No abstract

The presence of highly acetylated histone H4 during spermatogenesis was studied to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement. We utilized an antibody raised to a pentaacetylated synthetic peptide that preferentially recognizes highly (tetra- and tri-) acetylated forms of rat testis H4. Electrophoretic separation of histones from enriched fractions of spermatogenic cells followed by detection of these forms by staining and by immunoblotting using this antibody showed that the highly acetylated forms were limited almost exclusively to spermatids beginning at step 11 of development. Immunoflurescence also revealed a striking polarity in the progression of histone from the spermatid nucleus. Highly acetylated H4 was displaced from the anterior to the caudal portion of the spermatid nucleus during steps 11 and 12, along with other histones, prior to their displacement by transition proteins. Thus, while monoacetylated and low levels of diacetylated forms of H4 were associated with stages at which histone deposition and transcription occur, the more highly acetylated forms appeared in high levels only at the stage at which histone displacement occurs.

A study of manchette development during spermiogenesis in azh/azh mutant mice was carried out by thin-section transmission electron microscopy with the goal of determining which of the initial steps in spermatid development are aberrant. In the homozygous mutant, spermatogenesis was quantitatively normal; but 100% of the sperm nuclei produced had abnormal shapes. The first defect, observed in steps 8-9, was the abnormal positioning of many manchette microtubules. These microtubules were directed towards regions of the plasma membrane not normally associated with manchette formation, in addition to being located at the caudal rim of the acrosome in the normal region of manchette formation. At steps 10-12, sheets of manchette microtubules were often in ectopic positions along the plasma membrane, rather than in association with the nuclear membrane as well. The fine structural appearance of the manchette was generally normal; the defect appeared to be in its positioning within the cell. In many step 8-10 spermatids nuclear invaginations and evaginations were observed, always associated with irregularities in the position of some of the manchette microtubules; these illustrate the capacity of manchette microtubules to deform nuclear shape. The nuclear irregularities remained throughout spermiogenesis. These observations are consistent with the hypothesis that the manchette is involved in at least some aspects of sperm nuclear shaping and that the improper positioning of manchette formation is a likely candidate for the primary abnormality resulting from a defective allele at the azh locus.

Germinal cells or nuclei with attached cytoskeletal elements were prepared from the testes and epididymides of normal mice and mice homozygous for the recessive azh mutation, which results in abnormal sperm heads. To make observations, we utilized phase-contrast microscopy, immunofluorescence microscopy with antitubulin antibodies, and a direct-view stereo electron microscope system developed by A. Cole. Sperm nuclei, tails, manchettes, and other cytoskeletal structures were studied at various stages of development. The tail architectures were similar in the normal and mutant forms, but the shape of the heads at the attachment regions were markedly different. Normal sperm nuclei were very flat, whereas the posterior regions of mutant nuclei were tapered cylinders. The manchette, an organized microtubular structure that girdles the posterior region of the spermatid nucleus, differed in size and configuration between normal and mutant forms. In normal midstage spermatids, the manchette microtubules extended outward at a 45 degree angle from the long axis of the flattened head, whereas in mutant spermatids, the microtubules formed tapered cylinders around the long axis of the caudal part of the nucleus. Radical differences in head shapes between normal and mutant sperm could be related, in part, to the manner in which manchettes formed and matured on the spermatids.