James R. Trimarchi

Oxidative Phosphorylation-Dependent and -Independent Oxygen Consumption by Individual Preimplantation Mouse Embryos

Abstract The self-referencing electrode technique was employed to noninvasively measure gradients of dissolved oxygen in the medium immediately surrounding developing mouse embryos and, thereby, characterized changes in oxygen consumption and utilization during development. A gradient of depleted oxygen surrounded each embryo and could be detected >50 μm from the embryo. Blastocysts depleted the surrounding medium of 0.6 ± 0.1 μM of oxygen, whereas early cleavage stage embryos depleted the medium of only 0.3 ± 0.1 μM of oxygen, suggesting a twofold increase in oxygen consumption at the blastocyst stage. Mitochondrial oxidative phosphorylation (OXPHOS) accounted for 60–70% of the oxygen consumed by blastocysts, while it accounted for only 30% of the total oxygen consumed by cleavage-stage embryos. The amount of oxygen consumed by non-OXPHOS mechanisms remained relatively constant throughout preimplantation development. By contrast, the amount of oxygen consumed by OXPHOS in blastocysts is greater than that consumed by OXPHOS in cleavage-stage embryos. The amount of oxygen consumed by one-cell embryos was modulated by the absence of pyruvate from the culture medium. Treatment of one-cell embryos and blastocysts with diamide, an agent known to induce cell death in embryos, resulted in a decline in oxygen consumption, such that the medium surrounding dying embryos was not as depleted of oxygen as that surrounding untreated control embryos. Together these results validate the self-referencing electrode technique for analyzing oxygen consumption and utilization by preimplantation embryos and demonstrate that changes in oxygen consumption accompany important physiological events, such as development, response to medium metabolites, or cell death.

Involvement of Mitochondria in Oxidative Stress-Induced Cell Death in Mouse Zygotes

Abstract Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are “immature,” in contrast to “mature” mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 μM for 15 min) hydrogen peroxide (H2O2) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H2O2 induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.

Mitochondrial dysfunction leads to telomere attrition and genomic instability

Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging‐associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging‐associated pathologies.

Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux

Biological systems have very different internal ion compositions in comparison with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasma membrane, we can classify these mechanisms into three types, pumps, porters, and channels. Channels have been extensively studied, particularly since the advent of the patch clamp technique, which opened new windows into ion channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca2+‐ATPase, and porters are more illusive. The technique described in this paper, the self‐referencing, ion‐selective (or Seris) probe, has the ability to monitor the behavior of membrane transport mechanisms, such as the pumps and porters, in near to real‐time by non‐invasively measuring local extracellular ion gradients with high sensitivity and square micron spatial resolution.

Haploidy but Not Parthenogenetic Activation Leads to Increased Incidence of Apoptosis in Mouse Embryos

Abstract Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.

Increased Birefringence in the Meiotic Spindle Provides a New Marker for the Onset of Activation in Living Oocytes

Abstract The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

Noninvasive Measurement of Potassium Efflux as an Early Indicator of Cell Death in Mouse Embryos

Abstract Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 μM H2O2 exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 ± 0.1 μM. Pretreatment with tetraethylammonium inhibited this increase (0.2 ± 0.1 μM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

A non-invasive method for measuring preimplantation embryo physiology

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.

Characterization of oxygen and calcium fluxes from early mouse embryos and oocytes

Despite the importance of the mammalian embryo to clinical and biomedical sciences, the physiology of pre-implantation embryos and oocytes is largely unexplored. For example, although calcium is known to participate in the early events of fertilization (1) and also plays a brief but critical role at each cleavage (2, 3) the regulation of transmembrane calcium flux during the interim between cleavages or during blastocoel formation is unknown. One reason for this gap in our knowledge lies in the difficulty in studying a single oocyte or embryo. The goal of the project reported here is to take advantage of new techniques for monitoring physiological parameters from individual cells and to begin to characterize changes in embryo physiology during development. This work is part of larger study on the viability of the pre-implantation embryo. The self-referencing electrode technique (4) pioneered by the BioCurrents Research Center at the MBL is ideal for exploring the physiology of the early embryo. The technique allows the derivation of flux values by measuring the concentrations of free ions or dissolved oxygen at two probe positions micrometers apart, where the electrode is moved in a square wave oscillation. One pole of this oscillation is brought close to the zona pellucida (within about 1 pm). With this approach, physiological measurements can be obtained from individual oocytes or embryos. Being noninvasive, the self-referencing electrode can be used to record from oocytes or developing embryos without perturbation, an assumption recently confirmed by the successful birth of implanted, post-experimental blastocysts (Trimarchi and Liu, unpub. obs.). We employed self-referencing electrode techniques to characterize calcium and oxygen flux from a series of developmental stages of mouse embryos. The embryos were collected at stages ranging from unfertilized oocytes to expanded blastocysts and cultured in M2 medium according to standard techniques (5). Physiological experiments were conducted in M2 medium with reduced calcium (50 @Q at 37°C in a Faraday box mounted on an airtable. For the measurement of oxygen fluxes, oxygen microelectrodes, with a tip diameter of 2-3 pm, were purchased from Diamond General Development Corp. (Model 723, Ann Arbor, MI). These were calibrated in Nz and air-bubbled dH1O. The measured current was converted to a flux value as previously described (6). The ion-selective electrodes were constructed as described by Smith et al. (4) incorporating a short (30 pm) column of ion-selective resin (ETH 1001: Fluka). Unlike the oxygen electrode response, which is linear with concentration,

Comparing data mining and logistic regression for predicting IVF outcome

Design We utilized Quinlan’s C5.0 decision tree data mining algorithm to retrospectively investigate the predictive power of the 100 parameters that we track for each IVF cycle. The parameters investigated include patient demographics, stimulation regime, response properties, oocyte and embryo parameters and embryo transfer variables. To validate our findings from a statistical point of view we also constructed a statistical model based on logistic regression.