Germ Cell Protocols

All sexually reproducing organisms produce primordial germ cells, a small population of cells that differentiate into gametes of either sex and carry to- potency, an ability to develop into an entire new organism. The study of germ cells has undergone enormous advances in recent years and has entered into an explosive phase of new discoveries with the introduction of transgenic te- nologies and nuclear cloning. Basic knowledge and techniques developed for lower vertebrate and invertebrate systems have facilitated the study of higher vertebrates, including humans. Many experiments that have first been performed on lower vertebrates provided the tools and strategies that could later be applied to other less readily available mammalian systems. The discovery of centrosomes in ascidians and sea urchin eggs now benefits studies of fertility and infertility in mammals. External in vitro fertilization, now a common technique in assisted fertilization, has only been possible as a result of numerous studies in lower systems in which external fertilization is natural. Egg activation, first explored in sea urchin and ascidian eggs, now benefits techniques designed to increase cl- ing efficiency in farm and domestic animals. Gene manipulations and molecular methods have added to the possibilities of producing live offspring with en- mous biomedical, ecological, and economic implications. The two volumes on germ cells combine techniques in a variety of d- ferent systems; we have selected those systems that have provided landmarks in advancing our knowledge on germ cells.

Includes supplementary material: sn.pub/extras

Klappentext
Germ cell research has produced enormous advances in recent years and more recently has entered into an explosive phase of new discoveries with the introduction of transgenic technologies and nuclear cloning. In Germ Cell Protocols, expert laboratory investigators describe in step-by-step detail a series of powerful techniques for the molecular and genetic analysis of germ cells in a variety of different reproductive systems. Chosen for their proven success in advancing our understanding of germ cells, these methods cover sperm and egg activation, motility, fertilization, nuclear development, nuclear cloning, the molecular characterization of specific events, and the imaging of cell structures. Volume 1: Sperm and Oocyte Analysis focuses on sperm cells, oocyte analysis, oocyte maturation, fertilization, and preparation techniques. Highlights include in vitro maturation and fertilization of human, porcine, and canine oocytes; the cryopreservation of sperm cells; establishment of an in vitro spermatogenesis system; visualization of sperm accessory structures; and motility assays of spermatozoa. Each readily reproducible protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. An accompanying second volume, Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning, contains methods for molecular egg analysis, live egg imaging, nuclear cloning, oocyte preservation, and nuclear transfer.
Comprehensive and cutting-edge, Germ Cell Protocols offers both novice and established researchers a gold-standard collection of hard-to-find methods for high impact research, diverse procedures that are easy-to-follow, well-illustrated, and allow a cross-species transfer of knowledge from lower vertebrates to higher mammalian systems.

Inhalt
Cryopreservation of Semen of the Salmonidae with Special Reference to Large-Scale Fertilization.- Fertilization and Sperm Chemotaxis in Ascidians.- Assays for Vertebrate Sperm Chemotaxis.- Stallion Spermatozoa Viability.- Visualization of Sperm Accessory Structures in the Mammalian Spermatids, Spermatozoa, and Zygotes by Immunofluorescence, Confocal, and Immunoelectron Microscopy.- Localization of Zona Pellucida Receptors on Live Sperm by Fluorophore-Conjugated Solubilized Zona Pellucida Proteins.- Detection of Human Papillomavirus DNA in Sperm Using Polymerase Chain Reaction.- Spermatogonial Stem Cells.- Molecular and Cellular Approaches for Establishment of an In Vitro Spermatogenesis System.- Acrosin Activity Assay for the Evaluation of Mammalian Sperm Acrosome Reaction.- Isolation of Sea Urchin Sperm Plasma Membranes.- Detection of Centrosome Structure in Fertilized and Artificially Activated Sea Urchin Eggs Using Immunofluorescence Microscopy and Isolation of Centrosomes Followed by Structural Characterization with Field Emission Scanning Electron Microscopy.- Microscopic Techniques for Studying Sperm-Oocyte Interaction During Fertilization and Early Embryonic Development.- Immunocytochemical Staining of the Metaphase II Spindle in Mammalian Oocytes.- Spindle Imaging in Living Mammalian Oocytes Using the Liquid-Crystal Polarized Light Microscope and Its Practical Use.- In Vitro Maturation and Fertilization of Pig Oocytes.- In Vitro Maturation and In Vitro Fertilization of Human Oocytes.- In Vitro Maturation and Fertilization of Canine Oocytes.- Detection and Measurement of Membrane-Bound Protein Tyrosine Kinases in the Zebrafish Egg.- Single-Cell Method for Estimation of Protein Tyrosine Kinases in the Zebrafish Egg.- Activity of MAPK/p90rsk During Fertilization inMice, Rats, and Pigs.