Human oocyte cryopreservation: past, present and future.

Despite inferior results in the past compared with embryo freezing, oocyte cryopreservation has made great strides in recent years. In fact, it has become a necessity in assisted reproduction technology, providing alternatives to legal, moral and religious problems originating from embryo freezing. Recent advances in freezing technology, modifications of conventional protocols used and continuing optimization of vitrification have efficiently improved the method. A historical description of the method's progression over time, and a comparison of principles, procedures and results as reported in the literature are presented in this review.

Gelsolin binds to polymeric actin at a low rate.

Association of gelsolin with actin filament subunits was investigated by the decrease of the fluorescence intensity of a 7-nitro-2-oxa-1,3-diazole (NBD) label covalently linked to gelsolin. The rate constant of this reaction was found to be 4 x 10(3) M-1 s-1. Binding of NBD-labeled gelsolin to monomeric actin proceeds at a similar low rate. The rate of association of gelsolin that was unmodified to actin filament subunits was estimated too. Unmodified gelsolin was added to a mixture of actin filaments and actin-DNase I complex. The fractions of gelsolin that bound to actin filament subunits or to actin-DNase I complex depended on the relative rates of these two competing reactions. In this way it was possible to estimate the rate constant of association of unmodified gelsolin with actin filament subunits (2 x 10(4) M-1 s-1). Thus, gelsolin associates with actin filament subunits at a rate that is considerably slower than diffusion-controlled and similar to the rate of binding of gelsolin to monomeric actin.

Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin-actin complex.

The rate constant and equilibrium constant of association of an actin monomer with 1:1 gelsolin-actin complex isolated from chicken were measured by using fluorescently labeled actin. According to fluorescence stopped-flow experiments, the rate constant of formation of the 1:2 gelsolin-actin complex from 1:1 gelsolin-actin complex and actin was found to be about 2 x 10(7) M-1 s-1 under conditions where gelsolin binds Ca2+. The rate of dissociation of one actin molecule from the 1:2 gelsolin-actin complex was determined by exchange of actin for fluorescently labeled actin. The rate constant of dissociation was about 0.02 s-1. Thus, the equilibrium constant for association of actin with 1:1 gelsolin-actin complex can be calculated to be in the range of 10(9) M-1. The rate of dissociation of actin from 1:2 gelsolin-actin complex was independent of the Ca2+ concentration. Ca2+ affects only the rate of association of actin with 1:1 gelsolin-actin complex.

Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization.

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomere. Formed filaments keep a constant length of about 1 micron. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomeres and can stabilize the pointed ends of actin filaments. We observed that tropomyosin-troponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.