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.

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.

scholarly journals Inhibition of glycolytic enzymes by 2-phosphotartronate

1976 ◽  
Vol 153 (3) ◽  
pp. 741-744 ◽  
Author(s):  
M K Thomas ◽  
T G Spring
Keyword(s):  
Pyruvate Kinase ◽  
Competitive Inhibition ◽  
Phosphoglycerate Kinase ◽  
Triose Phosphate Isomerase ◽  
Glycolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Triose Phosphate ◽  
Permanganate Oxidation

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.

scholarly journals Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase.

1979 ◽  
Vol 254 (22) ◽  
pp. 11357-11359
Author(s):  
C.D. Fitch ◽  
R. Chevli ◽  
M. Jellinek
Keyword(s):  
Pyruvate Kinase ◽  
Rabbit Muscle
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