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Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at -196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

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Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

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A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

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Circadian rhythms are oscillations of physiological functions. The period of their oscillation is about 24 h, and can be synchronized by environmental periodic signals as night-day cycle. The endogenous periodical changes depend on various structural elements of the circadian system which consists of the effectors, the secondary oscillators, the synchronizers and the circadian pacemaker. In mammalian species, the physiological function better understood respect their oscillation pattern are the synthesis and release of several hormones (i.e. cortisol and melatonin), the body temperature, the sleep-awake cycle, the locomotive activity, cell proliferation, neuronal activity among other rhythms. The Suprachiasmatic nucleus is the main circadian pacemarker in mammals; its oscillation keeps the circadian system synchronized particularly with respect to the environment photo period. When light reaches the pigment melanopsin in ganglionar neurons in the retina, the photoperiod signal is sent to Suprachiasmatic nucleus, and its postsinaptic neurons distributes the temporal signal to pheripheral oscillators by nervous or humoral pathways. Among the oscillators, the pineal gland is a peripheral one modulated by Suprachiasmatic nucleus. At night, the indolamine melatonin is synthesized and released from pinealocytes, and reaches other peripheral oscillators. Melatonin interacts with membrane receptors on Suprachiasmatic nucleus pacemarker neurons, reinforcing the signal of the photoperiod. In mammals, exogenous melatonin synchronizes several circadian rhythms including locomotive activity and melatonin release. When this indolamine is applied directly into the Suprachiasmatic nucleus, it produces a phase advance of the endogenous melatonin peak and increases the amplitude of the oscillation. In humans, melatonin effect on the circadian system is evident because it changes the circadian rhythms phase in subjects with advanced sleep-phase syndrome, night workers or blind people. Also it reduces jet lag symptoms enhancing sleep quality and reseting the circadian system to local time. Melatonin effects on circadian rhythms indicate their role as a chronobiotic, since decreased daily melatonin levels that occur with age and in neuropsychiatric disorders are associated with disturbances in the sleep-awake cycle. In particular, it has been described that Alzheimer's disease patients have disturbed sleep-awake cycle and have decreased serum melatonin levels. Sleep disorders in Alzheimer's disease patients decrease when they are treated with melatonin. Moreover, sleep disturbances have been observed in bipolar disorder patients and often precede relapses of insomnia-associated mania and hypersomnia-associated depression. These disturbances are linked to delayed- and advanced- phases of circadian rhythms or arrhythmia; therefore, it has been suggested that bipolar disorder patients could be treated with light and dark therapy. In depressed patients, the levels of melatonin are low throughout the 24 hour period and have a delayed onset of the indolamine concentration and showed an advance of its peak. Schizophrenic patients have decreased levels in the plasmatic melatonin in both phases of the light-dark cycle. Melatonin administration to these patients increases their sleep efficiency. In addition, melatonin acts as a neuroprotector because of its potent antioxidant action and through its cytoskeletal modulation properties. In neurodegenerative animal models, its protector effect has been observed using okadaic acid. This neurotoxin is employed for reproducing cytoskeletal damage in neurons and increased oxidative stress levels, which are molecular events similar to those that occur in Alzheimer's disease. In N1E-115 cell cultures incubated with okadaic acid, the administration of melatonin diminishes hyperphosphorylated tau and oxidative stress levels, and prevents the neurocytoskeletal damage caused by the neurotoxin. Although it is known that melatonin plays a key role in the circadian rhythms entrainment, little is known about its synchronizing effects at molecular and structural level. In algae, it has been observed a link between morphological changes and the light-dark cycle and it is known that shape is determinated by the cytoskeletal structure. In particular, the alga Euglena gracilis changes its shape two times per day under the effect of a daily light-dark cycle. This alga has a long shape when there is a higher photosynthetic capacity at the half period of the day; on the contrary, it showed a rounded shape at the end of 24 h cycle. Also, the influence of the cell shape changes on the photosynthetic reactions was investigated by altering them with drugs that disrupt the cytoskeletal structure as cytochalasin B and colchicine. Both inhibitors blocked the rhythmic shape changes and the photo-synthetic rhythm. Moreover, there are some reports about cytoskeletal changes in plants targeted by circadian rhythms. Guarda cells of Vicia faba L. showed a diurnal cycle on the alpha and beta tubulin levels. In addition, it has been proposed that melatonin synchronizes different body rhythms through cytoskeletal rearrangements. In culture cells, nanomolar melatonin concentrations cause an increase in both the polimerization rate and microtubule formation through calmodulin antagonism. A cyclic pattern produced by melatonin in the actin microfilament organization has been demonstrated in canine kidney cells. Cyclic incubation of MDCK cells with nanomolar concentrations of melatonin, resembling the cyclic pattern of secretion and release to plasma produces a microfilament reorganization and the formation of domes. Studies in animals are controvertial regarding if the amount of microtubules in different tissues varies cyclically. In rats and baboons, melatonin administration or exposure of rats to darkness induced an increased number of microtubules in the pineal gland. However, in the hypothalamus, the exposure of rats to light resulted in an increase in the microtubular protein content. Similarly, (X-tubulin mRNA was augmented during the light phase in the hypothalamus, hippocampus and cortex. By contrast, in rats maintained in constant darkness, a decreased level in the tubulin content was observed in the visual cortex. Additional information on cycle variations observed in cytoskeletal molecules indicated that beta actin mRNA levels are lower during the day in the hippocampus and cortex. But no change was observed in actin protein levels in the cerebral cortex. However, increased levels of actin and its mRNA were observed in the hypothalamus. Exogenous melatonin administration at onset of night decreased the amount of actin in the hypothalamus, while the actin mRNA levels decreased when the administration was realized in the morning. In this review we will describe the synchronizer role of melatonin in the sleep-awake cycle and in the organization of cytoskeletal proteins and their mRNAs. Also, we will describe alterations in the melatonin secretion rhythm associated with a neuronal cytoskeleton disorganization in the neuropsychiatric diseases such as Alzheimer, depression, bipolar disorder and schizophrenia.

Los ritmos circadianos son patrones de oscilación con un periodo cercano a 24h que se observan en los procesos fisiológicos. En los mamíferos se han descrito funciones biológicas con regulación circádica tal como el ciclo sueño-vigilia. La administración de la melatonina, una indolamina secretada por la glándula pineal, sincroniza los ritmos circadianos. En los humanos, este efecto se ha estudiado en sujetos con síndrome de <

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Introdução: A qualidade da conservação obtida em um órgão e a duração do armazenamento seguro são dependentes do tipo de solução e tempo de conservação, estando sujeitas a intercorrências imprevisíveis. Em virtude disso, o parênquima renal e, particularmente, o túbulo contornado proximal (TCP), local onde ocorre necrose tubular aguda (NTA), são susceptíveis às mudanças na homeostase funcional do órgão, durante a conservação e durante a reperfusão pós-implante. O TCP reabsorve cerca de 70% de Na+ e H2O filtrados; sua membrana apical tem células especializadas com microvilosidades para aumentar a área de transporte unidirecional de solutos do lúmen para o sangue. O principal componente estrutural da bordadura em escova, filamentos de F-actina, interage com uma variedade de proteínas transmembranas, inclusive moléculas transportadoras de íons. Esta função é regulada por parâmetros fisiológicos e hormonais, sendo a Angiotensina II (AII) uma das principais envolvidas com o citoesqueleto de actina e proteínas associadas, mediada principalmente por seus receptores AT1. Métodos: Neste trabalho, túbulos isolados de fragmentos frescos e de fragmentos conservados por períodos de 1 e 24h nas soluções de Euro - Collins (ECO) e de Belzer (UW) foram tratados com AII, losartan e AII+losartan, para medida do volume de absorção de fluido (Jv=nl. min_1. mm-1 (microperfusão in vitro), e medida da intensidade (média) de pixel da fluorescência dos receptores AT1 e do citoesqueleto de actina (imunoistoquímica). Resultados: a) Demonstrou-se que AII (10-12M) estimula absorção fluida (Jv), que é diminuída com losartan (10-6M) em TCP frescos e também em TCP conservados em soluções...

Introduction: Depending on the preservation quality and duration, considering the solution composition and the preservation method, unexpected intercurrences may occur. In consequence, the renal parenchyma and particularly the proximal convoluted tubule (PCT), site where the acute tubular necrosis (NTA) occurs, are susceptible to changes in the organ functional homeostasis during preservation in the post implantation reperfusion period. The PCT reabsorbs approximately 70% of filtered Na+ and H2O, and its apical membrane has specialized cells with microvilli to increase the area of unidirectional solute transport from the lumen to the blood. The major structural component of brush border microvilli is the filamentous F-actin, which has been shown to interact with a variety of transmembrane proteins, including ion transport molecules. This function is regulated by physiological and hormonal parameters, such as angiotensin II (AII) interacting with actin cytoskeleton and associated proteins, mediated mainly through type I receptors (AT1R). After via different signaling pathways having been triggered, the varied functions are started, including fluid absorption (Jv), which is directly related to the actin cytoskeleton. Methods: Fresh (no preserved) and tubules preserved for 1h and 24hrs in Euro-Collins (EC) and Belzer (UW) solutions, treated with AII, losartan and AII+losartan, evaluated by "in vitro" microperfusion technique (fluid absorption (Jv=nl. min-1. mm-1), and by immunohistochemical technique (AT1 receptor measurement and actin cytoskeleton). Results: a) Our results showed that AII (10-12M) (physiological concentration) stimulates...

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The adhesive organs or “duo-gland adhesive organs” of platyhelminths are formed by a specialized epithelialcell and extensions of two gland cells. These organs are used for temporary fixation of the organism tosurfaces in aquatic habitats. The mechanisms involved in adhesion to and release from a given surfacedepend on secretions produced by the glands; less is known about the involvement of cytoplasmic filamentsin the anchoring cell itself. In this study, we examined the structure of the adhesive organs present in thetail plate of Macrostomum tuba Graff, 1882 (Platyhelminthes, Macrostomida), a freshwater, free-livingflatworm. Scanning and transmission electron microscopy allowed elucidation of the three-dimensionalorganization of the adhesive system, especially of the microvilli that formed the outer collar (or papilla),which was endowed with a fibrous core. Electrical stimulation caused the flatworms to extend their papillaeabove the ciliated surface. The use of tannin- and diamine-containing fixatives showed that the filamentousarray contained tonofilaments and actin filaments. Tonofilaments concentrate in the axis of each microvillus;actin filaments, about 7-8 nm thick, spread out towards the periphery. Scanning images demonstrated thefinger-like shape of the papillae, about 7-8 ìm high, with a terminal opening. Microvilli followed a straightcourse along the surface.

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