Cytochrome c, a protein found in the matrix between the inner and outer membrane of mitochondria plays a key role in the synthesis of Adenosine Triphosphate (ATP), the energy currency of the cell through the electron transport chain. The location and the oxidation state of cytochrome c play a vital role in deciding the fate of a cell. Besides its function in the electron transport chain (ETC) where cell survival is ensured, it also activates apoptotic proteins in the cytosol, upon cellular damage and stress leading to apoptosis otherwise known as programed cell death. Thus, cytochrome C in the right place with the right oxidation state possesses the ability to make or break the cell. The study of early stages of embryonic development provides insights into understanding processes during development and is important for applications in Assisted Reproductive Technology (ART). Embryos that look healthy morphologically could have molecular changes that could lead to cellular damage during the later course of development. We can suggest that these internal abnormalities can be determined by Raman spectroscopy which is a potential tool to estimate changes on a molecular scale. In the event of cellular damage by external factors like radiation or chemical treatment, there is a release of cytochrome c into the cytoplasm which triggers the process of apoptosis by the activation of cascade proteins. In healthy cells under stress, this activation is interrupted by the reduction of cytochrome c. Thus, the analysis of changes in the Raman peak at 1585 cm-1, which is one of the characteristic peaks of cytochrome c, in embryos damaged artificially by laser irradiation are compared and discussed. In this study, Raman spectroscopy is applied to study the changes in the oxidation states of cytochrome C. Immunostaining is applied to understand the distribution of cytochrome c post laser treatment. This in vitro analysis of developing and laser treated embryos can aid in the better understanding of parameters that could be used for the quality estimation of preimplantation embryos.

Table of contents ……………………………………………………………. iii
Index of figures ……………………………………………………………... v
Acknowledgements …………………………………………………………. vii
Abstract ……………………………………………………………………… viii
Aim …………………………………………………………………………..
Chapter 1: Introduction
1.1 Artificial reproductive technology (ART) ……………………………….. 1
1.2 Quality determination of preimplantation embryos ……………………… 1
1.3 Raman Spectroscopy ……………………………………………………... 2
1.3.1 Raman spectra of a murine (mouse) embryo ……………………… 3
1.4 Cytochrome c can make or break the cell ………………………………... 5
1.5 Research motivation ……………………………………………………... 8
Chapter 2: Materials and methods
2.1 Extraction and preparation of embryos
2.1.1 Extraction embryos ………………………………………………… 9
2.1.1 Animals ……………………………………………………………. 9
2.1.2 Embryo collection …………………………………………………. 9
2.2 Treatment and investigation
2.2.1 High power laser treatment ………………………………………… 10
2.2.2 Raman measurement ……………………………………………….. 10
2.2.3 Immunostaining …………………………………………………….. 11
Chapter 3: Results and discussion
3.1 Raman analysis of embryos exposed to laser ……………………………... 13
3.2 Localization of cytochrome c through immunocytochemistry ……………. 18
Chapter 4: Conclusion ………………………………………. 23
References ………………………………………………………… 24

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