Biomedical subjects

H Schatten

Publications and source records attributed to H Schatten.

At least 19 recordsLinked to original sources

PubMed2001

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals.

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Animals↗

PubMed2001

Cytoplasmic changes in relation to nuclear maturation and early embryo developmental potential of porcine oocytes: effects of gonadotropins, cumulus cells, follicular size, and protein synthesis inhibition.

Morphological and biochemical changes indicative of cytoplasmic maturation in relation to nuclear maturation progression and early embryo developmental potential was studied. Fluorescently labeled microfilaments and cortical granules were visualized by using laser scanning confocal microscopy. The mitogen-activated protein (MAP) kinase phosphorylation and cyclin B1 levels were revealed by Western blot. With the maturation of oocytes, cortical granules and microfilaments were localized at the cell cortex. A cortical granule-free domain (CGFD) and an actin-thickening area were observed over both the MII spindle of a mature oocyte and chromosomes of a nocodazole-treated oocyte, suggesting that chromosomes, but not the spindle, determined the localization of CGFD and actin-thickening area. In oocytes that are incompetent to resume meiosis, as indicated by the failure of germinal vesicle breakdown (GVBD), peripheral localization of cortical granules and microfilaments, phosphorylation of MAP kinase and synthesis of cyclin B1 did not occur after 44 hr in vitro. These cytoplasmic changes were also blocked when GVBD of meiotically competent oocytes was inhibited by cycloheximide. Culture of oocytes in a chemically defined medium showed that biological factors such as gonadotropins, cumulus cells and follicle size affected both nuclear and cytoplasmic maturation as well as embryo developmental potential. Absence of gonadotropins or removal of cumulus cells alone did not significantly influence GVBD or cyclin B1 levels, but decreased the final maturation and developmental ability of oocytes. A combination of gonadotropin absence and cumulus removal decreased GVBD, MAP kinase phosphorylation and embryo development. A high proportion of oocytes derived from small follicles were able to resume meiosis, synthesize cyclin B(1), phosphorylate MAP kinase and translocate CGs, but their maturation and embryo developmental ability were limited. Removal of cumulus cells from small follicle-derived oocytes severely affected their ability to undergo cytoplasmic and nuclear maturation.

Actin Cytoskeleton↗

PubMed2001

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy.

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.

Antibodies, Monoclonal↗

PubMed2001

Phosphorylation of p90rsk during meiotic maturation and parthenogenetic activation of rat oocytes: correlation with MAP kinases.

This paper reports on the activation of p90rsk during meiotic maturation and the inactivation of p90rsk after electrical parthenogenetic activation of rat oocytes. In addition, the correlation between p90rsk and MAP kinases after different treatments was studied. We assessed p90rsk activity by examining its electrophoretic mobility shift on SDS-PAGE and evaluated ERK1+2 activity by both mobility shift and a specific antibody against phospho-MAP kinase. The phosphorylation of p90rsk during rat oocyte maturation was a sequential process that may be divided into two stages: the first stage was partial phosphorylation, which was irrelevant with MAP kinases because p90rsk phosphorylation took place prior to activation of MAP kinases. The second stage inferred full activation occurred at the time when MAP kinases began to be activated (3 h after germinal visicle breakdown). Evidence for the involvement of MAP kinases in the p90rsk phosphorylation was further obtained by the following approaches: (1) okadaic acid (OA) accelerated the phosphorylation of both MAP kinases and p90rsk; (2) OA induced phosphorylation of both MAP kinases and p90rsk in the presence of IBMX; (3) when activation of MAP kinases was inhibited by cycloheximide, p90rsk phosphorylation was also abolished; (4) dephosphorylation of p90rsk began to take place at 3 h post-activation, temporally correlated with the completion of MAP kinase inactivation; (5) phosphorylation of both kinases was maintained in oocytes that failed to form pronuclei after stimulation; (6) OA abolished the dephosphorylation of both kinases after parthenogenetic activation. Our data suggest that MAP kinases are not required for early partial activation of p90rsk but are required for full activation of p90rsk during rat oocyte maturation, and that p90rsk dephosphorylation occurs following MAP kinase inactivation after parthenogenetic activation of rat oocytes.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Dynamic events are differently mediated by microfilaments, microtubules, and mitogen-activated protein kinase during porcine oocyte maturation and fertilization in vitro.

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

Actin Cytoskeleton↗

PubMed2001

Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3',5'-adenosine monophosphate, and protein phosphatase modulators during meiosis resumption in rat oocytes.

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.

Animals↗

PubMed2001

Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro.

The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

Actin Cytoskeleton↗

PubMed2000

Introduction

Explore the source record for details and available documents.

Journal Article↗

PubMed2000

From fertilization to cancer: the role of centrosomes in the union and separation of genomic material.

Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.

Animals↗

PubMed2000

Centrosome alterations induced by formamide cause abnormal spindle pole formations.

The formation of the bipolar mitotic apparatus depends on accurate centrosome organization which is crucial for the separation of the genome during cell division. While it has been shown that mutations and overexpression of centrosome proteins (Brinkley and Goepfert, 1998; Pihan et al., 1998) can cause abnormal spindle pole formation, here we report that damages to centrosome structure caused by the chaotropic agent formamide will cause multipolar mitoses upon recovery from the effect when applied at first cell division in sea urchin eggs. Formamide was used as a chemical tool to manipulate centrosome structure and to investigate the effects on microtubule organization. When 1-1.5 m formamide was administered for 30 min at prometaphase of first cell division, microtubules were disassembled and centrosomes compacted into dense spheres around highly condensed chromatin. Upon recovery from formamide, centrosomes decompacted and attempted to form various mitotic organizations. Normal recovery (and attempts of recovery) to bipolarity was possible in five percent of cells treated with 1-1.5 m formamide for 30 min, but abnormal patterns of spindle formation were observed in all other cells, which included mono- (20%), tri (45%), and multipolar (30%) formations organized by mono-, tri-, and multipolar centrosome clusters. When cells were treated with 1.5 m formamide for 90 min, centrosomes became pulverized and fragmented and only monopolar mitotic formations were observed upon recovery. These results are highly reproducible and reveal that abnormalities in centrosome structure can lead to abnormal mitosis which is not caused by mutation or overexpression of centrosome proteins.

Animals↗

PubMed2000

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model.

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.

Adenocarcinoma↗

PubMed2000

Antioxidants stimulate meiosis resumption, but inhibit mitogen-activated protein kinase phosphorylation and further cell cycle progression in porcine oocytes.

In the present study the effects of two cell-permeant antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behaviour and spindle assembly were investigated. The antioxidants BHA and NDGA stimulated meiosis resumption in a dose-dependent manner in both cumulus-enclosed and denuded porcine oocytes. After in vitro culture for 8 h, few oocytes underwent germinal vesicle breakdown (GVBD) in control groups, whereas GVBD occurred in high percentages of oocytes treated with BHA or NDGA at concentrations that inhibit GVBD in rodent oocytes, although mitogen-activated protein (MAP) kinase was not phosphorylated as revealed by Western immunoblots. Orcein staining and fluorescein isothiocyanate-anti-alpha-tubulin labelling showed that chromosome and spindle formation, respectively, and further meiosis progression were inhibited 20 and 25 h after culture. Instead, chromatin was highly condensed or existed in scattered condensed clusters. Correspondingly, MAP kinase phosphorylation was inhibited by both BHA and NDGA in a dose-dependent manner. The inhibitory effects of BHA on meiosis completion and MAP kinase phosphorylation was reversible. These results suggest that, unlike in rodent oocytes, antioxidants stimulate GVBD in the absence of MAP kinase activation, but inhibit MAP kinase phosphorylation, meiotic apparatus formation and thus the further progression of the meiosis of porcine oocytes.

Animals↗

PubMed2000

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells.

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.

Androgens↗

PubMed1999

Centrosome and microtubule instability in aging Drosophila cells.

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

Animals↗

PubMed1999

Activation of Sp1 and its functional co-operation with serum amyloid A-activating sequence binding factor in synoviocyte cells trigger synergistic action of interleukin-1 and interleukin-6 in serum amyloid A gene expression.

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between -254 and -226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6. The role of Sp1 in SAA induction was demonstrated by increased DNA binding activity, phosphorylation, and increased protein content of Sp1 during cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF. Sp1 heteromeric complex. Furthermore, using a phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in regulating SAA expression by acting in conjunction with SAF.

Animals↗

PubMed1999

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus.

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.

Animals↗

PubMed1999

Role of the MAPK cascade in mammalian germ cells.

The mitogen-activated protein kinase (MAPK) cascade is one of the most important of the intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation and cell cycle regulation. Since the first report in 1993 of MAPK's involvement in the functional regulation of mammalian oocytes, much work has been done on the role of the MAPK cascade in germ cells in different species of mammals. This review describes the possible involvement of the MOS/MEK/MAPK/RSK cascade in spermatogenesis, sperm function, oocyte meiotic re-initiation, spindle assembly, metaphase II arrest, pronuclear formation and the entry of first mitosis, as well as the cross-talk of this cascade to maturation-promoting factor (MPF) and other signal molecules in mammals.

Animals↗

PubMed1999

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.

Antimycin A↗