Search

Transmission electron microscopy of the spermatozoon of six species of intertidal sea urchins has shown that each species has a sperm with unique dimensions, indicating that spermatozoon morphology could be used as a taxonomic character. However, although the structure of the sperm is species-specific, all sperm have a similar appearance which is typical of the Echinoidea. The sperm head is composed of a conical nucleus capped by a small, spheroidal acrosome which is undifferentiated internally. The mid-piece consists of a single ring-shaped mitochondrion which surrounds the centrioles. An examination of spermatogenesis reveals that each spermatogonium and spermatocyte develops a flagellum, the flagellum being resorbed prior to cell division. In addition a few small vesicles which resemble proacrosomal vesicles form in primary spermatocytes, a feature which has not been reported for echinoids, although such vesicles are a common feature of pre-spermiogenic cells of ophiuroids and holothurians. Spermiogenesis in all species is similar to descriptions published by other workers.

SummaryGerm plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve molluscRuditapes philippinarum(Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene–pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of ‘germ plasm granule formation complex’, a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

An integrated microscopic study of the destruction of mouse Ehrlich ascites carcinoma (EAC) cells under starvation conditions has been carried out. It has been found that, in addition to apoptosis, necrosis, and apoptotic necrosis, already known for EAC, cell destruction can also occur through mitochondrial autolysis, which is proposed to be considered a new kind of mitoptosis. A mitoptosis in EAC is characterized by the appearance of many autolyzing mitochondria, the fusion of which leads to rupture of the cell membrane and the ejection of the nucleus from the cell. It is assumed that the polymorphism of EAC destruction patterns is explained by the different physiological state of the cells, which determines the "choice" of the cell death mechanism. This situation poses a challenge for researchers to develop complex inducers with the ability to stimulate all possible types of cancer cell death.

The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation and thus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fully functional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy and other mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying cell differentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCs during neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined by monitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Given that neural differentiation is also accompanied by synthetic processes and oxidative stress, this process was included in the scope of this work. We demonstrated an increased contribution of protein-bound NADH and NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes or oxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neural differentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and the enhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolic status of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount we registered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.

The differentiation of endothelial cells from human iPSC has incontestable advantages in diseases research and therapeutic applications. However, the safe use of iPSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Shifts in cellular metabolism associated with intracellular pH changes affect the enzymes that control epigenetic configuration, which impact chromatin reorganization and gene expression changes during reprogramming and differentiation. FLIM-based metabolic imaging of NADH and FAD is a powerful tool for measuring mitochondrial metabolic state and widely used diagnostic method for identification of neoplastic diseases, skin diseases, ocular pathologies and stem cells differentiation. Therefore, in this study, we used the potential of FLIM-based metabolic imaging and fluorescence microscopy of NADH and FAD to study the metabolic changes during iPSC differentiation in endothelial cells. The evaluation of the intracellular pH was carried out with the fluorescent pH-sensor SypHer-2 and fluorescence microscopy to obtain complete information about metabolic status of iPSC and their endothelial derivatives. Based on the FAD/NAD(P)H optical redox ratios increase and the contributions rise of the NAD(P)H fluorescence lifetime in iPSC during endothelial differentiation, we demonstrated an contribution increase of OXPHOS to cellular metabolism. Based on the shift toward more acidic intracellular pH in endothelial cell derived from iPSCs we verified their oxidative state.

Glutaredoxin 2 (GRX2), a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC) to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies., Graphical abstract fx1

An expression of calpain and caspase-1 as well as the concomitant ultrastructural alterations were investigated during necrosis of the mouse Ehrlich ascites carcinoma. The calpain expression was registered at 0 h and 1 h although caspase-1 did not induce any signals during these time periods. The rise of the cytoplasmic lytic zones contacted by calpain antibodies was identified as a morphologic event corresponding to the expression of calpain. Lytic zone's distribution followed by the appearance of the calpain/caspase-1 clusters assigned for lysis of the Golgi vesicles and ER. Also, the microapocrine secretion of the vesicles containing the calpain/caspase-1 clusters was detected. Further, the lysis of the plasma membrane occurred due to progression of intracellular lysis. Rupture of the plasma membrane resulted in the termination of secretion and dissemination of cell contents. The nuclei still had their normal shape. Nuclear lysis continued to rise with intranuclear lytic zones, of which the progression was accompanied with the presence of calpain/caspase-1 clusters. The data contribute to the concept of the initial role of calpain for tumor cell destruction, provide first evidence of the calpain/caspase-1 pathway in tumor cells, and highlight microapocrine secretion as a possible tumor cell death signalling mechanism.

The phenomenon of the cytoplasmic localisation of mitochondrial ribosomal subunits (12 S mitochondrial rRNA and 16 S mitochondrial rRNA) has been discovered by scientific teams working with spermatogenic cells of mice. Previous reports showed that the release of mitochondrial substance occurs during interaction of mitochondria with the germ plasm granules (GG). To determine if the interplay between the vasa-positive GG and the mitochondria is associated with cytoplasmic localisation of mtrRNAs, we studied the spermatogenic cells of zebrafish, Danio rerio. It was revealed that in type A undifferentiated spermatogonia the GG did not contact mitochondria, and the extra-mitochondrial localisation of the mtrRNAs was not found. In type A differentiated spermatogonia, the amount of GG in contact with mitochondria increased, but the extra-mitochondrial localisation of the mtrRNAs was not found either. In type B late spermatogonia, which are pre-meiotic cells, the GG/mitochondrion complexes were typically found in contact with the nucleus. This stage was associated with the intra-mitochondrial localisation of GG-originated vasa and extra-mitochondrial localisation of 12 S mtrRNA and 16 S mtrRNA. Until the onset of meiosis, which was determined by the observation of synaptonemal complexes in zygotene-pachytene spermatocytes I, the GG/mitochondrion complexes disappeared, but both types of mtrRNAs persisted in the cytoplasm of spermatids and spermatozoa.

It was shown that tobacco leaf treatment with 100 mM H₂0₂ increased their content ofendogenous H₂0₂ and activities of catalase and hydrolases (acid phosphatase, proteases, and RNase) and also caused'various chang- es in the cell structure. In this case, programmed cell death (PCD) occurred in some cells, which was ob- served as chromatin condensation, cytoplasm collapse, etc. In the meantime, many cells displayed organelle activation rather than PCD. It is suggested that cells that undergo H₂0₂-dependent PCD release signaling molecules inducing protective mechanisms against oxidative stress in neighboring cells not exhibiting PCD.

Formation of germinal granules during oogenesis was studied in sea cucumbers, Apostichopus japonicus, Selenka, 1867, using transmission electron microscopy. It was found that within the early oogenesis mitochondria interact with a Balbiani body, gradually become immersed into its substance and transformed into electron-dense structures, which are similar to the germinal granules of metazoans. In late oogenesis, formed germinal granules lose their contacts with a Balbiani body and are localized in the cortical portion of the oocyte, near the cell membrane. Vestigial mitochondrial cristae are a typical feature of germinal granules. The obtained data suggest that the process of transformation of mitochondria into germinal granules is one of the functions of the Balbiani body, whose role still remains unexplained.

Modern ideas on the biogenesis of plant peroxisomes, their involvement in the production and detoxification of reactive oxygen species, and the role in the processes they mediate are discussed. The data on the proliferation and degradation of these organelles in the cell during the oxidative stress and on their involvement in the generation of signaling molecules are considered.

SummaryMouseVasahomologue (MVH) antibodies were applied to mouse Graafian oocytes to clarify if mitochondrion-originated germinal body-like structures, described previously by conventional electron microscopy, were associated with the germ plasm. It was found that both the mitochondrion-like structures with cristae and the germinal body-like structures that lacked any signs of cristae were labelled specifically by the anti-MVH antibody. Moreover, some granules were MVH-positive ultrastructural hybrids of the mitochondria and germinal body-like structures, the presence of which clearly supported the idea of a mitochondrial origin for the germinal body-like structures. This finding is the first evidence that mitochondrion-originated germinal body-like granules represent mouse germ plasm.

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

The mesophyll ultrastructure and hydrolase activity in leaves of tobacco cv. Samsun exposed to chitosan were studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus size and amount of both mitochondria and membranes of rough endoplasmic reticulum increased). At the same time, we observed activation of the lytic compartment displayed as stimulated production of dictyosomes, smooth ER elements, and cytoplasmic vacuoles, which are prominent constituents of this compartment. Biochemical experiments showed that, in the leaves, chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) as compared to untreated leaves. In some cells, chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm, etc.) that can be classified as development of programmed cell death.

The patterns of secretion utilized by embryonic cells during early development in the starfish Pisaster ochraceus were studied by transmission electron microscopy and morphometry. In addition to exocytosis of cortical granules, exocytosis and micro-apocrine secretion-like blebbing were performed by secretory vacuoles and secretory vesicles. In 2-cell and 4-cell embryos, as well the 22-hour blastula, secretory vacuole exocytosis (VAE) was the most frequent of the secretory types. In the early to middle gastrula, VAE declined and secretory vacuole blebbing (VAB) appeared. Both VAE and VAB almost disappeared in 5-day gastrulae, and secretory vesicle exocytosis (VEE) as well as the secretory vesicle blebbing (VEB) became dominant. VEB was the only mechanism of secretion in bipinnaria. With regard to yolk granules, Y1, Y2, and Y3 granules underwent lysosome-induced utilization (LIU). In addition, Y3 yolk underwent lysosome-induced sparseness (LIS), followed by Y3-Y5B, the pathway that assumes the formation of ...

The study of destruction of mouse Ehrlich ascites carcinoma (EAC) cells demonstrated that the loss of cell viability occurred via apoptosis detected by AnnexinV staining of phosphatidylserine inverted from the inner monolayer in the outer layer of cyto� plasmic membrane and via necrosis identified by staining of nuclei of autolyzed cells with propidium iodide penetrating into the cells via membranes with disrupted barrier properties. However, in an experi� ment with cucumarioside A 2�2, we detected an increased number of cells destroyed by combined cel� lular destruction, including necrosis and apoptosis (8). Thus, the use of cucumarioside A2�2, probably involves stimulation of the socalled "apoptotic necrosis," which is one of the possible variants of cell death (9). For verification of this hypothesis, morpho� logical identification of all variants of EAC cell death induced by cucumarioside A2�2 is required.

The effect of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in mesophyll cells of Nicotiana tabacum L. var. Samsun leaves is studied in the early stage of the development of the infection (3 days after infection of leaves). In the cells of leaves treated with chitosan 24 h before infection, the virus accumulated to a lesser degree than in the control. With the use of chitosan, TMV-specific granular inclusions were often observed in infected cells, the presence of which is ascribed to the early stages of virus reproduction, whereas the control cells contained mainly tubular inclusions formed from granular inclusions at the late stages of the infectious processes. This shows that chitosan delays the development of the infection. In the phosphotungstic acid-treated juice preparation made from infected leaves, abnormal (swollen and thin), as well as normal, TMV particles were observed. The appearance of abnormal viral particles seems to result from the virus-induced activation of intracellular lytic processes. In chitosan-treated infected cells, the lytic activity was the highest and the number of abnormal viral particles increased compared to the control. It is suggested that the chitosan-mediated stimulation of lytic processes that cause the destruction of TMV particles may be one of the protective mechanisms that limit the accumulation of the virus in cells.

Ultrastructural examination of tobacco mosaic virus-induced local lesions growing in Datura stramonium leaves is carried out. It is demonstrated that, in the central area of the lesions, the cell response to viral invasion is not uniform. Most cells exhibited an acute hypersensitive reaction (HR) and underwent rapid and complete necrosis. However, some cells, despite considerable virus accumulation and immediate contact with completely collapsed cells, maintained a certain degree of structural integrity. Analysis performed showed that the proportion of collapsed and uncollapsed cells in the lesion centre 3 to 5 days after infection essentially did not change. These data suggest that the absence of HR in some cells in the lesion centre is not due to an early stage of infection but is likely caused by cell tolerance of the virus.

To highlight the diversity of extracellular embryonic matrix (ECM) secreting structures in the gastrulae of the starfish Pisaster ochraceus the localization of antibody generated to hyaline layer, was studied. It was shown that hyaline layer epitope takes place in the cortical granules. Moreover, this material was revealed in secretory vacuoles and secretory vesicles which are situated in the apical parts of ectoderm cells. Besides, the same molecules were found in the yolk granules having loosened content. It seems likely that in P. ochraceus the ECM is secreted by four types of secretory structures.

The alterations involved in oocyte atresia of the starfish Pisaster ochraceus were investigated using both light and electron microscopy. It was shown that atresia may be defined by three patterns of cell destruction. Initially, the small electron-lucent vesicles produced by the Golgi complex underwent amalgamation into groups. This was followed by loss of vesicle membranes and consequent formation of transparent necrotic zones in the cytoplasm. The second pattern, ultrastructurally comparable with autophagic cell death, was marked by apparent amalgamation of the morphologically similar electron-lucent vesicles into growing vacuoles, giving rise to a multibranched autophagic vacuole. This vacuole engulfed the cytosol granules and ultimately came to occupy the entire space within the oocyte. In addition, the cytosol insulation inside of the 'apoptotic body-like spheres' was regularly observed. Thus, it is supposed that oocyte destruction may occur by a complex mechanism that includes elements of necrosis, autophagic cell death and apoptosis.

Reunov, A.A., Yurchenko, O.V., Alexandrova, Y.N. and Radashevsky, V.I. 2009. Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision. —Acta Zoologica (Stockholm) 91: 477–456. To characterize novel features that will be useful in the discussion and validation of the spionid polychaete Boccardiella hamata from the Sea of Japan, the successive stages of spermatogenesis were described and illustrated. Spermatogonia, spermatocytes and early spermatids are aflagellar cells that develop synchronously in clusters united by a cytophore. At the middle spermatid stage, the clusters undergo disintegration and spermatids produce flagella and float separately in coelomic fluid as they transform into sperm. Spermatozoa are filiform. The ring-shaped storage platelets are located along the anterior nuclear area. The nucleus is cupped by a conical acrosome. A nuclear plate is present between the acrosome and nucleus. The nucleus is a cylinder with the implantation fossa throughout its length and with the anterior part of the flagellum inside the fossa. There is only one centriole, serving as a basal body of the flagellum, situated in close vicinity of the acrosomal area. A collar of four mitochondria is located under the nuclear base. The ultrastructure of B. hamata spermatozoa from the Sea of Japan appears to be close to that of B. hamata from Florida described by Rice (Microscopic Anatomy of Invertebrates, Wiley-Liss, Inc., New York, 1992), suggesting species identity of the samples from the two regions. However, more detailed study of Florida’s B. hamata sperm is required for a reliable conclusion concerning the similarity of these two polychaetes. In addition to sperm structure, features such as the cytophore-assigned pattern of spermatogenic cell development, the synchronous pattern of cell divisions, the non-flagellate early spermatogenic stages, and the vesicle amalgamation that drives meiotic cell cytokinesis and spermatid diorthosis will likely be useful in future testing of the validity of B. hamata and sibling species throughout the world.

The distribution of the yolk epitope, PY4F8, was studied in the testes of the starfish Pisaster ochraceus (Brandt, 1835). Western blots have revealed that testes only show one 180 kDa band, whereas ovaries exhibit four PY4F8 bands at 90, 110, 120, and 180 kDa. This finding suggests that male yolk is less complex than female yolk. It was shown that PY4F8-positive material abounds in the hemal sinus of testes. Moreover, this epitope was found in the basal lamina, in the lumen of the testes, and in the cytoplasm of auxiliary and spermatogenic cells. It is likely that a hemal testis barrier is absent in P. ochraceus, and this lack allows direct penetration of nutrition to sustain metabolism of both spermatogenic and auxiliary cells. Morphologically, this penetration is possible through the gaps between adjacent auxiliary cell bases. Yolk granules containing the PY4F8 epitope are located in spermatogonia and spermatocytes, but not in spermatids and sperm. Therefore, yolk may play an important role in early spermatogenesis, but it does not participate at the final stage of sperm formation.

Spermatogenesis in silver salmon Oncorhynchus kisutch and cherry salmon O. masou was studied by transmission electron microscopy. The spermatogenic cells of both species have been shown to have no considerable differences in their dimensional and ultrastructural parameters. The characteristic peculiarity of spermatogonia was the presence of a germ determinant substance, which is the cytoplasmic marker of sex line cells. The primary and secondary spermatocytes, as well as early spermatids, were arranged in gonads as cell clusters at the same stage of development. In spermiogenesis, the regular appearance of the electron-lucent vesicle was observed, which forms a structural complex with the apical condensation of the nuclear membrane. This complex is morphologically similar to the not-yet-formed spermatid acrosomes in many Metazoa and is referred to by the authors as an acrosome-like structure (AS). At the final stage of spermiogenesis, the disappearance of an AS allows one to considering it to be a temporarily existing structure that recapitulates the ancestral morphotype of salmonid sperm. Spermatozoa of the both species are typical, primitive, acrosome-lacking cells, which are characteristic of many representatives of Teleostei that have external insemination.

The accumulation of potato virus X (PVX) in mesophyll cells of detached Datura stramonium L. leaves treated with fucoidan from brown algae Fucus evanescens C. Ag. have been evaluated by electron microscopy during the early infection period (three days after infection). It was found that cells of the leaves treated with fucoidan 24 h before infection accumulated virus less than untreated control. Ultrastructure-morphometric assay showed that fucoidan treatment causes an increase in the protein-synthesizing capability of cells (nucleolus dimention and amount of both mitochondria and rough endoplasmic reticulum membranes increased). At the same time, fucoidan treatment slightly activates the lytic compartment, which leads to the destruction of virus particles. Therefore, it may be considered that fucoidan induces a cellular defense mechanism that limits virus accumulation. Stimulation by the fucoidan of the formation of PVX-specific laminar structures able to bind virus particles may be another antiviral cell defense mechanism induced by the virus that prevents PVX reproduction and its intra- and intercellular transport.

SummaryTo find more morphological characteristics useful for discussion on aralian or non-aralian Panax affinity, pollen morphological diversity was comparatively analysed in P. ginseng, Aralia elata and Oplopanax elatus collected during their pollination periods. In the anthers of both the buds and open flowers, the pollen average diameter varied between some species-specific maximum and minimal measurement. However, the larger pollen grains were typically found in the buds whereas the smaller pollen prevailed in the open flowers, testifying to the pollen size diminution during anther maturation. Based on this finding, the subsequent examination of pollen according to size decrease was put into operation as a method of pollen modification for the study. The structural mechanisms of pollen metamorphosis were identified as not being species specific but rather universal. These mechanisms are suggested to be the shrinkage of the pollen vegetative cytoplasm, the intine enlargement, the deepening of three colporate apertures provided by exine sunken into enlarged intine areas, the aperture accretion as well as the transformation of the exine from thick/sculptured into thin/less sculptured. During ‘size-reducing metamorphosis’, the pollen grains changed dramatically, going through a species-specific set of intermediate morphs to the final species-specific morphotype. In P. ginseng this morphotype is round (diameter is about 16 μm), in A. elata it is round with a single projection (diameter is about 15 μm) and in O. elatus it is ovoid with a single projection (average diameter is about 18 μm). In addition, every species is peculiar in having the unique vegetative cytoplasm inclusions and individual construction of the largest pollen exine. From a phylogenetic perspective, these findings presumably add support to the option of equal remoteness of P. ginseng from A. elata and O. elatus. The characteristics found seem to be suitable for examination of Panax affinity, by the subsequent study of more Araliaceae representatives.

Egg hull formation is described in detail for the first time in Rhyssoplax tulipa from South Africa. Although previous descriptions of hull formation in chitons provide some basis for comparison, the mechanisms we have discovered are totally different. The process begins with the release of clear vacuoles from the oocyte into the intercellular space, which separates it from a layer of follicle cells. Materials contributing to the layers of the hull are formed primarily by micro-apocrine secretion processes, in which extensions of the follicle cells and oocyte are budded off into the intercellular space, rather than by formation and release of vesicles from the Golgi bodies, as previously thought. Later in oogenesis, these secretions become organized into three functional layers, each with a characteristic substructure. Follicle cells mold the shape of the hull spines even down to the petalloid tips that are characteristic of this group. Late in oogenesis the oocyte secretes the vitelline layer be...

It was observed that in the female gametophyte of Undaria pinnatifida (Harv.) Suringar (Phaeophyta, Laminariales) gametangial initials and maturing oogonia demonstrated different levels of alkaline-phosphatase activity (APA). The oogonia exhibited a higher level of APA than in its initials. Electron-dense granular ovoid structures ∼0.5–0.6 μm were present in the cytoplasm of oogonia. These inclusions were not membrane bound and do not appear to be associated with any particular organelles. The number of the inclusions was 1 to 2 in a single section of the cell. In essential details, the specific APA and subcellular germinal body-like structure of the developing female gamete in U. pinnatifida were very similar to those in metazoan oocytes.

Some peculiarities of the lipid composition of the outer membrane of the marine Gram-negative bacterium Chryseobacterium indoltheticum CIP 103168T have been studied. It is found that C. indoltheticum cells do not contain phosphatidylglycerol. Lipopolysaccharide could not be extracted by conventional methods; therefore, lipid A was isolated by hydrolysis of whole bacterial cells with 10% acetic acid with subsequent treatment with a chloroform: methanol mixture. The isolated lipid A has an unusual monosaccharide structure and constitutes 1-phosphate-D-glucosamine acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids at C2 and C3 atoms, respectively. The C. indoltheticum cells are shown to have a capsule; its polysaccharide apparently fulfils the LPS function.

SummaryThis report presents data from ultrastructural and morphometric studies on the germinal-body-like structures, nuage, nuage–mitochondrial clusters and chromatoid bodies in 4.5-day embryo cells and spermatogenic cells of the laboratory mouseMus musculus. In the 4.5-day embryo cells the germinal-body-like structures that, according to previous data, arise by condensation of mitochondria in Graafian oocytes, were found not to undergo any ultrastructural alterations. In spermatogonia the germinal-body-like structures presumably were transformed into nuage that functioned as ‘intermitochondrial cement’ binding the mitochondrial clusters. In primary spermatocytes mitochondria aggregated by nuage were found with large vacuoles containing membraneous conglomerates that were obviously excreted by organelles into the cytoplasm. The chromatoid bodies that arose in spermatocytes and finally disintegrated in the posterior part of late spermatids seemed not to be implicated in the pathway of the germinal-body-like structure. The dispersion of chromatoid bodies was noted to be accompanied by excretion of membraneous conglomerates by late spermatid mitochondria. The spermatozoa were not found to contain either the germinal-body-like structures or any other germ-plasm-related structures.

SummaryTo highlight the ultrastructural features of transformation occurring with germplasm-related structures (GPRS), the spermatogenic cells of Xenopus laevis were studied by transmission electron microscopy and quantitative analysis. It was determined that in spermatogonia and spermatocytes, the compact germinal granules underwent fragmentation into particles comparable with inter-mitochondrial cement (IMC). Fragments of IMC agglutinated some cell mitochondria and resulted in the creation of mitochondrial clusters. Clustered mitochondria responded with loss of their membranes that occurred by the twisting of membranous protrusions around themselves until multi-layered membranes were formed. The mitochondrial affinity of multi-layered membranes was proven by an immunopositive test for mitochondrial dihydrolipoamide acetyltransferase. As a consequence of mitochondrial membrane twisting, the naked mitochondrial cores appeared and presumably underwent dispersion, which is the terminal stage of GPRS transformation. As no GPRS were observed in spermatids and sperm, it was assumed that these structures are functionally assigned to early stages of meiotic differentiation.

The ultrastructural mechanism of postspawning sperm resorption in the testes of the sea urchin Strongylocentrotus nudus is described. Two types of phagosomes (containing sperm and containing residual bodies) are formed in the cytoplasm of nutritive phagocytes. The phagosomes fuse with electron-dense globules, and their contents are gradually destroyed. Afterward, phagosomes are transformed into electron-transparent vacuoles, which are finally compressed by the surrounding cytoplasm.

Summary The interaction between chromatoid bodies and mitochondria in neoblasts of the asexually reproducing planarian Girardia (Dugesia) tigrina, as well as in oogonial cells of the spontaneously sexualized individual G. tigrina, was studied using transmission electron microscopy. Mitochondria-chromatoid body complexes (chromatoid bodies surrounded by several mitochondria) are typical of planarian neoblasts and oogonia. In many mitochondria contacting the chromatoid bodies in gonial cells, the outer mitochondrial membrane disappeared, and release of the mitochondrial matrix was observed. The mitochondial derivatives devoid of an outer membrane and initially containing cristae of the inner mitochondrial membrane apparently were integrated with the material of chromatoid bodies. These findings agree with our previous ultrastructural data on sea urchin, fish and holothurian gonial cells and also with the molecular evidence provided by other authors for the presence of mitochondrial ribosomal RNA in germline...

It was found that in the Graafian oocytes of laboratory mice Mus musculus the population of electron-dense bodies contains two patterns of structures. One of these, designated as cortical granules, originated from the Golgi complex and was surrounded by a membrane. The other was discovered as cristae-containing mitochondrial derivatives lacked an outer membrane. It was found that the mitochondrial derivatives underwent progressive condensation and transformed into electron-dense bodies similar to germinal bodies of metazoan animals. Based on examination of Graafian follicle oocytes from 5 female individuals, about 15% of electron-dense bodies were cortical granules. However, about 85% of electron-dense bodies were condensing mitochondrial derivatives transforming into electron-dense bodies.

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles ("zymogen granules") and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in "empty" vacuoles inside the remnant body. The "empty" vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of "cheese-hole"-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.

The process of sperm development in Phoronopsis harmeri was studied by electron microscopy. Developing spermatogenical cells are aggregated around the capillaries of the haemal plexus. The spermatogonia, which are situated around the capillary walls of the caeca, are remarkable for the presence of germ-line vesicles and contain their centrioles near the cell membrane. The spermatocytes and spermatids are flagellated cells arranged in clusters. During spermiogenesis the basal body/flagellum complex migrates to the apical pole of the spermatid. The acrosome-like structure arises from material produced by the Golgi complex. It lacks a surrounding membrane and has a fibrillar content. The nucleus elongates and the condensation of chromatin is caused by an activation of 'initiation centres'. The late spermatid and the spermatozoon appear as two-armed 'V'-shaped cells in which one arm contains the nucleus and posteriorly located mitochondria, and the other one is the axoneme. Spermatogenesis of P. harmeri is an interesting example of gamete differentiation where advanced sperm structure is combined with a plesiomorphic pattern of sperm development characterized as 'flagellate spermatogenesis'.

Summary The phenomenon of “flagellate spermatogenesis” typically known among marine invertebrates with “primitive” sperm and external or external-internal fertilization is discussed. It is suggested that “flagella bearing” in early germinative cells might be explained by plesiomorphic similarity between these cells and flagellate somatic epithelial cells. The early germ cells of more apomorphic multicellular animals using internal fertilization with “modified” and “aberrant” sperm typically have no flagella and this organelle, as the sperm tail, first appears in spermatids. It is speculated that the “flagellate” pattern typifies the basal level and that the transition between “flagellate” and “specialized” spermatogenesis constitutes a significant step in evolution.

Literature data on the structure and origin of material of the germ cell line determinants and on the presence of products of nuclear and mitochondrial genomes in the structured germ determinants (nuage) are reviewed. The personal data, obtained on spermatogenic cells of sea urchin and of other marine invertebrates, evidence the transformation of the mitochondrial matrix into the nuage material. The presence of matrix structures of mitochondrial origin in the germ plasm, which ensure reproduction and the function of the structured macromolecular complex of germ determinants relatively independent from the nuclear genome, is suggested.

The process of sperm development in the sea urchin Anthocidaris crassispina was studied by light and electron microscopy. Similar to other echinoids studied, a single flagellum, striated rootlet and nuage-like materials were present in spermatogonia of A. crassispina. Spermatocytes near the diplotene stage showed intracellular localization of the axoneme which appeared to be a retracted flagellum prior to cell division. Fibrous filaments were associated with a proximal centriole in spermatocytes and spermatids and might be involved in movement of the proximal centriole. An acrosomal vesicle was developed and a residual body was formed in spermatids. The special development patterns in A. crassispina attributed to the presence of two patterns of tail development and two patterns of mitochondrial development during spermiogenesis. These four lines of spermiogenesis resulted in the formation of four morphological types of sperm cell, i.e. sperms with: (1) a symmetrical midpiece and posterior tail, (2) an asymmetrical midpiece and posterior tail, (3) a symmetrical midpiece and bent tail and (4) an asymmetrical midpiece and bent tail. Sperm cells with bent tails (type 3+4) were probably still at the late spermatid stage because results of scanning electron microscopy demonstrated gradual detachment and eventual straightening of the bent tail, and their percentage occurrence in the sperm population decreased significantly (P

Accessory cells were studied in early spermatogenesis of flatfishes Glyptocephalus stelleri and Pleuronectes pinnifasciatus using transmission electron microscopy. The morphological organization of accessory cells in G. stelleri was similar to that of Sertoli cells. In P. pinnifasciatus, these cells had morphological organization, which had not been previously described.

The relationships between germinal bodies and mitochondria were studied in the holothurian Apostichopus japonicus and the flounder Pleuronectes asper using TEM. In the gonial cells of both species the mitochondria are arranged around germinal bodies and are in contact with the latter. A gradual disappearance of the outer membrane is found in the mitochondria that interact with the germinal substance. Later on, dispersion of the globules of the mitochondrial matrix containing mitochondrial cristae occurs. It is supposed that the substance of the mitochondrial matrix takes part in the development and functioning of the germinal plasm in both invertebrates and vertebrates.

Summary In the holothurian, Cucumaria japonica, deep invaginations of the germinal epithelium of the testis are formed during the period of active spermatogenesis (January-February) but are reduced in prespawning animals (August). The invaginations contain basal lamina which envelope the testis and separate the haemal sinus from the gonad lumen. They are lined with germinal epithelium. Nurse cells are not present within regions of the germinal epithelium associated with invaginations of the lamina. These regions are invested by spermatogenic cells only.

Phagocytic resorption during spermatogenesis was studied in the sea urchin Anthocidaris crassispina. Nutritive phagocytes in gonad absorbed both waste sperm cells and residual bodies discarded from maturing spermatids, and these materials were subsequently compartmented in heterophagosomes. Based on 180 heterophagosomes examined by transmission electron microscopy, over 99% of heterophagosomes contained either residual bodies or sperm cells only. Simultaneous resorption of sperm cells and residual bodies in a heterophagosome was uncommon, with only ∼0.56% occurrence, suggesting that heterophagosomes have a selective resorption ability in nutritive phagocytes.

Summary The spermiogenesis of the brachiopod Rhynchonella psittacea was studied. The acrosome develops from cup-shaped acrosomal vesicles near the Golgi complex in the basal part of the spermatid. The acrosome vesicle then migrates to the apical region of the cell. The periacrosomal substance appears between the acrosomal vesicle and the nucleus. A ring-shaped mitochondrion is localized around the basal part of the nucleus. During spermiogenesis the proximal centriole is in contact with the nucleus via a single pericentriolar element. A distal centriole of the late spermatid has nine elements in a pericentriolar complex. The tail flagellum has a normal axonemal complex (9 + 2).

An ultrastructural study of nuage-mitochondria complexes in spermatogonia of the sea urchin, Anthocidaris crassispina, was carried out. Release of mitochondrial contents into the cytoplasm was observed. The mitochondrial derivatives persisted as cristae-containing globules of friable material that subsequently contacted and integrated with nuage. The present ultrastructural findings agree with the results of other researchers who proposed that germ plasm substance probably produced by the nucleus is supplemented by the mitochondrial genome.