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

2012 ◽  
Vol 197 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Anna D. Burkart ◽  
Bo Xiong ◽  
Boris Baibakov ◽  
Maria Jiménez-Movilla ◽  
Jurrien Dean
Keyword(s):  
Zona Pellucida ◽  
Cleavage Site ◽  
Single Copy ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Successful Development ◽  
Mouse Sperm ◽  
Subcellular Organelles ◽  
Sperm Binding ◽  
Definitive Role

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.

scholarly journals Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

1993 ◽  
Vol 123 (6) ◽  
pp. 1431-1440 ◽  
Author(s):  
D J Miller ◽  
X Gong ◽  
G Decker ◽  
B D Shur
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Binding Activity ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Sperm Binding ◽  
Competitive Inhibitors ◽  
Glycosidase Inhibitor ◽  
Egg Coat

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

scholarly journals Receptor function of mouse sperm surface galactosyltransferase during fertilization.

1985 ◽  
Vol 101 (4) ◽  
pp. 1501-1510 ◽  
Author(s):  
L C Lopez ◽  
E M Bayna ◽  
D Litoff ◽  
N L Shaper ◽  
J H Shaper ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Dorsal Surface ◽  
Mouse Sperm ◽  
Sperm Surface ◽  
Sperm Binding ◽  
Total Inhibition ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Plasma Membrane Domain ◽  
Dose Dependent

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads

1989 ◽  
Vol 92 (4) ◽  
pp. 713-722
Author(s):  
M.H. Vazquez ◽  
D.M. Phillips ◽  
P.M. Wassarman
Keyword(s):  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Solid Phase ◽  
Galactose Oxidase ◽  
Flow Cytofluorometry ◽  
Mouse Sperm ◽  
Sperm Binding ◽  
Silica Beads ◽  
Covalently Linked ◽  
Solid Phase Assay

We describe a solid-phase assay that has permitted further analysis of zona pellucida glycoprotein, ZP3, as sperm receptor and acrosome reaction-inducer during fertilization in mice. The assay employs silica beads that contain epoxy groups to which purified, mouse oocyte ZP3 is covalently linked (ZP3-beads). ZP3-beads were characterized, e.g. by whole-mount autoradiography and flow cytofluorometry, incubated with capacitated mouse sperm under a variety of conditions, and the extent of sperm binding determined by light microscopy. Results of experiments presented suggest the following: (1) sperm bind specifically to ZP3-beads, but not to silica beads either exposed to 2-aminoethanol or derivatized with oocyte ZP2, fetuin or bovine serum albumin. (2) In nearly all cases, only one sperm binds per ZP3-bead and binding occurs via the sperm head. (3) The extent of sperm binding to ZP3-beads is dependent on ZP3 and sperm concentrations, as well as on incubation time and temperature. (4) Sperm binding to ZP3-beads is unaffected by antibodies directed against ZP3, but is inhibited in a reversible manner by treatment of ZP3-beads with galactose oxidase. (5) Only acrosome-intact sperm bind to ZP3-beads but, once bound, sperm can undergo the acrosome reaction, which results in their release from ZP3-beads. (6) Islet-activating protein and 3-quinuclidinyl benzilate, two inhibitors of the zona pellucida-induced acrosome reaction, prevent sperm bound to ZP3-beads from undergoing the acrosome reaction. These results confirm and extend previous studies of sperm-egg interaction in mice, and suggest that the solid-phase assay will be useful for both cellular and biochemical analyses of mammalian fertilization.

scholarly journals A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida.

1982 ◽  
Vol 95 (2) ◽  
pp. 574-579 ◽  
Author(s):  
B D Shur ◽  
N G Hall
Keyword(s):  
Zona Pellucida ◽  
Milk Protein ◽  
Preceding Paper ◽  
Transferase Activity ◽  
Mouse Sperm ◽  
Sperm Surface ◽  
Galactosyl Transferase ◽  
Sperm Binding ◽  
Beta Galactosidase

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N-acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha-lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta-N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.

Early Reactions of the Rodent Egg to Spermatozoon Penetration

1956 ◽  
Vol 33 (2) ◽  
pp. 358-365 ◽  
Author(s):  
C. R. AUSTIN ◽  
A. W. H. BRADEN
Keyword(s):  
Zona Pellucida ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Cortical Granules ◽  
Rats And Mice ◽  
Mouse Eggs

In the rat, mouse and hamster the spermatozoon passes rapidly through the thick, homogeneous zona pellucida surrounding the egg and the head almost immediately becomes attached to the surface of the inner cytoplasmic mass or vitellus. As a result of this attachment a block to polyspermy is developed in rat and mouse eggs. In the hamster a block is apparently not formed. It seems likely, therefore, that the disappearance of cortical granules in the hamster egg, also an outcome of contact with the spermatozoon head, could signal the release of an agent that is responsible, after crossing the perivitelline space, for bringing about the zona reaction, reducing the penetrability of the zona pellucida to spermatozoa. Data suggest that this mechanism exists also in rats and mice, although a cortical granule response has not been distinguished in these animals. Thus, attachment of the spermatozoon head to the vitellus probably elicits both the zona reaction and the block to polyspermy. These changes appear to be specific to spermatozoon penetration and to be initiated before the spermatozoon head passes through the surface of the vitellus and before the resumption of the second meiosis.

183 CALRETICULIN, A 60-kDa PROTEIN, IS EXOCYTOSED AFTER CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 203
Author(s):  
R. Romar ◽  
M. D. Saavedra ◽  
H. González-Márquez ◽  
Y. Ducolomb ◽  
R. Fierro ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Control Group ◽  
Cortical Granule ◽  
Cortical Region ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
Rabbit Igg

Following gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. The specific cortical granule-derived proteins responsible for these post-fertilization events are not fully characterized. Calreticulin, a highly conserved ubiquitous protein of 60 kDa, was exocytosed from activated hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Preliminary results from our laboratory have shown that calreticulin is located in the cortical area of pig oocytes (data not shown). This study was designed to test whether calreticulin is exocytosed after oocyte activation with calcium ionophore. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with 0.5% pronase in Ca2+-free PBS. After washing, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM) for 2min, transferred to a 100-μL droplet of exudate medium (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst) and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. After incubation, the medium containing the oocyte exudate (n = 1000) was carefully aspirated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The gel was then electro transferred onto a polyvinylidene fluoride (PVDF) membrane, incubated with an anti-calreticulin rabbit polyclonal antibody (1:1000) and finally conjugated to horseradish peroxidase (1:20 000) for 1 h with a monoclonal anti-rabbit IgG. Membrane visualization was accomplished using the ECL plus method and Typhoon 9410. A control group was performed with exudate collected from non-activated ZP-free oocytes. To verify cortical reaction and calreticulin exocytosis, an aliquot of activated ZP-free oocytes (n = 18) were fixed (3.7% paraformaldehyde for 30 min), permeabilized (0.1% Triton X-100 for 10 min), incubated with anti-calreticulin antibody (1:10 for 1 h) and conjugated to tetramethyl rhodamine isothiocyanate (1:400 for 1 h) with an anti-rabbit IgG. Finally, samples were incubated with peanut agglutinin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min), mounted and examined under a confocal microscope. No statistical analysis was made because the observations were purely qualitative. A Western blot analysis showed an immunoreactive band of ∼60 kDa, consistent with the expected size of calreticulin, in the lane containing the exudate from activated oocytes. No band was observed in the lane with the exudate collected from non-activated oocytes. Observation under confocal microscopy showed no PNA or anti-calreticulin fluorescence in the cortical region, indicating that the activated pig oocytes displayed full cortical reaction and calreticulin exocytosis during incubation time. These results show that calreticulin protein is exocytosed after the chemical activation of ZP-free pig oocytes as well as the disappearance of the cortical granule monolayer. The possible role of calreticulin on preventing polyspermy should be further investigated. Supported by MEC and FEDER (AGL2009-12512-C02-01) and CONACYT (0105961/I0110/194/09).

scholarly journals Expression of genes encoding zona pellucida glycoproteins and cortical granule distribution in porcine oocytes isolated from small and medium follicles in relation to puberty status of donors

2017 ◽  
Vol 62 (No. 6) ◽  
pp. 234-241 ◽  
Author(s):  
M. Jeseta ◽  
J. Budna ◽  
W. Kranc ◽  
S. Hanulakova ◽  
A. Bryja ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zona Pellucida ◽  
Marker Gene ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Expression Of Genes ◽  
Gamete Recognition ◽  
Genes Encoding ◽  
Central Concentration

The zona pellucida proteins belong to a group of proteins that regulate the processes of gamete recognition, interaction, and fusion, since they are recognized as primary and secondary sperm receptors. It is suggested that cortical granule distribution is significantly associated with the zona pellucida structure and membrane preparation to cortical and acrosome reaction. Therefore, this study investigated the zona pellucida marker gene expression (ZP2 and ZP4 protein) in relation to cortical granules distribution in oocytes and puberty status of donors. Oocytes were collected from adult cyclic sows (isolated from medium and small follicles) and juvenile gilts (isolated only from small follicles). The oocytes were examined by RT-qPCR and by confocal microscopy. The expression of genes for ZP2 and ZP4 protein in oocytes collected from small follicles from cycling sows was higher in comparison to oocytes from medium follicles or oocytes collected from small follicles from juvenile gilts (P < 0.001). We also observed increased expression of both ZP2 and ZP4 mRNA in oocytes collected from small follicles (juvenile gilts) as compared to medium follicles (cycling sows) (P < 0.001). Moreover, we found a difference in the distribution of cortical granules. In oocytes from medium follicles, the peripheral localization of cortical granules was twice higher than their central concentration. However, in oocytes derived from small follicles (cyclic sows) cortical granules in comparison to oocytes from medium follicles were more centrally localized (P < 0.05). It has been suggested that the donor puberty status and the size of follicles significantly influenced the zona pellucida gene expression and cortical granule localization in porcine oocytes. This is accompanied by fertilization specificity and fertilizability of porcine oocytes in vitro.

Cold lability of mouse sperm binding to zona pellucida

1982 ◽  
Vol 219 (2) ◽  
pp. 155-161 ◽  
Author(s):  
Linda J. Heffner ◽  
Bayard T. Storey
Keyword(s):  
Zona Pellucida ◽  
Mouse Sperm ◽  
Sperm Binding

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

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