Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity.

BACKGROUND: In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART). RESULTS: In this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development. CONCLUSIONS: Our observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus-oocyte complexes with lissamine green B.

We evaluated the usefulness of lissamine green B (LB) staining of cumulus-oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Analyzing and Modeling the Kinetics of Amyloid Beta Pores Associated with Alzheimer's Disease Pathology.

Amyloid beta (Aß) oligomers associated with Alzheimer's disease (AD) form Ca2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca2+) homeostasis. The resultant up-regulation of intracellular Ca2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca2+ permeability of Aß pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aß pores. The fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aß pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. Towards the end, we demonstrate the up-regulation of gating of various Ca2+ release channels due to Aß pores and show that the extent and spatial range of such up-regulation increases as Aß pores with low open probability and Ca2+ permeability transition into those with high open probability and Ca2+ permeability.

[Genotoxicity risk assessment and oocytes: basis of genetic toxicology and application in reproductive science]. / Risque génotoxique et ovocytes : principes de toxicologie génétique et applications.

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line and on mouse oocytes.

Cryopreservation of germinal vesicle stage porcine oocytes based on intracellular ice formation assessment.

This study aimed at evaluating the feasibility of slow freezing for cryopreservation of germinal vesicle (GV) stage porcine oocytes. In this study, intracellular ice formation (IIF) characteristics of GV porcine oocytes were investigated by using a thermoelectric cooling (TEC) cryomicroscope system. This cryomicroscope system used a thermoelectric cooling (TEC) chip in its cold stage as a heat sink and employed a PID control algorithm to achieve accurate temperature control. The temperature was controlled to a range between 70 degree C and -55 degree C with an accuracy of +/- 0.5 degree C. Five constant cooling rates of 24, 12, 6, 3 and 1.5 degree C/min were tested in experiments in freezing GV porcine oocytes from 20 degree C to -50 degree C in an NCSU-23 medium plus 2.0 M DMSO. The IIF temperature of each individual oocyte was recorded and cumulative IIF probabilities were calculated for each cooling rate. The total cumulative probabilities of IIF temperature distribution were 100 percent, 100 percent, 50.0 percent, 54.3 percent and 58.6 percent at cooling rates of 24, 12, 6, 3 and 1.5 degree C/min, respectively. A Weibull distribution model was found to adequately describe the distribution of IIF temperatures of GV porcine oocytes for the cooling rates tested (R2 = 0.858 +/- 0.09). The IIF experimental results indicate that cooling rates of 6, 3 and 1.5°C/min could be considered as possible cryopreservation protocols. Further experiments were performed to examine the feasibility of using these protocols to cryopreserve GV porcine oocytes. After 44 h of in-vitro maturation in NCSU-23, the survival of thawed oocytes was checked. Porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining, followed by Lacmoid staining as a secondary check. Normalized survival rates of 37.7 +/- 4.6 percent, 45.0 4.4 percent and 45.4 +/- 5.9 percent were obtained for GV oocytes frozen at 1.5, 3 and 6 degree C/min, respectively. The experimental results indicate that slow freezing is a feasible approach for cryopreservation of GV porcine oocytes when cooling rate is properly selected. This study also demonstrated an efficient approach for investigating optimal cooling rates by assessing the IIF characteristics of GV porcine COCs.

Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes.

Using reverse transcription and real-time quantitative PCR analysis we evaluated the transcript levels of integrins (alphaL, alphaM, beta1, and beta6), CD9 and CD18 antigens as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes isolated from puberal gilts (n=20) and multiparous sows (n=20). We found significantly (p<0.05) higher transcript contents of alphaL, alphaM, beta1, and beta integrins, CD9 antigen, and pZP2 and pZP3 in puberal gilt oocytes compared to multiparous sow oocytes. Our results suggest that a decrease in the level of oocyte transcripts encoding essential proteins involved in oocyte fertilization may be associated with increased porcine female age.

A physical model of potassium channel activation: from energy landscape to gating kinetics.

We have developed a method for rapidly computing gating currents from a multiparticle ion channel model. Our approach is appropriate for energy landscapes that can be characterized by a network of well-defined activation pathways with barriers. To illustrate, we represented the gating apparatus of a channel subunit by an interacting pair of charged gating particles. Each particle underwent spatial diffusion along a bistable potential of mean force, with electrostatic forces coupling the two trajectories. After a step in membrane potential, relaxation of the smaller barrier charge led to a time-dependent reduction in the activation barrier of the principal gate charge. The resulting gating current exhibited a rising phase similar to that measured in voltage-dependent ion channels. Reduction of the two-dimensional diffusion landscape to a circular Markov model with four states accurately preserved the time course of gating currents on the slow timescale. A composite system containing four subunits leading to a concerted opening transition was used to fit a series of gating currents from the Shaker potassium channel. We end with a critique of the model with regard to current views on potassium channel structure.

Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei.

The nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically. In vitro fertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.