Effects of proteolytic enzymes on the zona pellucida of fertilized and unfertilized mammalian eggs

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.

Download Full-text

ZP2 and ZP3 Traffic Independently within Oocytes prior to Assembly into the Extracellular Zona Pellucida

Molecular and Cellular Biology ◽

10.1128/mcb.00904-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 7991-7998 ◽

Cited By ~ 25

Author(s):

Tanya Hoodbhoy ◽

Manuel Avilés ◽

Boris Baibakov ◽

Olga Epifano ◽

María Jiménez-Movilla ◽

...

Keyword(s):

Zona Pellucida ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Egg Recognition ◽

Endomembrane System ◽

Glycosylation Sites ◽

Physical Interactions ◽

Green Fluorescent ◽

Mammalian Eggs

ABSTRACT The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-μm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.

Download Full-text

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.

The Journal of Cell Biology ◽

10.1083/jcb.102.4.1363 ◽

1986 ◽

Vol 102 (4) ◽

pp. 1363-1371 ◽

Cited By ~ 186

Author(s):

J D Bleil ◽

P M Wassarman

Keyword(s):

Plasma Membrane ◽

Zona Pellucida ◽

Present Report ◽

Fusion Event ◽

Acrosomal Membrane ◽

Specific Receptors ◽

Species Specific ◽

Mammalian Eggs ◽

Silver Grains ◽

Sperm Receptor

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

Download Full-text

Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm

Reproduction Fertility and Development ◽

10.1071/rd05122 ◽

2006 ◽

Vol 18 (2) ◽

pp. 53 ◽

Cited By ~ 68

Author(s):

Allison J. Gardner ◽

Janice P. Evans

Keyword(s):

Plasma Membrane ◽

Zona Pellucida ◽

Animal Species ◽

Mammalian Species ◽

Experimental Studies ◽

Present Review ◽

Cellular Signalling ◽

Classical Studies ◽

Vitelline Envelope ◽

Mammalian Eggs

To inhibit fertilisation by more than one sperm (a condition known as polyspermy), eggs have developed preventative mechanisms known as blocks to polyspermy. The block at the level of the egg extracellular coat (the zona pellucida in mammals, the vitelline envelope in non-mammals) has been well characterised in many different animal species and the block at the level of the egg plasma membrane is understood in some non-mammalian species. However, virtually nothing is known about the membrane block to polyspermy in mammalian eggs, despite data dating back 50–90 years that provide evidence for its existence. In the present review, we will discuss the background on blocks to polyspermy used by animal eggs and then focus on the membrane block to polyspermy in mammalian eggs. This will include a summary of classical studies that provide evidence for this block in mammalian eggs, assays used to study the mammalian membrane block and what has been elucidated from recent experimental studies about the cellular signalling events that lead to membrane block establishment and the mechanism of how the membrane block may prevent additional fertilisation.

Download Full-text

The role of the zona pellucida in the development of mouse eggs in vivo

Development ◽

10.1242/dev.23.3.539 ◽

1970 ◽

Vol 23 (3) ◽

pp. 539-547

Author(s):

Jacek A. Modliński

Keyword(s):

Zona Pellucida ◽

Blastocyst Stage ◽

Cell Stage ◽

Development In Vitro ◽

Single Blastomeres ◽

Mammalian Eggs ◽

Mouse Eggs

Up to the present time the function and significance of the zona pellucida in the development of mammalian eggs has not been fully explained. Zona-free mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964) showed that in this species also cleavage can occur when the zona pellucida is absent, although the blastomeres exhibit a tendency to fall away from each other. Tarkowski's observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct. A few hours after transplanting the naked eggs none could be recovered by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered.

Download Full-text

Conserved Furin Cleavage Site Not Essential for Secretion and Integration of ZP3 into the Extracellular Egg Coat of Transgenic Mice

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3111-3120.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3111-3120 ◽

Cited By ~ 28

Author(s):

Ming Zhao ◽

Lyn Gold ◽

Ann M. Ginsberg ◽

Li-Fang Liang ◽

Jurrien Dean

Keyword(s):

Transgenic Mice ◽

Zona Pellucida ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Green Fluorescent ◽

Mammalian Eggs ◽

Mouse Eggs

ABSTRACT The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR → ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.

Download Full-text

ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.626679 ◽

2021 ◽

Vol 8 ◽

Author(s):

Mª José Izquierdo-Rico ◽

Carla Moros-Nicolás ◽

Míriam Pérez-Crespo ◽

Ricardo Laguna-Barraza ◽

Alfonso Gutiérrez-Adán ◽

...

Keyword(s):

Animal Model ◽

Zona Pellucida ◽

Acrosome Reaction ◽

Functional Roles ◽

Sperm Binding ◽

The Status ◽

Murine Rodents ◽

Mouse Species ◽

Mammalian Eggs

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Download Full-text

294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab294 ◽

2006 ◽

Vol 18 (2) ◽

pp. 254

Author(s):

H.-H. Rhee ◽

S.-J. Sa ◽

H.-T. Cheong ◽

B.-K. Yang ◽

C.-K. Park

Keyword(s):

In Vitro Fertilization ◽

Zona Pellucida ◽

Acrosome Reaction ◽

Proteolytic Enzymes ◽

Extracellular Proteins ◽

Control Group ◽

Sperm Viability ◽

Vitro Fertilization ◽

Sperm Binding

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P < 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P < 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P < 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.

Download Full-text

Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation

Reproduction ◽

10.1530/rep-06-0304 ◽

2007 ◽

Vol 133 (2) ◽

pp. 383-393 ◽

Cited By ~ 36

Author(s):

Allison J Gardner ◽

Carmen J Williams ◽

Janice P Evans

Keyword(s):

Plasma Membrane ◽

Zona Pellucida ◽

Egg Activation ◽

Membrane Function ◽

Independent Event ◽

Calcium Dependent ◽

Normal Sperm ◽

Egg Membrane ◽

Mammalian Eggs ◽

Mouse Eggs

One crucial result of egg activation is the establishment of blocks on the zona pellucida and the egg plasma membrane to prevent fertilization by additional sperm. The mechanism(s) by which a mammalian egg regulates the establishment of the membrane block to polyspermy is largely unknown. Since Ca2+ signaling regulates several egg activation events, this study investigates how sperm-induced Ca2+ transients affect the membrane block to polyspermy, building on our previous work (Biology of Reproduction 67:1342). We demonstrate that mouse eggs that experience only one sperm-induced Ca2+ transient establish a membrane block that is less effective, than in eggs that experience normal sperm-induced Ca2+ transients but that is more effective than in eggs with completely suppressed [Ca2+]cyt increases. Sperm-induced increases in [Ca2+]cyt regulate the timing of membrane block establishment, as this block is established more slowly in eggs that experience one or no sperm-induced Ca2+ transients. Finally, our studies produce the intriguing discovery that there is also a Ca2+-independent event that is associated with fertilization in the pathway leading to membrane block establishment. Taken together, these data indicate that Ca2+ plays a role in facilitating membrane block establishment by regulating the timing with which this change in egg membrane function occurs, and also that the membrane block differs from other post-fertilization egg activation responses as Ca2+ is not the only stimulus. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple post-fertilization events that together modify the egg membrane’s receptivity to sperm.

Download Full-text