Analysis of Biological Development (K. Kalthoff)

Updates to Topic 04: Fertilization


Answers to Questions in Text

Role of Bindin in Sea Urchin Sperm-Egg Adhesion (p. 81)

  1. Why were the contents of the acrosomal vesicle a logical place to start the search for a bindin-like component? Answer: Because the sperm adheres with the acrosomal process, and the plasma membrane covering the acrosomal process is derived from the inner acrosomal membrane.
  2. If bindin from species A would agglutinate eggs from species A and S equally well, what should have been the relative frequencies of A-A, A-S, and S-S egg associations in Fig. 4.6? Answer: 1:2:1

Bioassay for Mouse Zona Pellucida Protein (p.87)

  1. In the bioassay outlined in Fig. 4.11, why was it important that the sperm be present in limited supply rather than in excess? Answer: The basic idea of the bioessay is to test defined molecules for their ability to compete with eggs for binding sites on sperm. If sperm were in excess, no competition would be detected over a wide concentration range of molecules added because there would always be enough sperm to adhere to - and fertilize - all eggs present.
  2. In the course of preparing zona proteins from eggs and two-cell embryos, it became apparent that zona pellucida from embryos is much harder to dissolve in aqueous media. What does this difference in solubility suggest? Answer: That zona proteins have become crosslinked or otherwise modified after fertilization.

Comments

See Movies on Fertilization on Movies page.

Clarifications and Corrections

On page 85, the label in Figure 4.10b should read "... and outer acrosomal membrane". The membrane is labeled correctly in Figure 4.9 on page 84.

New Review Articles

Runft L.L., Jaffe L.A. and Mehlmann L.M. (2002) Egg activation at fertilization: Where it all begins. Devel.Biol. 245: 237-254

New Research Articles

Inoue N., Ikawa M., Isotani A., and Okabe M. (2205) The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434: 234-238
Using a fusion-inhibiting monoclonal antibody and gene cloning, the authors identify a mouse sperm membrane protein required for sperm egg fusion. They terned the protein Izumo. Males lacking the Izumo gene produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

Runft L.L. and Jaffe L.A. (2000) Sperm extract injection into ascidian eggs signals Ca2+ release by the same pathway as fertilization. Development 127: 3227-3236

Yamamoto S., Kubota H.Y., Yoshimoto Y. and Iwao Y. (2001) Injection of a sperm extract triggers egg activation in the newt Cynops pyrrhogaster. Devel. Biol. 230: 89-99

Egg activation entails a transient rise in cytoplasmic free Ca2+, which is observed after normal fertilization and - in several species - after injection of sperm extract. Ca2+ is thought to be released from the endoplasmatic reticulum in response to IP3 (see Fig. 2.27 in text p. 45). IP3 in turn is generated by the phospholipase C (PLC) family of enzymes, which includes the cytoplasmic PLCgamma. The latter is activated when its two Src-homolgy 2 (SH2) domains interact with an activated tyrosine kinase (TK). When Runft and Jaffe (2000) injected eggs of the ascidian Ciona intestinalis with an excess of PLCgamma SH2 domains the release of Ca2+ after normal fertilization was inhibited, presumably due to competition between the injectant and endogenous PLCgamma for TK binding sites. The release of Ca2+ upon injection of sperm extract was similarly inhibited, suggesting that fertilization and sperm extract injection release Ca2+ by a pathway requiring PLCgamma and a Src family kinase. In this regard, Ciona eggs behaved like sea urchin eggs and unlike mouse and frog eggs (see Runft et al., 1999 reference in text). The latter do not require SH2 domain-mediated activation of PLCgamma for Ca2+ release. In contrast, Yamamoto et al. (2001) found that eggs of the newt Cynops pyrrhogaster showed Ca2+ release and other signs of activation in response to injection with sperm extract. Pretreatment of the sperm extract with heat or proteinase K abolished the activating effect, suggesting that the activating sperm component may be a protein.

Miller MA, Nguyen VQ, Lee MH, Kosinski M, Schedl T, Caprioli RM, Greenstein D. (2001) A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation. Science 291: 2144-2147

Caenorhabditis elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of smooth muscle-like gonadal sheath cells, which are required for ovulation. Using a bioassay (sperm-conditioned medium introduced into the uterus of spermless hermaphrodites), the investigators show that the major sperm cytoskeletal protein (MSP) is a bipartite signal for oocyte maturation and sheath contraction. MSP synthesized in recombinant bacteria elicits the same responses, indicating that the activity of MSP purified from sperm-conditioned medium does not result from a contaminant. The result is unexpected because MSP also functions in sperm locomotion, playing a role analogous to actin. Thus, MSP has both an extracellular signaling and an intracellular cytoskeletal function in C. elegans reproduction. Proteins with MSP-like domains are also found in plants, fungi, and other animals, suggesting that similar signaling functions may exist in other phyla.


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