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)

Distinct mechanisms controlling rough and smooth endoplasmic reticulum contacts with mitochondria.

Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

Molecular characterisation of the smooth endoplasmic reticulum Ca(2+)-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle.

The anterior sternal epithelial cells of the terrestrial isopod Porcellio scaber transport large amounts of calcium during the formation and resorption of intermittent calcium carbonate deposits. Recent investigations on epithelia involved in mineralisation processes suggest a role of the smooth endoplasmic reticulum Ca(2+)-ATPase (SERCA) in transcellular calcium transport. We present the first molecular characterisation of a SERCA within a crustacean mineralising epithelium. We cloned the SERCA from a cDNA library of the anterior sternal epithelium and used in situ hybridisation to compare the expression of the SERCA mRNA between three different moulting stages. The full-length SERCA cDNA has an open reading frame of 3006 nucleotides. The deduced 1002 amino-acid polypeptide has a predicted molecular mass of 109.7 kDa and 87% identity to the SERCA of Procambarus clarkii axial muscle isoform. In situ hybridisation confirmed expression within the anterior sternal epithelium and revealed an increase in SERCA mRNA abundance from the non-transporting, early premoult stage to the calcium transporting, late premoult and intramoult stage. The results support previous indications of a contribution by the smooth endoplasmic reticulum to transcellular calcium transport and suggest a transcriptional regulation of SERCA activity.

Postprandial induction of chaperone gene expression is rapid in mice.

Molecular chaperones assist in the biosynthesis and processing of proteins. Most chaperones are induced by physiological stresses. We have shown that dietary energy restriction decreases the mRNA and protein levels of many endoplasmic reticulum chaperones in the livers of mice. Here, we have investigated the response of chaperone mRNA to feeding. Control and 50% energy-restricted C3B10RF1 mice were deprived of food for 24 h, fed, and killed 0, 1.5, 5 or 12 h after feeding. Chaperone mRNAs were strongly induced as early as 1.5 h after feeding in control and energy-restricted mice. The integrated levels of these mRNA over 24 h were significantly lower in energy-restricted mice. The mRNA response to energy intake was mirrored over the course of days in the level of chaperone protein. A similar but smaller response to feeding was found in kidney and muscle. Puromycin and cycloheximide failed to inhibit the feeding response, suggesting that feeding releases chaperone expression from an unstable inhibitor. Studies with dibutyryl-cAMP- and glucagon-supplemented, normal and streptozotocin-diabetic mice suggest that glucagon and insulin may be mediators of the feeding response. Adrenalectomy enhanced the feeding induction, but dexamethasone administration had no effect. Thus, postprandial changes in insulin and glucagon may link chaperone gene expression to feeding, possibly in several tissues including liver.