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

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 α-helices to the β-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 α-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.

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus‐oocyte complexes: A biological and spectroscopic approach

“…In contrast, the addition of ɛ-ACA resulted in a significant decrease in cleavage (59.20 ± 3.10%) and blastocyst rates (21.85 ± 4.46%; p < .05, Figure 4). and phenylalanine (Phe,~1605 and~1000 cm −1 ) described by other authors (Bogliolo et al, 2013(Bogliolo et al, , 2014Rizo et al, 2016;Rusciano et al, 2017) were detected (Table 2). In a global analysis, it should be noticed that the spectral profile obtained from oocytes in the SK group showed high intense protein bands similar to the control spectrum.…”

“…Figure 6 shows the spectral profiles of the four groups truncated in the region between 1800 and 800 cm −1 . Characteristic bands of proteins such as amide I (AmI, ~1656 cm −1 ) mode, amide III (AmIII, region 1320–1260 cm −1 ) mode and the signals assigned to the vibrations of aromatic amino acids of the aromatic rings of tyrosine (Tyr, ~1616 cm −1 ) and phenylalanine (Phe, ~1605 and ~1000 cm −1 ) described by other authors (Bogliolo et al, 2013, 2014; Rizo et al, 2016; Rusciano et al, 2017) were detected (Table 2). In a global analysis, it should be noticed that the spectral profile obtained from oocytes in the SK group showed high intense protein bands similar to the control spectrum.…”

“…First, CCs were removed by pipetting in warm H-TALP medium containing 1 mg/ml hyaluronidase. Only denuded oocytes were selected, washed three times in PBS-BSA and fixed in 4% (w/v) paraformaldehyde for 30 min (Bogliolo et al, 2014;Davidson et al, 2013;Jimenez et al, 2019;Rizo et al, 2016;Rusciano et al, 2017 was acquired by accumulating 40 expositions with an exposure time of 3 s each. The Raman spectrum of the PBS/BSA medium was also acquired.…”

Serum albumin concentration of donor cows as an indicator of developmental competence of oocytes

“…Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Johannesburg, South Africa). Mediums used for IVF culture procedures were based on protocols used by the Department of Veterinary Medicine and Animal Reproduction, Federico II University, Naples, Italy [21,22]. In short, the aspiration medium was TCM 199 medium consisted of Synthetic Oviduct Fluid (SOF) medium [24] with 30 µL/mL essential amino acids and 10 µL/mL non-essential amino acids.…”

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

“…It has been demonstrated that vitrification solution could induce zona hardening and biochemical changes in zona pellucida (ZP) in mammalian oocytes (Bogliolo et al, ; Larman, Sheehan, & Gardner, ). Oocyte structure and competence can be also influenced by high concentrations of cryoprotective agents (CPAs) required to achieve a glass‐like state in vitrification (Devi & Goel, ; Ezoe et al, ; Liu et al, ; Rusciano et al, ). Since the time when the first live offspring was obtained from cryopreserved domestic oocytes (Hamano, Koikeda, Kuwayama, & Nagai, ), a limited number of studies has reported high developmental rate of vitrified‐warmed ovine oocytes.…”