The changing biochemical composition and organisation of the murine oocyte and early embryo as revealed by Raman spectroscopic mapping

Abstract

Despite an exponential uptake in recent years of assisted reproductive techniques, such as in vitro fertilisation, much is still not fully understood about the biochemical modifications that take place during the development and maturation of the egg and embryo. As such, in order to improve the efficiency of these techniques, furthering our understanding of the processes that underpin oocyte and embryo development is necessary. Raman spectroscopic mapping as a technique enables the investigation of biochemical variation within intact cells without the need for labelling. Here, Raman maps of fixed immature and mature oocytes along with early stage embryos were collected using 785 nm excitation and a step size of 2 µm. The results were analysed using both univariate and multivariate techniques. It was found that significant macromolecular accumulation took place during oocyte maturation, while a decrease in total lipid content consistent with the formation of new cellular membranes is observed upon embryo cleavage. Furthermore, an observed asymmetrical localisation of macromolecules in the mature oocyte may indicate the existence of cytoplasmic polarisation, a phenomenon that has been observed in the eggs of lower organisms. As such, these results indicate that Raman spectroscopic mapping may present an alternative analytical tool for investigating the biochemistry of egg and embryo development. In particular, these results indicate that temporal Raman analysis may help to reveal the existence of cytoplasmic polarisation in the murine egg.

Raman Micro-Spectroscopy Can Be Used to Investigate the Developmental Stage of the Mouse Oocyte

“…To achieve this will require a significant reduction in the time required for spectral acquisition on the sample [33]. For the present work we sequentially reduced our spectral sampling to 101 positions across the cell’s midline from the 1610 we have used in previous imaging studies [19]; we regard 101 positions to be a minimum sample set to give a robust averaging of the cell’s biomolecular composition. Alternately, we can speed Raman data acquisition at each point by sampling only the Raman peaks of interest (namely 1605 and 1447 cm −1 ): indeed, this could be further accelerated through the use of resonant Raman excitation employed in techniques such as coherent anti-Stokes Raman scattering microscopy (CARS) [34].…”

“…This approach is particularly powerful when used in combination with multivariate statistical methods. Indeed, vibrational spectroscopy used in combination with principal component analysis (PCA) has been described for the discrimination of oocytes with intact germinal vesicles and those at MII collected after stimulated ovulation cycles [18], whilst Raman mapping informed by hierarchical cluster analysis has been used to image biochemical redistribution in maturing oocytes [19]. Raman offers the advantage of acquiring information regarding the biochemical constituents of a sample without labelling, on both fixed and unfixed samples.…”

Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy

“…The power of this technique for the examination of the oocyte has been reported by the pivotal study of Wood et al [28] which was addressed to investigate the difference in the molecular architecture of immature and mature mouse oocytes. Later studies focused on the biochemical variation within oocytes and embryos at different stage of development [29,30] and on the aging-related oxidative damage of mouse oocytes [32].…”

“…This technique has been, widely, used to study the biological changes of various cellular components during cell cycle progression, apoptosis, and response to stress [27]. On the other hand, studies focused on female gamete are restricted to a few works [28][29][30][31][32]. Specifically, changes of the molecular architecture of the oocyte at different maturation stages have been revealed by RMS [28][29][30].…”

“…On the other hand, studies focused on female gamete are restricted to a few works [28][29][30][31][32]. Specifically, changes of the molecular architecture of the oocyte at different maturation stages have been revealed by RMS [28][29][30]. Recently, our group demonstrated that RMS is able to assess the structural modifications of the zona pellucida of ovine oocytes following vitrification/warming [31].…”