Translocation Of Granules Research Articles

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils.

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

Rab44 regulates murine mast cell–driven anaphylaxis through kinesin-1–dependent secretory granule translocation

Mast cells (MCs) are key effectors of the allergic response. Following the cross-linking of IgE receptors (FcεRIs), they release crucial inflammatory mediators through degranulation. Although degranulation depends critically on secretory granule (SG) trafficking toward the plasma membrane, the molecular machinery underlying this transport has not been fully characterized. This study analyzed the function of Rab44, a large, atypical Rab guanosine triphosphatase highly expressed in MC, in the MC degranulation process. Murine knockout (KO) mouse models (KORab44 and DKOKif5b/Rab44) were used to perform passive cutaneous anaphylaxis experiments and analyze granule translocation in bone marrow-derived MCs during degranulation. This study demonstrate that mice lacking Rab44 (KORab44) in their bone marrow-derived MCs are impaired in their ability to translocate and degranulate SGs at the plasma membrane on FcεRI stimulation. Accordingly, KORab44 mice were less sensitive to IgE-mediated passive cutaneous anaphylaxis invivo. Alack of Rab44 did not impair early FcεRI-stimulated signaling pathways, microtubule reorganization, lipid mediator release, or cytokine secretion. Mechanistically, Rab44 appears to interact with and function as part of the previously described kinesin-1-dependent transport pathway. These results highlight a novel role of Rab44 as a regulator of SG transport during degranulation and anaphylaxis acting through the kinesin-1-dependent microtubule transport machinery. Rab44 can thus be considered a potential target for modulating MC degranulation and inhibiting IgE-mediated allergic reactions.

Glycine and Melatonin Improve Preimplantation Development of Porcine Oocytes Vitrified at the Germinal Vesicle Stage

Lipid-rich porcine oocytes are extremely sensitive to cryopreservation compared to other low-lipid oocytes. Vitrification has outperformed slowing freezing in oocyte cryopreservation and is expected to improve further by minimizing cellular osmotic and/or oxidative stresses. In this study, we compared the effects of loading porcine cumulus-oocyte complexes with glycine (an organic osmolyte) or glycine plus melatonin (an endogenous antioxidant) during vitrification, thawing and subsequent maturation to mitigate osmotic injuries or osmotic and oxidative damages on the developmental potential of porcine oocytes. Our data demonstrated that glycine treatment significantly increased the vitrification efficiency of porcine oocytes to levels comparable to those observed with glycine plus melatonin treatment. It was manifested as the thawed oocyte viability, oocyte nuclear maturation, contents of reactive oxygen species, translocation of cortical granules and apoptotic occurrence in mature oocytes, levels of ATP and transcripts of glycolytic genes in cumulus cells (markers of oocyte quality), oocyte fertilization and blastocyst development. However, the latter was more likely than the former to increase ATP contents and normal mitochondrial distribution in mature oocytes. Taken together, our results suggest that mitigating osmotic and oxidative stresses induced by vitrification and thawing can further enhance the developmental competency of vitrified porcine oocytes at the germinal vesicle stage.

NanoSIMS Imaging of Bioaccumulation and Subcellular Distribution of Manganese During Oyster Gametogenesis

Many bivalve mollusks display remarkable sex differentiation of gonadal accumulation of manganese (Mn), but the underlying processes responsible for such differences have seldom been explored. In this study, the accumulation of Mn in male and female gonads during the reproductive cycle of oysters was first examined, and the distributions of Mn in oocytes and sperm cells at different developmental stages were imaged by the nanoscale secondary ion mass spectrometry (NanoSIMS) at the subcellular level. We found that the distribution and accumulation of Mn during oogenesis were closely associated with the formation and translocation of cortical granules. This is the first time that the enrichment of Mn was directly visualized in cortical granules, which was identified as the major storage site of Mn in oocytes of oysters. Yolk granules were revealed as another storage pool of Mn in oyster oocytes with lower accumulation. In contrast, Mn was mainly distributed in the nucleus of sperm cells with accumulation levels much lower than those in cortical and yolk granules of oocytes. These results demonstrated great differences of the subcellular localization and accumulation capacity of Mn between oocytes and sperm cells in oysters, implying the sex differentiation in susceptibility of reproductive response to Mn stress. Our study also highlights the importance of gender difference in future biomonitoring and ecotoxicological studies of Mn in marine bivalves.

AMPing up killing by cytotoxic T cells

Immunology Cytotoxic T cells form immune synapses at the interface formed with their target cells and then reorient intracellular lytic granules toward the contact site to ensure efficient killing. Zurli et al. found that stimulation of the receptor CD2 on cytotoxic T cells by target B cells enhanced T cell receptor signaling during the formation of immune synapses. CD2 stimulation led to activation of the metabolism-regulating kinase adenosine monophosphate (AMP)–activated protein kinase on lysosomes, which promoted lytic granule translocation to the immune synapse. Boosting either of these factors may thus enhance cytotoxic T cell efficacy. Sci. Signal. 12 , eaaz1965 (2020).

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase.

Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued "in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17's role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

THE NEW LOOK AT NEUTROPHILIC GRANULOCYTES: RETHINKING OLD DOGMAS. PART 2

Numerous modern basic research done undeniable fact that neutrophilic granulocytes (NG) are key effector and regulatory circuits both innate and adaptive immunity, and play a crucial role in the pathogenesis of a wide range of diseases. NG have potent receptor repertoire, providing a connection between them, cells of the immune system, as well as communication with endothelial cells, epithelial and other tissues. NG inducing stimuli activate and promote the translocation of cytoplasmic granules and vesicles surface molecules on the cytoplasmic membrane the secretion of a large spectrum of pro-and anti-inflammatory, immunoregulatory cytokines, colony, angiogenic factors and fibrogenic, TNF superfamily members, chemokines, regulatory protein, etc. Chromatin nuclei NG capable of restructuring under the influence of inducing stimuli, which is associated with the expression of multiple cytokine genes. NG receiving complex cytokine influence not only acquire new features, but also in various stages of activation and differentiation processes involved in intracellular intraphagosomalis degranulation and killing of implementing elimination microorganisms and extracellular neutrophil degranulation in the formation neutrophil extracellular traps (NET), while this dying through NETosis. Features NG phenotype and their functional properties, demonstrate the existence of subpopulations of NG with different capabilities: equipment of different receptor, the ability to restructure chromatin expressing cytokine genes and secrete cytokines to implement the contents of the granular system, produce reactive oxygen species, implement cytotoxicity form NET. In our opinion, there subpopulation NG: regulatory; suppressor; proinflammatory — initiating an inflammatory response; inflammation with a positive potential microbicidal (antibacterial, antiviral, antifungal); inflammatory cytotoxic potential of the negative — «aggressive»; anti-inflammation regulating regression; antitumoral — TAN1; pro-tumoral — TAN2; hybrid, combining the characteristics of NG and dendritic cells. The absence of adequate response, or hyperactivation blockade NG functions leads to the development of low-intensity infectious and inflammatory diseases, do not respond to conventional therapy of autoimmune diseases/chronic immune-dependent processes. Remo deling dysfunctions NG — the key to new immunotherapeutic strategies.

THE NEW LOOK AT NEUTROPHILIC GRANULOCYTES: RETHINKING OLD DOGMAS. PART 1

Numerous modern basic research done undeniable fact that neutrophilic granulocytes (NG) are key effector and regulatory circuits both innate and adaptive immunity, and play a crucial role in the pathogenesis of a wide range of diseases. NG have potent receptor repertoire, providing a connection between them, cells of the immune system, as well as communication with endothelial cells, epithelial and other tissues. NG inducing stimuli activate and promote the translocation of cytoplasmic granules and vesicles surface molecules on the cytoplasmic membrane the secretion of a large spectrum of pro-and anti-inf lammatory, immunoregulatory cytokines, colony, angiogenic factors and fibrogenic, TNF superfamily members, chemokines, regulatory protein, etc. Chromatin nuclei NG capable of restructuring under the influence of inducing stimuli, which is associated with the expression of multiple cytokine genes. NG receiving complex cytokine inf luence not only acquire new features, but also in various stages of activation and differentiation processes involved in intracellular intraphagosomalis degranulation and killing of implementing elimination microorganisms and extracellular neutrophil degranulation in the formation neutrophil extracellular traps (NET), while this dying through NETosis. Features NG phenotype and their functional properties, demonstrate the existence of subpopulations of NG with different capabilities: equipment of different receptor, the ability to restructure chromatin expressing cytokine genes and secrete cytokines to implement the contents of the granular system, produce reactive oxygen species, implement cytotoxicity form NET. In our opinion, there subpopulation NG: regulatory; suppressor; proinf lammatory — initiating an inf lammatory response; inf lammation with a positive potential microbicidal (antibacterial, antiviral, antifungal); inf lammatory cytotoxic potential of the negative — “aggressive”; anti-inf lammation regulating regression; antitumoral — TAN1; pro-tumoral — TAN2; hybrid, combining the characteristics of NG and dendritic cells. The absence of adequate response, or hyperactivation blockade NG functions leads to the development of low-intensity infectious and inf lammatory diseases, do not respond to conventional therapy of autoimmune diseases/chronic immune-dependent processes. Remodeling dysfunctions NG — the key to new immunotherapeutic strategies.

The Regulatory Roles of Mitogen-Activated Protein Kinase (MAPK) Pathways in Health and Diabetes: Lessons Learned from the Pancreatic β-Cell.

Glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cell involves several intracellular metabolic events which lead to the translocation of insulin granules towards the membrane for fusion and release. It is well established that loss of β-cell function and decreased GSIS underlie the pathogenesis of diabetes. Evidence from several laboratories, including our own, demonstrated requisite roles of Rac1 and phagocyte-like NADPH oxidase (Nox2)-derived reactive oxygen species (ROS) in optimal function of the pancreatic β-cell, including GSIS. However, it is becoming increasingly clear that prolonged exposure of β-cells to hyperglycemic conditions, leads to sustained activation of Rac1-Nox2 signaling axis culminating in excessive generation of intracellular ROS (oxidative stress) and β-cell dysregulation and demise. Such "cytotoxic" effects of ROS appear to be mediated via the stress-activated protein kinases/mitogen-activated protein kinases (SAPK/MAPK) signaling pathways. This review discusses our current understanding of regulation and functions of the conventional MAPKs, namely, ERK1/2, JNK1/2 and p38MAPK. The MAPK pathways are activated in the presence of various stress stimuli including intracellular ROS, via distinct signaling cascades. Once activated, MAPKs participate in specific intracellular signaling processes via interaction with several downstream kinases including the MAPKactivated protein kinases (MAPKAPKs) and transcription factors including c-jun and p53. We have provided an overview of existing evidence in the islet β-cell on the regulatory roles of these MAPKs in mediating cellular responses to alterations in intracellularly generated ROS, which is mediated by the Rac1-Nox2 signaling module. Additionally, we enlisted recent patents developed to improve β-cell function in diabetes and novel pharmacological agents that target oxidative stress and MAPK pathways.

Components of RNA granules affect their localization and dynamics in neuronal dendrites.

In neurons, RNA transport is important for local protein synthesis. mRNAs are transported along dendrites as large RNA granules. The localization and dynamics of Puralpha and Staufen1 (Stau1), major components of RNA transport granules, were investigated in cultured hippocampal neurons. Puralpha-positive granules were localized in both the shafts and spines of dendrites. In contrast, Stau1-positive granules tended to be localized mainly in dendritic shafts. More than 90% of Puralpha-positive granules were positive for Stau1 in immature dendrites, while only half were positive in mature dendrites. Stau1-negative Puralpha granules tended to be stationary with fewer anterograde and retrograde movements than Stau1-positive Puralpha granules. After metabotropic glutamate receptor 5 activation, Stau1-positive granules remained in the dendritic shafts, while Puralpha granules translocated from the shaft to the spine. The translocation of Puralpha granules was dependent on myosin Va, an actin-based molecular motor protein. Collectively our findings suggest the possibility that the loss of Stau1 in Puralpha-positive RNA granules might promote their activity-dependent translocation into dendritic spines, which could underlie the regulation of protein synthesis in synapses.

A flavanone derivative from the Asian medicinal herb (Perilla frutescens) potently suppresses IgE-mediated immediate hypersensitivity reactions

Perilla frutescens is a dietary leafy herb consumed as a traditional Japanese condiment as well as used for Chinese medicine with anti-inflammatory activity. Here we report a hitherto-unrecognized P. frutescens phytochemical that potently suppresses IgE-mediated type I hypersensitivity reactions. Structural analysis reveals that the purified anti-allergic compound (Perilla-derived methoxyflavanone, PDMF) is identified as 8-hydroxy-5,7-dimethoxyflavanone. PDMF significantly inhibits IgE-mediated histamine release from RBL-2H3 rat basophilic leukemia cells as compared with those seen in known P. frutescens-derived anti-inflammatory polyphenols. We also show that oral administration of PDMF not only suppresses passive cutaneous anaphylaxis, but also prevents allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis. Mechanistically, PDMF negatively regulates Akt phosphorylation and intracellular Ca2+ influx, both of which are essential for mast cell secretory granule translocation and its exocytosis upon high-affinity IgE receptor (FcεRI) cross-linking. These results represent PDMF as a new potent anti-allergic phytochemical useful for prevention of IgE-driven hypersensitivity reactions.

Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes.

An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems.

Chinese herbs have long been used to treat allergic disease, but recently the development was greatly impeded by the lack of good methods to explore the mechanism of action. Here, we showed the effects of Chinese herb Radix Paeoniae alba were identified and characterized by a mast cell activation assay that involves electronic impedance readouts for dynamic monitoring of cellular responses to produce time-dependent cell responding profiles (TCRPs), and the anti-allergic activities were further confirmed with various conventional molecular and cell biology tools. We found Radix P. alba can dose-dependently inhibit TCPRs, and have anti-allergic function in vitro and in vivo. Radix P. alba suppressed mast cell degranulation not only inhibiting the translocation of granules to the plasma membrane, but also blocking membrane fusion and exocytosis; and that there may be other anti-allergic components in addition to paeoniflorin. Our results suggest that Radix P. alba regulated mast cell activation with multiple targets, and this approach is also suitable for discovering other mast cell degranulation-targeting Chinese herbs and their potential multi-target mechanisms.

Post-ovulatory aging of mouse oocytes in vivo and in vitro: Effects of caffeine on exocytosis and translocation of cortical granules.

The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150 mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6 h, 12 h and 18 h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10 mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6 h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18 h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Cellular and molecular mechanisms of heat stress related to bovine ovarian function.

In light of the intensive genetic selection for high milk production and the onset of global warming, it seems that the reduced fertility of lactating cows during the summer will worsen in coming years. Although not entirely clear, the mechanism appears to be multifactorial in nature. It includes alterations in follicular development, depression of follicular dominance, and impairment of steroidogenesis and gonadotropin secretion. Heat-induced perturbations in the physiology of the follicle-enclosed oocyte have also been documented, expressed by impaired cleavage rate and reduced developmental competence. With respect to the oocyte, alterations include an increase in PUFA in the membrane, reactive oxygen species, ceramide formation and caspase activity, and induction of apoptosis via the sphingomyelin and/or mitochondrial pathways. New insight into cellular and molecular alterations has revealed that heat induces perturbations in both nuclear and cytoplasmic maturation events, such as resumption of meiosis, metaphase II plate formation, cytoskeleton rearrangement, and translocation of cortical granules. Alterations in mitochondrial distribution (i.e., low proportion of category I mitochondria) and function (i.e., low membrane potential) have recently been reported for oocytes collected during the summer. These were associated with impaired expression of both nuclear (succinate dehydrogenase subunit [SDHD], adenosine triphosphate [ATP] synthase subunit beta [ATP5B]), mitochondrially NADH dehydrogenase subunit 2 (ND2), and mitochondiral (cytochrome c oxidase subunit II [MT-CO2] and cytochrome b [MT-CYB]) genes that are crucial in the mitochondrial respiratory chain. In addition, season-induced alteration in the stored maternal mRNA has been documented, expressed by reduced transcript levels (oocyte maturation factor MOS [C-MOS], growth differentiation factor 9 [GDF9], POU domain class 5 transcription factor 1 [POU5F1], and glyceraldehyde-3-phosphate dehydrogenase []GAPDH) in metaphase II stage oocytes and embryos before (i.e., 2-, 4-, and 8-cell stages) and after (i.e., 8- to 16-cell stage) embryonic genome activation. Taken together, the findings indicate an association between cellular and molecular modifications and reduced developmental competence during the hot seasons. Such knowledge is essential for the development of new approaches to cope with this unsolved problem.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): Functional roles for the cytoskeleton and its molecular motors

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca2+/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

IGF-I slightly improves nuclear maturation and cleavage rate of bovine oocytes exposed to acute heat shock in vitro.

An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus-oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.