细胞功能测定

Protein phosphorylation plays a pivotal role in cell cycle regulation. MPF (M-phase Promoting Factor) and MAPK (Mitogen-activated protein kinase) are two major kinases driving oocyte maturation and early embryonic divisions. Their activities can be measured experimentally wi ...

The development of primordial germ cells into oocytes within primordial follicles involves a complex sequence of proliferation, developmental commitment, entry and arrest in meiosis, and association with surrounding somatic cells. These processes occur over the first few mont ...

Distinct meiotic cohesin complexes play fundamental roles in various meiosis-specific chromosomal events in spatiotemporally different manners during mammalian meiotic prophase. Immunostaining is one of the essential methods to study meiotic cohesin dynamics. For the s ...

The nuclear transfer (NT) technique in the mouse has enabled us to generate cloned mice and to establish NT embryonic stem (ntES) cells. Direct nuclear injection into mouse oocytes with a piezo impact drive unit can aid in the bypass of several steps of the original cell fusion procedure. It is important ...

Meiosis is the specialized cell division in sexually reproducing organisms in which haploid gametes are produced. Meiotic prophase I is the defining stage of meiosis, when pairing and synapsis occur between homologous chromosomes, concurrent with reciprocal recombination (or c ...

The cytogenetic analysis of single cells, such as oocytes and polar bodies, is extremely challenging. The main problem is low probability of obtaining a metaphase preparation in which all of the chromosomes are sufficiently well spread to permit accurate analysis (no overlapping chrom ...

Mouse oocytes and zygotes are semitransparent and large cells approximately 80 μm in diameter. Bisection is one of the easiest ways for performing micromanipulations on such cells. It allows living sister halves or smaller fragments to be obtained, which can be cultured and observed for long ...

Chromosomal spreads are an established method to assess ploidy in different cell types. However, many traditional chromosome-spreading techniques require dissolution of the cell and can only be used to assess hyperploidy because of potential chromosome loss inherent in the proce ...

At fertilization mammalian eggs are activated by a prolonged series of oscillations in the intracellular free Ca2+ concentration. These oscillations can be monitored with any number of Ca2+-sensitive fluorescent dyes. The oscillations last for several hours at fertilization and so ...

The visualization of subcellular organelles and structures is a valuable tool for understanding cellular changes that occur in oocytes and early embryos as a result of genetic alterations, incubation conditions, drug treatments, and many other manipulations. Preparing oocytes f ...

This chapter describes a technique for performing chromosome spreads from mouse oocytes. It is based on a previously described protocol (Hodges and Hunt, Chromosoma 111: 165–169, 2002), which we have modified. Chromosomes are stained with either Propidium Iodide or Hoechst. This spread ...

We previously isolated Aurora-C (Aurkc/Aie1) in a screen for kinases expressed in mouse sperm and eggs. Aurora-C kinase was reported to be a chromosomal passenger protein that plays critical roles in chromosome alignment, segregation, kinetochore-microtubule attachment, and cyt ...

Understanding how human oocytes execute chromosome segregation is of paramount importance as errors in this process account for the overwhelming majority of human aneuploidies and increase exponentially with advancing female age. The spindle is the cellular apparatus respon ...

Radiolabeled compounds that are substrates for transmembrane transporters can be used to study transport and metabolism in mammalian oocytes and preimplantation embryos. Because even very small amounts of radioisotopes can be detected, these techniques are feasible to use with o ...

Gene silencing techniques have brought new insights into mammalian oocyte and embryo development. More specifically, the use of Morpholino oligonucleotides which sterically inhibit translation from target mRNAs thereby compromising gene function, allowed the identifi ...

Memory is a high-level brain function that enables organisms to adapt their behavioral responses to the environment, hence increasing their probability of survival. The Ras–ERK pathway is a key molecular intracellular signalling cascade for memory consolidation. In this chapter we ...

Several essential cellular functions are critically influenced by the Ras-Raf-MEK-ERK cascade, and pathological problems have been implicated with loss as well as gain of its activity. Therefore, there is a great demand for chemical probes that act as modulators of the cascade in order to cor ...

Since its original description, the yeast two-hybrid system has been extensively used to identify protein–protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the ...

Cysteine is one of the most reactive amino acids and is modified by a number of oxidants. The reactivity of cysteines is dependent on the thiol pK a; however, measuring cysteine pK a values is nontrivial. Ras family GTPases have been shown to contain a free cysteine that is sensitive to oxidation, and free ra ...

Redox agents have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that mammalian cells can rapidly respond to ligand stimulat ...