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Comparison of the Oocyte Quality Derived from Two-Dimensional Follicle Culture Methods and Developmental Competence of In Vitro Grown and Matured Oocytes.

ABSTRACT: In vitro follicle growth (IVFG) is an emerging fertility preservation technique, which can obtain fertilizable oocytes from an in vitro culture system in female. This study aimed to compare efficiency of the most widely used two-dimensional follicle culture methods [with or without oil layer (O+ or O- group)]. Preantral follicles were isolated from mice and randomly assigned. Follicles were cultured for 10 days and cumulus-oocyte complexes harvested 16-18 hours after hCG treatment. Follicle and oocyte growth, hormones in spent medium, meiotic spindle localization, expression of reactive oxygen species (ROS), mitochondrial activity, and gene expression were evaluated. In follicle growth, survival, pseudoantral cavity formation, ovulation, and oocyte maturation were also significantly higher in O+ group than O- group. Hormone production was significantly higher in follicles cultured in O+ than O-. There were no significant differences in mRNA expression related to development. On the other hand, the level of ROS was increased while the mitochondrial activity of in vitro grown matured oocyte was less than in vivo matured oocytes. In conclusion, follicle culture with O+ group appears to be superior to the culture in O- group in terms of follicle growth, development, oocyte growth, maturation, and microorganelles in oocyte.

SUBMITTER: Lee J

PROVIDER: S-EPMC5904821 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparison of the Oocyte Quality Derived from Two-Dimensional Follicle Culture Methods and Developmental Competence of In Vitro Grown and Matured Oocytes.

Lee Jaewang J Kim Eun Jung EJ Kong Hyun Sun HS Youm Hye Won HW Kim Seul Ki SK Lee Jung Ryeol JR Suh Chang Suk CS Kim Seok Hyun SH

BioMed research international 20180404

In vitro follicle growth (IVFG) is an emerging fertility preservation technique, which can obtain fertilizable oocytes from an in vitro culture system in female. This study aimed to compare efficiency of the most widely used two-dimensional follicle culture methods [with or without oil layer (O+ or O- group)]. Preantral follicles were isolated from mice and randomly assigned. Follicles were cultured for 10 days and cumulus-oocyte complexes harvested 16-18 hours after hCG treatment. ...[more]

PMID: 29850567

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Project description:The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.

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:BackgroundIn vertebrates, late oogenesis is a key period during which the oocyte acquires its ability to resume meiosis (i.e. maturational competence) and to develop, once fertilized, into a normal embryo (i.e. developmental competence). However, the molecular mechanisms involved in these key biological processes are far from being fully understood. In order to identify key mechanisms conserved among teleosts and amphibians, we performed a comparative analysis using ovarian tissue sampled at successive steps of the maturational competence acquisition process in the rainbow trout (Oncorhynchus mykiss) and in the clawed toad (Xenopus laevis). Our study aimed at identifying common differentially expressed genes during late oogenesis in both species. Using an existing transcriptomic analysis that had previously been carried out in rainbow trout, candidate genes were selected for subsequent quantitative PCR-based comparative analysis.ResultsAmong the 1200 differentially expressed clones in rainbow trout, twenty-six candidate genes were selected for further analysis by real-time PCR in both species during late oogenesis. Among these genes, eight had similar expression profiles in trout and Xenopus. Six genes were down-regulated during oocyte maturation (cyp19a1, cyp17a1, tescalcin, tfr1, cmah, hsd11b3) while two genes exhibited an opposite pattern (apoc1, star). In order to document possibly conserved molecular mechanisms, four genes (star, cyp19a1, cyp17a1 and hsd11b3) were further studied due to their known or suspected role in steroidogenesis after characterization of the orthology relationships between rainbow trout and Xenopus genes. Apoc1 was also selected for further analysis because of its reported function in cholesterol transport, which may modulate steroidogenesis by regulating cholesterol bioavailability in the steroidogenic cells.ConclusionsWe have successfully identified orthologous genes exhibiting conserved expression profiles in the ovarian follicle during late oogenesis in both trout and Xenopus. While some identified genes were previously uncharacterized during Xenopus late oogenesis, the nature of these genes has pointed out molecular mechanisms possibly conserved in amphibians and teleosts. It should also be stressed that in addition to the already suspected importance of steroidogenesis in maturational competence acquisition, our approach has shed light on other regulatory pathways which may be involved in maturational and developmental competence acquisitions that will require further studies.

Project description:BackgroundDNA methylation is one the epigenetic mechanisms, which is critically involved in gene expression. This phenomenon is mediated by DNA methyl-transferases and is affected by environmental stress, including in vitro maturation (IVM) of oocytes. Melatonin, as an antioxidant, may theoretically be involved in epigenetic regulation via reductions of reactive oxygen species. This study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin.Materials and methodsThis experimental study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin. For this purpose, oocytes with granulated cytoplasm were selected and co-cultured with at least two layers of cumulus cells in maturation medium with 10-6 M, 10-9 M, 10-12 M and 0-M (as control group) of melatonin. Nucleus status, glutathione content and developmental competence of the oocytes in each experimental group were assessed. Also, expression of genes associated with DNA methylation, including DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and DNA methyltransferase 3a (DNMT3a) was evaluated by quantitative real time-polymerase chain reaction (RT-PCR).ResultsAccording to our findings, the percentage of oocytes that reached the M-II stage significantly increased in the 10-12 M group (P<0.05). Also, a significant elevation of glutathione content was observed in melatonin-treated oocytes (P<0.05). Analysis of blastocyst formation revealed that developmental competence of the oocytes was higher than the control group (P<0.05). It was observed that melatonin treatment decreased expression levels of DNA methyltransferases (DNMTs) and global DNA methylation (P<0.05). In addition, the expression of melatonin receptor1A (MTNR1A) was detected in both cumulus and oocyte by RT-PCR.ConclusionThe results suggested that in goat model melatonin affects DNA methylation pattern, leading to an improvement in the developmental competence of the oocytes.

Dataset Information

Comparison of the Oocyte Quality Derived from Two-Dimensional Follicle Culture Methods and Developmental Competence of In Vitro Grown and Matured Oocytes.

Publications

Comparison of the Oocyte Quality Derived from Two-Dimensional Follicle Culture Methods and Developmental Competence of <i>In Vitro</i> Grown and Matured Oocytes.

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