Spermatological characteristics and effects of cryopreservation in Lebranche mullet spermatozoa (Mugil liza Valenciennes, 1836): First report of ultra-rapid freezing.

The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL-1 Trehalose dehydrate (n = 15). Two treatments - diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) - were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 109 sperm mL-1; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.

Biomechanical regulation of drug sensitivity in an engineered model of human tumor.

Predictive testing of anticancer drugs remains a challenge. Bioengineered systems, designed to mimic key aspects of the human tumor microenvironment, are now improving our understanding of cancer biology and facilitating clinical translation. We show that mechanical signals have major effects on cancer drug sensitivity, using a bioengineered model of human bone sarcoma. Ewing sarcoma (ES) cells were studied within a three-dimensional (3D) matrix in a bioreactor providing mechanical loadings. Mimicking bone-like mechanical signals within the 3D model, we rescued the ERK1/2-RUNX2 signaling pathways leading to drug resistance. By culturing patient-derived tumor cells in the model, we confirmed the effects of mechanical signals on cancer cell survival and drug sensitivity. Analyzing human microarray datasets, we showed that RUNX2 expression is linked to poor survival in ES patients. Mechanical loadings that activated signal transduction pathways promoted drug resistance, stressing the importance of introducing mechanobiological cues into preclinical tumor models for drug screening.

Vascularized Tissue-Engineered Model for Studying Drug Resistance in Neuroblastoma.

Neuroblastoma is a vascularized pediatric tumor derived from neural crest stem cells that displays vasculogenic mimicry and can express a number of stemness markers, such as SOX2 and NANOG. Tumor relapse is the major cause of succumbing to this disease, and properties attributed to cancer stem-like cells (CSLC), such as drug-resistance and cell plasticity, seem to be the key mechanisms. However, the lack of controllable models that recapitulate the features of human neuroblastoma limits our understanding of the process and impedes the development of new therapies. In response to these limitations, we engineered a perfusable, vascularized in vitro model of three-dimensional human neuroblastoma to study the effects of retinoid therapy on tumor vasculature and drug-resistance. METHODS: The in vitro model of neuroblastoma was generated using cell-sheet engineering and cultured in a perfusion bioreactor. Firstly, we stacked three cell sheets containing SKNBE(2) neuroblastoma cells and HUVEC. Then, a vascular bed made of fibrin, collagen I and HUVEC cells was placed onto a collagen-gel base with 8 microchannels. After gelling, the stacked cell sheets were placed on the vascular bed and cultured in the perfusion bioreactor (perfusion rate: 0.5 mL/min) for 4 days. Neuroblastoma models were treated with 10µM isotretionin in single daily doses for 5 days. RESULTS: The bioengineered model recapitulated vasculogenic mimicry (vessel-like structure formation and tumor-derived endothelial cells-TECs), and contained CSLC expressing SOX2 and NANOG. Treatment with Isotretinoin destabilized vascular networks but failed to target vasculogenic mimicry and augmented populations of CSLCs expressing high levels of SOX2. Our results suggest that CSLCs can transdifferentiate into drug resistant CD31+-TECs, and reveal the presence of an intermediate state STEC (stem tumor-derived endothelial cell) expressing both SOX2 and CD31. CONCLUSION: Our results reveal some roles of SOX2 in drug resistance and tumor relapse, and suggest that SOX2 could be a therapeutic target in neuroblastoma.

Mimicking biophysical stimuli within bone tumor microenvironment.

In vivo, cells reside in a complex environment regulating their fate and function. Most of this complexity is lacking in standard in vitro models, leading to readouts falling short of predicting the actual in vivo situation. The use of engineering tools, combined with deep biological knowledge, leads to the development and use of bioreactors providing biologically sound niches. Such bioreactors offer new tools for biological research, and are now also entering the field of cancer research. Here we present the development and validation of a modular bioreactor system providing: (i) high throughput analyses, (ii) a range of biological conditions, (iii) high degree of control, and (iv) application of physiological stimuli to the cultured samples. The bioreactor was used to engineer a three-dimensional (3D) tissue model of cancer, where the effects of mechanical stimulation on the tumor phenotype were evaluated. Mechanical stimuli applied to the engineered tumor model activated the mechanotransduction machinery and resulted in measurable changes of mRNA levels towards a more aggressive tumor phenotype.

Soluble vascular endothelial-cadherin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of ovarian stimulation, and the pathophysiological mechanisms that trigger the syndrome remain unknown. HCG increases serum vascular endothelial growth factor (VEGF) concentrations, and VEGF modulates transendothelial permeability via endothelial adherens junctions, a downstream target for VEGF signalling. We examined whether women with severe OHSS have altered serum levels of soluble vascular endothelial (sVE)-cadherin. METHODS: We conducted a prospective, case-control study of 28 women with severe OHSS and 34 women undergoing controlled ovarian hyperstimulation (COH) for IVF without developing OHSS. We collected serum samples from both groups on the day of ovum retrieval (Day 0), and on Days 3, 6, 9 and 15. Samples were assayed for sVE-cadherin by enzyme-linked immunosorbent assay. RESULTS: Women with severe OHSS had significantly higher levels of sVE-cadherin than patients without OHSS (P = 0.001). sVE-cadherin serum levels decreased with clinical improvement; however, they did not reach normal levels in the resolution phase. A positive correlation was demonstrated between sVE-cadherin and serum estradiol levels at the time of HCG administration (r = 0.621; P < 0.001). Serum sVE-cadherin levels were more closely chronologically correlated with corpus luteum function than with biological and clinical aspects of severe OHSS. CONCLUSIONS: sVE-cadherin may be involved in the pathogenesis of severe OHSS and may possibly serve as an indicator of corpus luteum function after COH.

Vascular endothelial cadherin regulates vascular permeability: Implications for ovarian hyperstimulation syndrome.

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. It is characterized by increased vascular permeability and simultaneous overexpression of vascular endothelial growth factor (VEGF) in ovarian cells. OBJECTIVE: We tested the hypothesis that the endothelium and endothelial cell-to-cell junctions are downstream targets of VEGF during OHSS pathogenesis. We investigated the potential involvement of vascular endothelial (VE)-cadherin, an interendothelial adhesion molecule, in the capillary hyperpermeability in OHSS. DESIGN: Human endothelial cells from umbilical veins (HUVEC) were used as an in vitro model of OHSS. INTERVENTION: Cell cultures were treated with varying doses of estradiol (E2), human chorionic gonadotropin (hCG), VEGF, and antihuman VEGF antibodies, either alone or in combination, and the effect on VE-cadherin release was evaluated at different time points. Permeability assays were performed using fluoresceinisothiocyanate-labeled albumin, and actin filaments rearrangement was evaluated by fluorescent microscopy. RESULTS: Culturing of HUVEC with high doses of E2 produced no significant changes in VE-cadherin concentration, but hCG and VEGF produced a significant increase in VE-cadherin release. Time-course experiments showed that VE-cadherin was secreted 12 h after VEGF addition. Antihuman VEGF antibodies prevented these changes. Permeability assays demonstrated that, although E2 did not alter the arrangement of HUVEC in vitro, hCG and VEGF caused changes in the actin fibers indicative of increased capillary permeability. VEGF also induced an increase in paracellular permeability of HUVEC at the same doses used in the previous experiments. CONCLUSIONS: Adhesion molecules like VE-cadherin may play a role in the development and progression of increased capillary permeability in severe OHSS.

Pericentromeric regions containing 1.688 satellite DNA sequences show anti-kinetochore antibody staining in prometaphase chromosomes of Drosophila melanogaster.

A striking characteristic of the centromeric heterochromatin of Drosophila melanogaster is that each chromosome carries different satellite DNA sequences. Here we show that while the major component of the 1.688 satellite DNA family expands across the centromere of the X chromosome the rest of the minor variants are located at pericentromeric positions in the large autosomes. Immunostaining of prometaphase chromosomes with the kinetocore-specific anti-BUB1 antibody reveals the transient presence of this centromeric protein in all the regions containing the 1.688 satellite.

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation.

Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability. By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences. Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time. In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes. The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process. The involvement of telomere-related sequences in centromere function is discussed.

Searching for a common centromeric structural motif: Drosophila centromeric satellite DNAs show propensity to form telomeric-like unusual DNA structures.

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophila centromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation.

The analysis of Circe, an LTR retrotransposon of Drosophila melanogaster, suggests that an insertion of non-LTR retrotransposons into LTR elements can create chimeric retroelements.

Circe is a transposable element recently identified in Drosophila melanogaster which appears to be mostly associated with the constitutive heterochromatin. This element shows the structural features of a long terminal repeat (LTR)-containing retrotransposon: It is flanked by 240-bp-long terminal repeats, and its two open reading frames encode putative proteins resembling the gag and pol polyproteins of retroviruses. However, Circe displays striking similarities of both LOA and Ulysses, a non-LTR element and an LTR element, respectively. The result of its phylogenetic and structural analysis has allowed us to propose a new mechanism for non-LTR retrotransposon evolution.

Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences.

Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster. We have identified a YAC clone that originated from this region. Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18. Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans. That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere. The existence of common determinants for the function of both centromeres and telomeres is discussed.

The 3' non-coding region of the Drosophila melanogaster HeT-A telomeric retrotransposon contains sequences with propensity to form G-quadruplex DNA.

HeT-A elements are non-long terminal repeat retrotransposons added onto the Drosophila chromosome ends. We have investigated the formation in vitro of higher order structures by oligonucleotides derived from the 3' non-coding region of HeT-A elements and found that they are capable of forming G-quadruplex DNA. These results suggest that the 3' repeat region of HeT-A may structurally behave as the telomeric repeats common to a majority of eukaryotes. The presence of structural motifs shared by telomeres and centromeres and the implications of these findings for chromosome evolution are discussed.

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3.

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retro-transposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed.

Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome.

The structural analysis of a yeast artificial chromosome clone from Drosophila melanogaster enriched in dodecasatellite sequences has led us to find a new retrotransposon that we have called Circe. Moreover, this retrotransposon has allowed the isolation of a contig encompassing approximately 200 kb near the centromere of the Y chromosome, providing an entry point into a region from which very little sequence information has been obtained to date. The molecular characterization of the contig has shown the presence of HeT-A telomeric retrotransposons close to the centromere of the Y chromosome, suggesting a telocentric origin for this submetacentric chromosome.

Autosomal location of a new subtype of 1.688 satellite DNA of Drosophila melanogaster.

During the screening of a Drosophila melanogaster YAC library with DNA from the minichromosome Dp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.

Centromeric dodeca-satellite DNA sequences form fold-back structures.

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G.A pairs as well as regular Watson-Crick G.C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75 degrees C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

The Drosophila melanogaster dodecasatellite sequence is closely linked to the centromere and can form connections between sister chromatids during mitosis.

We have used fluorescence in situ hybridisation to wild-type and rearranged mitotic chromosomes to map the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric heterochromatin of the right arm of the third chromosome, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as one or two dots throughout the cell cycle. This distribution can be altered in a cell cycle-dependent manner in two ways. Firstly, in interphase cells, hypotonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string-like structure. Secondly, some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space connecting the main dodecasatellite signals of each chromatid. The distribution of dodecasatellite seems to be rather constant between individuals of the same species, as indicated by the lack of any detectable variations in its pattern amongst individuals from six geographically distant strains of D. melanogaster. On the other hand, the distribution of dodecasatellite shows a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from a non-detectable signal in Drosophila yakuba and Drosophila teissieri, to staining in the X, second and third chromososomes of Drosophila mauritiana.

Dodeca satellite: a conserved G+C-rich satellite from the centromeric heterochromatin of Drosophila melanogaster.

To identify sequences from the centromeric region, we have constructed a Drosophila melanogaster yeast artificial chromosome (YAC) library and screened it with purified DNA from the minichromosome Dp(1;f)1187 derived from the X chromosome. We describe the structure of one clone isolated in this way. This YAC is structurally unstable and contains tandemly repeated G+C-rich 11-mer and 12-mer units, which we call dodeca satellite. Most of this satellite is located near the centromere of an autosome. Cross-hybridizing sequences are found in the genomes of organisms as distant as Arabidopsis thaliana and Homo sapiens.

Structure and polymorphism of human telomere-associated DNA.

We have analyzed the DNA sequences associated with four different human telomeres. Two are members of distinct repeated sequence families which are located mainly but not exclusively at telomeres. Two are unique in the genome, one deriving from the long arm telomere of chromosome 7 and the other from the pseudoautosomal telomere. One telomere-associated repeated sequence has a polymorphic distribution among the chromosome ends, being present at a different combination of ends in different individuals. These data thus identify a new source of human genetic variation and indicate that the canonical features of the organization of telomere-associated DNA are widely conserved in evolution.