ABSTRACT OF THE THESIS
Ovarian Follicle Toxicity Due to Low Dose Gamma Radiation: Determination of Persistence of Oxidative Protein, Lipid, and DNA Damage
By
Stephen Craft
Master of Science in Environmental Health Sciences
University of California, Irvine, 2023
Professor Ulrike Luderer, Chair
In November 2022, Artemis I successfully tested transport and automated docking systems for future missions that will ultimately usher in a new era for the United States National Aeronautics and Space Administration (NASA) and deep space exploration to Mars and beyond (1,2,3). Exposure to radiation in the form of galactic cosmic radiation (GCR) is likely one of the most dangerous of the five categories of hazards to be encountered by astronauts as we begin exploration beyond the Earth’s protective geomagnetic fields (7). GCR is known to cause damage to ovarian follicles resulting in sterility and other related reproductive health problems, and with forty percent of the current 2021 Class of NASA astronauts consisting of female astronauts, a large portion of the team would be at risk (5,7).
In this study, we sought to build on previous research by quantifying the effects of dose-dependent gamma (γ) radiation on persistence of ovarian follicular oxidative lipid, protein, and DNA damage. This data will provide valuable comparison data for future studies looking at an overall mechanistic model and investigations into mixed-ion exposure effects like those encountered by astronauts in deep space exploration.
Methods: Three-month-old C57BL/6J female mice were exposed to γ-radiation at doses of 0, 15, or 50cGy at the UCI Department of Radiation Oncology cesium 137 source. Subject mice were euthanized one week after exposure and one ovary was fixed and stained for ovarian follicle counts. The other ovary was fixed for immunostaining using 4-Hydroxy-2-Nonenal (4-HNE), phosphorylated histone H2AFX (γ-H2AX), and nitrotyrosine (NTY) antibodies to identify oxidative lipid, double-strand DNA breaks, and protein damage respectively. Follicles with immuno-positive and negative oocytes and granulosa cells were counted and the percentages of positive follicles were calculated. We hypothesized that γ-radiation will cause a positive linear dose-dependent persistence of ovarian follicular oxidative stress at all follicular maturation stages.
Results: 137Cs y-radiation induced a general trend towards significance for dose-dependent persistence of oxidative lipid damage in granulosa cell positive primary and secondary follicles when directly comparing the 0cGy and 15cGy dose groups and oocyte positive primordial and primary follicles when directly comparing the 0cGy and 15cGy dose groups. Positive dose-dependent persistence of double-strand DNA damage also trended towards significance in the 15cGy dose group for oocyte immunofluorescence staining when directly comparing primordial, primary, and secondary follicle maturation stages and primordial to secondary follicles for positive granulosa cell staining. Nitrotyrosine immunofluorescence staining for oxidative protein damage was ultimately inconclusive due to technical issues with the dual-antibody protocol.
Conclusions: Our data indicate that in vivo γ-radiation of mice does not result in dose-dependent increased persistence of dsDNA breaks and oxidative lipid damage in the ovaries at one week after irradiation. These findings are inconsistent with published data from heavy oxygen and charged iron studies. Ultimately, further investigation could be completed with varied mouse genetic strains, increased testing iterations to increase the power of the study data, and additional dose groups to elucidate any possible dose-response relationship. Finally, the newly developed immunofluorescence protocol will greatly increase the speed and reliability of data acquisition for these types of studies in the future.
