NMR characterization of animals’ follicular fluids

Follicular Fluid (FF) provides a special environment to the oocyte during its maturation in vivo. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall that contain a large variety of metabolites (1). These metabolites are probably involved in the physiology of the oocytes (1). The chemical composition of follicular fluids is important because it is an indicator of the secretory activities and metabolism of follicular cells and thus could be related to the follicular quality. It could also provide a useful indication of the oocyte growth and maturation (2). High Resolution Nuclear Magnetic Resonance (NMR) spectroscopy provides a unique tool for studying metabolites. Initially, NMR spectroscopy was used mainly in biomedicine but it is found now in many physiological applications (3). As the NMR spectroscopy provides opportunities for obtaining qualitative and quantitative data from body fluids, it was hypothesized that this technique could provide information on mammals’ follicular fluid and on intrafollicular changes that occur during follicular growth and ovulation. As some of these changes are probably of crucial importance for oocyte developmental competence, a better knowledge of the mammals’ follicular fluid composition by 1H NMR analysis should help to resolve some of the problems encountered during in vitro procedures in the mammals. The characterization of the chemical composition of mammals follicular fluids, namely sheep, cattle, mare and pork, and the study of the changes observed during follicular growth and maturation using NMR spectroscopy will be presented. FF samples were collected from antral follicle of different dimensions. One-dimensional (1D) 1H experiments (CPMG, DOSY) were obtained for all the FF samples. In addition, several two dimensional (2D) (homo and heteronuclear) experiments (DQF-COSY, TOCSY, JRES, 1H-13C HSQC) were performed to aid in the assignment of the signals and in the identification of the metabolites in FF. A direct evaluation of the lipids, carbohydrates and metabolites were obtained from the combination of the 1D and 2D NMR experiments

Methylation dynamics during folliculogenesis and early embryo development in sheep

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Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans

Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season

The domestic cat is commonly used as a model for the development of assisted reproductive technologies, including in vitro embryo production (IVEP) in felid species. Seasonal reproduction is a feature of domestic cats as well as of several species of wild feline. Likewise, the number and the quality of blastocysts produced in in vitro systems is linked to season. Maintaining stable in vitro embryo production throughout the year is crucial not only for research purposes but also for programs aimed at protecting endangered felines. We assess whether using Brilliant Cresyl Blue (BCB) selection in addition to the classical morphological selection could improve the IVEP outcomes during non-breeding season. Blastocyst yield and quality of the embryos (hatching rate and blastocyst cell numbers) were higher after IVM/IVF in oocytes defined as BCB+ (colored cytoplasm) based on the BCB test than in oocytes only morphologically selected. Furthermore, no adverse effects on bioenergetic/oxidative status were observed in oocytes subjected to BCB staining. In conclusion, BCB test implementation in IVEP programs might ensure a steady output of domestic cat blastocysts throughout the year. In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season

A (Cyber)ROAD to the Future: A Methodology for Building Cybersecurity Research Roadmaps

We describe the roadmapping method developed in the context of the CyberROAD EU FP7 project, the aim of which was to develop a research roadmap for cybercrime and cyberterrorism. To achieve this aim we build on state-of-the-art methodologies and guidelines, as well as related projects, and adapt them to the specific characteristics of cybercrime and cyberterrorism. The distinctive feature is that cybercrime and cyberterrorism co-evolve with their contextual environment (i.e., technology, society, politics and economy). This poses specific challenges to a roadmapping effort. Our approach could become a best practice in the field of cybersecurity, and could also be generalised to phenomena that exhibit a similar, strong co-evolution with their contextual environment. In this chapter, we define our route to developing the CyberROAD research roadmap and contextualise it with an example of Enterprise 2.0

Roscovitina para o atraso da progressão meiótica em oócitos de ovelhas pré-púberes

The objective of this work was to evaluate the efficiency of roscovitine on reversibly inhibiting oocytes from prepubertal sheep at the germinal vesicle (GV) stage, and to investigate the kinetics of meiosis progression after inhibitor removal. Cumulus-oocyte complexes, recovered from Sarda breed lambs aged 30–40 days, were cultured for 6 hours in a maturation medium (control) containing 75 µmol L-1 roscovitine (Rosco) at 38.5°C and 5% CO2. Then, the complexes were subjected to in vitro maturation (IVM) for 18 or 23 hours, in an inhibitor-free medium supplemented with gonadotropins. The evaluation of nuclear configuration by Hoescht staining, under a fluorescence-inverted microscope, showed that 88.7% of the lamb oocytes treated with roscovitine remained at the GV stage, as observed for the immature ones (97.3%) stained after collection. The inhibitory action was reversible; however, the proportion of oocytes (83.3%) at the metaphase-II stage, after 23 hours of IVM, was significantly higher than that observed after 18 hours (29.5%), in which meiosis was still in progression with 34.2% oocytes at metaphase-I, 11.6% oocytes at anaphase-I, and 18.5% oocytes at telophase-I. Roscovitine is efficient to arrest the nuclear maturation in oocytes from prepubertal sheep; however, despite the reversibility, meiosis progression is delayed, requiring more time to be completed.O objetivo deste trabalho foi avaliar a eficiência da roscovitina na inibição reversível de oócitos de ovelhas pré-púberes, no estádio de vesícula germinativa (VG), e investigar a cinética da progressão da meiose após a remoção do inibidor. Complexos cumulus-oócito, recuperados de cordeiras da raça Sarda com 30–40 dias, foram cultivados por 6 horas em meio de maturação (controle) contendo 75 µmol L-1 de roscovitina (Rosco) a 38,5°C e 5% de CO2. Em seguida, os complexos foram submetidos à maturação in vitro (MIV) por 18 ou 23 horas, em meio isento de inibidor, suplementado com gonadotrofinas. A avaliação da configuração nuclear em coloração Hoescht, sob microscópio invertido de fluorescência, revelou que 88,7% dos oócitos tratados permaneceram no estágio VG, conforme observado para os imaturos (97,3%) corados após a coleta. Essa inibição foi reversível; contudo, a proporção de oócitos (83,3%) em metáfase-II, após 23 horas de MIV, foi significativamente maior do que a observada após 18 horas (29,5%), em que a meiose ainda estava em progressão com 34,2% de oócitos em metáfase-I, 11,6% de oócitos em anáfase-I e 18,5% de oócitos em telófase-I. A roscovitina é eficiente no bloqueio da maturação nuclear em oócitos de ovelhas pré-púberes; no entanto, apesar da reversibilidade, a progressão da meiose é retardada e requer mais tempo para ser concluída

Raman microspectroscopy as a tool to detect molecular modifications induced by aging-related oxidative stress in mouse oocytes

The conditions of oxidative stress that can be generated during physiological events, such as post-ovulatory aging and reproductive aging, as well as by the PMA procedures, can seriously degrade the oocyte developmental competence. The ability to identify predictive markers of oxidative stress using noninvasive techniques may provide a useful diagnostic tool for the assessment of oocyte quality. The aim of the present work is to evaluate the potential of Raman spectroscopy (RMN) as a tool to detect molecular modifications induced by aging-related oxidative stress in mouse oocytes. The research was carried out using CD-1 mice; at the age of 4 to 8 weeks (young mice) and 48 to52 weeks (old mice), females were superovulated and oocytes at metaphase II stage were recovered from oviducts. The MII oocytes from young animals were divided into 3 experimental groups: (A) young oocytes, processed immediately after collection; (B) in vitro aged oocytes, cultured in vitro for 10 h before processing; (C) oxidative-stressed oocytes, exposed to 10 mM hydrogen peroxide for 2 min before processing (oocytes with a fully oxidized status). Oocytes from reproductively old mice were referred to as old oocytes (D). After fixation in 3.7% paraformaldehyde, oocytes (n = 10 for each group) were immersed in a 50-µL drop of PBS on quartz windows and analyzed using a Bruker Senterra confocal Raman microscope. Measurements were performed by recording 3 line scans across the oocyte with 5-µm step size, totalling 32 point spectra for each oocyte. The spectra were statistically analyzed using principal component analysis. Principal component analysis showed a clear discrimination between the spectra of young oocytes (A), in vitro aged oocytes (B), oxidative-stressed oocytes (C), and old oocytes (D). Compared with the control group (A), B, C, and D groups revealed significant differences in the bands attributable to lipid components; specifically, a reduction in the intensity of the peaks at 1653 and 1602 cm–1 (stretching of the C = C bond) and of the peaks at 1485, 1462, 1437, 1396 cm–1 (CH3-CH2 vibration) was recorded. With regard to the protein component, spectra of B, C, and D groups showed modifications in the intensities of peaks 1297 and 850 cm–1, which refer respectively to amide III and to CNC symmetric stretching compared with group A. Principal component analysis also revealed an overlap between the spectra of in vitro aged oocytes, old oocytes, and oxidative-stressed oocytes, suggesting that the molecular damage caused by ageing has similar characteristics to chemically induced oxidative damage. In conclusion, the results of our study show that Raman spectroscopy is a valuable tool for the identification of molecular biochemical markers of oxidative stress. This technique could represent a highly informative method of investigation to evaluate the oocyte quality in response to various stress conditions (in vitro maturation, aging, cryopreservation, and so on) that may negatively affect its potential development

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

Purpose: Investigation of the changes induced by vitrification on the cortical F-actin of in vitro matured ovine oocytes by Raman microspectroscopy (RMS). Methods: Cumulus-oocyte complexes, recovered from the ovaries of slaughtered sheep, were matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. The cortical region of metaphase II (MII) oocytes (1) exposed to vitrification solutions but not cryopreserved (CPA-exp), (2) vitrified/warmed (VITRI), and (3) untreated (CTR) was analyzed by RMS. A chemical map of one quadrant of single CPA-exp, VITRI and CTR oocytes was, also, performed. In order to identify the region of Raman spectra representative of the cortical F-actin modification, a group of in vitro matured oocytes were incubated with latrunculin&#8212;A (LATA), a specific F-actin destabilizing drug, and processed for RMS analysis. Thereafter, all the oocytes were stained with rhodamine phalloidin and evaluated by fluorescence confocal microscopy. Raman spectra of the oocytes were, statistically, analyzed using Principal Component Analysis (PCA). Results: The PCA score plots showed a marked discrimination between CTR oocytes and CPA-exp/ VITRI groups. The main differences, highlighted by PCA loadings, were referable to proteins (1657, 1440 and 1300 cm−1) and, as indicated by LATA experiments, also included the changes of the F-actin. Analysis by confocal microscopy revealed a clear alteration of the cortical F-actin of CPA-exp and VITRI oocytes confirming RMS results. Conclusions: Raman microspectroscopy may represent an alternative analytical tool for investigating the biochemical modification of the oocyte cortex, including the F-actin cytoskeleton, during vitrification of in vitro matured ovine oocytes