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 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.

Ultrastructural localization of dystropin in human muscle by using gold immunolabelling

1990 ◽  
Vol 240 (1297) ◽  
pp. 197-210 ◽  
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Muscle ◽  
Muscle Fibres ◽  
Mouse Muscle ◽  
Transverse Tubular System ◽  
Cytoplasmic Face ◽  
Tubular System ◽  
Gold Probe

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al . 1989 a ; England et al . 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin mole­cule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

scholarly journals Visualization of anti-sperm plasma membrane IgG and Fab as a method for localization of boar sperm membrane antigens.

1982 ◽  
Vol 30 (12) ◽  
pp. 1217-1227 ◽  
Author(s):  
L D Russell ◽  
R N Peterson ◽  
T A Russell
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Localization ◽  
Surface Antigens ◽  
Ultrastructural Level ◽  
Simple Method ◽  
Sperm Surface ◽  
Sperm Plasma Membrane ◽  
Sperm Antigens

A simple method for ultrastructural localization of sperm surface antigens by direct visualization of bound antibodies is presented. Anti-sperm plasma membrane (ASPM) immunoglobulin (Ig) G, visualized in tissues treated with an osmium:ferrocyanide mixture, projected 11-13 nm from the surface and ASPM Fab fragments projected 8-10 nm from the surface. The density of IgG labeling, as subjectively estimated, corresponded to indirect immune fluorescein isothiocyanate, indirect immunoferritin, and sperm-vesicle labeling patterns. Agglutination of sperm vesicles and sperm were demonstrated and the linking antibody visualized. A second antibody on protein A directed against ASPM IgG made the immunologic tag more apparent and indicated, in disrupted sperm preparations, labeling of both sides of the plasma membrane. The method provides for easy and sensitive localization of sperm surface antigens at the ultrastructural level and is presently being used to localize specific sperm antigens.

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.

Characterisation of the progesterone receptor on canine spermatozoa

2005 ◽  
Vol 17 (7) ◽  
pp. 733 ◽  
Author(s):  
Jui-Te Wu ◽  
Pei-Shiue Tsai ◽  
Shuang-Lin Lee ◽  
Feng-Pang Cheng
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Progesterone Receptor ◽  
Seminal Plasma ◽  
High Capacity ◽  
Fluorescein Isothiocyanate ◽  
Dependent Manner ◽  
Sperm Plasma Membrane ◽  
Pre Treatment ◽  
Dose Dependent

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone–bovine serum albumin–fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 ± 4%), but this labelling was markedly reduced (33 ± 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 ± 10% (onset) to 59 ± 7% by 5 h, where it plateaued. Progesterone (P4) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 µg/mL P4 corresponding to 10 ± 5%, 16 ± 9%, 23 ± 7% and 30 ± 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P4-induced AR (12 ± 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P4-induced AR (10 µg/mL) in capacitated ejaculated spermatozoa from 19 ± 6% to 11 ± 4% (h151, 1 : 10) and 12 ± 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognise 200x103 Mr glycoproteins stored within large peripheral vesicles

1990 ◽  
Vol 95 (2) ◽  
pp. 199-206
Author(s):  
M. TERESA ESTRADA-GARCIA ◽  
JAMES A. CALLOW ◽  
JONATHAN R. GREEN
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Immunogold Labelling ◽  
Pythium Aphanidermatum ◽  
Cell Coat ◽  
Adhesive Material ◽  
Ultrastructural Studies ◽  
Carbohydrate Epitopes ◽  
A Cell

During encystment the motile zoospores of the plant pathogen Pythium aphanidermatum secrete an adhesive cell coat that is involved in their attachment to roots. Previous ultrastructural studies have indicated that the adhesive material is pre-packaged within large peripheral vesicles underlying the zoospore plasma membrane. In the present study, four monoclonal antibodies (MAbs) designated PA3-6, which were raised against zoospores and cysts of P. aphanidermatum, were used to re-examine the formation of the adhesive cell coat and to study its molecular nature. Immunogold labelling of zoospores and cysts shows that all the antibodies recognise material contained within the large peripheral vesicles of zoospores. These structures are morphologically distinct from the microbodies, which also underly the plasma membrane, and the latter are not labelled by the antibodies. During encystment the material recognised by the MAbs is secreted to form a cell coat around the zoospores and cysts and this can be seen to be separated from the cyst plasma membrane by a distinct layer (cyst wall). The MAbs also label material within vesicles that are located towards the centre of the cyst cytoplasm. Western blotting and antigen-modification techniques have shown that all the MAbs bind to carbohydrate epitopes of a set of high molecular weight glycoproteins (> 200xl03Mr). One of the antibodies, PA6, also binds to several lower molecular weight components. Overall, the results show that the adhesive material secreted by P. aphanidermatum zoospores is stored within large peripheral vesicles and is composed of several glycoproteins. The results are discussed in the context of studies on the secretion of adhesive material by zoospores of related oomycete fungi.

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

2005 ◽  
Vol 17 (4) ◽  
pp. 467 ◽  
Author(s):  
H. D. Guthrie ◽  
G. R. Welch
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Calcium Ionophore A23187 ◽  
Membrane Phospholipid ◽  
Ionophore A23187 ◽  
Liquid Storage ◽  
Boar Spermatozoa ◽  
Plasma Membrane Phospholipid

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin–fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3–8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.

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.

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Zygote ◽  
1998 ◽  
Vol 6 (4) ◽  
pp. 329-339 ◽  
Author(s):  
John D. Baldwin ◽  
Fred J. Griffin ◽  
Wallis H. Clark
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Actin Filaments ◽  
Acrosome Reaction ◽  
Ultrastructural Level ◽  
Unique Sequence ◽  
Amino Acid Sequencing ◽  
Marine Shrimp ◽  
Vesicle Exocytosis ◽  
New Type

During sperm/egg interaction, the sperm of Sicyonia ingentis undergoes a unique biphasic acrosome reaction. After acrosomal vesicle exocytosis, the sperm generates an acrosomal filament, over a period of 4-6 min, that is approximately 10 μm in length. Neither actin filaments nor normal microtubules have been demonstrated at the ultrastructural level of this unusual filament. Using a battery of cytoskeleton-directed antibodies the biochemical nature of this filament has been investigated. Antibodies to actin and tubulin do not label the subacrosome or acrosomal filament, but do recognise actin and tubulin in other shrimp tissues. Antibodies to tau, MAP2, and neurofilament medium and heavy subunits were all localised to the subacrosomal region of the sperm. It is interesting, however, that only the two clones of neurofilament monoclonal antibodies recognised the acrosomal filament. Electrophoretic and Western blot analysis in conjunction with amino acid sequencing revealed that the proteins localised to the acrosomal filament are of a unique sequence and may represent a new type of protein of centrosomal origin.

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