3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors.
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ABSTRACT:
SUBMITTER: Bebbere D
PROVIDER: S-EPMC8614943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeThe aim of this work was to develop a microbioreactor using liquid marble (LM) as a novel system for oocyte in vitro maturation (IVM) in small volumes.MethodsCumulus-oocyte complexes (COCs) obtained from slaughterhouse sheep ovaries were in vitro matured in a LM system prepared by placing a drop (30 μl containing 10 COCs) suspended in TCM 199 supplemented with 10 % (v/v) oestrus sheep serum (OSS) and 0.1 IU FSH and LH onto a polytetrafluoroethylene (PTFE) particle bed (LM group). As a control group (CTRL group), COCs were in vitro matured in standard volume and conditions (600 μl of IVM medium in a four-well dish). After 24-h culture at 38.5 °C in 5 % CO2 in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes, (b) oocyte developmental competence following in vitro fertilization (IVF) or parthenogenetic activation (PA) and embryo culture for 8 days in synthetic oviductal fluid (SOF) medium at 38.5 °C in 5 % O2, 5 % CO2, and 90 % N2.ResultsThe results indicated similar percentage of MII oocytes in LM and CTRL groups (88.0 vs. 92.0 %). No differences were observed in blastocyst rate after IVF (LM 47.5 % vs. CTRL 50.2 %, P=0.637) or PA (LM 44.4 % vs. CTRL 48.3 %, P=0.426).ConclusionsThe results indicate that LM microbioreactor is a viable technique that provides a suitable microenvironment to induce oocyte in vitro maturation.
| S-EPMC4818634 | biostudies-literature
Project description:Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.
| S-EPMC3690804 | biostudies-literature
Project description:High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.
| S-EPMC4771806 | biostudies-literature
Project description:PurposeOocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence.MethodsWe tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts.ResultsIn summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts.ConclusionsOur results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.
| S-EPMC6439068 | biostudies-literature
Project description:ObjectiveTo determine the multidrug-resistant transporter (MDR) activity in oocytes and their potential role in oocyte susceptibility to chemotherapy.DesignExperimental laboratory study.SettingUniversity and academic center for reproductive medicine.Subject(s)Women with eggs retrieved for intracytoplasmic sperm injection cycles and adult female FVBN and B6C3F1 mouse strains.Intervention(s)Inhibition of MDR activity in oocytes.Main outcome measure(s)Efflux activity of MDRs with the use of quantitative fluorescent dye efflux, and oocyte cell death when exposed to chemotherapy.Result(s)Oocytes effluxed fluorescent reporters, and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. Geminal vesicle oocytes were more efficient at efflux than metaphase 2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death with the use of two different viability assays compared with control samples and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes, with the greatest accumulation in the geminal vesicle stage.Conclusion(s)Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis and that these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up-regulation of these transporters during gonadotoxic therapy.
| S-EPMC3818338 | biostudies-literature
Project description:BackgroundHeavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 μmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 μmol/L) exposure. Therefore, the concentration of 1 μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes.ResultsWe found that 1 μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes.ConclusionsTaken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.
| S-EPMC9306212 | biostudies-literature
Project description:In contrast to most other mammals, canine oocytes are ovulated in an immature state and undergo oocyte maturation within the oviduct during the estrus stage. The aim of the study was to investigate whether oviduct cells from the estrus stage affect the maturation of oocytes and show gene expression patterns related to oocyte maturation.We analyzed MAPK1/3, SMAD2/3, and BMP6/15 expression in oviduct cells, cumulus cells, and oocytes from anestrus, estrus, and diestrus stages. Next, we investigated the effect of co-culture with oviduct cells derived from the estrus stage upon in vitro maturation (IVM) of canine oocytes.There was significantly higher MAPK1/3 (1.42 ± 0.02 and 2.23 ± 0.06), SMAD2/3 (0.77 ± 0.03 and 2.39 ± 0.07), and BMP15 (2.21 ± 0.16) expression in oviduct cells at the estrus stage (P < 0.05). In cumulus cells, MAPK1 (1.26 ± 0.07), SMAD2/3 (0.82 ± 0.01, 1.04 ± 0.01), and BMP6 (13.09 ± 0.11) expression was significantly higher in the estrus stage (P < 0.05). In oocytes, significant upregulation of MAPK1/3 (14,960 ± 3121 and 1668 ± 253.4), SMAD3 (774.6 ± 79.62), and BMP6 (8500 ± 895.4) expression was found in the estrus stage (P < 0.05). After 72 h of IVM culture, a significantly higher maturation rate was observed in oocytes co-cultured with oviduct cells (10.0 ± 1.5%) than in the control group (3.2 ± 1.4%).We demonstrate that oviduct cells at the estrus stage highly expressed MAPK1/3, SMAD2/3, and BMP15. Furthermore, canine oviduct cells from the estrus stage enhance the culture environment for canine oocyte maturation.
| S-EPMC5476537 | biostudies-literature
Project description:Messenger RNA is remarkably stable during oocyte growth, thus enabling mRNAs to accumulate during the growth phase and thereby provide mRNAs that support early embryonic development. MSY2, a germ cell-specific RNA-binding protein, is implicated in regulating mRNA stability. MSY2 is essential for development because female Msy2(-/-) mice are infertile. We describe here the characterization of Msy2(-/-) oocytes. Mutant oocytes grow more slowly during the first wave of folliculogenesis, and maturation to and arrest at metaphase II is severely compromised because of aberrant spindle formation and chromosome congression. Consistent with MSY2 conferring mRNA stability is that the amount of poly(A)-containing RNA is reduced by ~25% in mutant oocytes. Stability of an exogenous mRNA injected into mutant oocytes is lower than when compared to their wild-type counterparts, and moreover, expression of wild-type MSY2 in mutant oocytes increases mRNA stability, whereas injection of a mutant form of MSY2 not capable of binding RNA does not. Transcription quiescence that normally occurs during the course of oocyte growth is not observed in mutant oocytes, and the transcriptome of mutant oocytes is markedly perturbed. These results, and those of previous studies, strongly implicate a central role of MSY2 in regulating mRNA stability.
| S-EPMC3159539 | biostudies-literature
Project description:AimsThe mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro maturation (IVM) oocytes, remain to be elucidated. Here we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression.ResultsThree key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Furthermore, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP+ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy.InnovationThe present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence.ConclusionIn vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.
| S-EPMC6338670 | biostudies-literature
Project description:ObjectiveAlpha-lipoic acid (ALA) as a strong antioxidant has a protective effect. This study was designed to assess whether supplementation of maturation medium with ALA during in vitro maturation (IVM) can attenuate the toxic effect of ethanol.Materials and methodsIn this experimental study, to assess the antioxidant capacity of ALA challenged by 1% ethanol during in vitro maturation, immature ovine oocytes were exposed to 1% alcohol in the presence or absence of 25 μM ALA during oocyte maturation. The cumulus expansion index, intracellular reactive oxygen species (ROS), and thiol content levels were assessed in matured oocytes of various treatment groups. Consequently, the blastocyst formation rate of matured oocytes in various treatment groups were assessed. In addition, total cell number (TCN), cell allocation, DNA fragmentation, and relative gene expression of interested genes were assessed in resultant blastocysts.ResultsThe results revealed that alcohol significantly reduced cumulus cells (CCs) expansion index and blastocyst yield and rate of apoptosis in resultant embryos. Addition of 25 μM ALA to 1% ethanol during oocyte maturation decreased ROS level and elevated Thiolcontent. Furthermore, supplementation of maturation medium with ALA attenuated the effect of 1% ethanol and significantly increased the blastocyst formation and hatching rate as compared to control and ethanol groups. In addition, the quality of blastocysts produced in ALA+ethanol was improved based on the low number of TUNEL positive cells, the increased expression level of mRNA for pluripotency, and anti-oxidant markers, and decreased expression of apoptotic genes.ConclusionThe current findings demonstrate that ALA can diminish the effect of ethanol, possibly by decreasing the ROS level and increasing Thiolcontent during oocyte maturation. Using the ALA supplement may have implications in protecting oocytes from alcohol toxicity in affected patients.
| S-EPMC8196229 | biostudies-literature