Aneuploidy in human eggs is the leading cause of pregnancy loss

Aneuploidy in human eggs is the leading cause of pregnancy loss and Downs syndrome. and merotelic attachments. Chromosome arm cohesion was weakened, and the fraction of bivalents that precociously dissociated into univalents was Angiotensin II increased. Together, our data reveal multiple age-related changes in chromosome architecture that could explain why oocyte aneuploidy increases with advanced maternal age. DOI: 1:1000). As secondary antibodies, Alexa-Fluor-488/564/647 labelled anti-rat/anti-human/anti-mouse (all Molecular Probes; 1:400) were used. DNA was stained with 0.5 g/ml Hoechst 33342 (Molecular Probes). Immunostained oocytes were imaged on a Zeiss LSM710 confocal microscope equipped with a 63x C Apochromat 1.2 NA water immersion objective at a spatial resolution of 0.3 m optical sections (xy pixel 1024×1024). For analysis, images were deconvolved using Huygens Professional (Scientific Volume Imaging). Data analysis Kinetochore counting and assessment of sister pair configurations Sister kinetochore pair configurations were determined by 3D analysis (Imaris, Bitplane) of high resolution deconvolved images encompassing the entire meiosis I spindle. First, all kinetochores belonging to the same bivalent were identified by comparing CREST and Hoechst staining in consecutive z-planes spanning the entire bivalent. Then, the two most proximal kinetochores within a bivalent were defined as a sister pair. The center of each kinetochore was detected with Angiotensin II subpixel accuracy using the automated spot detection function based on local maxima in Imaris (Bitplane). All automated spot detections were confirmed by visible inspection and corrected when software program mistake was apparent manually. Kinetochore configurations had been defined as comes after: indistinguishablea one CREST spot noticeable by inspection of the utmost intensity projection as well as computerized single spot recognition by Imaris; overlappinga markedly extended CREST signal together with computerized recognition of two areas; distincttwo discrete CREST areas that contact but usually do not overlap with automated recognition of two areas jointly; separatedtwo discrete non-touching CREST areas identified by visual inspection with automated recognition of two areas jointly. 98% of kinetochore pairs could possibly be resolved confidently and were contained in the following evaluation. The distances between your sister kinetochores had been assessed in Microsoft Excel using the xyz coordinates of kinetochore centers described by the computerized spot recognition function in C10rf4 Imaris (Bitplane). Sister kinetochores had been frequently discovered in various z-sections as well as the Pythagorean Theorem was utilized to estimate the ranges between all kinetochore pairs. The parting length between all indistinguishable and overlapping sister kinetochores which were discovered as single areas by Imaris was established to 0 m (Body 2C). All ranges and kinetochore configurations were decided blindly to donors age. The quantifications were further confirmed by an independent second count. Total chromosome and kinetochore counts were performed in each oocyte to exclude the possibility that any two bivalents with indistinguishable kinetochore configurations are in fact two univalents originating from a precociously dissociated bivalent. Any bivalents that were located at the spindle poles and hence were not under tension were excluded from this analysis. Modes of kinetochore-microtubule attachment Microtubule-kinetochore attachments were determined by 3D analysis of high resolution deconvolved images of the whole spindle volume. Only oocytes in which the spindle was oriented in parallel to the imaging plane were included in this analysis. For quantification of K-fiber attachment Angiotensin II modes, first the number of discrete K-fibers attaching to each sister kinetochore pair was decided. Then, if two or more K-fiber attachments were identified, the type (A-C) of the attachment was evaluated. Attachment modes were classified as: type A – two individual K-fibers that run in parallel to each other; type B – two individual K-fibers that originate from distant locations around the spindle; type C – a K-fiber that initially appears as one bundle but then branches out to form separate attachments to each Angiotensin II of the sister kinetochores. In Physique 2FCG, only kinetochores with end-on attachments originating from opposite spindle poles were included in.

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