Amrane, S. | bioRxiv Science

bioRxiv2025

Low nucleus to cytoplasmic ratio compromises cytokinesis, genome stability and development in haploid embryonic cells

Mammalian species exist as diploid organisms, with only gametes existing as stable, nonproliferating haploids. Haploid mammalian pluripotent stem cells, in all derived species to date - mouse, human and rat - undergo spontaneous diploidization. Here, we investigated the mechanisms affecting the stability of the haploid state using the mouse and human embryo, as well as a human embryonic stem cells. We demonstrate that diploidization occurs early in preimplantation development and is often unproductive, with haploid embryos exhibiting decreased developmental potential. Haploid embryos show increased chromosome segregation errors at the first mitosis, failure to align chromosomes on the metaphase plate in the second mitosis. Delayed mitotic progression and failure to form a central spindle and a midbody are followed by cytokinesis failure, diploidization, increased DNA damage marked by {gamma}H2AX and RPA32 foci, and low developmental potential. By increasing the nucleo-cytoplasmic ratio in diploids, or reducing this ratio in haploids, these phenotypes can be induced in diploids or reduced in haploids. Changing ploidy alone from haploid to diploid without also adjusting nucleo-cytoplasmic ratio does not improve mitotic phenotypes and developmental outcomes. Thus, the most upstream driver responsible for the instability of the haploid state is a stage- specific ratio of nucleus to cell size.

cell biology↗

bioRxiv2018

Early detection of impending cryostorage tank failure using a weight-based monitoring system

ObjectTo study the ability of custom-built, web-enabled scales to monitor liquid nitrogen (LN2) levels in cryostorage dewars.\n\nDesignLaboratory study\n\nSettingA large academic fertility center in New York City.\n\nInterventionsCryostorage dewars were placed on top of the custom-engineered scales with continuous real-time monitoring, and weight and temperature data were recorded in the setting of slow, medium, and fast rate-loss of LN2 designed to mimic models of tank failures.\n\nMain Outcome MeasuresWeights were continuously monitored and recorded, with a calculated alarm trigger set at 10% weight loss. Temperature within the tanks was simultaneously monitored with probes placed near the top of the tanks, with calculated alarms using a -185 {degrees}C as the threshold. For the \"slow rate-loss\" simulations, tanks were left intact and closed in usual operating conditions, and LN2 was allowed to evaporate at the normal rate. For the \"medium rate-loss\" simulation, the foam core of the tank neck was removed and the insulating vacuum was eliminated by making a 1/16 inch hole in the outer tank wall. For the \"fast rate-loss\" simulation, a 1/16\" hole was made through the outer tank wall and LN2 was released at a rate of 0.15 L/second. All simulations were performed in duplicate.\n\nResultsWith an intact and normally functioning tank, a 10% loss in LN2 occurred in 4.2-4.9 days. Warming to -185 {degrees}C occurred in 37.8 - 43.7 days, over 30 days after the weight-based alarm was triggered. Full evaporation of LN2required 36.8 days. For the medium rate-loss simulation, a 10% loss in LN2 occurred in 0.8 h. Warming to -185 {degrees}C occurred in 3.7 - 4.8 hours, approximately 3 hours after the weight-based alarm was triggered. For the fast rate-loss simulation, a 10% weight loss occurred within 15 seconds and tanks were completely depleted in under 3 minutes. Tank temperatures began to rise immediately and at a relatively constant rate of 43.9 {degrees}C/hour and 51.6 {degrees}C/hour. Temperature alarms would have sounded within 0.37 and 0.06 hours after the breech.\n\nConclusionsThis study demonstrates that a weight-based, automated alarm system can detect tank failures prior to a temperature-based alarm system, in some cases over a month in advance. In combination with existing safety mechanisms such as temperature probes, a weight-based monitoring system could serve as a redundant safety mechanism for added protection of cryopreserved reproductive tissues.

developmental biology↗