Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture

Sun F, Betzendahl I, Van Wemmel K, et al. Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS. 2008;651(1-2):114-124.Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 mu g/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 mu g/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 mu M) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression. at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization. (c) 2007 Elsevier B.V. All rights reserved

Cryopreserved subcutaneous adipose tissue for fat graft

Cryopreservation of subcutaneous white adipose tissue (sWAT) avoids multiple surgeries in patients subjected to reconstructive procedure. Fat grafts were performed subcutaneously on 26 mice treated with fresh (13 mice) or cryopreserved (13 mice) human sWAT. Cytofluorometry for CD marker expression of stem cells, differentiation capability, and in vivo survival of fat grafts were evaluated. In vitro analysis evidenced that cryopreservation did not affect the stem potential of samples. In vivo MRI showed that grafts were well preserved in 13 mice treated with fresh sWAT, whereas in 13 animals treated with thawed fat, graft volumes were strongly reduced after 1\ua0week. Ultrastructural studies performed both on fresh and thawed specimens demonstrated that grafts performed with thawed sWAT are able to store lipids more slowly with respect to grafts performed with fresh sWAT and adipocytes maintained a multilocular appearance. Collected data demonstrated that the protocol of cryopreservation could maintain the regenerative capability of the sWAT, but the rate of reabsorption after fat grafting is higher using cryopreserved sWAT. Maintaining the stem potential of sWAT after cryopreservation is a very important aspect for reconstructive and regenerative medicine. The employment of cryopreserved sWAT represents an interesting goal for surgeons. Surely there is the necessity to improve the protocol of cryopreservation

DNA integrity, growth pattern, spindle formation, chromosomal constitution and imprinting patterns of mouse oocytes from vitrified pre-antral follicles

Trapphoff T, El Hajj N, Zechner U, Haaf T, Eichenlaub-Ritter U. DNA integrity, growth pattern, spindle formation, chromosomal constitution and imprinting patterns of mouse oocytes from vitrified pre-antral follicles. Human Reproduction. 2010;25(12):3025-3042.Cryopreservation of follicles for culture and oocyte growth and maturation in vitro provides an option to increase the number of fertilizable oocytes and restore fertility in cases where transplantation of ovarian tissue poses a risk for malignant cell contamination. Vitrification for cryopreservation is fast and avoids ice crystal formation. However, the influences of exposure to high concentrations of cryoprotectants on follicle development, oocyte growth and maturation, and particularly, on the DNA integrity and methylation imprinting has not been studied systematically. Follicle survival and development, DNA damage, oocyte growth patterns, maturation, spindle formation and chromosomal constitution were studied after Cryo-Top vitrification of mouse pre-antral follicles cultured to the antral stage and induced to ovulate in vitro. Methylation of differentially methylated regions (DMRs) of two maternally (Snrpn and Igf2r) and one paternally (H19) imprinted genes was studied by bisulfite pyrosequencing. Vitrification results in partial or total loss of oocyte-granulosa cell apposition and actin-rich transzonal projections, a transient increase in DNA breaks and a delay in follicle development. However, the oocyte growth pattern, maturation, spindle and chromosomal constitution are not significantly different between the vitrified and the control groups. Vitrification is not associated with elevated levels of imprinting mutations (aberrant methylation of the entire DMR), although the distribution of sporadic CpG methylation errors in the Snrpn DMR appears to differ slightly between control and vitrified oocytes. DNA breaks appear to be rapidly repaired and vitrification of oocytes inside pre-antral follicles by the Cryo-Top method does not appear to increase risks of abnormal imprinting or disturbances in spindle formation and chromosome segregation

Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected

Demant M, Trapphoff T, Froehlich T, Arnold GJ, Eichenlaub-Ritter U. Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected. Human Reproduction. 2012;27(4):1096-1111.Vitrification is a fast and effective method to cryopreserve ovarian tissue, but it might influence mitochondrial activity and affect gene expression to cause persistent alterations in the proteome of oocytes that grow and mature following cryopreservation. In part one of the study, the inner mitochondrial membrane potential ((mit)) of JC-1 stained oocytes from control and CryoTop vitrified pre-antral follicles was analyzed by confocal microscopy at Day 0, or after culture of follicles for 1 or 12 days. In part two, proteins of in vivo grown germinal vesicle (GV) oocytes were subjected to proteome analysis by SDS polyacrylamide gel electrophoresis, tryptic in-gel digestion of gel slices, and one-dimensional-nano-liquid chromatography of peptides on a multi-dimensional-nano-liquid chromatography system followed by mass spectrometry (LC-MS/MS) and Uniprot Gene Ontology (GO) analysis. In part three, samples containing the protein amount of 40 GV and metaphase II (MII) oocytes, respectively, from control and vitrified pre-antral follicles cultured for 12 or 13 days were subjected to 2D DIGE saturation labeling and separated by isoelectric focusing and SDS gel electrophoresis (2D DIGE), followed by DeCyder(Tm) analysis of spot patterns in three independent biological replicates. Statistical and hierarchical cluster analysis was employed to compare control and vitrified groups. (i) Mitochondrial inner membrane potential differs significantly between control and vitrified GV oocytes at Day 0 and Day 1, but is similar at Day 12 of culture. (ii) LC-MS/MS analysis of SDS gel fractionated protein lysates of 988 mouse GV oocytes revealed identification of 1123 different proteins with a false discovery rate of 1. GO analysis assigned 811 proteins to the obiological process' subset. Thirty-five percent of the proteins corresponded to metabolic processes, about 15 to mitochondrion and transport, each, and close to 8 to oxidation-reduction processes. (iii) From the 2D-saturation DIGE analysis 1891 matched spots for GV-stage and 1718 for MII oocyte proteins were detected and the related protein abundances in vitrified and control oocytes were quantified. None of the spots was significantly altered in intensity, and hierarchical cluster analysis as well as histograms of p and q values suggest that vitrification at the pre-antral stage does not significantly alter the proteome of GV or MII oocytes compared with controls. Vitrification appears to be associated with a significant transient increase in (mit) in oocyte mitochondria, which disappears when oocyte/cumulus cell apposition is restored upon development to the antral stage. The nano-LC-MS/MS analysis of low numbers of oocytes is useful to obtain information on relevant biological signaling pathways based on protein identifications. For quantitative comparisons, saturation 2D DIGE analysis is superior to LC-MS/MS due to its high sensitivity in cases where the biological material is very limited. Genetic background, age of the female, and/or stimulation protocol appear to influence the proteome pattern. However, the quantitative 2D DIGE approach provides evidence that vitrification does not affect the oocyte proteome after recovery from transient loss of cellcell interactions, in vitro growth and in vitro maturation under tested conditions. Therefore, transient changes in mitochondrial activity by vitrification do not appear causal to persistent alterations in the mitochondrial or overall oocyte proteome