Comparison and Avoidance of Toxicity of Penetrating Cryoprotectants

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well

Dormant Pathogenic CD4(+) T Cells Are Prevalent in the Peripheral Repertoire of Healthy Mice

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3− T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3− cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including selfpeptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage

Self and Microbiota-Derived Epitopes Induce CD4⁺ T Cell Anergy and Conversion into CD4⁺Foxp3⁺ Regulatory Cells

The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4+CD44+Foxp3−CD73+FR4+ anergic (Tan) cells expands the CD4+Foxp3+ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4+CD44-Foxp3− T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation

Optimization of Cryoprotectant Loading into Murine and Human Oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me₂SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me₂SO exposure time, revealing that neither shrinkage nor Me₂SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me₂SO addition appears to result from interactions between the effects of Me₂SO toxicity and osmotic stress. We also investigated Me₂SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me₂SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me₂SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.Keywords: Propane-1,2-diol, Freezing, Propylene glycol, DMSO, Vitrification, Cryopreservation, Mouse, Cryoprotectant, Dimethyl sulfoxide, Simplex optimization, Oocyte, Human, Me₂S

Toxicity of penetrating CPAs at room temperature.

<p>Ovulated mouse oocytes were exposed to a 1.5 M solution of each CPA at RT (∼23°C) for 15 minutes and evaluated for their (A) post-exposure survival, fertilization, and embryonic development, as well as for their (B) ploidy and (C) parthenogenetic activation. Data shown are mean±SEM except for the ploidy rates, which represent percentage of total number of euploid eggs. The total number of oocytes (n) used in each group was also shown. * denotes significant differences in survival and parthenogenetic activation (<i>p</i><0.05).</p

Dose-dependence of the toxicity of PROH.

<p>Ovulated mouse oocytes were exposed to a 0.75 M solution of PROH at both RT and 37°C for 15 minutes and then evaluated for their (A) post-exposure survival, fertilization, and embryonic development, as well as for their (B) parthenogenetic activation rate. Data shown are mean±SEM. The total number of oocytes (n) used in each group was also shown. There was no significant difference between the groups (<i>p</i>>0.05).</p

Time- and temperature-dependence of CPA toxicity.

<p>Ovulated mouse oocytes were exposed to a 1.5 M solution of each CPA at 37°C for 0, 15, and 30 minutes and evaluated for their (A) post-exposure survival, (B) fertilization, (C) embryonic development, (D) parthenogenetic activation, and (E) ploidy. Data shown are mean±SEM except for the ploidy rates, which represent percentage of total number of euploid eggs. The total number of oocytes (n) used in each group was also shown. The differences between the control, DMSO and EG groups were not significant while only a few oocytes survived exposure to 1.5 M PROH at 37°C. Therefore, parthenogenetic activation and ploidy were not evaluated in the PROH group. N/A: not applicable.</p

Avoidance of CPA toxicity and improvement of the cryosurvival.

<p>(A) Post-exposure survival, fertilization, and embryonic development. PROH and DMSO were combined using half (i.e., 0.75 M) of their typical concentrations to bring the total CPA concentration to a cryoprotective level without a significant toxic effect. Subsequently, ovulated mouse oocytes were exposed to a 1.5-M mixture of PROH and DMSO at both RT and 37°C for 15 minutes, and then were evaluated for their post-exposure survival, fertilization, and embryonic development rates. Data shown are mean±SEM. The total number of oocytes (n) used in each group was also shown. There was no significant difference between the groups (<i>p</i>>0.05). (B) Post-thaw survival rates. Ovulated mouse oocytes were loaded with either 0.75 M PROH+0.75 M DMSO or 1.5 M DMSO at RT for 15 minutes, and then subjected a freeze-thaw cycle to evaluate the cryoprotection of the combined 0.75-M concentrations of PROH and DMSO with respect to a commonly used concentration of DMSO alone. Data shown are mean±SEM. The total number of oocytes (n) used in each group was also shown. * denotes significant difference in the cryosurvival (<i>p</i><0.001).</p

Probing lasting cryoinjuries to oocyte-embryo transcriptome.

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods