Initiation and Organization of Events During the First Cell Cycle in Mammals: Applications in Cloning

Abstract

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca²⁺]_i) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca²⁺ channel in the endoplasmic reticulum and release Ca²⁺ into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca²⁺]_i increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca²⁺]_i activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca²⁺]_i oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca²⁺]_i and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca²⁺]_i elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.