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LECTURE 32

LECTURE 32 GLYCOBIOLOGY OF ANIMAL FERTILIZATION # 2 MAY 23, 2002 V.D. VACQUIER The use of plant lectins to study cell-cell interaction exploded about

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LECTURE 32

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  1. LECTURE 32 GLYCOBIOLOGY OF ANIMAL FERTILIZATION # 2 MAY 23, 2002 V.D. VACQUIER The use of plant lectins to study cell-cell interaction exploded about 1970. From that time on there developed a literature of hundreds of papers on the effects lectins on sperm, eggs and their interaction during in vitro fertilization. Data implicating glycoproteins in fertilization: 1. Lectins blocked fertilization in many animal species. (WGA is a potent inhibitor of sea urchin fertilization.) 2. The pattern of lectin staining of mammalian sperm cells changed as the sperm traveled down the epididymis. The sperm “matured” as they moved down the epididymis. 3. Protease digests (glycopeptides) of gamete surfaces blocked fertilization in a variety of animals. 4. Analysis of gamete surface proteins showed they are almost always heavily glycosylated.

  2. The years went by and very little satisfying information was obtained by the lectin / glycopeptide approach. However, in some non-mammalian species (fish, frog, sea urchin, abalone, ascidian), gametes could be obtained in large quantities to permit biochemical isolation of gamete recognition proteins. (We know most about abalone fertilization compared to any other animal.) There is very little work on model species such as Drosophila because they have internal fertilization and small numbers of gametes. There is a little recent work on C.elegans, but in total very little is known. Most of the papers are phenomenological with very little chemical data. In many of the recent experiments, peptides synthesized based on cloned genes have been used to block steps in fertil- ization. Very little knowledge of the role of carbohydrates has been gained from this approach.

  3. A review (Glycobiology 11, 37R-43R, 2001) summarizes all the recent information on the glycobiology of fertilization in: -Sea urchins (you heard it already) -Starfish (somewhat similar to urchins) -Ascidians (self-sterile hermaphrodites) -Amphibia (Xenopus) -Teleost fish (Rainbow trout very interesting and new) -Mammals (hopefully I will get to them) In rainbow trout, the most recent paper shows that a deaminated neuraminyl ganglioside on the sperm “(KDN)GM3”, (KDN23Galß4Glcß1Cer) (90% of total sperm gangliosides) binds to a glycolipid, “Gg3Cer” (GalNAcß4Galß4Glcß1Cer), on the egg. PNAS 99 (5), 2854, 2002.

  4. Fertilization in the mouse Photo by P. Wassarman

  5. Perivitelline space Zona pellucida Egg plasma membrane (= egg) Mammalian Sperm Egg interaction

  6. The mouse egg ZP is made of 3 glycoproteins: ZP1 = 623 aa = 68 kDa All 3 arose by gene duplication. ZP2 = 713 aa = 80 kDa ZP3 = 424 aa = 46 kDa All 3 glycoproteins are made and secreted by the growing oocyte to form a fibrous meshwork = the ZP. ZP3 binds the acrosome intact sperm & induces the sperm AR. Soluble ZP3 will induce the AR. It will also block sperm from binding to the ZP. ZP2 binds the sperm to the ZP after the AR has occurred. ZP1 crosslinks the fibrous molecules of ZP2 & ZP3 together. If ZP3 is “K.O.ed”, a ZP does not form and the mouse is sterile. ZP2 and ZP3 are two of the most rapidly evolving genes between rodents and humans (Nature, Rev. Gen. 3: 137, 2002).

  7. NH2 Zhao et al, Mol. & Cell. Biol. 22(9), 3111, 2002

  8. Facts About ZP3 -Soluble ZP3 blocks sperm binding to eggs, it will also induce the AR. (= ZP3’s “biological activities”) -Glycopeptide digests of ZP block sperm binding to eggs, but will not induce the AR. -Mild alkaline hydrolysis of ZP liberates oligosaccharide chains that block sperm binding to eggs, but will not induce the AR. -Inject Xenopus oocytes with mouse ZP3 mRNA and one gets incorporation of mouse ZP3 into the Xenopus vitelline envelope. -N-linked oligosaccharides are not involved in ZP3’s biological activity. (PNGase treatment does decrease the Mr on SDS-PAGE.) -ZP3 is sulfated, but removal of SO4 does not decrease activity. -Neuraminidase treatment lowers the Mr on SDS-PAGE, but does not alter the biological activity.

  9. ZP3 Facts-- Continued -Soluble ZP3 will activate G-proteins Gi-1 and Gi-2. The activation in pertusis toxin sensitive. -There are no receptors, or 2nd messengers known for the mouse spermG protein pathway leading to the AR. -Oligosaccharides of ZP3 stimulate voltage activated T-type Ca channels in sperm. The PM resting potential goes from -60 mV to -30 mV.

  10. Positive Selection in ZP3 Maximum likelihood analysis of ZP3 from an alignment of ZP3s from eight mammalian species shows that several positions in the N- and C- terminal regions are subjected to adaptive evolution (positive Darwinian selection). The evolution of the sperm combining site has been especially influenced by positive selection. The oligosaccharide chains on Ser 332 & Ser 334 are critical for ZP3 “biological activity”. Swanson et al, PNAS 98(5), 25, 2001

  11. PNAS 95, 6193, 1998 Ser 332 and Ser 334 are critical to the Sperm binding activity of mZP3

  12. Site-directed mutagenesis of Ser 332 or 334 destroys the ability of soluble ZP3 to block sperm binding to the ZP of intact mouse eggs. Nothing is known about the oligosaccharide chains of these two critical Ser residues. It’s these chains that matter. Mutated all indicated residues to alanine. Chen, Litscher & Wassarman

  13. The AR

  14. To what sperm membrane protein does ZP3 bind? People thought that only one sperm surface protein would be found to be the partner for egg ZP3. However, during the past 20 years about 2 dozen sperm PM proteins have been claimed to be the real sperm ZP3 partner. Many friendships have been lost due to conflicting data coming out of different labs. The situation reminds me of a quote from my own professor, the late Danial Mazia, who said in his wisdom, “It’s not worth loosing a good friend over something that in five years will be shown to be wrong anyway”. It also reminds me of the warning I try to teach my own students, “never become wedded to your own hypothesis, always be ready to accept the fact that your hypothesis will turn out to be wrong”.

  15. Sperm proteins identified as potential ligands for ZP receptors Galactosyl transferase (GalTase) Miller et al 1992 Zona Receptor Kinase (p95) Leyton & Saling 1989 Sp56 Bleil & Wassarman 1990 M42 Saling et al 1985 Zonadhesin Hardy & Garbers 1995 Mannose binding protein Cornwall et al 1991 Sp17 Richardson et al 1994 FA-1 Naz 1988 PLA2 Riffo and Parraga Sperm agglutinin 1 Diekman et al 1997 PH20 Cherr et al 2001 Acrosin Howes et al 2001 Spermadhesin Topfer-Petersen 2001 Well what happened? Why are there so many sperm Proteins claimed to be important for AR induction?

  16. MOUSE KNOCKOUTS The major thing that happened was that mouse knockouts were developed. To the great surprize of many, knocking out all candidate genes either had no effect on male fertility, or decreased fertility. No single gene knockout resulted in infertile male mice. Possible reasons: 1. There are many different egg binding proteins in different species. (We know gamete recognition system evolve fast.) 2. Complexes of several different proteins are involved in ZP binding. No one protein is indispensable. 3. In vivo results might be different from in vitro results. 4. Like many other systems, there is degeneracy built into mechanism of acrosome reaction induction. See article on degeneracy in biological mechanisms by Edelman & Gally, PNAS 98 (24), 2001.

  17. Conclusion: In the mouse, the most widely worked on mammal for fertilization studies, oligosaccharide chains on Ser 332 and Ser 334 of ZP3 are crucial for interaction with receptors on the sperm.The exact chemical nature of these chains remains unknown.To my knowledge no one has attempted to sequence them and then perform a synthesis. Glycoprotein recognition events are undoubtedly involved in every step of the fertilization cascade---chemo- attraction, binding to the ZP, induction of the AR, passing through the ZP, attachment to the egg PM, fusion of sperm and egg PMs. The field of fertilization is ready for some well trained young glycobiologists to get involved and produce some really good data that will move the field forward.

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