That Signals Oocyte
Meiotic Maturation and OvulationBy Michael
Miller et al.

• Alison Walkden
• Melissa Capan

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Introduction

• Maturation cell cycle progression
• Breakdown of nuclear envelope
• Cortical rearrangement
• Ovulation exit of oocyte from gonad arm
• to spermatheca
• - requires contraction of sheath cells
• - dilation of distal spermatheca

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Purpose

• Oocytes of unmated females arrest at diakinesis
• Sperm promote the resumption of meiosis and the
  contraction of sheath cells
• Show that MSP is the bipartite signal

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ResultsIn Vivo Bioassay

• Inject SCM into uterus of fog-2 females
• Resulted in an increase in maturation and
  contraction

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Figure 1. A
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SDS Polyacrylamide Gel Electrophoresis

• Compared SCM to sperm lysates
• SCM is enriched with a single protein

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Figure 1. B

• Sperm lysates
• 2. SCM

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Reverse Phase Liquid Chromatography

• Mobile Phase polar
• Stationary Phase non polar, C4 C18 columns
• Separate factors according to their affinity for
  both phases
• Activities are likely found in same protein

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Figure 1. Diagram of HPLC Source
http//www.biotech.iastate.edu/facilities/protein/
hplc.html
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Figure 1. C
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MALDI-TOF Mass Spectrometry

• Factor is mixed in a solution and dried
• Pulsed with UV and becomes ionized
• Put in electric field and flight tube
• Separated by mass to charge ratio
• Found polypeptide 14,121 daltons

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Figure 1. Principle of ionisation and
volatilisation Source http//www.metabion.com/t
echinfo/z-maltof.html
(M matrix A analyte)
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Figure 2. Schematical illustration of the
principle of MALDI-TOF mass spectrometry
Source http//www.metabion.com/techinfo/z-maltof
.html
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Figure 1. D
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Further Analysis

• MSP 77 and 38 promoted maturation and ovulation
• Antibodies caused reduction in ovulation
• MSP promoted maturation and ovulation in C.
  ramanei

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Figure 2. AMaturations/hr
6His-MSP-77 6His-MSP-38 Sperm MSP
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Figure 2. BContractions/Min
6His-MSP-77 6His-MSP-38 Sperm MSP
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Figure 2. CAverage Displacement
6His-MSP-77 6His-MSP-38 Sperm MSP
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Alignment of COOH-terminal 20 amino acids of MSP
Figure 3. A

• Taken from Ascaris suum (intestinal roundworm),
  Globodera rostochiensis (potato cyst), C. elegans
  and Onchocerca volvulus (a nemoatode parasite)

• Highly conserved among nematodes

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Figure 3
3. B-D C. elegans fog-2(q71) females were
injected with buffer, Sperm MSP, 6His-MSP-77,
MSP(1-106), and MSP(106-126)
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Results

• MSP mutant lacking MSP(1-106) promotes oocyte
  maturation, but not sheath cell contraction
• A peptide corresponding to MSP(106-126) promotes
  sheath cell contraction, but not oocyte
  maturation
• MSP may act in two separate pathways in oocytes
  and sheath cells

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Role of MAPK (Mitogen-activated protein kinase)

• MAPK activation is critical in oocyte maturation
  in vertebrates
• MSP signaling activates the conserved MAPK
  cascade in oocytes and results in a number of
  cellular responses
• M-Phase entry
• Cortical cytoskeleton rearrangement
• Histone H3 phosphorylation
• Meiotic spindle assembly
• Gonadal sheath cell contraction

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Is MAPK activated in C. elegans oocytes during
maturation?
Figure 4

• Results
• Activated MAPK (red) is observed in females that
  were mated (A) or injected with 100nM
  6-His-MSP-77 (E and G).
• Not observed in unmated females which lack sperm
  (C).
• Not observed in mpk-1(ga117) (I).

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VAPs and MSP

• MSP folds into a seven-layered ß sandwich, the
  MSP domain
• MSP domain is structurally related to other
  NH2terminal domains found in fungi, plants and
  animals
• These domains are called VAPs (VAMP-associated
  proteins)

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VAPs and MSP

• VAMP-associated proteins (VAPs) have functions
  in somatic cells
• Functions in germ cells?
• VAP interacts with SNARE proteins, which have
  been linked to fertilization in sea urchins and
  mammals
• What is the relationship between VAPs and MSP?

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MSP domain predates Nematodes
Supplementary Figure 1 Evolutionary relationship
between MSP and VAP protein families
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Results of Phylogenetic Tree Analyses

• From phylogenetic analyses, VAP and MSP families
  form distinct monophyletic groups
• MSP domain may predate nematodes
• But

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BUT

• Clear orthologs have not been found in other
  organisms
• Sexual reproductive proteins can evolve very
  quickly
• So

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Two Alternate Suggestions

• MSP arose from VAP
• MSP was a common ancestor and lost in some
  lineages

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MSP and Locomotion

• Nematodes use a pseudopod packed with MSP, not
  actin, to crawl
• MSP forms filaments in pseudopod crawling is
  thought to be dependent on MSP
• Pseudopod formation is not needed for MSP
  signaling

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Summary

• MSP functions include extracellular signaling,
  and intracellular cytoskeletal functions
• Phylogenetics revealed possibility that MSP
  functions were conserved during evolution on
  multi-cellular organisms

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Why Study This?
Human beings are really bad at meiosis. We hope
that by studying how eggs mature meiosis in a
simple system like C. elegans, we will be able to
glean general principles about the signals
important to reproduction. We need to understand
the basic biology before we can attempt to fix
the problem. - Michael A. Miller
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