The distribution and speciation of trace elements in bovine ovaries using synchrotron techniques

Abstract

When studying the relationship of trace elements with human health, it becomes increasingly evident that keeping a balanced level of minerals in every cell of the human body may be crucial to optimising health and preventing disease. Dietary deficiencies of trace elements have been reported to alter various aspects of reproductive physiology, however, there is an alarming weighting towards studies focusing on their involvement in male reproductive function. Furthermore, those studies which are female focused have a tendency to investigate the role of trace elements in maintaining a healthy pregnancy, and the impact on offspring, as opposed to addressing preconception ovarian function. This research predominantly used a combination of two synchrotron-based X-ray techniques, X-ray absorption spectroscopy (XAS) and X-ray fluorescence (XRF) imaging, to investigate the role that trace elements play in female reproductive function. More specifically, by probing the in situ bioaccumulation of trace elements in bovine ovaries, important findings were made regarding the distribution of iron (Fe), selenium (Se), and zinc (Zn) which were localised to specific structures. Additionally, widespread high levels of bromine (Br) were observed across all imaged tissues. By classifying the ovarian follicles according to their health status, as well as their size (follicular diameter), and extracting average elemental concentrations for these follicles from the XRF images, it was possible to quantitatively analyse the cohort of 97 imaged follicles. Statistically significant differences were found for the elements Fe and Br (health status), and for Se (size). Se appeared to be the element which most greatly distinguished large (> 10 mm) antral follicles from smaller counterparts and PCA scores plots supported this notion with the large follicles deviating from the rest of the cohort. Owing to the statistically significant and very precise localisation of Se to the granulosa cells of large healthy follicles, a suite of additional biologically-based experiments were performed (quantitative real-time reverse transcription polymerase chain reactions (qRTPCR), immunohistochemical staining, and Western immunoblotting) in order to identify the Se species as glutathione peroxidase 1 (GPx1). Taking into account what is already known about this selenoprotein, it was concluded that Se and selenoproteins may play a critical role as an antioxidant during late follicular development. There has been much debate about the essentiality of Br to human and animal life. Owing to its high levels across all ovarian tissues imaged, subsequent experimentation was carried out to probe the chemical form of this element. Analyses of the X-ray absorption near-edge structure (XANES) spectra of a variety of mammalian tissues and fluids led to the conclusion that the form of Br in all samples, detected under normal physiological conditions, was bromide. The application of synchrotron radiation to measure trace elemental distributions in bovine ovaries at such high resolutions has provided new insights into this organ. While this research was intended to form a baseline study for healthy ovaries, if extended to disease states in the future, our understanding of the biochemical mechanisms occurring in this complex organ could be significantly enhanced.