Mouse oocytes develop in cysts with the help of nurse cells

Mammalian oocytes develop initially in cysts containing many more germ cells than the primordial oocytes they generate. We identified abundant nurse cells with reduced unique molecular identifiers (UMI)/cell from ovaries aged E14.5 to P1. Low UMI nurse cells are found in cysts and express the same major meiotic genes as pro-oocytes of the same stage, suggesting they are oocyte sisters that are signaled to transfer cytoplasm at different times and only subsequently diverge. Oocyte vs nurse cell selection occurs in cysts with a robust microtubule cytoskeleton, that closely interact with somatic cells and that develop a dense actin cytoskeleton around nurse cell nuclei that are held back from cytoplasmic transfer. Mouse and Drosophila nurse cells undergo programmed cell death by acidification from adjacent somatic pre-granulosa cells that express V-ATPases and cathepsin proteins. Disrupting acidification in cultured mouse ovaries blocked nurse cell turnover. About 200 genes are induced in mouse dictyate oocytes as previously reported, including Tuba1c and Tubb2b, genes that we find contribute to Balbiani body formation. Thus, mouse oocytes are specified within germline cysts and develop with the assistance of nurse cells using highly conserved mechanisms.

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory “aged” oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

Cytoplasmic Transfer Improves Human Egg Fertilization and Embryo Quality: an Evaluation of Sibling Oocytes in Women with Low Oocyte Quality

The aim of this study was to evaluate if cytoplasmic transfer can improve fertilization and embryo quality of women with oocytes of low quality. During ICSI, 10–15% of the cytoplasm from a fresh or frozen young donor oocyte was added to the recipient oocyte. According to the embryo quality, we defined group A as patients in which the best embryo was evident after cytoplasmic transfer and group B as patients in which the best embryo was evident after a simple ICSI. We investigated in the period of 2002–2018, 125 in vitro fertilization cycles involving 1011 fertilized oocytes. Five hundred fifty-seven sibling oocytes were fertilized using ICSI only and 454 oocytes with cytoplasmic transfer. Fertilization rates of oocytes were 67.2% in the cytoplasmic transfer and 53.5% in the ICSI groups (P < 0.001). A reduction in fertilization rate was observed with increased women age in the ICSI but not in the cytoplasmic transfer groups. The best embryo quality was found after cytoplasmic transfer in 78 cycles (62.4%) and without cytoplasmic transfer in 40 cycles (32%, P < 0.001). No significant differences were detected between the age, hormonal levels, dose of stimulation drugs, number of transferred embryos, pregnancy rate and abortion rate between A and B groups. Cytoplasmic transfer improves fertilization rates and early embryo development in humans with low oocyte quality. All 28 children resulting from cytoplasmic transfer are healthy.

Potential hydrodynamic cytoplasmic transfer between mammalian cells: Cell-projection pumping

ABSTRACTWe earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fib) and malignant cells (MC). Others report similar transfer via either tunneling nanotubes (TNT) or shed membrane vesicles and this changes the phenotype of recipient cells. Our current time-lapse microscopy showed most exchange was from Fib into MC, with less in the reverse direction. Although TNT were seen, we were surprised transfer was not via TNT, but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless, by their organellar cargo and the grooves they formed indenting MC, while this was consistent with holotomography. Discrete, rapid and highly localized transfer events, evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell-projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell-projections normally returns cytoplasm to the cell body. We hypothesize ‘cell-projection pumping’ (CPP), where cytoplasm in retracting cell-projections partially equilibrates into adjacent recipient cells via micro-fusions that form temporary inter-cellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modelling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure towards least resistance. Predictions from the model were satisfied when Fib were co-cultured with MC, and fluorescence exchange related with cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MC or Fib was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian inter-cellular cytoplasmic transfer and communication.SIGNIFICANCETime-lapse observations of co-cultured cells led us to hypothesize what we believe to be a novel hydrodynamic mechanism transferring cytoplasm between cells. Similar transfer by other mechanisms markedly affects cell behavior. Combined mathematical modelling, satisfaction of predictions from the mathematical model in cell culture experiments, and separate computer simulations that generate outcomes similar to experimental observations, support our hypothesized mechanism.