Light-Induced Smooth Endoplasmic Reticulum Rearrangement in a Unique Interlaced Compartmental Pattern in Macaca mulatta RPE.

Purpose: To investigate light-induced modifications of the smooth endoplasmic reticulum of the RPE in primates. Methods: Eyes of three terminally anesthetized Rhesus monkeys were exposed to 5000 lux for 10 minutes or kept in the dark. Transmission electron microscopy and electron tomography were conducted on small fragments of retina sampled from different regions of the retina. Results: RPE cells smooth endoplasmic reticulum shows a previously unknown arrangement characterized by an interlaced compartmental pattern (ICP). Electron tomograms and 3D-modelling demonstrated that the smooth endoplasmic reticulum with an ICP (ICPSER) consisted of four parallel, independent and interwoven networks of tubules arranged as interconnected coiled coils. Its architecture realized a compact labyrinthine structure of tightly packed tubules stabilized by intertubular filamentous tethers. On average, the ICPSER is present in about 14.6% of RPE cells. Although ICPSER was preferentially found in cells located in the peripheral and in the para/perifoveal retina, ICPSER cells significantly increased in number upon light exposure in the para/perifovea and in the fovea. Conclusions: An ICPSER is apparently a unique feature to primate RPE. Its rapid appearance in the area centralis of the retina upon light exposure suggests a function related to the foveate structure of primate retina or to the diurnal habits of animals that may require additional protection from photo-oxidation or enhanced requests of visual pigments regeneration.

IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.

C-type synaptic boutons (C-boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)-related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C-boutons, including M2 muscarinic receptors, potassium channels, and σ-1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C-boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. C-bouton-mediated regulation of MN excitability has been implicated in MN disease, but NRG1-mediated functions and the impact of various pathologic conditions on C-bouton integrity have not been studied in detail. Here, we investigated changes in C-boutons after electrical stimulation, pharmacological treatment, and peripheral nerve axotomy. SSC-linked NRG1 clusters were severely disrupted in acutely stressed MNs and after tunicamycin-induced ER stress. In axotomized MNs, C-bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal. Activated microglia displayed a positive chemotaxis to C-boutons. Analysis of transgenic mice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC-like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals. Moreover, MN-derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C-boutons in MN injury and suggest that distinct NRG1 isoform-mediated signaling functions regulate the complex matching between pre- and postsynaptic C-bouton elements.-Salvany, S., Casanovas, A., Tarabal, O., Piedrafita, L., Hernández, S., Santafé, M., Soto-Bernardini, M. C., Calderó, J., Schwab, M. H., Esquerda, J. E. Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.

Local resources of polyribosomes and SER promote synapse enlargement and spine clustering after long-term potentiation in adult rat hippocampus.

Synapse clustering facilitates circuit integration, learning, and memory. Long-term potentiation (LTP) of mature neurons produces synapse enlargement balanced by fewer spines, raising the question of how clusters form despite this homeostatic regulation of total synaptic weight. Three-dimensional reconstruction from serial section electron microscopy (3DEM) revealed the shapes and distributions of smooth endoplasmic reticulum (SER) and polyribosomes, subcellular resources important for synapse enlargement and spine outgrowth. Compared to control stimulation, synapses were enlarged two hours after LTP on resource-rich spines containing polyribosomes (4% larger than control) or SER (15% larger). SER in spines shifted from a single tubule to complex spine apparatus after LTP. Negligible synapse enlargement (0.6%) occurred on resource-poor spines lacking SER and polyribosomes. Dendrites were divided into discrete synaptic clusters surrounded by asynaptic segments. Spine density was lowest in clusters having only resource-poor spines, especially following LTP. In contrast, resource-rich spines preserved neighboring resource-poor spines and formed larger clusters with elevated total synaptic weight following LTP. These clusters also had more shaft SER branches, which could sequester cargo locally to support synapse growth and spinogenesis. Thus, resources appear to be redistributed to synaptic clusters with LTP-related synapse enlargement while homeostatic regulation suppressed spine outgrowth in resource-poor synaptic clusters.

Nanoscale enrichment of the cytosolic enzyme trichodiene synthase near reorganized endoplasmic reticulum in Fusarium graminearum.

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.

Presence of aggregates of smooth endoplasmic reticulum in MII oocytes affects oocyte competence: molecular-based evidence.

STUDY QUESTION: Does the presence of aggregates of smooth endoplasmic reticulum (SERa) impact the transcriptome of human metaphase II (MII) oocytes?. SUMMARY ANSWER: The presence of SERa alters the molecular status of human metaphase II oocytes. WHAT IS KNOWN ALREADY: Oocytes presenting SERa are considered dysmorphic. Oocytes with SERa (SERa+) have been associated with reduced embryological outcome and increased risk of congenital anomalies, although some authors have reported that SERa+ oocytes can lead to healthy newborns. The question of whether or not SERa+ oocytes should be discarded is still open for debate, and no experimental information about the effect of the presence of SERa on the oocyte molecular status is available. STUDY DESIGN, SIZE, DURATION: This study included 28 women, aged <38 years, without any ovarian pathology, and undergoing IVF treatment. Supernumerary MII oocytes with no sign of morphological alterations as well as SERa+ oocytes were donated after written informed consent. A total of 31 oocytes without SERa (SERa-) and 24 SERa+ oocytes were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pools of 8-10 oocytes for both group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package. MAIN RESULTS AND THE ROLE OF CHANCE: The expression profiles of SERa+ oocytes significantly differed from those of SERa- oocytes in 488 probe sets corresponding to 102 down-regulated and 283 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics disclosed that genes involved in three main biological processes were significantly down-regulated in SERa+ oocytes respective to SERa- oocytes: (i) cell and mitotic/meiotic nuclear division, spindle assembly, chromosome partition and G2/M transition of mitotic cell cycle; (ii) organization of cytoskeleton and microtubules; and (iii) mitochondrial structure and activity. Among the transcripts up-regulated in SERa+ oocytes, the most significantly (P = 0.002) enriched GO term was 'GoLoco motif', including the RAP1GAP, GPSM3 and GPSM1 genes. LARGE SCALE DATA: Raw microarray data are accessible through GEO Series accession number GSE106222 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106222). LIMITATIONS, REASONS FOR CAUTION: Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change >3 or <1/3 and FDR < 0.1). Surveys on clinical outcomes, malformation rates and follow-up of babies born after transfer of embryos from SERa+ oocytes are necessary. WIDER IMPLICATIONS OF THE FINDINGS: We provide information on the molecular status of SERa+ oocytes, highlighting possible associations between presence of SERa, altered oocyte physiology and reduced developmental competence. Our study may offer further information that can assist embryologists to make decisions on whether, and with what possible implications, SERa+ oocytes should be used. We believe that the presence of SERa should be still a 'red flag' in IVF practices and that the decision to inseminate SERa+ oocytes should be discussed on a case-by-case basis. STUDY FUNDING/COMPETING INTEREST(s): This study was partially supported by Ferring Pharmaceuticals. The authors have no conflicts of interest to declare.

Dysmorphic patterns are associated with cytoskeletal alterations in human oocytes.

Study question: Are specific morphological anomalies in human mature oocytes, as revealed by transmitted light microscopy, associated with intrinsic damage to the meiotic spindle and actin cytoskeleton? Summary answer: Aggregates of smooth endoplasmic reticulum (SER) and domains of centrally localized granular cytoplasm (GC) reflect intrinsic damage to the oocyte cytoskeleton, namely alterations in spindle size, chromosome misalignment and cortical actin disorganization. What is known already: In preparation for ICSI, oocytes are often selected for use in treatment by morphological criteria, but the rationale and implications of this practice are controversial. Very little information is available on the relationship between oocyte morphology and intrinsic cellular characteristics, such as the actin cytoskeleton, meiotic spindle and chromosome alignment. Study design, size, duration: A total of 170 metaphase II (MII) oocytes were donated by consenting IVF patients and analysed; 62 were classified as morphologically normal (control), 54 had SER clusters and 54 had centrally localized GC. Participants/materials, setting, methods: Supernumerary oocytes were fixed within 3 h from recovery and stained for tubulin, chromatin and actin. Spindles were analysed for 1D and 2D characteristics by high-performance confocal microscopy. Chromosomes were classified as scattered or aligned and the conformation and intensity of cortical actin was evaluated. Main results and the role of chance: In comparison with control oocytes, both SER and GC oocytes showed greater spindle length (P = 0.033 and 0.003, respectively) and GC oocytes also showed greater spindle width (P= 0.049) and area (P= 0.036). Control and SER oocytes had statistically comparable rates of chromosome displacement from the metaphase plate, unlike GC oocytes where chromosome displacement occurred at higher rate (P = 0.013). In situations where a complete Z-stack was reconstructed from a polar angle, chromosome disposition was classified as being normal when two sets of concentric arrays were visible. Based on these parameters, the proportions of oocytes with normal chromosomal arrangement or partial/total disarrangement was not statistically different between control and SER oocytes. Conversely, in GC oocytes, chromosome disarrangement was higher (P = 0.002). All control oocytes displayed a continuous meshwork of suboolemmal actin, which appeared as an uninterrupted ring in thin optical sections. In contrast, in SER and  GC groups, integrity of suboolemmal actin was observed in only 66.7 and 42.9% of oocytes, respectively (P = 0.0001). Large scale data: N/A. Limitations reason for caution: Only two of several known oocyte dysmorphisms were investigated, while oocyte quality was assessed only by cytoskeletal criteria. Wider implications of the findings: This study represents a significant step toward a more objective assessment of oocyte morphology, offering information that can assist embryologists to make a more aware and rationally founded decision on whether, and with what possible implications, oocytes with certain dysmorphic characters should be used for treatment or discarded. More generally, it also demonstrates that morphometric parameters of the cytoskeleton and chromosome organization can be used as biomarkers of oocyte quality. Study funding and competing interest(s): This study was funded by Biogenesi Reproductive Medicine Centre (Monza, Italy). All authors declare no conflict of interests.

Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII.

New ultrastructural observations of human oocyte smooth endoplasmic reticulum tubular aggregates and cortical reaction: update on the molecular mechanisms involved / Nuevas observaciones ultraestructurales en ovocitos humanos de los agregados tubulares de retículo endoplásmico liso y de la reacción cortical: actualización sobre los mecanismos moleculares implicados

Introduction. The ultrastructure of the human mature metaphase-II oocyte before and after the cortical reaction has been previously described. However, we found a new vesicular aggregate associated with smooth endoplasmic reticulum tubular aggregates (aSERT) and new observations regarding the cortical reaction. Objectives. To describe at the ultrastructural level a novel vesicle aggregate associated with aSERT and new observations regarding the cortical reaction.Materials and methods. Donated, surplus mature oocytes were processed for transmission electron microscopy immediately after recover. Calcium detection was performed using the pyroantimonate technique. The cortical reaction was artificially induced by ionophore treatment. Results. We observed an accumulation of small vesicles at the periphery of aSERT. At high magnification these were composed by small pale vesicles coated by tiny vesicles, with associated dense materials, giving a rosette-like appearance. Adjacent to these there was another group of small dense vesicles incompletely coated by similar tiny vesicles. Using calcium detection at the ultrastructural level, antimonate deposits were observed in the tubules of aSERT and in the surrounding mitochondria but not in the rosette-like structures. Regarding cortical vesicles, although most previous studies described their contents as homogeneous dense, others referred the presence of another type of cortical vesicles whose contents were moderate dense. Using ionophore oocyte activation, we observed that moderate dense cortical vesicles may correspond to a progressive swelling of the dense cortical vesicles prior to exocytosis. Conclusions. We describe a novel vesicle aggregate associated to aSERT and new observations on the remodeling of cortical vesicles before exocytosis (AU)

Introducción. La ultraestructura del ovocito humano maduro en metafase-II humana antes y después de la reacción cortical se ha descrito previamente. Sin embargo, encontramos un nuevo agregado vesicular asociado a los agregados tubulares de retículo endoplásmico liso (aSERT) y nuevas observaciones con respecto a la reacción cortical. Objetivos. Describir a nivel ultraestructural un nuevo agregado vesicular asociado a los agregados tubulares de retículo endoplásmico liso y nuevas observaciones con respecto a la reacción cortical. Material y métodos. Se procesaron ovocitos maduros excedentes de ciclos de donante para microscopia electrónica de transmisión inmediatamente después de recuperarse. La detección de calcio se realizó mediante la técnica de piroantimoniato. La reacción cortical fue inducida artificialmente mediante tratamiento ionóforo. Resultados. Se observó una acumulación de vesículas pequeñas en la periferia de aSERT. A mayor aumento, estas estaban compuestas por pequeñas vesículas pálidas recubiertas por diminutas vesículas, con materiales densos asociados, dando una apariencia de roseta. Junto a ellas había otro grupo de pequeñas vesículas densas, incompletamente recubiertas por similares vesículas diminutas. Con el uso de la detección de calcio a nivel ultraestructural se observaron depósitos de antimoniato en los túbulos de aSERT y en las mitocondrias de los alrededores, pero no en las estructuras de roseta. En cuanto a las vesículas corticales, aunque la mayoría de los estudios anteriores describen su contenido como denso homogéneo, se refirió la presencia de otro tipo de vesículas corticales cuyo contenido era denso moderado. Con el uso de la activación de ovocitos con ionóforo, se observó que las vesículas corticales de densidad densa moderada pueden corresponder a una hinchazón progresiva de las vesículas corticales densas antes de la exocitosis. Conclusiones. Describimos una nuevo agregado vesicular asociado a aSERT y nuevas observaciones sobre la remodelación de vesículas corticales antes de la exocitosis (AU)

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.

A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy.

An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 µm or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.

Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism: Light and electron microscopy approach.

Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with altered morphology in both groups of hypothyroid pups was increased. Apoptosis markers have not shown significant difference between groups but PCNA had an increased expression in the oocytes and granulosa cells in primordial follicles of hypothyroid rats. Light and electron microscopy analysis indicate that previously detected premature ovarian follicular atresia in pre-pubertal and pubertal hypothyroid rats is preceded with premature formation of primordial follicles followed by slight changes on sER and mitochondria in examined oocytes, and increased expression of PCNA.

Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

A stereological study on organelle distribution in human oocytes at prophase I.

The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.

Policy of IVF centres towards oocytes affected by Smooth Endoplasmic Reticulum aggregates: a multicentre survey study.

PURPOSE: The presence of Smooth Endoplasmic Reticulum aggregates (SERa) has been reported to be associated with adverse outcomes. An Alpha-ESHRE Consensus was published in 2011, strongly recommending to not inseminating affected oocytes. On the other hand, healthy babies have been born from oocytes presenting this dysmorphism. We surveyed several European IVF centres, to assess their attitudes concerning affected oocytes. METHODS: This survey is based on a computer format and includes questions regarding the fate of affected oocytes. RESULTS: About 14 % of centres who answered our survey discard SERa+ oocytes. 43 % of centres that do not discard the oocytes, register and follow up neonatal data. About a quarter of centres inform their patients about this dysmorphism. Half of them require an informed consent prior to transferring affected embryos. Twenty-one centres reported having SERa+ births, with one reporting a malformation. 48 % of centres declared having been influenced by the Alpha-ESHRE Consensus, in their management policy of SERa+ oocytes. CONCLUSIONS: Few centres scrupulously respect the recommendations of the Alpha-ESHRE Consensus and discard affected oocytes. Since it is essential to determine if there truly is an impact of this dysmorphism and whether the guidelines are still valid, transfer of affected embryos should only be done when accompanied with data recording and monitoring of all foetal malformations from IVF. Clarifying the situation will allow IVF centres to correctly inform patients about the risk of birth malformations as well as whether a decreased chance of pregnancy exists.

Disruption of paranodal axo-glial interaction and/or absence of sulfatide causes irregular type I inositol 1,4,5-trisphosphate receptor deposition in cerebellar Purkinje neuron axons.

Paranodal axo-glial junctions (PNJs) play an essential role in the organization and maintenance of molecular domains in myelinated axons. To understand the importance of PNJs better, we investigated cerebroside sulfotransferase (CST; a sulfatide synthetic enzyme)-deficient mice, which partially lack PNJs in both the central nervous system (CNS) and the peripheral nervous system (PNS). Previously, we reported that axonal mitochondria at the nodes of Ranvier in the PNS were large and swollen in CST-deficient mice. Although we did not observed significant defects in the nodal regions in several areas of the CNS, myelinated internodal regions showed many focal swellings in Purkinje cell axons in the cerebellum, and the number and the size of swellings increased with age. In the present analysis of various stages of the swellings in 4-12-week-old mutant mice, calbindin-positive axoplasm swellings started to appear at an early stage. After that, accumulation of neurofilament and mitochondria gradually increased, whereas deposition of amyloid precursor protein became prominent later. Ultrastructural analysis showed accumulations of tubular structures closely resembling smooth endoplasmic reticulum (ER). Staining of cerebellar sections of the mutant mice for type I inositol 1,4,5-trisphosphate receptor (IP3 R1) revealed high immunoreactivity within the swellings. This IP3 R1 deposition was the initial change and was not observed in development prior to the onset of myelination. This suggests that local calcium regulation through ER was involved in these axonal swellings. Therefore, in addition to the biochemical composition of the internodal myelin sheath, PNJs might also affect maintenance of axonal homeostasis in Purkinje cells.

Light and electron microscopic studies on the pigmented epithelium and photoreceptors of the retina of common buzzard (Buteo buteo).

The current study is essentially carried out to reveal the histological and ultra-structural details of the retinal pigmented epithelium (RPE) and photoreceptors cell layers of a common buzzard (Buteo buteo). The recorded results revealed that the neural retina of common buzzard consisted of seven distinct cell layers. The inner nuclear layer was markedly revealed as the thickest one among these layers. A highly melanized RPE was recorded in between the choroid and neural retina. Histologically, the RPE was represented by a single layer of cuboidal epithelial cells with centrally located nucleus. Ultrastructurally, the RPE cells showed numerous melanosomes, mitochondria, phagosomes, myeloid bodies, smooth endoplasmic reticulum (SER), but very rare rough endoplasmic reticulum (RER). The photoreceptor cell layer was represented by three categories of photoreceptor cells: few single rods, numerous single and double cones. Each double cone consisted of a short accessory cone and a long principle cone. The photoreceptor outer segment consisted of bi-membranous discs that are enclosed by outer membrane. Moreover, the inner segment of rods consisted of an ellipsoid and an inner hyperboloid. The hyperboloid was rich with RER, polysomes, Golgi apparatus and autophagic vacuoles. Furthermore, the inner segment of single cone and accessory cone consisted of an ellipsoid, paraboloid and myoid regions, while, the inner segment of principle cone lacked the paraboloid regions. At the proximal end of each inner segment for all types of cones, there was a large heterogeneous oil droplet. The paraboloid region was markedly rich with glycogen granules. The myoid region exhibited the same organelles but with little glycogen granules when compared with hyperboloid.

Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis.

BACKGROUND: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. METHODS: We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. RESULTS: Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 µm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. CONCLUSIONS: Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.

Deliveries of babies with normal health derived from oocytes with smooth endoplasmic reticulum clusters.

PURPOSE: To examine the impact on development of derived embryos from smooth endoplasmic reticulum clusters (SERC) in human metaphase II (MII) oocytes. METHODS: Retrospective analysis at Kyono ART Clinic. Comparison of embryological development, pregnancy, live birth and fetal malformation between oocytes with SERC (the SERC(+) group) and those without (the SERC(-) group) in 2,158 patients (3,758 cycles) after ICSI. RESULTS: Fertilization and implantation rate were significantly lower in SERC(+) MII oocytes than in SERC(-) MII oocytes. After the transfer of fresh and vitrified embryos derived from SERC(+) oocytes, 14 pregnancies resulted in 14 healthy babies, including 2 from fresh embryo transfer (ET) and 12 from vitrified-warmed ET, with no malformations. CONCLUSION(S): The presence of SERC in MII oocytes was associated with significantly lower fertilization rates and implantation rates than seen in SERC(-) MII oocytes within SERC (+) cycles. However, SERC had no impact on post-implantation development as well as neonatal outcome.

El retículo endoplasmático liso en hepatocitos estimulados con distintas dosis de láser infrarrojo / The smooth endoplasmic reticulum in hepatocytes stimulated with different doses of infrared laser

Veinticuatro ratas hembras Sprague Dawley de 4 meses de vida con peso aproximado de 250 g, fueron divididas en cuatro grupos (A, B, C y D), donde el grupo A (control) no recibió estimulación infrarroja, B se irradió con láser infrarrojo 4 J/cm², C con dosis de 8 J/cm² y D con 16 J/cm². La estimulación infrarroja se realizó diariamente, por 15 días ininterrumpidos. Las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado normal (control) como estimulado con las distintas dosis infrarrojas, las que fueron procesadas para microscopía electrónica de transmisión. De los hepatocitos normales y estimulados, se obtuvieron microfotografías con aumentos finales de hasta 36.500 X, que fueron sometidas a estudios morfométricos para determinar fracciones volumétricas con especial énfasis en el retículo endoplásmico liso (REL) y de los siguientes componentes celulares: retículo endoplasmático rugoso (RER), mitocondrias, glicógeno, eu y heterocromatina. De igual manera se cuantificaron las áreas celulares y nucleares. Del análisis de los resultados entre hepatocitos normales y estimulados con diferentes dosis infrarrojas, se visualiza que existen notables diferencias en todos los componentes celulares cuantificados particularmente el REL. Se concluye que las estimulaciones infrarrojas provocan una drástica transformación en la ultraestructura y morfología de los hepatocitos, lo que provocaría una variación funcional, representando de esta manera el efecto que estas estimulaciones provocan en este tipo celular.

A total of 24 female Sprague-Dawley rats aged 4 months and weighing approximately 250 g, were divided into four groups labeled A, B, C and D. Group A received no infrared stimulation and served as control. Group B was radiated with a dose of 4 J/cm² of infrared laser, Group C with doses of 8 J/cm² and Group D with 16 J/cm². This infrared stimulation was carried out daily for 15 days uninterrupted. The rats were then sacrificed and samples of both normal-control liver and liver stimulated with the different infrared doses were extracted for immediate processing via transmission electron microscopy. Transmission electron microphotographs were obtained at magnifications of 21300X from both normal and stimulated hepatocytes; these were subjected to morphometric studies to determine volumetric fractions with special emphasis on the smooth endoplasmic reticulum (SER) and the following cell components: rough endoplasmic reticulum (RER), mitochondria, glycogen, eu and heterochromatin. Likewise, cell and nuclear areas were quantified. Analysis of the results of normal and stimulated hepatocytes with different infrared doses showed considerable differences in all the quantified cell components and particularly from the SER it is concluded that the effects of these stimulations bring about a drastic transformation in the ultrastructure and morphology of the hepatocytes, which may ultimately translate into a functional variation, thus representing the effect that these stimulations cause in this cell type.