Egg jelly triggers a calcium influx which inactivates and is inhibited by calmodulin antagonists in the sea urchin sperm

doi:10.1016/0005-2736(89)90206-X

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 980, Issue 1, 27 March 1989, Pages 109-116

https://doi.org/10.1016/0005-2736(89)90206-X Get rights and content

Abstract

Sea urchin sperm must undergo the acrosome reaction to fertilize eggs. The natural inducer of this reaction is the most external coat of the egg, named ‘jelly’. The ionic composition of the extracellular and intracellular media and the permeability properties of the sperm plasma membrane are fundamental in this reaction. As Ca²⁺ is required for the acrosome reaction to occur, its intracellular concentration ([Ca²⁺]_i) was measured with fura-2. In 10 mM Ca²⁺, egg jelly induced the acrosome reaction and an increase in [Ca²⁺]_i that lasted for several minutes. However, at 0.5 or 2 mM Ca²⁺, it became evident that the Ca²⁺-influx pathway activated by jelly opened only for a few seconds; this prevented both the full increase in [Ca²⁺]_i and the acrosome reaction even after the concentration of Ca²⁺ was raised to 10 mM. In the presence of jelly, the time this permeability pathway remained open was inversely related to the extracellular concentration of Ca²⁺ ([Ca²⁺]_c). Using Bisoxonol (a permeant fluorescent membrane potential probe), it was found that the jelly-induced depolarization depended on [Ca²⁺]_e and was proportional to the increase in [Ca²⁺]_i. Since [Ca²⁺]_i could affect the jelly-induced Ca²⁺ influx through calmodulin, two of its antagonists, trifluoperazine and W-7, were tested. Both compounds blocked the acrosome reaction by inhibiting the jelly-induced increase in [Ca²⁺]_i. W-5 at the same concentration had no effect. The results suggest that one of the jelly-activated Ca²⁺-influx pathways, probably a channel, is the target of the calmodulin antagonists.

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T-type Ca2+ channels in sperm function
2006, Cell Calcium
Citation Excerpt :
Possibly this channel could be a CaV channel. Five seconds later, a second channel, insensitive to the latter blockers, that does not inactivate and is sensitive to pHi, opens and leads to the AR [61]. The second channel appears to belong to the family of SOCs, possibly a member of the transient receptor potential (TRP) channel family [62].
Intracellular Ca²⁺ regulates many fundamental physiological processes in excitable and non-excitable cells. Certainly this is the case of sperm where the local concentration of intracellular Ca²⁺ ([Ca²⁺]i) is significantly influenced by Ca²⁺ permeable channels present in the cell plasma membrane. Amongst these channels, the voltage dependent Ca²⁺ channels (Ca_V) of the T-type (Ca_V3) appear to have an eminent role in the acrosome reaction (AR) of some sperm species, though they may participate in other important functions like motility and capacitation. The AR is an exocytotic event where the acrosome vesicle in the posterior region of the head fuses with the plasma membrane. This reaction allows sperm to fuse and fertilize the egg. Here we summarize our present knowledge regarding Ca_V3 channels in sperm, show the first direct electrophysiological evidence for their presence in maturing mouse sperm and discuss some of the relevant unanswered questions.
When a sperm meets an egg: Block to polyspermy
2006, Molecular and Cellular Endocrinology
Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a series of events known as egg activation. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. CGE is an evolutionary developed mechanism that causes modification of the zona pellucida to prevent penetration of additional spermatozoa, ensuring successful egg activation and embryo development. The egg CGE is a unique and convenient mammalian model for studying the different proteins participating at the membrane fusion cascade, which, unlike other secretory cells, occurs only once in the egg's lifespan. This article highlights a number of proteins, ascribed to participate in CGE and thus the block to polyspermy. CGE can be triggered either by a calcium dependent pathway, or via protein kinase C (PKC) activation that requires a very low calcium concentration.
In a recent study, we suggested that the filamentous actin (F-actin) at the egg's cortex is a dynamic network. It can be maneuvered towards allowing CGE by activated actin associated proteins and/or by activated PKC and its down stream proteins, such as myristoylated alanine-rich C kinase substrate (MARCKS). MARCKS, a protein known to cross-link F-actin in other cell types, was found to be expressed and colocalized with actin in non-activated MII eggs. We further demonstrated MARCKS dissociation from actin after activation by ionomycin, a process that can lead to the breakdown of the actin network, thus allowing CGE. The more we know of the intricate process of CGE and of the proteins participating in it, the more the assisted reproductive procedures might benefit from that knowledge.
Calcium channels and Ca2+ fluctuations in sperm physiology
2005, International Review of Cytology
Citation Excerpt :
Some examples of the use of fluorescent probes in live sperm are as follows: [Ca2+]i and pHi: Arnoult et al. (1999), Babcock et al. (1983), Baldi et al. (1991), Blackmore (1993), Florman et al. (1989, 1992), Fukami et al. (2003), Guerrero and Darszon (1989a,b), Lee (1984), Meizel et al. (1997), Sase et al. (1995), Schackmann and Chock (1986), Wood et al. (2003), and Zeng et al. (1996). It has proved difficult to directly record sperm ion channels in situ.
Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca²⁺ is probably the key messenger in this information exchange. It is therefore not surprising that different Ca²⁺-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca²⁺, membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.
Measuring ion fluxes in sperm
2004, Methods in Cell Biology
The chapter discusses different strategies to study ion fluxes in sperm. Ion channels and transporters in the sperm plasma membrane participate crucially in fertilization. Ion-permeability changes are deeply involved in how sperm sense environmental cues and signals from the outer envelope of the egg to achieve fertilization. Combining the in vivo measurements of intracellular ions and membrane potential in sperm populations and single cells with electrophysiological approaches, such as the smart patch-clamp, and reconstitution strategies in planar bilayers reveal how sperm ion channels participate in sperm motility and the acrosome reaction. This chapter describes ion transport protocols for sea urchin sperm. Egg ligands immediately induce sperm responses. It is important to measure the rapid kinetics of their responses to understand the underlying signaling mechanisms. To perform a successful time-resolved measurement, it is essential that sperm be exposed to egg ligands as rapidly as possible. There are two different methods to achieve this: (1) rapid mixing (stopped-flow fluorometry) and (2) photolysis of caged (photoactivatable) ligands. The chapter presents techniques used in the study of ion fluxes in single sea urchin sperm.
Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle
2003, Biochemical and Biophysical Research Communications
The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the ∼1 μm long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca²⁺ entry through two distinct Ca²⁺ channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca²⁺ entry into sperm. These data indicate that the second Ca²⁺ channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca²⁺ elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH₄Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca²⁺ channel of sea urchin sperm is a SOC that triggers AV exocytosis.
High molecular mass egg fucose sulfate polymer is required for opening both Ca2+ channels involved in triggering the sea urchin sperm acrosome reaction
2002, Journal of Biological Chemistry
Citation Excerpt :
For example, seawater is ∼10 mm Ca2+. Treatment of sperm with EJ in 2 mm Ca2+ makes the cells refractory to AR induction and to increases in [Ca2+]i when the Ca2+ is returned to 10 mm (40, 41). This AR inactivation is hypothesized to result from uncoupling the linkage between the sperm EJ receptors and Ca2+ channels.
A linear fucose sulfate polymer (FSP), >10⁶ daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca²⁺channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca²⁺channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of ∼60 kDa blocks intact FSP from inducing the AR. At 44 μg/ml hydrolyzed FSP, Ca²⁺ entry into sperm is almost equal to that occurring in 3.8 μg/ml intact FSP; however the AR is not induced. The shape of the [Ca²⁺]_i increase curve and use of the Ca²⁺ channel blockers nifidipine and Ni²⁺ indicate that hydrolyzed FSP opens the second Ca²⁺ channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca²⁺ channels involved in triggering the AR.

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Biochimica et Biophysica Acta (BBA) - Biomembranes

Abstract

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Cited by (41)

T-type Ca<sup>2+</sup> channels in sperm function

When a sperm meets an egg: Block to polyspermy

Calcium channels and Ca<sup>2+</sup> fluctuations in sperm physiology

Measuring ion fluxes in sperm

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

High molecular mass egg fucose sulfate polymer is required for opening both Ca<sup>2+</sup> channels involved in triggering the sea urchin sperm acrosome reaction

Regular paperEgg jelly triggers a calcium influx which inactivates and is inhibited by calmodulin antagonists in the sea urchin sperm

Abstract

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Regular paper
Egg jelly triggers a calcium influx which inactivates and is inhibited by calmodulin antagonists in the sea urchin sperm