Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation

2008 ◽  
Vol 75 (9) ◽  
pp. 1396-1407 ◽  
Author(s):  
Hiroshi Harayama ◽  
Kazumi Nakamura
Keyword(s):  
Camp Analog ◽  
A Cell ◽  
Principal Piece ◽  
Boar Spermatozoa

Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa

1997 ◽  
Vol 110 (15) ◽  
pp. 1821-1829 ◽  
Author(s):  
D. Westhoff ◽  
G. Kamp
Keyword(s):  
Glycolytic Enzyme ◽  
Sperm Maturation ◽  
Short Term ◽  
Fibrous Sheath ◽  
Typical Structure ◽  
Immunogold Staining ◽  
Atp Production ◽  
Principal Piece ◽  
Boar Spermatozoa ◽  
Mammalian Spermatozoa

Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3-phosphate dehydrogenase to be a hexamer. Rabbit polyclonal antibodies raised to purified glyceraldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3-phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label. During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis. The inverse compartmentation of the glycolytic enzyme and mitochondria in the mammalian sperm flagella suggests that ATP-production in the principal piece mainly occurs by glycolysis and in the midpiece by respiration.

Surface views of spermatozoa as revealed by fracture-flip

1989 ◽  
Vol 92 (3) ◽  
pp. 415-426
Author(s):  
C.A. Forsman ◽  
P. Pinto da Silva
Keyword(s):  
Plasma Membrane ◽  
Random Distribution ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
High Density ◽  
Surface Textures ◽  
Principal Piece ◽  
Boar Spermatozoa ◽  
Posterior Ring

We have used fracture-flip to produce new, macromolecular-resolution images of the surface of boar spermatozoa. Over the head, acrosomal and postacrosomal regions display sharply demarcated, subtly different surface textures. The rim is particle-poor, as well as a region above the oblique cords over the posterior ring. The tail shows two morphologically distinct domains: (1) the principal piece is covered by a high density of parallel-helical strands and a high density of large globular particles; (2) the midpiece and the neck are covered by smaller particles with apparent random distribution. Rectangular surface specializations frequently seen near the annulus display a waffle-like texture. With the notable exception of the parallel-helical strands of the principal piece the fracture-flip images of the boar spermatozoon can be directly related to the freeze-fracture morphology of its plasma membrane.

scholarly journals Elucidating the Role of K+ Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

2019 ◽  
Vol 20 (24) ◽  
pp. 6330 ◽  
Author(s):  
Marc Yeste ◽  
Marc Llavanera ◽  
Guillermo Pérez ◽  
Fabiana Scornik ◽  
Josep Puig-Parri ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Channel Blocker ◽  
Physiological Role ◽  
Membrane Lipid ◽  
K Channel ◽  
Sperm Capacitation ◽  
Lipid Disorder ◽  
Principal Piece ◽  
Boar Spermatozoa

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.

scholarly journals Effect of ligands that increase cAMP on caerulein-induced zymogen activation in pancreatic acini

2003 ◽  
Vol 285 (5) ◽  
pp. G822-G828 ◽  
Author(s):  
Z. Lu ◽  
T. R. Kolodecik ◽  
S. Karne ◽  
M. Nyce ◽  
F. Gorelick
Keyword(s):  
Acinar Cell ◽  
Pancreatic Acinar Cell ◽  
Zymogen Activation ◽  
Pancreatic Acini ◽  
Camp Analog ◽  
Pituitary Adenylate Cyclase ◽  
Protease Activation ◽  
A Cell ◽  
Camp Levels ◽  
Stimulation Of

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 μM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.

scholarly journals Molecular demarcation of surface domains as established by label-fracture cytochemistry of boar spermatozoa.

1987 ◽  
Vol 35 (10) ◽  
pp. 1069-1078 ◽  
Author(s):  
F W Kan ◽  
P Pinto da Silva
Keyword(s):  
High Resolution ◽  
Cell Surface ◽  
Lateral Diffusion ◽  
Surface Receptors ◽  
Free Zone ◽  
Receptor Sites ◽  
Principal Piece ◽  
Surface Domains ◽  
Membrane Components ◽  
Boar Spermatozoa

We used "label-fracture" (J Cell Biol 99:1156, 1984) to establish high-resolution maps of wheat germ agglutinin (WGA) and concanavalin A (ConA) receptor sites on the cell surface of boar spermatozoa and to investigate the possible association of these receptors to integral membrane components. Label-fracture reveals intense WGA labeling over the region of the plasma membrane that overlies the acrosome, including the equatorial segment. The density of WGA receptors decreases from the post-acrosomal area to the posterior ring. The WGA receptor domain changes abruptly into a microdomain with an unusually high density of WGA receptors over a sharply delimited, particle-free zone at the base of the head. Over the tail, the density of WGA receptors in the tail is high and uniform over the midpiece, annulus, and principal piece, but narrow patches of rectilinear arrays of pits close to the annulus are not labeled. Labeling of the entire sperm head by ConA is both intense and uniform, except for the particle-free zone at the base of the head, which is barren of receptors. Over the tail, ConA labeling is strong over the midpiece, absent over the annulus, and sparse over the principal piece and the end piece. In contrast to WGA, ConA receptors co-distribute with the intramembrane particles. Our results confirm the potential of label-fracture for high-resolution mapping of the distribution of cell surface receptors. They show that in this specialized cell membrane domains can be sharply defined, i.e., apparent without free lateral diffusion of components.

Expression and compartmentalisation of the glycolytic enzymes GAPDH and pyruvate kinase in boar spermatogenesis

2008 ◽  
Vol 20 (6) ◽  
pp. 713 ◽  
Author(s):  
Sandra Feiden ◽  
Uwe Wolfrum ◽  
Gerhard Wegener ◽  
Günter Kamp
Keyword(s):  
Pyruvate Kinase ◽  
Developmental Stages ◽  
Immunofluorescence Microscopy ◽  
Glycolytic Enzymes ◽  
Rabbit Muscle ◽  
Atp Production ◽  
Sperm Flagellum ◽  
Cell Structures ◽  
Principal Piece ◽  
Boar Spermatozoa

Boar spermatozoa contain isoforms of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) and pyruvate kinase (PK, EC 2.7.1.40). The sperm-specific forms, GAPDH-S and PK-S, are tightly bound to cell structures. By immunofluorescence microscopy GAPDH-S and PK-S were localised in the principal piece of the boar sperm flagellum as well as in the acrosomal region of the sperm head and at the head–midpiece junction. The midpiece of the flagellum, however, contains isoforms of GAPDH and PK that were only recognised by antibodies against somatic GAPDH and PK, respectively, but not by the antibodies against GAPDH-S and PK-S. In sections of boar testis, GAPDH-S and PK-S were first detected in elongating spermatids when both the developing flagellum and the head were labelled with antibodies against GAPDH-S and PK-S. In contrast, antibodies against rabbit muscle GAPDH and PK labelled all developmental stages of germ cells and also neighbouring contractile cells. Thus, the structure-bound sperm-specific enzymes, GAPDH-S and PK-S, appeared only late in spermatogenesis simultaneously with the development of the structures to which they are bound. Anchoring glycolytic enzymes to structures in these mitochondria-free regions may secure ATP-production for both motility and acrosome function.

scholarly journals A novel pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and the acrosome

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 81-95 ◽  
Author(s):  
Sandra Feiden ◽  
Heike Stypa ◽  
Uwe Wolfrum ◽  
Gerhard Wegener ◽  
Günter Kamp
Keyword(s):  
Pyruvate Kinase ◽  
Immunofluorescence Microscopy ◽  
Immunogold Labelling ◽  
Sperm Head ◽  
Relative Molecular Mass ◽  
Rabbit Muscle ◽  
Fibrous Sheath ◽  
Cell Structures ◽  
Principal Piece ◽  
Boar Spermatozoa

Boar spermatozoa contain a novel pyruvate kinase (PK-S) that is tightly bound at the acrosome of the sperm head and at the fibrous sheath in the principal piece of the flagellum, while the midpiece contains a soluble pyruvate kinase (PK). PK-S could not be solubilized by detergents, but by trypsin with no loss of activity. Purified PK-S as well as PK-S still bound to cell structures and soluble sperm PK have all kinetics similar to those of rabbit muscle PK-M1. The PK-S subunit had a relative molecular mass of 64 ± 1 × 103(n= 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH2-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is encoded by thePKMgene. Antibodies produced against the N-terminus of purified PK-S (NH2-TSEAMPKAHMDAG-COOH) were specific for PK-S as they did not react with somatic PKs or soluble sperm PK, while anti-PK-M1 recognized both sperm PKs. Immunofluorescence microscopy showed anti-PK-S to label the acrosome and the flagellar principal piece, whereas the midpiece containing the mitochondria was labelled only by anti-PK-M1. Immunogold labelling confirmed the localization of PK-S at the acrosome. In the principal piece, both polyclonal anti-PK-M1 and anti-PK-S were found at the fibrous sheath. Our results suggest that PK-S is a major component in the structural organization of glycolysis in boar spermatozoa.

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