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.

LECTURE 32 GLYCOBIOLOGY OF ANIMAL FERTILIZATION # 2 MAY 23, 2002 V.D. VACQUIER

The use of plant lectins to study cell-cell interaction exploded about1970. From that time on there developed a literature of hundreds ofpapers on the effects lectins on sperm, eggs and their interactionduring 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 blockedfertilization in a variety of animals.

4. Analysis of gamete surface proteins showed they are almost always heavily glycosylated.

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 permitbiochemical isolation of gamete recognition proteins. (We knowmost about abalone fertilization compared to any other animal.)

There is very little work on model species such as Drosophilabecause they have internal fertilization and small numbers of gametes. There is a little recent work on C.elegans, but in totalvery little is known.

Most of the papers are phenomenological with very little chemicaldata. In many of the recent experiments, peptides synthesizedbased on cloned genes have been used to block steps in fertil-ization. Very little knowledge of the role of carbohydrates hasbeen gained from this approach.

A review (Glycobiology 11, 37R-43R, 2001) summarizesall 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”,(KDN23Galß4Glcß1Cer) (90% of total sperm gangliosides)binds to a glycolipid, “Gg3Cer” (GalNAcß4Galß4Glcß1Cer),on the egg.

PNAS 99 (5), 2854, 2002.

Fertilization in the mouse

Photo by P. Wassarman

Perivitelline space

Zona pellucidaEgg plasma membrane

Mammalian Sperm Egg interaction

(= egg)

The mouse egg ZP is made of 3 glycoproteins:ZP1 = 623 aa = 68 kDa All 3 arose by gene duplication.ZP2 = 713 aa = 80 kDaZP3 = 424 aa = 46 kDaAll 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 spermfrom 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 genesbetween rodents and humans(Nature, Rev. Gen. 3: 137, 2002).

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

NH2

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 theAR.

-Inject Xenopus oocytes with mouse ZP3 mRNA and one gets incorporation of mouse ZP3 into the Xenopus vitellineenvelope.

-N-linked oligosaccharides are not involved in ZP3’sbiological activity. (PNGase treatment does decreasethe 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.

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 goesfrom -60 mV to -30 mV.

Positive Selection in ZP3Maximum likelihood analysisof ZP3 from an alignment of ZP3sfrom eight mammalian species shows that several positions in theN- and C- terminal regions are subjected to adaptive evolution (positive Darwinian selection). The evolutionof the sperm combining sitehas been especially influenced by positive selection.

The oligosaccharide chains onSer 332 & Ser 334 are critical forZP3 “biological activity”.

Swanson et al, PNAS 98(5), 25, 2001

Ser 332 and Ser 334 are critical to theSperm binding activity of mZP3

PNAS 95, 6193, 1998

Site-directed mutagenesisof Ser 332 or 334 destroysthe ability of soluble ZP3to block sperm binding tothe ZP of intact mouse eggs.

Nothing is known about theoligosaccharide chainsof these two critical Serresidues. It’s these chainsthat matter.

Chen, Litscher & Wassarman

Mutated all indicated residuesto alanine.

The AR

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 proteinshave been claimed to be the real sperm ZP3 partner. Manyfriendships have been lost due to conflicting data coming out ofdifferent 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 infive years will be shown to be wrong anyway”.

It also reminds me of the warning I try to teach my ownstudents, “never become wedded to your own hypothesis,always be ready to accept the fact that your hypothesiswill turn out to be wrong”.

Sperm proteins identified as potential ligands for ZP receptorsGalactosyl transferase (GalTase) Miller et al 1992Zona Receptor Kinase (p95) Leyton & Saling 1989Sp56 Bleil & Wassarman 1990M42 Saling et al 1985Zonadhesin Hardy & Garbers 1995Mannose binding protein Cornwall et al 1991Sp17 Richardson et al 1994FA-1 Naz 1988PLA2 Riffo and ParragaSperm agglutinin 1 Diekman et al 1997PH20 Cherr et al 2001Acrosin Howes et al 2001Spermadhesin Topfer-Petersen 2001

Well what happened? Why are there so many sperm Proteins claimed to be important for AR induction?

MOUSE KNOCKOUTSThe major thing that happened was that mouse knockoutswere developed. To the great surprize of many, knocking out allcandidate genes either had no effect on male fertility, ordecreased 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 evolvefast.)

2. Complexes of several different proteins are involved in ZPbinding. 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. Seearticle on degeneracy in biological mechanisms by Edelman & Gally, PNAS 98 (24), 2001.

Conclusion:In the mouse, the most widely worked on mammal for

fertilization studies, oligosaccharide chains on Ser 332 andSer 334 of ZP3 are crucial for interaction with receptors onthe 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 andegg PMs.

The field of fertilization is ready for some well trainedyoung glycobiologists to get involved and produce some really good data that will move the field forward.