Advances in the fundamental cryobiology of mammalian oocytes

"May 2007"The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita.Thesis (Ph. D.) University of Missouri-Columbia 2007.Having effective means to cryopreserve mammalian oocytes could increase the efficiency of managing populations of laboratory animals, increase the effectiveness of breeding programs for livestock, and improve the means by which assisted reproductive therapy is applied to human patients. Unfortunately, for most mammals oocyte cryopreservation suffers from inefficiencies. The work completed in this dissertation was directed at advancing our knowledge of the fundamental cryobiological properties of oocytes from cows, pigs, and humans. The first series of experiments was designed to determine the likelihood of damage to the metaphase II spindle from osmotic stress. Increasing levels of hypotonic and hypertonic stress resulted in an increased proportion of oocytes displaying a damaged spindle as assessed by immunocytochemical staining. Human oocytes appeared more sensitive to hypertonic stress compared to oocytes from cows and pigs. Hypotonic stress caused more damage to cow oocytes compared to human and pig oocytes. Pig oocytes were also shown to lose in vitro developmental potential, and the proportion damaged was greater compared to the proportion showing damage to the spindle. The permeability of mature human oocytes to ethylene glycol and water was also determined. It was shown that the permeability was temperature dependent. The results from the osmotic tolerance and membrane permeability studies for human oocytes was used to develop a theoretically-optimized procedure for vitrifying human oocytes in standard 0.25 cc straws.Includes bibliographical reference

Vitrification: Fundamental Principles and Its Application for Cryopreservation of Human Reproductive Cells

The fundamental understanding of cryobiology through experimentation in the 1960s, 1970s, and 1980s has led to the development of today’s vitrification technology. Although human embryo and oocyte vitrification was slow to evolve, it has become an invaluable technology in the field of reproductive medicine. The aim of this chapter is to discuss some of the underlying basic principles behind forming a metastable glass phase during rapid cooling in liquid nitrogen (LN2) and the prevention of recrystallization events upon warming. We then highlight how this understanding has led to its highly effective and reliable usage in clinical IVF. Furthermore, we describe how quality control factors (e.g., ease of use, repeatability, reliability, labeling security, and cryostorage safety) can vary between vitrification device systems, potentially influencing clinical outcomes and creating possible liability issues. An open-minded approach to continued experimentation is a necessity, especially pertaining to oocyte freeze preservation, if we are to optimize the vitrification of reproductive cells and tissue in the future

Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity

SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability

Membrane permeability of Bovine Ooctyes to Propylene Glycol and the application to the improvement of Cryopreservation

Abstract only availableIn this study, the goal was to determine the permeability parameters of bovine oocytes for water (Lp) and Propylene Glycol (PPG) at temperatures of 30, 20, 10, and 5°C. By determining permeability parameters, we can model cell volume changes during addition and removal of cryoprotectants to determine a method that will prevent osmotic damage to the cells. Individual oocytes were held stationary by a holding pipette in a Petri dish on a Nikon inverted microscope. The oocytes were initially equilibrated in propylene glycol (PG) and 0.1M Sucrose for 20 minutes and then a solution of TL-Hepes with 0.1M Sucrose was added to a drop of 1.5M PG containing the oocyte. The specific initial concentration of PG and volumes of added solutions were modified for each temperature. Then digital images were captured on a regular time scale using a Spot RT Cooled CCD Digital camera in order to record shrinking and swelling. Morphometrical analysis was then performed on each image using Adobe Photoshop to measure the radius of each oocyte at the various time points during the volume excursions. Using Microsoft Excel, we were able to fit the experimental data to a best fit curve of a theoretical model for volume change, which allowed the determination of the values of Lp and PPG. These values were used to model the cell volume changes using MLAB (Civilized Software, Inc., Bethesda, MD) to developing optimized addition and removal procedures for 3.0M CPA that would minimize potential damage of the oocyte due to shrinking and swelling, and toxicity effects of the CPA due to excessive exposure. Currently, our results for the mean values of the permeability parameters Lp and PPG at 20°C are 0.3 ± 0.03 µm-min.atm and 15 ± 7.2 µm/min, respectively (mean ± SD, n=2). Further data acquisition and analysis is in progress.NSF-REU Program in Biosystems Modeling and Analysi

Bovine oocyte permeability in 1, 2-propanediol

Abstract only availableCryopreservation of oocytes is of great importance both in medical research and in the agricultural industry. Cryopreservation of oocytes allows for future breeding of selected genetic lines of animals. Effective cryopreservation of bovine oocytes has drawn considerable attention due to its application in the agricultural industry. The reproductive cells of genetic lines of cattle can now be cryopreserved and used in the future to breed superior cattle through in vitro fertilization (IVF). Currently, the preservation of bovine oocytes and other cells has had marginal success due to damage sustained to the cell during freezing and thawing due to volume fluctuations (Mazur et al; Experimental Cell Research 71(1972) 345-355). The Kedem and Katchalsky model (Biochem Biophys Acta, 1958, 27:229-246) can be utilized to model changes in cellular volume during freezing and thawing in cryoprotectant solutions. This model takes into account the specific plasma membrane permeability of the cell that is exposed to a particular solution. The purpose of this study was to determine the hydraulic conductivity, Lp, and the permeability coefficient for the cryoprotective agent, 1, 2-propandiol (PrOH), PCPA, for bovine oocytes. The activation energies of each of these parameters can be determined under the assumption that the plasma membrane permeability parameters follow an Arrhenius relationship. Experimental trials were conducted at temperatures of 30°C, 20°C, 10°C, and 4°C. In order to study the response of a single bovine oocyte to 1.5 M 1, 2-propanediol, a micro-pipette holding device (Gao et al.; Biophysics J, 71:443-450) was used to immobilize the oocyte in a small drop of TL-Hepes media. The oocyte was then abruptly exposed to 1.5 M PrOH media. The volume change of the oocyte (dv/dt) was recorded with a digital camera that was mounted to an inverted light microscope. The area of the cell in each image was calculated with a Fovea Pro Software plug-in to Adobe Photoshop. The volume of the cell was calculated from the calculated area, assuming that the cell was spherical. The constants, Lp and PCPA, were numerically approximated assuming that the cellular volume dynamics followed the Kedem and Katchalsky model. It was found that the oocytes underwent osmotically driven volume changes upon exposure to the cryoprotectant, 1, 2-propanediol. The bovine oocytes contracted more rapidly at higher temperatures. The oocytes also regained their isosmotic volume faster at higher temperatures. The permeability parameters found in this study along with there activation energies will be used in the future to develop an optimal cryopreservation protocol for bovine oocytes through computer-based modeling.NSF-REU Biosystems Modelin

Rifaximin has the potential to prevent complications of cirrhosis

Background: Cirrhosis-related complications are associated with poor prognosis. With our analyses, we examined the potential benefit of rifaximin in reducing the risk of developing cirrhosis-related complications. Methods: Adults with cirrhosis and hepatic encephalopathy (HE) in remission were randomly assigned to receive rifaximin 550 mg twice daily or placebo for 6 months with concomitant lactulose permitted. Post hoc analyses examined time to cirrhosis-related complications (HE, spontaneous bacterial peritonitis (SBP), variceal bleeding, acute kidney injury/hepatorenal syndrome). Subgroup analyses evaluated efficacy for select baseline disease characteristics. Results: Of patients receiving rifaximin (n = 140) and placebo (n = 159), 53.6% and 49.1%, respectively, had baseline Model for End-Stage Liver Disease (MELD) score ⩾ 12 and international normalized ratio (INR) ⩾ 1.2. Baseline ascites was observed in 36.4% (rifaximin) and 34.6% (placebo) of patients. In patients with MELD score ⩾ 12 and INR ⩾ 1.2, rifaximin reduced the relative risk (RR) of any first complication experienced during trial by 59% [hazard ratio (HR) = 0.41, 95% confidence interval (CI): 0.25–0.67; p \u3c 0.001] versus placebo. For patients with baseline ascites, rifaximin reduced the RR of any first complication experienced during trial by 42% versus placebo (HR = 0.58, 95% CI: 0.34–1.0; p = 0.045). For some subgroups, there was a decrease in RR of complications of SBP, variceal bleeding, and acute kidney injury/hepatorenal syndrome with rifaximin versus placebo, although there were few events reported in the study. Conclusion: Rifaximin may reduce the incidence of cirrhosis-related complications and the recurrence of overt HE. [ClinicalTrials.gov identifier: NCT00298038.