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In this study, we investigated in an androgenized rat model the involvement of autophagy and mitochondrial dynamics in granulosa cells in the pathogenesis of polycystic ovarian syndrome (PCOS) and its modulation by exogenous gonadotropin (eCG). We found 5α-dihydrotestosterone (DHT) treatment reduces ovarian length and weight with predominantly late antral and/or preovulatory stage follicles and no corpora lutea. DHT increased the population of large lysosomes (>50 micron) and macroautophagy, an event associated with granulosa cell apoptosis. Increased granulosa cell Dynamin Related Protein 1 (Drp1) content in the DHT group was accompanied by increased circular and constricted, but reduced rod-shaped, mitochondria. eCG eliminated all atypical follicles and increased the number of late antral and preovulatory follicles with less granulosa cell apoptosis. eCG-treated rats had a higher proportion of connected mitochondria, and in combination with DHT had a lower proportion of circular and constricted mitochondria than rats treated with DHT alone, suggesting that eCG induces mitochondrial fusion and attenuates fission in granulosa cells. In summary, we observed that DHT-induced up-regulation of Drp1 is associated with excessive mitochondrial fission, macroautophagy and apoptosis in granulosa cells at the antral stage of development in an androgenized rat model for PCOS, a response partially attenuated by exogenous gonadotropin.

Ovarian cancer (OVCA) is the most lethal gynecological cancer, due predominantly to late presentation, high recurrence rate and common chemoresistance development. The expression of the actin-associated protein cytosolic gelsolin (GSN) regulates the gynecological cancer cell fate resulting in dysregulation in chemosensitivity. In this study, we report that elevated expression of plasma gelsolin (pGSN), a secreted isoform of GSN and expressed from the same GSN gene, correlates with poorer overall survival and relapse-free survival in patients with OVCA. In addition, it is highly expressed and secreted in chemoresistant OVCA cells than its chemosensitive counterparts. pGSN, secreted and transported via exosomes (Ex-pGSN), upregulates HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as confers cisplatin resistance in otherwise chemosensitive OVCA cells. These findings support our hypothesis that exosomal pGSN promotes OVCA cell survival through both autocrine and paracrine mechanisms that transform chemosensitive cells to resistant counterparts. Specifically, pGSN transported via exosomes is a determinant of chemoresistance in OVCA.

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.

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.

Conventional techniques are insufficient to precisely describe the internal structure, heterogeneous cell populations and the dynamics of biological processes of the diseased liver during and after surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease to plan surgery, efficacy of regeneration and avoid postoperative liver failure. Here, we analyze acute and chronic liver pathology (cholestasis, cirrhosis, fatty liver disease) during progression by multiphoton microscopy with FLIM and SHG modes and chemical analysis by TOF-SIMS to obtain new data about hepatocyte pathological changes at the cellular and molecular level. All of these techniques enable to study cellular metabolism, lipid composition and collagen structure without sample staining and incorporation of fluorescent or another markers that may allow to use these methods in clinic. Complex of multiphoton microscopy and mass spectrometry provides complete information about the liver structure and function that could not be assessed using each method individually. The data can be used for both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique of liver quality analysis to plan surgery and avoid postoperative liver failure in clinic.

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.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

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.

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic.

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.

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper., Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes.

Transmission electron microscopy of the spermatozoa of five species from three families of bivalves has shown that each species has a sperm with unique morphology. However, the morphology of the acrosomes of each species is typical of the subclass of bivalve to which they belong. An examination of spermatogenesis in the five species, along with a re-examination of material from six other species of bivalves, has revealed that pre-spermiogenic cells possess flagella. In addition, acrosome formation begins in the spermatocytes with the formation of proacrosomal vesicles in the Golgi body. During spermiogenesis the proacrosomal vesicles coalesce at the presumptive posterior of the spermatid, with a larger vesicle produced by the Golgi body. The single acrosomal vesicle eventually migrates to the anterior of the spermatid where it assumes its mature form. © 1994 Wiley-Liss, Inc.

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.

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.

In young systemically infected leaves of Datura stramonium L., a severe strain of Potato virus X (PVX) accumulated to a lower degree than a mild strain. Infected leaves had increased protease and RNase activities in comparison with those of healthy controls. The highest hydrolase activities were found in leaves infected with the severe strain. Negative-staining electron microscopy of dips from the infected leaves indicated that PVX virions underwent destructive changes, which resulted in the appearance of abnormal (swollen and ‘thin’) particles. Immuno-electron microscopic assays showed that thin PVX particles, in contrast to those of normal diameter, lost the ability to bind with specific antiserum. The relative number of thin virions in leaves infected with the severe PVX strain was considerably higher than in leaves infected with the mild strain. This shows that a correlation exists between increased protease activity and intracellular destruction of virions. In abnormal virions, the viral RNA appears to be available for RNase attack. Therefore, it seems that high RNase activity together with increased generation of abnormal virions in the leaves infected with the severe strain promote inactivation of the viral RNA with RNase. We suppose that the enhanced hydrolase activities in the leaves infected with severe PVX strain, on the one hand, limit viral accumulation and thus play a defensive role and, on the other hand, cause considerable intracellular pathological changes resulting in severe symptoms.

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.

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 activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-β-D-glucan, fucoidan, and κ/β-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

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.

Egg hull formation during oogenesis in the chiton Callochiton dentatus does not follow the typical model of merocrine secretion involving Golgi vesicle exocytosis. Instead, microapocrine secretions are primarily responsible for egg hull formation, although merocrine secretions contribute “areolae” and the vitelline layer. Microapocrine secretion mechanisms are poorly understood, involving a different cellular pathway than is typical. Egg hull formation in C. dentatus involves two types of microapocrine secretions released by the oocyte, one of which is described here for the first time. The plesiomorphic jelly-like egg hull of chitons, as exemplified by the eggs of members of the basal order Lepidopleurida and present also in eggs of C. dentatus (Chitonida: Callichitonidae), may have evolved solely as an oocyte secretion, whereas members of some other families in the order Chitonida form their egg hulls with considerable secretory input from the follicle cells as well.

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.

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.

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.

Summary The somatic accessory cells in the testes of the sea cucumber, Chiridota pellucida (Holothuroidea: Apoda), are flattened flagellated epithelial cells covering the basal lamina of the germinal epithelium. Their nucleus is irregular or elongated with a prominent nucleolus. The cytoplasm contains the Golgi complex, cisternae of rough and smooth endoplasmic reticulum, numerous electron-lucent vesicles, and lipid droplets. C. pellucida accessory cells differ in ultrastructure from those in other holothurians.

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.

Difficulties in characterization of spermatozoa of the Metazoa are now related to insufficiency of the traditional terms “primitive spermatozoon,” “modified spermatozoon,” and “aberrant spermatozoon” introduced by G. Retzius and A. Franzen for description of intermediate forms discovered in the 20th century. In this respect, some authors propose to reject the Retzius' and Franzen's terms and turn to the terminological system of G. Rouse and B. Jamieson, the terms of which (“ectaquaspermatosoa,” “entaquaspermatozoa,” and “introspermatozoa”) determine specific features of insemination, but do not reflect the structure of spermatozoa. The assertion of “helplessness” of the traditional structural Retzius' and Franzen's terms is unfounded. Their preservation is desirable if one wishes to preserve the comparative-morphological approach to the characterization of male gametes of the Metazoa. The terms “primitive spermatozoon,” “modified spermatozoon,” and “aberrant spermatozoon” will become more universal if the method of their combined utilization is applied.

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.

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'.

It is established that the central area of TMV-induced local lesions developed in detached Datura stramonium leaves, along with the completely collapsed cells (types I and II), contains cells (type III) conserving to a certain degree integrity of their structural components. A characteristic of the type III cells was the accumulation of considerable amount of virus and formation of TMV-specific granular and tubular inclusions. The study of lesion development showed that a proportion of the collapsed cells and cells of type III did not essentially change in the period up from 3 to 5 days after infection of the leaves. These data suggest that the disease development in cells of type III does not lead to a hypersensitive response and is very similar to that in the systemically infected cells.

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.

Background:Injury to the mesothelial layer of the peritoneal membrane during peritoneal dialysis (PD) is implicated in loss of ultrafiltration capacity, but there are no validated biomarkers for me...

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

Spermatogenesis and sperm structure of the nemertine wormProcephalothrix sp. were studied by transmission electron microscopy. It is shown that a flagellum and proacrosomal vesicles are common in spermatogonia and spermatocytes as in spermatogenesis of a number of marine invertebrates with external fertilization. Originally, the animals were collected asProcephalothrix spiralis but they were found to have a type of spermatozoon different from that ofP. spiralis as described by Turbeville & Ruppert (1985). The re-identification of the material collected in the Japan Sea has shown that the features are characteristic, ofP. spiralis (Coe, 1930). This finding suggests thatP. spiralis shows variations in different parts of the world.

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.

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.

Summary The structure of the spermatozoon and spermatogenesis of Discinisca tenuis (Inarticulata) and Kraussina rubra (Articulata) are described by transmission electron microscopy. The spermatozoon of D. tenuis has a similar morphology to that of other inarticulate brachiopods. The head (about 1.9 μm long) is composed of a nucleus which is invaginated anteriorly and posteriorly, the anterior invagination partially housing the pyriform acrosome. The acrosome is differentiated internally and is separated from the nucleus by subacrosomal material. The mid-piece is composed of a ring of four or five spherical mitochondria which surround the proximal and distal centrioles which are arranged orthogonally. By contrast the spermatozoon of K. rubra has a head which is 2 μm long × 1.1 μm in diameter. The nucleus which is rounded anteriorly is capped by a small (0.5 μm diameter) disc-shaped acrosome. The mid-piece has a single ring-shaped mitochondrion which surrounds the centrioles which are almost in line with ea...