Pre-implantation mouse embryos cultured In vitro under different oxygen concentrations show altered ultrastructures

Abstract Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes

Purpose: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Methods: Samples were studied by light and transmission electron microscopy. Results: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. Conclusions: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism

Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner

Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides

Facility for validating technologies for the autonomous space rendezvous and docking to uncooperative targets

We present the latest advancements in the air-bearing facility installed at La Sapienza’s GN Lab in the School of Aerospace Engineering. This facility has been utilized in recent times to validate robust control laws for simultaneous attitude control and vibration active damping. The instrumentation and testbed have been restructured and enhanced to enable simulations of close proximity operations. Relative pose determination, accomplished through visual navigation as either an auxiliary or standalone system, is the first building block. Leveraging the acquired knowledge, optimal guidance and control algorithms can be tested for contactless operations (e.g. on-orbit inspection), as well as berthing and docking tasks

Methylglyoxal-dependent glycative stress and deregulation of SIRT1 functional network in the ovary of PCOS mice

Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEA ovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1 alpha), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response

on the fluorine nucleosynthesis in agb stars in the light of the 19f p α 16o and 19f α p 22ne reaction rate measured via thm

In the last years the [Formula: see text]O and the [Formula: see text]F([Formula: see text],p)[Formula: see text]Ne reactions have been studied via the Trojan Horse Method in the energy range of interest for astrophysics. These are the first experimental data available for the main channels of [Formula: see text]F destruction that entirely cover the energy regions typical of the stellar H- and He- burning. In both cases the reaction rates are significantly larger than the previous estimations available in the literature. We present here a re-analysis of the fluorine nucleosynthesis in Asymptotic Giant Branch stars by employing in state-of-the-art models of stellar nucleosynthesis the THM reaction rates for [Formula: see text]F destruction

Effects of the pesticide lindane on granulosa cell ultrastructure

The excessive exposure to pesticides in the Aral Sea area was correlated to the increased reproductive pathologies in those regions. One of the principal chemical employed was the gamma-hexachlorocycloexane herbicide Lindane (L), a persistent organochlorine that may induces alterations in granulosa cell (GCs) survival. However, a comprehensive experimental study on the L-induced dose-effect morphological alterations, has not yet addressed. Therefore, we studied by means of transmission and scanning electron microscopy, the morphological changes of mouse GCs, matured in vitro with increasing concentrations of L. GCs showed several dose-dependent changes, in respect to controls. In particular, we observed significant reduction of GC microvilli and decrease of cytoplasmic processes between adjacent GCs. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants and cellular debris were also present. Mitochondria, endoplasmic reticula and Golgi apparatuses did not show significant changes. In conclusion, our results showed a dose-dependent toxicity of L on GCs, associated to morphological signs of apoptosis. Alterations of GCs may be associated to impaired oocyte competence and sterility

THE RGB AND AGB STAR NUCLEOSYNTHESIS IN LIGHT OF THE RECENT 17O(p, α)14N AND 18O(p, α)15N REACTION-RATE DETERMINATIONS

In recent years, the Trojan Horse Method (THM) has been used to investigate the low-energy cross sections of proton-induced reactions on A = 17 and A = 18 oxygen isotopes, overcoming extrapolation procedures and enhancement effects due to electron screening. In particular, the strengths of the 20 keV and 65 keV resonances in the {sup 18}O(p, {alpha}){sup 15}N and {sup 17}O(p, {alpha}){sup 14}N reactions, respectively, have been extracted, as well as the contribution of the tail of the broad 656 keV resonance in the {sup 18}O(p, {alpha}){sup 15}N reaction inside the Gamow window. The strength of the 65 keV resonance in the {sup 17}O(p, {alpha}){sup 14}N reaction, measured by means of the THM, has been used to renormalize the corresponding resonance strength in the {sup 17}O + p radiative capture channel. As a result, more accurate reaction rates for the {sup 18}O(p, {alpha}){sup 15}N, {sup 17}O(p, {alpha}){sup 14}N, and {sup 17}O(p, {gamma}){sup 18}F processes have been deduced, devoid of systematic errors due to extrapolation or the electron screening effect. Such rates have been introduced into state-of-the-art red giant branch and asymptotic giant branch (AGB) models for proton-capture nucleosynthesis coupled with extra-mixing episodes. The predicted abundances have been compared with isotopicmore » compositions provided by geochemical analysis of presolar grains. As a result, an improved agreement is found between the models and the isotopic mix of oxide grains of AGB origins, whose composition is the signature of low-temperature proton-capture nucleosynthesis. The low {sup 14}N/{sup 15}N found in SiC grains cannot be explained by the revised nuclear reaction rates and remains a serious problem that has not been satisfactorily addressed.« les

Indirect Measurements of n- and p-Induced Reactions of Astrophysical Interest on Oxygen Isotopes

Observations of abundances and isotopic ratio determinations in stars yield powerful constraints on stellar models. In particular, the oxygen isotopic ratios are of particular interest because they are affected not only by nucleosynthesis but also by mixing processes, which are not very well-understood yet. This review is focused on the measurements via the Trojan Horse Method (THM) that have been carried out to investigate the low-energy cross sections of proton and neutron-induced reactions on 17O as well as the proton-induced reaction on 18O, overcoming extrapolation procedures and enhancement effects due to electron screening. The (p,α) reactions induced on these oxygen isotopes are of paramount importance for the nucleosynthesis in a number of stellar sites, including red giants (RGs), asymptotic giant branch (AGB) stars, massive stars, and classical novae. In detail, the indirect measurement of the low-energy region of 17O(p,α)14N was performed. The strength of the narrow resonance at 65 keV was evaluated, and it was used to renormalize the corresponding resonance strength in the 17O+p radiative capture channel. The reaction rate was then evaluated for both the 17O(p,α)14N and the 17O(p,γ)18F reactions, and significant differences of 30 and 20% with respect the literature data were found, respectively, in the temperature range relevant for RG, AGB, and massive stars nucleosynthesis. Regarding the 18O(p,α)15N reaction, the strength of the 20 keV resonance was extracted, which is the main contribution to the reaction rate for astrophysical applications. This approach has allowed us to improve the data accuracy of a factor 8.5, as it is based on the measured strength instead of educated guesses or spectroscopic measurements. Finally, the 17O(n,α)14C reaction was studied because of its role during the s-process nucleosynthesis as a possible neutron poison reaction. This study represents the extension of THM to resonant neutron-induced reactions. In this measurement, the subthreshold level centered at −7 keV in the center-of-mass system, corresponding to the 8.039 MeV 18O excited level, was observed. Moreover, the THM measurements showed a clear agreement with the available direct measurements and the additional contribution of the 8.121 MeV 18O level, strongly suppressed in direct measurements because of its l = 3 angular momentum. The contributions of those levels to the total reaction rate were than evaluated for future astrophysical applications

Magnetic Mixing in Red Giant and Asymptotic Giant Branch Stars

The available information on isotopic abundances in the atmospheres of low-mass Red Giant Branch (RGB) and Asymptotic Giant Branch (AGB) stars requires that episodes of extensive mixing occur below the convective envelope, reaching down to layers close to the hydrogen burning shell (Cool Bottom Processing). Recently \cite{Busso:2007jw} suggested that dynamo-produced buoyant magnetic flux tubes could provide the necessary physical mechanisms and also supply sufficient transport rates. Here, we present an $\alpha-\Omega$ dynamo in the envelope of an RGB/AGB star in which shear and rotation drain via turbulent dissipation and Poynting flux. In this context, if the dynamo is to sustain throughout either phase, convection must resupply shear. Under this condition, volume-averaged, peak toroidal field strengths of $\simeq3\times10^3$ G (RGB) and $\simeq5\times10^3$ G (AGB) are possible at the base of the convection zone. If the magnetic fields are concentrated in flux tubes, the corresponding field strengths are comparable to those required by Cool Bottom Processing.Comment: Replaced to correct small error in published version: In \S 2.1, paragraphs 2 and 3 incorrectly refer to the poloidal field when qualitatively discussing magnetic diffusion in the shear zone. The correct physical interpretation is that the toroidal field diffuses through the shear zone consistent with the value of $\beta_\phi