Dataset Information

Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

ABSTRACT: To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.

SUBMITTER: Chen L

PROVIDER: S-EPMC4999887 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

Chen Lingsheng L Zhai Linhui L Qu Chunfeng C Zhang Chengpu C Li Sheng S Wu Feilin F Qi Yingzi Y Lu Fenghua F Xu Ping P Li Xiangping X Shi Deshun D

Scientific reports 20160826

To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KE ...[more]

PMID: 27561356

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Project description:BackgroundIn livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Project description:BACKGROUND: Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. METHODOLOGY/PRINCIPAL FINDING: Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. CONCLUSIONS/SIGNIFICANCE: Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Project description:OBJECTIVE:Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. METHODS:Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and 200 ?M), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. RESULTS:Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the 50 ?M EGCG-treated group compared with the control group. Adding 50 ?M EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the 50 ?M EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the 50 ?M EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the 50 ?M EGCG-treated oocytes. CONCLUSION:In conclusion, 50 ?M EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

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Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

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Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

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