CEDIE   05498
CENTRO DE INVESTIGACIONES ENDOCRINOLOGICAS "DR. CESAR BERGADA"
Unidad Ejecutora - UE
capítulos de libros
Título:
Exploring the cytoskeleton during Intracytoplasmic sperm injection in humans
Autor/es:
RAWE V; CHEMES H.E.
Libro:
Methods in Molecular Biology
Referencias:
Año: 2006;
Resumen:
The interaction of mammalian spermatozoa with the oocyte after gamete fusion is a complex and meticulously orchestrated cascade of events. While intracytoplasmic sperm injection (ICSI) is an efficient treatment for male infertility (Palermo et al., 1992), there is a significant number of clinical cases of fertilization failure that remain unclear (Asch et al., 1995; Rawe et al., 2000). The cytoskeleton has numerous and diverse  functions  during fertilization (Longo, 1989). The mouse oocyte and zygote is the most useful and popular model for studying in vitro gametes development and fertilization events but there are huge differences in terms of cytoskeletal organization compared with humans (Menezo and Herubel, 2002). During human fertilization, the microtubules, but not the actin filaments (rodents), are required for pronuclear migration and apposition as well as the formation of the first mitotic spindle. Additionally, the centrosome degenerates in the oocyte and is retained in the sperm during the maturation process. Contrarily to rodents, human spermatozoa contribute the centrosome during fertilization (Schatten, 1994), which serves as the dominant microtubule-organizing center in the zygote, and nucleates microtubules from a central structure known as the g-tubulin ring complex. In summary, attention must be taken to avoid extrapolations between animal  models and humans. The direct study of the internal organization of the human egg and sperm cytoskeleton and the topographical view of the whole flagellum provides new knowledge in the improvement of infertility treatments in humans. Despite extensive research in the area of human reproductive biology, much is still to be understood at the cellular level of how eggs control the assembly and disassembly of sperm’s components. Understanding the role of the cytoskeleton during ICSI is an important step to improve fertilization. The aim of this chapter is to show methods for studying cytoskeletal features during in vitro fertilization after ICSI among humans. The following protocols are going to provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes and sperm’s components by fluorescence (Confocal and Epifluorescence) and electron microscopy.