Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the azh/azh mutant mouse.

The tubulin-containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule-associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotype-specific tubulin antibodies as immunoprobes. Manchettes from wild-type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage-specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild-type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, alpha-3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild-type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase-contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso-like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation.