All-trans retinoic acid induces free radical generation and modulate antioxidant enzyme activities in rat sertoli cells

In this work we investigated the effects of retinoic acid (RA) in Sertoli cells. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with RA for 24 h. RA at high doses (1–10 μM) increased TBARS levels and induced a decrease in cell viability. At low doses (0.1–100 nM) RA did not increase TBARS level. RA also did not increase cell death at these doses. In order to investigate changes in antioxidant defenses we measured the CAT, SOD and GPx activities in Sertoli cells treated with RA. Compared to control, RA increased around 200% SOD activity in all doses tested (0.1–100 nM); GPx activity was increased 407.49, 208.98 and 243.88% (0.1, 1 and 10 nM, respectively); CAT activity was increased 127% with RA 1 nM. To clarify if RA induces ROS production per se, we performed experiments in vitro using 2-deoxyribose as specific substrate of oxidative degradation by ^•OH radical as well as TRAP assay. RA at 10 μM increased 2-deoxyribose degradation, suggesting that some of the RA-induced effects are mediated via ^•OH formation. Furthermore, the total reactive antioxidant potential (TRAP) of the RA was determined. At low concentrations RA has induced no redox activity. Conversely, higher concentration of RA (1–10 μM) increased chemiluminescence. The chemiluminescence produced was directly proportional to radical generation. We provide, for the first time, evidence for a free radical generation by RA. Our results demonstrated that RA plays an important role in Sertoli cells and these effects appear to be mediated by ROS.     

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence

The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.