Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm

Computer assisted semen analyzers in andrology research and veterinary practice

The mechanism of C- and G-banding of chromosomes

The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.

/pdf/ovastacin-a-cortical-granule-protease-cleaves-zp2-in-the-1vvz7x180f.pdf

Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

Biochemical and genetic studies have produced a vast fund of knowledge concerning gene action and regulation in prokaryotes In these organisms the DNA is exposed rather nakedly to the world, protected primarily by the cell membrane In eukaryotes the DNA seems far better shielded, being enmeshed in histone and nonhistone proteins and sequestered behind both the cell and the nuclear membrane These differences have led to a considerable degree of caution in the application of this knowledge of prokaryotes to problems of gene regulation in eukaryotes, and rightly so There are, however, several observations which suggest that higher organisms may have picked up a number of fundamental genetic tricks from their lowly predecessors It has frequently been suggested that eukaryotes must do things differently from prokaryotes, until proven otherwise It may be prudent to reverse this line of thought and suggest that they do things the came until proven different The following similarities suggest this (1) T he basic genetic dogmas concerning DNA replication, transcription, andlation are similar, (2) The genetic code is the same (3) Both systems appear to make use of cyclic AMP as a basic mediator for, humoral or diffusible, signals (4) In both systems DNA synthesis may be controlled at membranes (5) Both make use of different types of RNA polymerase and RNA polymerase cofactors (6) Recent studies of polylysine binding to chromatin suggest the eukaryotic DNA may not be so thoroughly enmeshed in Protein as once thought (7) The visualization of genes in action by electron microscopic techniques intimates that genes are spaced and read in a similar manner And finally, (8) merely because the clustering of related genes is unusual in higher organisms is no reason in itself to totally discard the promoter-operator-repressor concept as a way of regulating single structural genes This system has provided an immense amount of data concerning the manner in which proteins interact with specific DNA sequences to control the attachment and utilization of RNA polymerase It is hard to imagine that eukaryotes, being presented with such a superb mechanism for controlling DNA transcription, would totally discard it and opt for something different It is far more likely that they would build on to this solid foundation

The structure and function of chromatin.

BRADEN et al.1 have shown that the zona pellucida of mammalian eggs changes after fertilization so that penetration of further spermatozoa is greatly reduced. Austin and Braden2 inferred that this zona reaction was due to the action of the cortical granule material which is released from the egg into the perivitelline space shortly after sperm entry into the egg cytoplasm. We wish to present experimental evidence to support this inference.

Induction of zona reaction in golden hamster eggs by cortical granule material.

The zona pellucida of a fertilized egg recovered from an artificially inseminated female reacts to capacitated spermatozoa differently from the zona pellucida of an unfertilized egg. The zona of a fertilized egg shows a sperm-refractory response, “zona reaction,” so that capacitated spermatozoa neither firmly attach to nor penetrates it. On the other hand, the zona of an unfertilized egg shows no such sperm-refractory response and many spermatozoa attach to and penetrate it. The zona reaction was observed not only in recently penetrated eggs but also throughout cleavage.



Judging from the earliest fertilization stage at which we observed a complete zona reaction, it is estimated that this reaction is completed in less than 15 minutes after sperm attachment to the vitellus.



One hundred per cent of the fertilized eggs recovered from females five hours after artificial insemination were refertilized in vitro after removal of the zona pellucida by trypsin-treatment. The incidence of refertilization decreased when the eggs were recovered at later times. Only 3% of the eggs recovered at nine hours after artificial insemination were refertilized. Judging from the fertilization stage at which eggs were no longer refertilizable, it has been estimated that the completion of the vitelline surface block to polyspermy is established in not less than two nor more than three to three and one-half hours.



Possible causes of polyspermy encountered with in vitro fertilized eggs are discussed.

Polyspermy-preventing mechanisms in the golden hamster egg.

/pdf/failure-of-zona-penetration-of-hamster-spermatozoa-after-1cdgd75xgn.pdf

Failure of zona penetration of hamster spermatozoa after prolonged preincubation in a blood serum fraction.

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Induction of zona reaction in golden hamster eggs by cortical granule material.

Polyspermy-preventing mechanisms in the golden hamster egg.

Failure of zona penetration of hamster spermatozoa after prolonged preincubation in a blood serum fraction.