Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions

Abstract

To explore primate fertilization, oocytes and zygotes from fertile rhesus monkeys were imaged throughout fertilization, polyspermy, and artificial activation using confocal microscopy for microtubules and DNA, as well as ratiometric computer-enhanced video microscopy for intracellular calcium. Unfertilized oocytes displayed microtubules only in the radially oriented meiotic spindles. At insemination, a large calcium transient was followed by a series of smaller oscillations, and sperm astral microtubules had assembled from the sperm centrosome by 2.5 h after transient onset. This aster enlarged, and later duplicated, as the pronuclei converged near the cortex. Pronuclear apposition was prevented by microtubule inhibitors. At mitotic prophase, microtubules ensheathed both sets of condensing chromosomes. At metaphase, the spindle was barrelshaped and eccentrically positioned with two small asters at the pole with the sperm tail. Microtubules emanating from the telophase spindle interacted with the adjacent cortex and displaced the spindle toward the cell center as first cytokinesis ensued. During polyspermy, each sperm nucleated an aster, and the frequency of calcium oscillations increased. Activation resulted initially in disarrayed microtubules that eventually organized into functional mitotic spindles. These kinetic results demonstrate that rhesus monkeys accomplish fertilization in a fashion nearly identical to that of humans and are, therefore, ideal models in which to investigate cytoskeletal events during human reproduction.