Wan-Jong Kim

Cytochemical localization of nuclear actin of sperm and spermatids in urechis unicinctus

Abstract In this study, we found that sperm ball of Urechis unicinctus consisted of a somatic cell and spermatogenic cells. After separation from the sperm ball, individual spermatid floated freely in the coelomic fluid and differentiated into a mature sperm. Because of many nuclear vacuoles, spermatid nucleus was observed to be heterogeneous. Later, the spermatid nucleus condensed into the homogeneous round nucleus of the mature sperm. Perinuclear microtubules could be seen but did not seem to be organized into manchette microtubules. To understand the nature of nuclear condensation during spermiogenesis, the sperm and spermatids (spermiogenic cells) were treated with FITC‐phalloidin, or anti‐actin‐FITC, or labeled with anti‐actin immunogold particles (AAIP; 10 nm) followed by transmission electron microscopy or confocal laser scanning microscopy. The anti‐actin‐FITC and FITC‐phalloidin reactions occurred distinctly in the nuclei of both spermiogenic cells. FITC‐phalloidin reacted more intensely with acrosomes. The AAIP were incorporated mainly into nuclei of both cells sometimes showing local distribution in the nucleus. Nuclear vacuoles of spermatids disappeared progressively with condensation of the nucleus, as the number of incorporated AAIP/μm2 increased. These results suggest that nuclear actin microfilaments might be closely related to nuclear condensation.

Attribution of Cortical Granules to Formation of Fertilization Envelopes and Polyspermy Block in Urechis unicinctus

Abstract Cortical reaction and polyspermy block are well defined in most marine invertebrates. In Urechis species, the function of cortical granules (CGs) is not yet known, and there is controversy on whether the cortical reaction occurs, or the fertilization envelope (FE) is attributed to CG releases or functions to prevent polyspermy. This study was carried out to determine the cortical reactions and functions of the FE in Urechis unicinctus. Artificial insemination of the eggs revealed that CG release occurred to give rise to perivitelline space (PS) into the final FE. Both PS and final FE effectively blocked polyspermy. The final FE was accomplished within 10 min after sperm‐egg initial binding. No massive release of CGs occurred within the early phase of 5 min after the initial binding, initially and the PS seemed to play a role to prevent polyspermy. The CG massively released its content into the PS in late phase of FE formation, and differentiated PS into five intermediate layers. The layers opened into each other by anastomosis, so that the final FE consisted of two layers, the inner layer (15 μm in thickness) and the outer layer (1 μm in thickness). The outer layer derived from vitelline layer and the inner layer consisted of PS and CG secretions. Immunofluorescence and confocal laser microscopy revealed that the spermatozoon took up residence in the egg cortex during FE formation and successive meioses of the fertilized egg. These results suggest that both PS and final FE of U. unicinctus were equivalent to the early and late block, respectively, of other marine animals.

Metallothionein Induction in Liver Regeneration Stimulated by Partial He patectomy

Metallothionein (MT) is induced in the regenerating rat liver. We have investigated expression of MT gene by RT PCR as well as specific localization of MT by immunocytochemistry in regenerating rat liver after partial hepatectomy (PH). MT mRNA level started to increase from 1h and reached the peak at 8 h after PH. The level decreased gradually by 24 h, and became similar to that of control group. In the immunocytochemical study, in all groups treated with primary antibody, immunogold particles indicating the presence of MT were evenly distributed throughout both cytoplasm and nucleus of the rat hepatocytes. Within the nucleus, the gold particles appeared to be intensely localized in the areas of euchromatin and nucleolus. Within the cytoplasm, gold particles did not seem to adhere to mitochondria or lysosomes, but were freely distributed. However, rough endoplasmic reticulum was the obvious compartment on which the gold particles were localized. Time course of MT immuno re activity revealed that distribution of gold particles in hepatocytes increased gradually by 24 h, and decreased at 48 h after PH. Briefly, PH resulted in the sharpest increase in the expression of MT mRNA at 8 h and in the immuno re activity of MT at 24 h, respectively. It is suggested that the increase of MT mRNA expression, the intensity of immuno re activity and the specific localization of MT may be associated with the compensatory cell proliferation followed by PH.