Brown, Jeremy John Gibson; (1991) Cleavage arrest during mouse preimplantation development in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Concommitant with in vitro cleavage arrest at the 2-cell stage, the mitochondria of embryos from random-bred mice became clustered around the nuclei and the region of the cytocortex immediately adjacent to the plasma membrane. Injection of small aliquots of phase cytoplasm from normally cleaving embryos of an inbred mouse strain restored the homogeneous mitochondrial organisation characteristic of such embryos in a proportion of injected recipients. Subsequently, mitosis was reinitiated in these embryos and they proceeded to the blastocyst stage in vitro. Injection of cytoplasm from oocytes, after breakdown of the germinal vesicle, failed to reinitiate cleavage but led to nuclear membrane breakdown and partial condensation of the chromatin. It was concluded that the ''G2 cytoplasmic rescue factor" exhibits no functional homology with Maturation/Mitosis Promoting Factor (MPF). One-cell random-bred embryos developed to the blastocyst stage within explanted oviducts maintained in organ culture. However, whilst embryos flushed from in vivo oviducts at the mid to late 2-cell stage developed to the blastocyst stage in vitro, embryos from organ cultured oviducts were only capable of similar development when flushed at the 4-cell stage. The metabolic inhibitors KAs (potassium arsenate) and iodoacetamide induced mitochondrial aggregation in 2-cell inbred embryos, similar to that observed in cleavage arrested random-bred embryos. Culture in medium containing glucose as the sole energy source had a similar effect. Investigation of the substrate requirements of inbred embryos revealed that lactate alone supported first cleavage in a majority of embryos, and lactate + pyruvate supported complete development to the morula stage. Exposure to medium containing glucose for 24h at any stage before Day 4 of development was required to support the morula to blastocyst transition. In random-bred embryos, maximal development from the 1-cell stage was obtained in medium lacking lactate on Day 1 of culture, and glucose on Day 2, but including lactate, pyruvate and glucose from Day 3 onwards.

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