Raman spectroscopic investigation of the murine oocyte
Davidson, Bryony Kathryn
Over recent years, the application of assisted reproductive techniques in the treatment of infertility has increased exponentially, yet these methodologies still remain inherently inefficient. It has long been established that the single greatest obstacle to improving the success of these treatments is determining the quality of the oocytes used. However, currently the methods available for oocyte assessment are mainly qualitative, and suffer due to a lack of standardisation. As such, the efficiency of fertility treatments could benefit from the introduction of a rigorous quantitative measure of oocyte quality and maturation. The principal aim of this thesis was to determine the potential of Raman spectroscopy when applied to the field of oocyte biology. Consequently, this thesis addressed three main areas of investigation: I. the intra-oocyte biochemical variation; II. the biochemistry of oocyte maturation; and finally, III. the effect of environment on the mature oocyte in vivo and in vitro. I. To investigate the presence of intra-oocyte biochemical variation, oocytes from various stages of development were analysed using high resolution Raman mapping, in combination with univariate and multivariate analysis. Images revealed variation between the germinal vesicle and ooplasm in immature oocytes, as well as intra-ooplasmic variation in all oocytes. II. The spectral analysis of oocytes derived from pre-antral and in vitro cultured follicles revealed significant variation: It was found that Raman spectroscopy could successfully discriminate between immature and mature oocytes. III. Finally, the spectral analysis of oocytes derived from unstimulated and stimulated ovulation cycles, as well as those derived from in vitro follicle cultures, revealed that although biochemically similar, in vitro matured oocytes demonstrated reduced protein content. Furthermore, greater spectral variation was observed in superovulated oocytes, which was found to describe the corresponding morphological quality. In conclusion, this thesis has demonstrated the effective application of Raman spectroscopy to the study of fixed murine oocytes. Raman mapping experiments have demonstrated this technique for the visualisation of biochemical variation which exists within the oocyte. Furthermore, using Raman spectroscopy, the identification of the biochemical variation resulting from different maturation mechanisms has been achieved, as has the discrimination of immature and mature oocytes. These results indicate that Raman spectroscopy holds promise as a quantitative analysis method in the field of fertility treatment.