scholarly journals Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton

Reproduction ◽  
2010 ◽  
Vol 140 (5) ◽  
pp. 673-684 ◽  
Author(s):  
Yadira Bastián ◽  
Ana L Roa-Espitia ◽  
Adela Mújica ◽  
Enrique O Hernández-González
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Mammalian Species ◽  
Physiological Changes ◽  
Signal Transducer ◽  
Mammalian Sperm ◽  
Important Player ◽  
Calpain 2 ◽  
Spectrin Cytoskeleton

Research on fertilization in mammalian species has revealed that Ca2+is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca2+is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca2+plays a pivotal role. Calpain-1 and calpain-2 are Ca2+-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca2+. Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

Interactions between gametes leading to fertilization: the sperm's eye view

1995 ◽  
Vol 7 (4) ◽  
pp. 927 ◽  
Author(s):  
BT Storey
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Mammalian Species ◽  
Sperm Chromatin ◽  
Peptide Backbone ◽  
Mouse Sperm ◽  
Gamete Interactions ◽  
Sperm Plasma Membrane ◽  
Sperm Penetration ◽  
Strong Candidate

Sexual reproduction requires that the gamete carrying the male-derived haploid chromatin join with the gamete carrying the female-derived haploid chromatin during fertilization to produce the diploid zygote. To accomplish this feat, the sperm must not only meet the egg, it must recognize the egg and be recognized in turn by the egg, and in the end must enter and be engulfed by the egg. In this selective overview of gamete interactions that lead to fertilization, encounters of three kinds, followed by the finale of gamete fusion, are considered from the sperm's viewpoint, with particular emphasis on the mammalian species with the mouse as the principal model. The first encounter is with the zona pellucida of the egg, to whose surface the sperm must bind. Mouse sperm appear to have four binding sites for zona ligands. Three interact with sugar moieties of the oligosaccharide chains of the mouse zona glycoprotein ZP3; the fourth binds a peptide backbone arginine. Capacitation is not required for this encounter, but is obligate for the second encounter--induction of the acrosome reaction in the bound sperm. The acrosome reaction is an exocytotic process that makes available the enzymatic machinery needed for sperm penetration the zona which is the end point of a sequence of reactions directed by intracellular signalling systems. In mouse sperm, these systems are presumed to be activated by ligands on ZP3 binding to ligand-specific sperm receptors with consequent aggregation of receptors. No receptor has been identified with certainty, nor have candidates for putative ZP3 ligands been identified. Completion of the acrosome reaction allows the sperm to penetrate the zona and, bind to the egg plasma membrane, thereby completing the third encounter. In the mouse, a 94-kDa protein appears essential for this binding. In the guinea-pig, a sperm plasma membrane protein (formerly PH-30, now fertilin), is a strong candidate for the mediator of the fusion process by which the egg engulfs the sperm. Decondensation of the sperm chromatin reverses the remarkable packing of DNA organized by sperm protamines. Mitochondrial DNA is also engulfed by the egg; the question of whether this DNA makes a small finite, or null, contribution to cytosolic inheritance is still in debate. The puzzles attending these encounters are presented as reminders of the intricacy and fascination, as well as of the vital necessity, of gamete interaction.

scholarly journals Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

1987 ◽  
Vol 104 (4) ◽  
pp. 917-923 ◽  
Author(s):  
AE Cowan ◽  
DG Myles ◽  
DE Koppel
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Domain Boundary ◽  
Lateral Diffusion ◽  
Fold Increase ◽  
Membrane Domains ◽  
Mammalian Sperm ◽  
Acrosomal Membrane ◽  
Percent Recovery ◽  
Lipid Probe

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.

scholarly journals Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

1977 ◽  
Vol 74 (2) ◽  
pp. 561-577 ◽  
Author(s):  
DS Friend ◽  
L Orci ◽  
A Perrelet ◽  
R Yanagimachi
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Phosphatidylcholine Liposomes ◽  
Acrosomal Membrane ◽  
Large Particles ◽  
Fracture Appearance ◽  
Preparatory Stage

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

scholarly journals Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm

Antioxidants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 100 ◽  
Author(s):  
Marc Llavanera ◽  
Ariadna Delgado-Bermúdez ◽  
Samuel Olives ◽  
Yentel Mateo-Otero ◽  
Sandra Recuero ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mitochondrial Function ◽  
Sperm Quality ◽  
Mammalian Species ◽  
Membrane Stability ◽  
Sperm Function ◽  
Cell Protection ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) are essential sperm antioxidant enzymes involved in cell protection against oxidative stress and toxic chemicals, preserving sperm function and fertilising ability. Artificial insemination (AI) in pigs is commonly carried out through the use of liquid-stored semen at 17 °C, which not only reduces sperm metabolic activity but also sperm quality and AI-farrowing rates within the 72 h of storage. While one may reasonably suggest that such enzymes are implicated in the physiology and maintenance of mammalian sperm function during liquid-storage, no previous studies conducted on any species have addressed this hypothesis. Therefore, the objective of the present work was to characterise the presence and function of sperm GSTs in mammalian sperm, using the pig as a model. In this regard, inhibition of such enzymes by ethacrynic acid (EA) during semen storage at 17 °C was performed to evaluate the effects of GSTs in liquid-preserved boar sperm by flow cytometry, immunofluorescence, and immunoblotting analysis. The results of this study have shown, for the first time in mammalian species, that the inhibition of GSTs reduces sperm quality and functionality parameters during their storage at 17 °C. These findings highlight the key role of such enzymes, especially preserving mitochondrial function and maintaining plasma membrane stability. In addition, this study has identified and localised GSTM3 in the tail and equatorial subdomain of the head of boar sperm. Finally, this study has set grounds for future investigations testing supplementation of semen extenders with GSTs, as this may improve fertility outcomes of swine AIs.

Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm

1995 ◽  
Vol 108 (2) ◽  
pp. 767-777 ◽  
Author(s):  
C.A. Allen ◽  
D.P. Green
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
De Novo ◽  
Convex Surface ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Level ◽  
Immunogold Labelling ◽  
Cytoplasmic Face

Acrosome-intact mammalian sperm can adhere to zona pellucida-free oocytes but are only capable of fusing if they have previously undergone the acrosome reaction. This suggests that the acrosome reaction results in presentation of at least one novel epitope which plays a role in sperm-oocyte fusion. Monoclonal antibodies were raised against unfixed acrosome-reacted guinea pig sperm and screened by indirect immunofluorescence for binding to the equatorial segment. They were back-screened against unfixed acrosome-intact sperm for absence of binding. Using this approach, two antibodies, G11 and M13, were identified which detect equatorial segment epitopes presented de novo by sperm following an A23187-induced acrosome reaction. The localization of these epitopes to the equatorial segment was confirmed at the ultrastructural level by indirect immunogold-labelling. Fluorescein isothiocyanate-labelled Fab fragments of these two antibodies also localized to the equatorial segment. Affinity chromatography and western blotting established that the two mAbs recognize the same proteins, which have M(r)s of 34, 46, 48 and 51 × 10(3). When sperm were induced to undergo the acrosome reaction with A23187 and incubated with their discharged acrosomal contents, a further band was produced with an M(r) of 30 × 10(3). Production of this band was inhibited in the combined presence of 100 microM phenylmethylsulphonyl fluoride and 100 microM p-aminobenzamidine even though these compounds do not inhibit acrosomal exocytosis. Neuraminidase and O-glycosidase were without effect on the proteins detected by antibodies G11 and M13. Endoglycosidase F, however, eliminated the bands of M(r) 46, 48 and 51 × 10(3) and replaced them with a strong band of M(r) 44 × 10(3) and two minor bands of M(r) 43 and 45 × 10(3). Formaldehyde fixation of acrosome-intact sperm caused partial rupture of the acrosome with loss of the characteristic rouleaux (stacks) of guinea pig sperm. Indirect labelling of these formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm.

1982 ◽  
Vol 92 (3) ◽  
pp. 604-615 ◽  
Author(s):  
E L Bearer ◽  
D S Friend
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Membrane Fusion ◽  
Acrosome Reaction ◽  
Lipid Extraction ◽  
Surface Coat ◽  
Lipid Domains ◽  
Membrane Function ◽  
Anionic Lipid ◽  
Sperm Membrane

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin-phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.

Lectin binding to guinea-pig sperm zipper particles

1983 ◽  
Vol 60 (1) ◽  
pp. 303-329
Author(s):  
G.C. Enders ◽  
Z. Werb ◽  
D.S. Friend
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Lectin Binding ◽  
Mammalian Species ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Binding Study ◽  
Detergent Solubilization ◽  
Triton X

Zipper particles are morphologically distinct transmembrane specializations of sperm tails. In freeze-fracture replicas of the guinea-pig sperm, they appear as an interdigitating double row of intramembranous particles running longitudinally within the plasma membrane of the principal piece. In thin section, zipper particles appear as an increase in electron density both above and below the bilayer. Zipper particles have been observed on a variety of both mammalian and non-mammalian species, suggesting that they have been conserved to serve an essential sperm function. As a first step towards biochemically characterizing guinea-pig zipper particles and towards developing a zipper isolation procedure, we performed an in situ lectin-binding study. Examination of nine gold- or ferritin-conjugated lectins revealed that three lectins, concanavalin A, Ricinus communis agglutinin I and wheatgerm agglutinin, bound to zipper particles. These lectin binding results suggest the presence of N-linked oligosaccharides within zipper particles. The results of the lectin binding study were then used in conjunction with a detergent solubilization procedure to identify potential zipper components. Detergent solubilization involved two non-ionic detergents: digitonin, which solubilized most of the plasma membrane, but left approximately two thirds of the zipper particles attached to the cytoskeleton, and Triton X-100, which solubilized the remaining zipper particles while leaving most other sperm structures intact. Within sodium dodecyl sulphate/polyacrylamide gels of the Triton-X-100-soluble fraction potential zipper particle components with the same lectin-binding characteristics as in situ zipper particles were identified.

scholarly journals Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction

Reproduction ◽  
2005 ◽  
Vol 129 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Haim Breitbart ◽  
Gili Cohen ◽  
Sara Rubinstein
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Mammalian Sperm ◽  
Acrosomal Membrane ◽  
Close Proximity ◽  
Sperm Plasma Membrane ◽  
To Come

In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can undergo the acrosome reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is important for the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the occurrence of the acrosome reaction, the F-actin should undergo depolymerization, a necessary process which enables the outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing proteins which break down the actin fibers, and allows the acrosome reaction to take place.

scholarly journals Molecular physiology and pathology of Ca2+-conducting channels in the plasma membrane of mammalian sperm

Reproduction ◽  
2005 ◽  
Vol 129 (3) ◽  
pp. 251-262 ◽  
Author(s):  
Ricardo Felix
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Current Evidence ◽  
Molecular Physiology ◽  
Mammalian Sperm ◽  
Fertilizing Ability ◽  
Intracellular Ca ◽  
Gene Defects ◽  
Sperm Fertilizing Ability ◽  
Sperm Functions

Current evidence indicates that mechanisms controlling the intracellular Ca2+concentration play pivotal roles in determining sperm fertilizing ability. Multiple Ca2+-permeable channels have been identified and characterized in the plasma membrane and in the acrosome membrane of mammalian sperm. This review summarizes the recent findings and assesses the evidence suggesting that these channels play roles in controlling a host of sperm functions ranging from motility to the acrosome reaction, and describes recent advances in the identification of the underlying gene defects of inherited sperm Ca2+channelopathies.

scholarly journals Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus

2021 ◽  
Vol 22 (23) ◽  
pp. 12646
Author(s):  
Marc Yeste ◽  
Sandra Recuero ◽  
Carolina Maside ◽  
Albert Salas-Huetos ◽  
Sergi Bonet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Species ◽  
Physiological Role ◽  
Membrane Lipid ◽  
Equatorial Region ◽  
Lipid Disorder ◽  
Head Displacement ◽  
Mammalian Sperm ◽  
Few Data

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

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