Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

doi:10.1016/0012-1606(87)90058-3

Developmental Biology

Volume 119, Issue 2, February 1987, Pages 551-559

https://doi.org/10.1016/0012-1606(87)90058-3 Get rights and content

Abstract

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.

References (31)

J.D. Bleil et al.
Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida
Dev. Biol
(1980)
M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem
(1976)
G.N. Cherr et al.
In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zonae pellucidae
Dev. Biol
(1986)
H.M. Florman et al.
Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro
Dev. Biol
(1982)
L. Forni
Reagents for immunofluorescence and their use for studying lymphoid cell products
F. Kuzan et al.
Successful fertilization in vitro of fresh intact oocytes by perivitelline (acrosome-reacted) spermatozoa of the rabbit
Fertil. Steril
(1984)
M.G. O'Rand
Restriction of a sperm surface antigen's mobility during capacitation
Dev. Biol
(1977)
M.G. O'Rand et al.
Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa
Dev. Biol
(1981)
J.E. Welch et al.
Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit
Dev. Biol
(1985)
J.M. Bedford et al.
Membrane fusion events in fertilization of vertebrate eggs

J.D. Bleil et al.

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

J. Cell Biol

(1986)

B.G. Brackett et al.

In vitro sperm capacitiation and in vitro fertilization with normal development in the rabbit

J. Androl

(1982)

J. DiGuiseppi et al.

Applications of digitized fluorescence microscopy to problems in cell biology

BioTechniques

(1985)

D.W. Drell et al.

Immunological comparison of antibodies to porcine zonae pellucidae in rats and rabbits

Biol. Reprod

(1984)

B.S. Dunbar et al.

Identification of the three major proteins of porcine and rabbit zonae pellucidae by high resolution two-dimensional gel electrophoresis: Comparison with serum, follicular fluid, and ovarian cell proteins

Biol. Reprod

(1981)

Cited by (64)

The biological networks in studying cell signal transduction complexity: The examples of sperm capacitation and of endocannabinoid system
2014, Computational and Structural Biotechnology Journal
Citation Excerpt :
In sperm head plasma membrane (PM) it is possible to recognize different domains, separated by diffusion barriers, characterized by different chemical–physical and functional proprieties: the apical ridge area, the pre-equatorial area, the equatorial area and the post-equatorial area. The apical ridge is involved in sperm-ZP binding and contains specific zona binding proteins [17], in the pre-equatorial surface the fusion between PM and outer acrosome membrane (OAM) occurs during AR, and the equatorial surface area is involved in the fusion between spermatozoa and oocyte at the moment of fertilization [18,19]. Each domain, in turn, contains specialized areas, known as microdomains or detergent resistant membranes (DRMs).
Cellular signal transduction is a complex phenomenon, which plays a central role in cell surviving and adaptation. The great amount of molecular data to date present in literature, together with the adoption of high throughput technologies, on the one hand, made available to scientists an enormous quantity of information, on the other hand, failed to provide a parallel increase in the understanding of biological events. In this context, a new discipline arose, the systems biology, aimed to manage the information with a computational modeling-based approach. In particular, the use of biological networks has allowed the making of huge progress in this field.
Here we discuss two possible application of the use of biological networks to explore cell signaling: the study of the architecture of signaling systems that cooperate in determining the acquisition of a complex cellular function (as it is the case of the process of activation of spermatozoa) and the organization of a single specific signaling systems expressed by different cells in different tissues (i.e. the endocannabinoid system). In both the cases we have found that the networks follow a scale free and small world topology, likely due to the evolutionary advantage of robustness against random damages, fastness and specific of information processing, and easy navigability.
Defending the Zygote: Search for the Ancestral Animal Block to Polyspermy
2005, Current Topics in Developmental Biology
Citation Excerpt :
The absence of a taxon‐specific zona binding by SED1 reminds us that the purpose of some ligands may simply be to retain sperm attachment to the egg ECM rather than for recognition or conspecificity. Another generic oligosaccharide‐binding family prefers the sulfated fucose‐rich proteins found within the zona of mammalian eggs (O'Rand, 1988; O'Rand et al., 1985, 1988; O'Rand and Fisher, 1987) (Table IV). These ligands were first identified in a screen of autoimmune serum raised against a cluster of 13–15 kDa rabbit sperm autoantigens (RSAs) (O'Rand et al., 1988).
Fertilization is the union of a single sperm and an egg, an event that results in a diploid embryo. Animals use many mechanisms to achieve this ratio; the most prevalent involves physically blocking the fusion of subsequent sperm. Selective pressures to maintain monospermy have resulted in an elaboration of diverse egg and sperm structures. The processes employed for monospermy are as diverse as the animals that result from this process. Yet, the fundamental molecular requirements for successful monospermic fertilization are similar, implying that animals may have a common ancestral block to polyspermy. Here, we explore this hypothesis, reviewing biochemical, molecular, and genetic discoveries that lend support to a common ancestral mechanism. We also consider the evolution of alternative or radical techniques, including physiological polyspermy, with respect to our ability to describe a parsimonious guide to fertilization.
Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein
1999, Developmental Biology
In this study we have demonstrated that the native rabbit sperm protein, Sp17, is a 22- to 24-kDa triplet of proteins in washed ejaculated rabbit spermatozoa and is unaffected by capacitation. However, during the acrosome reaction, Sp17 is processed from a 22- to 24-kDa triplet of proteins to a triplet of proteins at 17–19 kDa by the removal of amino acids from the C-terminal. Recombinant rabbit Sp17 (rRSp17) can also be proteolytically processed by acrosome-reacted spermatozoa in a similar manner. Protease inhibitors prevent the proteolytic processing of Sp17. Both forms of native Sp17 remain associated with acrosome-reacted spermatozoa and are solubilized by ionic detergents. Previously, sequence analysis of Sp17 revealed that Sp17 amino acids 108–137 were 52% identical to the calmodulin binding domain of neuromodulin and contained an IQ motif found in other calmodulin binding proteins. In this study, a truncated recombinant Sp17, rRSp17CB, which lacks amino acids 118–146, including the potential calmodulin binding site, was made. Recombinant rabbit Sp17, but not rRSp17CB, binds to calmodulin in the presence of Ca²⁺or EDTA, under reduced or nonreduced conditions in biotinylated-calmodulin overlay assays. In DSS crosslinker experiments, calmodulin bound to rRSp17 in a 1:1 ratio but not to rRSp17CB. Additionally, biotinylated rRSp17 interacts with native sperm calmodulin. We propose that the processing of native Sp17, by removing a C-terminal fragment during the acrosome reaction, might be a mechanism to regulate the calmodulin binding activity of Sp17 and provide calmodulin at specific sites after the acrosome reaction.
Energy requirement of bovine spermatozoa for in vitro capacitation
1995, Theriogenology
The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.
Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm
2020, Frontiers in Cell and Developmental Biology
Update on mammalian sperm capacitation: How much does the horse differ from other species?
2019, Reproduction

View all citing articles on Scopus

^☆: Supported by NIH Grant HD-14232.

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Developmental Biology

Abstract

References (31)

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

Dev. Biol

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal. Biochem

In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zonae pellucidae

Dev. Biol

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

Dev. Biol

Reagents for immunofluorescence and their use for studying lymphoid cell products

Successful fertilization in vitro of fresh intact oocytes by perivitelline (acrosome-reacted) spermatozoa of the rabbit

Fertil. Steril

Restriction of a sperm surface antigen's mobility during capacitation

Dev. Biol

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa

Dev. Biol

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Dev. Biol

Membrane fusion events in fertilization of vertebrate eggs

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

J. Cell Biol

In vitro sperm capacitiation and in vitro fertilization with normal development in the rabbit

J. Androl

Applications of digitized fluorescence microscopy to problems in cell biology

BioTechniques

Immunological comparison of antibodies to porcine zonae pellucidae in rats and rabbits

Biol. Reprod

Identification of the three major proteins of porcine and rabbit zonae pellucidae by high resolution two-dimensional gel electrophoresis: Comparison with serum, follicular fluid, and ovarian cell proteins

Biol. Reprod

Cited by (64)

The biological networks in studying cell signal transduction complexity: The examples of sperm capacitation and of endocannabinoid system

Defending the Zygote: Search for the Ancestral Animal Block to Polyspermy

Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein

Energy requirement of bovine spermatozoa for in vitro capacitation

Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm

Update on mammalian sperm capacitation: How much does the horse differ from other species?

Full paperLocalization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae☆

Abstract

Dev. Biol

Anal. Biochem

Dev. Biol

Dev. Biol

Fertil. Steril

Dev. Biol

Dev. Biol

Dev. Biol

Membrane fusion events in fertilization of vertebrate eggs

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

J. Cell Biol

In vitro sperm capacitiation and in vitro fertilization with normal development in the rabbit

J. Androl

Applications of digitized fluorescence microscopy to problems in cell biology

BioTechniques

Immunological comparison of antibodies to porcine zonae pellucidae in rats and rabbits

Biol. Reprod

Identification of the three major proteins of porcine and rabbit zonae pellucidae by high resolution two-dimensional gel electrophoresis: Comparison with serum, follicular fluid, and ovarian cell proteins

Biol. Reprod

Full paper
Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae☆