Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis.

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules.

Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B.

Cytochemical studies on histone-type and protamine-type proteins during spermiogenesis in Chara vulgaris and Chara tomentosa.

Comparative studies concerning detection of histone-type and protamine-type proteins were carried out on Chara species (C. vulgaris, C. tomentosa). Analysis of antheridia during spermiogenesis (stages I-X) of both Charta species showed very similar staining patterns obtained after reactions revealing the examined proteins. Cytochemical studies showed a replacement of lysine-rich histone proteins by more basic arginine-rich ones during medium spermiogenesis (st. VI-VIII) in two Charta species, while late spermiogenesis (st. IX) and mature spermatozoids (st. X) were characterised by the presence of protamine-like proteins only.

Occurrence of ubiquitin during spermiogenesis in Chara vulgaris.

Experiments with an anti-ubiquitin antibody proved the presence of ubiquitin in spermatids at all spermiogenesis stages in Charta vulgaris. Its level increased before marked ultrastructural changes of spermatids correlated with disappearance of somatic proteins (histones) and appearance of protamine-type generative proteins. The obtained results seem to confirm our earlier hypotheses concerning a significant role of ubiquitin-proteasome system in Chara spermatozoid differentiation.

Disappearance of 3H-leucine incorporation circadian rhythm induced by symplasmic isolation of Chara vulgaris antheridium from thallus.

Autoradiographic research with the use of 3H-leucine demonstrates that circadian rhythm of protein synthesis characteristic of manubria at the proliferative phase of spermatogenesis in Chara vulgaris disappears after symplasmic isolation of antheridium from thallus during the time preceding block of DNA endoreplication in manubria following initiation of spermiogenesis insensitive to light.

GA3 content in antheridia of Chara vulgaris at the proliferative stage and in spermiogenesis estimated by capillary electrophoresis.

In male sex organs of Chara vulgaris L., the gibberellic acid (GA3), was identified by capillary zone electrophoresis. The antheridia at cell division stage of antheridial filaments leading to formation of spermatids contain 0.09 microg GA3 per antheridium, i.e. 5.3 times more than antheridia at differentiation stage of spermatozoids (spermiogenesis). Spermiogenesis is not regulated by gibberellins.