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

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.

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

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 x 10(3) (n = 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH(2)-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is encoded by the PKM gene. Antibodies produced against the N-terminus of purified PK-S (NH(2)-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.