A diploid genome is necessary for normal mammalian development, thus haploid parthenogenetic embryos undergo frequent self-diploidization during preimplantation development; however, the underlying mechanism is unclear. In this study, time-lapse recording revealed that human haploid parthenotes (HPs) undergo self-diploidization via failed cytokinesis (FC) and endomitosis (EM). The frequencies of FC/EM were significantly higher in HPs than in normal fertilized embryos (26.3% vs. 1.6%, P < 0.01; 19.7% vs. 0, P < 0.01), and above 90% of FC/EM occurred at the first cell cycle in HPs. Fluorescent in situ hybridization of chromosome 16,18 and X in HPs identified diploid recovery after the appearance of FC/EM, and FC/EM HPs showed improved blastocyst formation compared with non-FC/EM HPs (18.8% and 40.0% vs. 15.4%, P > 0.05). In 66.7% of the 1-cell stage HPs, furrow ingression was not observed during the time for normal cleavage, and both immunostaining and gene expression analysis of 1-cell stage HPs revealed the absence or down-regulation of several key genes of the Rho pathway, which regulates cytomitosis. Our results suggested that the major mechanism for self-diploidization is Rho pathway inhibition leading to FC/EM in the first cell cycle, and fine-tuning of this signalling pathway may help to generate stable haploid embryos for stem cell biology studies.

In this report, we establish a straightforward method for estimating the equilibrium constant for the creatine kinase reaction (CK K_eq″) over wide but physiologically and experimentally relevant ranges of pH, Mg²⁺ and temperature. Our empirical formula for CK K_eq″ is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we were able to obtain improved estimates of ΔG_ATP, necessitating the reinvestigation of previously reported ADP- and ΔG_ATP-dependent processes. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies and pH is not altered, the maximum Ca²⁺-activated isometric force depends on ΔG_ATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis around the role of submicromolar ADP in force generation.

In this report, we establish a straightforward method for estimating the equilibrium constant for the CK reaction over wide but physiologically and experimentally relevant ranges of pH, Mg2+ and temperature. Our empirical formula for CK Keq” is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells, and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism, and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we could estimate ΔGATP. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies but not when pH is altered, maximum Ca2+-activated isometric force depends on ΔGATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis for the role of submicromolar ADP in force generation.