Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes

Year: 2017

Authors: Rusciano G., De Canditiis C., Zito G., Rubessa M., Roca MS., Carotenuto R., Sasso A., Gasparrini B.

Autors Affiliation: Univ Naples Federico II, Dept Phys E Pancini, Via Cintia, I-80126 Naples, Italy; CNR, Natl Inst Opt INO, Via Campi Flegrei 34, I-80078 Pozzuoli, NA, Italy; Univ Naples Federico II, Dept Vet Med & Anim Prod, Via F Delpino 1, I-80137 Naples, Italy; CNR, Inst Prot Biochem IBP, Via Pietro Castellino 111, I-80131 Naples, Italy; Univ Illinois, Inst Genom Biol, Urbana, IL 61801 USA; Ist Nazl Tumori Fdn G Pascale IRCCS, Expt Pharmacol Unit, Naples, Italy; Univ Naples Federico II, Dept Biol, Via Cintia, I-80126 Naples, Italy

Abstract: Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the alpha-helices to the beta-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with alpha-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

Journal/Review: PLOS ONE

Volume: 12 (5)      Pages from: e0177677-1  to: e0177677-19

DOI: 10.1371/journal.pone.0177677

Citations: 15
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