ABSTRACT
Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.
Subject(s)
Fibroblast Growth Factor 2 , In Vitro Oocyte Maturation Techniques , Animals , Mice , Female , In Vitro Oocyte Maturation Techniques/veterinary , Fibroblast Growth Factor 2/metabolism , Insulin-Like Growth Factor I/metabolism , Endothelial Growth Factors/analysis , Endothelial Growth Factors/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Oocytes/metabolism , TOR Serine-Threonine Kinases/metabolism , Dietary Supplements , Glucose/pharmacology , Glucose/metabolism , Cumulus Cells/metabolismABSTRACT
Granulocyte colony-stimulating factor (G-CSF), a pleiotropic cytokine, is secreted by the reproductive tract. Furthermore, our previous study indicated that human recombinant G-CSF (hrG-CSF) supplementation during porcine oocyte in vitro maturation (IVM) or during embryo in vitro culture (IVC) improved their quality and development potential when using cumulus-oocyte complexes (COCs) with more than three cumulus cell layers (CCL >3). Thus, in this study, we investigate the optimal conditions of hrG-CSF supplementation throughout the in vitro production (IVP: IVM + IVC) system to improve the embryo production efficiency of "poor-quality (CCL ≤3)" oocytes. COCs were classified into two groups according to the number of CCL (>3 and ≤3) and embryonic viability was analyzed after treatment with hrG-CSF during IVC. The mRNA transcription levels of G-CSF in COCs were compared based on their type and the period of IVM. Finally, developmental capacity and quality were evaluated after treatment with hrG-CSF for different periods of IVP. No marked effects on the developmental potential of embryos when using CCL ≤3 type COCs were observed after supplementing hrG-CSF only during IVC. Moreover, the mRNA transcription level of G-CSF increased gradually with IVM culture time and was higher in CCL ≤3 COCs than in >3. Supplementing hrG-CSF only during the IVM period resulted in the best embryo developmental potential, while supplementing hrG-CSF during the IVP period resulted in the best quality embryos, reflected in the increased total cell number and decreased apoptotic nuclei index of blastocysts. These findings indicate that "poor-quality" COCs may have a greater demand for G-CSF than "good-quality", meanwhile hrG-CSF supplementation throughout IVP improves resource utilization efficiency in poor-quality COCs.
Subject(s)
In Vitro Oocyte Maturation Techniques , Oocytes , Female , Humans , Animals , Swine , In Vitro Oocyte Maturation Techniques/veterinary , In Vitro Oocyte Maturation Techniques/methods , Embryonic Development , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte Colony-Stimulating Factor/metabolism , Cumulus Cells/metabolism , Blastocyst , RNA, Messenger/metabolism , Dietary Supplements , GranulocytesABSTRACT
Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.
Subject(s)
Buffaloes , In Vitro Oocyte Maturation Techniques , Animals , Female , Cumulus Cells/metabolism , Dietary Supplements , Fibroblast Growth Factor 10/pharmacology , In Vitro Oocyte Maturation Techniques/veterinary , In Vitro Oocyte Maturation Techniques/methods , OocytesABSTRACT
RESEARCH QUESTION: The mechanism of Myo-Inositol, as an adjuvant, on key signaling pathways related to oocyte maturation, fertilization rate, and embryo quality as well as ovarian steroidogenesis in cumulus cells of PCOS patients, is still unclear. DESIGN: Infertile patients who were candidates for ART cycles were divided into three groups (n = 30 in each group), including group 1: PCOS patients only receiving folic acid, group 2: PCOS patients receiving daily Myo-Inositol combined with folic acid, and a control group (group 3): normal ovulatory women without PCOS receiving only folic acid from 1 month prior to IVF cycle until the day of ovum pick up. During the ART procedure, oocytes maturation, fertilization rate, and embryo quality were assessed. The gene expressions of FSHR, LHR, CYP11A1, CYP19A1, 3ß-HSD2, and StAR were also analyzed using qRT-PCR. Western blot analysis was performed for the evaluation of AKT, ERK, CREB, and AMPK phosphorylation. RESULT: Despite equal number of retrieved oocytes, the percentages of MII oocytes, fertilization rate, and embryo quality were found to be significantly higher in group 2 due to the administration of inofolic. The expressions of all the studied genes were significantly higher in the cumulus cells of group 1 compared to the group 2. Higher phosphorylation of ERK1/2 was found in the groups 2 and 3 compared to the group 1. On the other hand, p-Akt has significantly decreased in the group 2 compared to the group 1. CONCLUSION: Our study provides new insight into the molecular mechanism underlying the positive effect of Myo-Inositol on intrinsic ovarian defects in PCOS, steroidogenesis, oocyte maturation, fertilization rate, and embryo quality.
Subject(s)
Fertilization in Vitro/methods , Inositol/pharmacology , Polycystic Ovary Syndrome/drug therapy , Adult , Cumulus Cells/metabolism , Dietary Supplements , Female , Folic Acid/pharmacology , Gonadal Steroid Hormones/metabolism , Humans , Infertility, Female , Iran , Oocytes/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Polycystic Ovary Syndrome/complications , Polycystic Ovary Syndrome/physiopathology , Reproductive Techniques, AssistedABSTRACT
Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.
Subject(s)
Asparagus Plant/chemistry , Cumulus Cells/cytology , Cumulus Cells/metabolism , HSP70 Heat-Shock Proteins/metabolism , Plant Extracts/pharmacology , Animals , Cattle , DNA Damage , Gene Expression Regulation/drug effects , Glutathione/metabolism , HSP70 Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Response/drug effects , Heat-Shock Response/genetics , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Oxidative Stress/genetics , Plant Stems/chemistry , Progesterone/biosynthesis , Reactive Oxygen Species/metabolism , Reference StandardsABSTRACT
Female reproductive aging is an irreversible process associated with a decrease in oocyte quality, which is a limiting factor for fertility. Previous studies have shown that dehydroepiandrosterone (DHEA) has been shown to improve in vitro fertilization (IVF) outcomes in older women. Herein, we showed that the decline in oocyte quality with age is accompanied by a significant decrease in the level of bioenergetic metabolism genes. We compared the clinical characteristics between groups of infertile women who either received DHEA or did not. Treatment with DHEA may enhance oocyte quality by improving energy production and metabolic reprogramming in cumulus cells (CCs) of aging women. Our results showed that compared with the group without DHEA, the group with DHEA produced a large number of day-three (D3) embryos, top-quality D3 embryos, and had improved ongoing pregnancy rate and clinical pregnancy rate. This may be because DHEA enhances the transport of oxidative phosphorylation and increases mitochondrial oxygen consumption in CCs, converting anaerobic to aerobic metabolism commonly used by aging cells to delay oocyte aging. In conclusion, our results suggest that the benefit of DHEA supplementation on IVF outcomes in aging cells is significant and that this effect may be mediated in part through the reprogramming of metabolic pathways and conversion of anaerobic to aerobic respiration.
Subject(s)
Dehydroepiandrosterone/administration & dosage , Dietary Supplements , Energy Metabolism , Infertility, Female/metabolism , Oocytes/metabolism , Organelle Biogenesis , Adult , Aging , Cellular Senescence , Cumulus Cells/metabolism , Female , Fertilization in Vitro , Humans , Mitochondria/metabolism , Oxidative Phosphorylation , Oxygen Consumption , Pregnancy , Pregnancy OutcomeABSTRACT
Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.
Subject(s)
Cytoplasm/metabolism , Extracellular Matrix Proteins/metabolism , Oocytes/cytology , Sepharose/pharmacology , Adenosine Triphosphate/metabolism , Animals , Blastocyst/drug effects , Bone Morphogenetic Protein 15 , Cumulus Cells/cytology , Cumulus Cells/drug effects , Cumulus Cells/metabolism , Cytoplasm/drug effects , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , In Vitro Oocyte Maturation Techniques , Integrins/metabolism , Oocytes/drug effects , Oocytes/metabolism , Parthenogenesis/drug effects , Protein Subunits/metabolism , SwineABSTRACT
This study aims to evaluate the effect of melatonin supplementation on the outcomes of in vitro fertilization (IVF) and mitochondrial adenosine triphosphate production (MT-ATP6) gene expression in Iranian infertile couples. A single-blind nonrandomized controlled trial was conducted, recruiting 90 infertile couples who underwent IVF at an infertility center in Tehran, Iran. Patients who were assigned to the intervention group received melatonin as a supplementation to the standard controlled ovarian stimulation (COS). The control group received a COS protocol only. Primary outcome was the mRNA level of the MT-ATP6 gene in cumulus cells of ovarian follicles. Secondary outcomes were the mean number of mature oocytes retrieved, the embryo quality, and biochemical and clinical pregnancy rates. The mRNA level of the MT-ATP6 gene in cumulus cells between intervention and control groups was not statistically different (0.931 vs.1; P Ë 0.05). The mean number of poor-quality embryos was significantly lower in the intervention group than that in the control group (0.27 vs. 0.80; P = 0.028). The biochemical and clinical pregnancy rates were higher in the intervention group (24% vs. 14%, P = 0.089, and 14% vs. 7%, P = 0.302, respectively); however, the difference was not significant. Melatonin supplementation did not increase the odds of clinical pregnancy and the number of mature oocytes retrieved, but significantly reduced the number of low-quality embryos. More extensive studies focusing on the level of MT-ATP6 gene expression in the oocyte or blastomere cells may further elucidate the effect of supplementation with melatonin in infertile couples who have poor clinical outcomes. Trial registration: Current Controlled Trials: IRCT2015042912307N4.
Subject(s)
Fertilization in Vitro/trends , Infertility/metabolism , Infertility/therapy , Melatonin/administration & dosage , Mitochondrial Proton-Translocating ATPases/biosynthesis , Pregnancy Rate/trends , Administration, Oral , Adult , Antioxidants/administration & dosage , Cumulus Cells/drug effects , Cumulus Cells/metabolism , Female , Fertilization in Vitro/methods , Gene Expression , Humans , Infertility/epidemiology , Iran/epidemiology , Male , Mitochondrial Proton-Translocating ATPases/genetics , Pregnancy , Single-Blind Method , Treatment OutcomeABSTRACT
The pig oocyte maturation protocol differs from other mammalian species due to dependence on follicular fluid (FF) supplementation. One of the most abundant components of the porcine follicular fluid are fatty acids (FAs). Although evidence from other mammalian models revealed a negative impact of saturated fatty acids (SFA) on developmental competence of oocytes, pig has not yet been widely analyzed. Therefore, we aimed to investigate whether supplementation of IVM medium with 150 µM of stearic acid (SA) and oleic acid (OA) affects lipid content and expression of genes related to fatty acid metabolism in porcine cumulus-oocyte complexes and parthenogenetic embryo development. We found significant influence of fatty acids on lipid metabolism in cumulus cells without affecting the oocyte proper. The expression of ACACA, SCD, PLIN2, FADS1, and FADS2 genes was upregulated (P < 0.01) in cumulus cells, while their expression in oocytes did not change. The increase in gene expression was more pronounced in the case of OA (e.g., up to 30-fold increase in PLIN2 transcript level compared to the control). The number of lipid droplets and occupied area increased significantly in the cumulus cells and did not change in oocytes after SA treatment. Oleic acid improved the blastocyst rate (48 vs 32% in control), whereas stearic acid did not affect this parameter (27%). Additionally, we have discovered a phenotypic diversity of LD in cumulus cells in response to FA supplementation, suggesting extensive lipolysis in response to SA. Stearic acid excess in maturation media led to the formation of multiple micro lipid droplets in cumulus cells.
Subject(s)
Cumulus Cells/metabolism , Embryonic Development/physiology , Fatty Acids/pharmacology , Lipid Droplets/metabolism , Lipolysis/physiology , Swine/embryology , Animals , Apoptosis/drug effects , Blastocyst/drug effects , Embryonic Development/drug effects , Fatty Acids/metabolism , Female , Gene Expression/drug effects , Gene Expression/physiology , In Vitro Oocyte Maturation Techniques/veterinary , Lipid Droplets/drug effects , Lipid Metabolism/genetics , Lipolysis/drug effects , Oleic Acid/pharmacology , Oocytes/drug effects , Oocytes/metabolism , RNA, Messenger/analysis , Stearic Acids/pharmacologyABSTRACT
Endometriosis an important cause of female infertility and seriously impact physical and psychological health of patients. Endometriosis is now considered to be a public health problem that deserves in-depth investigation, especially the etiopathogenesis of endometriosis-associated infertility. We aimed to illuminate the etiopathogenesis of endometriosis-associated infertility that involve excessive oxidative stress (OS) induced pathological changes of ovary cumulus granulosa cell (GCs). Senescence-associated ß-galactosidase (SA ß-gal) activity in GCs from endometriosis patients, soluble isoform of advanced glycation end products receptor (sRAGE) expression in follicular fluid from endometriosis patients and differentially expressed senescence-associated secretory phenotype factors (IL-1ß, MMP-9, KGF and FGF basic protein) are all useful indexes to evaluate oocyte retrieval number and mature oocyte number. RNA-sequencing and bioinformatics analysis indicated senescent phenotype of endometriosis GCs and aggravated endoplasmic reticulum (ER) stress in endometriosis GCs. Targeting ER stress significantly alleviated OS-induced GCs senescence as well as mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) reduction in GCs. Moreover, melatonin administration rescued OS-enhanced ER stress, cellular senescence, and MMP and ATP abnormities of endometriosis GCs in vitro and in vivo. In conclusion, our results indicated excessive reactive oxygen species induces senescence of endometriosis GCs via arouse ER stress, which finally contributes to endometriosis-associated infertility, and melatonin may represent a novel adjuvant therapy strategy for endometriosis-associated infertility.
Subject(s)
Antigens, Neoplasm/genetics , Cumulus Cells/cytology , Endometriosis/drug therapy , Infertility, Female/drug therapy , Melatonin/administration & dosage , Mitogen-Activated Protein Kinases/genetics , Oxidative Stress/drug effects , Animals , Cell Line , Cellular Senescence/drug effects , Cumulus Cells/metabolism , Disease Models, Animal , Endometriosis/complications , Endometriosis/genetics , Female , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Infertility, Female/etiology , Infertility, Female/genetics , Melatonin/pharmacology , Mice , Ovulation Induction , Sequence Analysis, RNAABSTRACT
In vitro maturation (IVM) can impair the balance between antioxidant capacity and oxidative stress, and jeopardize embryo development by increasing oxidative stress, reducing energy metabolism, and causing improper meiotic segregation. Balancing the energy production and reduction of oxidative stress can be achieved by supplementation with coenzyme Q10 (CoQ10), an electron transporter in the mitochondrial inner membrane. To improve the in vitro production of ovine embryos, we studied the effect of CoQ10 supplementation during the maturation of sheep oocytes. A minimum of 100 cumulus-oocyte complexes (COCs) were matured in the presence of 15, 30, or 50 µM CoQ10 in three to five replicates; next, in vitro fertilization and culture in a subset of oocytes were done. Our data revealed that compared to control oocytes or other concentrations of CoQ10, supplementation with 30 µM CoQ10 resulted in a significant increase in blastocyst formation and hatching rates, improved the distribution, relative mass and potential membrane of mitochondria, decreased the levels of reactive oxygen species and glutathione and lessened the percentage of oocytes with misaligned chromosomes after spindle assembly. The relative expression levels of apoptosis markers CASPASE3 and BAX were significantly reduced in CoQ10-treated oocytes and cumulus cells whereas the relative expression level of GDF9, an oocyte-specific growth factor, significantly increased. In conclusion, supplementation with CoQ10 improves the quality of COCs and the subsequent developmental competence of the embryo.
Subject(s)
Mitochondria/metabolism , Oocytes/growth & development , Oocytes/metabolism , Signal Transduction/drug effects , Ubiquinone/analogs & derivatives , Vitamins/pharmacology , Animals , Blastocyst/metabolism , Cumulus Cells/metabolism , Embryonic Development/drug effects , Energy Metabolism/drug effects , Female , Fertilization in Vitro/methods , Glutathione/metabolism , In Vitro Oocyte Maturation Techniques/methods , Membrane Potential, Mitochondrial/drug effects , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Sheep , Ubiquinone/administration & dosage , Ubiquinone/pharmacology , Vitamins/administration & dosageABSTRACT
BACKGROUND: Detrimental exposures during pregnancy have been implicated in programming offspring to develop permanent changes in physiology and metabolism, increasing the risk for developing diseases in adulthood such as hypertension, diabetes, heart disease and obesity. This study investigated the effects of protein restriction on the metabolism of amino acids within the oocyte, liver, and whole organism in a rat model as well as effects on mitochondrial ultrastructure and function in the cumulus oocyte complex. METHODS: Wistar outbred female rats 8-11 weeks of age (n = 24) were assigned to three isocaloric dietary groups, including control (C), low protein (LP) and low protein supplemented with folate (LPF). Animals were superovulated and 48 h later underwent central catheterization. Isotopic tracers of 1-13C-5C2H3-methionine, 2H2-cysteine, U-13C3-cysteine and U-13C3-serine were administered by a 4 h prime-constant rate infusion. After sacrifice, oocytes were denuded of cumulus cells and liver specimens were obtained. RESULTS: Oocytes demonstrated reduced serine flux in LP vs. LPF (p < 0.05), reduced cysteine flux in LP and LPF vs. C (p < 0.05), and a trend toward reduced transsulfuration in LP vs. C and LPF. Folic acid supplementation reversed observed effects on serine flux and transsulfuration. Preovulatory protein restriction increased whole-body methionine transmethylation, methionine transsulfuration and the flux of serine in LP and LPF vs. C (p = 0.003, p = 0.002, p = 0.005). The concentration of glutathione was increased in erythrocytes and liver in LP and LPF vs. C (p = 0.003 and p = 0.0003). Oocyte mitochondrial ultrastructure in LP and LPF had increased proportions of abnormal mitochondria vs. C (p < 0.01 and p < 0.05). Cumulus cell mitochondrial ultrastructure in LP and LPF groups had increased proportions of abnormal mitochondria vs. C (p < 0.001 and p < 0.05). Preovulatory protein restriction altered oocyte expression of Drp1, Opa-1, Mfn1/2, Parl and Ndufb6 (p < 0.05) and Hk2 (p < 0.01), which are genes involved in mitochondrial fission (division) and fusion, mitochondrial apoptotic mechanisms, respiratory electron transport and glucose metabolism. CONCLUSIONS: Preovulatory protein restriction resulted in altered amino acid metabolism, abnormal cumulus oocyte complex mitochondrial ultrastructure and differential oocyte expression of genes related to mitochondrial biogenesis.
Subject(s)
Amino Acids/metabolism , Diet, Protein-Restricted , Folic Acid/pharmacology , Mitochondria/metabolism , Oocytes/drug effects , Animals , Cumulus Cells/cytology , Cumulus Cells/metabolism , Female , Follicular Phase , Gene Expression/drug effects , Kinetics , Microscopy, Electron, Transmission , Mitochondria/ultrastructure , Oocytes/metabolism , Rats, Wistar , Vitamin B ComplexABSTRACT
Aflatoxin B1 (AFB1) is a major food and feed contaminant that threaten public health. Previous studies indicate that AFB1 exposure disrupted oocyte maturation. However, an effective and feasible method is unavailable for protecting oocytes against toxicity of AFB1. In the present study, using in vitro matured porcine oocytes and parthenogenetic embryos as model, we confirmed that AFB1 exposure during in vitro oocyte maturation (IVM) significantly impaired both nuclear and cytoplasmic maturation in a dose- and time-dependent manner. The different concentrations of melatonin were also tested for their protective effects on oocytes against the AFB1-induced toxicity. Our results showed that supplementation of a relative high concentration of melatonin (10-3 mol/L) during IVM efficiently reversed the impaired development rate and blastocyst quality, to the levels comparable to those of the control group. Further analysis indicated that melatonin application efficiently alleviated reactive oxygen species accumulation and initiation of apoptosis induced by AFB1 exposure. In addition, disrupted GSH/GPX system, as well as inhibited mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis in AFB1-treated oocytes, can be notably reversed by melatonin application. Furthermore, cumulus cells may be important in mediating the toxicity of AFB1 to oocytes, and the metabolism of AFB1 in cumulus cells can be depressed by melatonin. To the best of our knowledge, this is the first report to confirm that melatonin application can efficiently protect oocytes from AFB1-induced toxicity. Our study provides a promising and practical strategy for alleviating or reversing AFB1-induced female reproductive toxicity in both clinical treatment and domestic reproductive management.
Subject(s)
Aflatoxin B1/pharmacology , In Vitro Oocyte Maturation Techniques , Melatonin/pharmacology , Oocytes/cytology , Oocytes/drug effects , Animals , Apoptosis/drug effects , Cells, Cultured , Cumulus Cells/cytology , Cumulus Cells/drug effects , Cumulus Cells/metabolism , DNA Copy Number Variations/genetics , DNA Copy Number Variations/physiology , DNA, Mitochondrial/drug effects , Female , Glutathione/metabolism , In Situ Nick-End Labeling , Oocytes/metabolism , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , SwineABSTRACT
Poor-quality oocytes (those with 1-2 layers of cumulus cells) typically possess low meiotic competence and development. Prolonging the duration of in vitro maturation (IVM; 52 hr) can enhance the maturation rate of poor-quality oocytes, but it does not improve subsequent embryonic development. This likely reflects the increased reactive oxygen species (ROS) production and apoptosis seen in these oocytes compared with the non-prolonged IVM (44 hr) group. Melatonin is a free radical scavenger, anti-oxidant and anti-apoptotic agent that reported to enhance the quality of embryos by inhibiting ROS generation and apoptosis. Therefore, we herein investigated whether melatonin combined with prolonged IVM (52 hr) could improve the quality and development of poor-quality oocytes. We supplemented IVM and/or in vitro culture (IVC) media with various concentrations (0, 10-7 , 10-6 , 10-5 M) of melatonin, and estimated parameters related to oocyte quality and development. The addition of melatonin (10-6 M) to a prolonged IVM system improved the oocyte quality and development compared with those of the melatonin-free poor-quality oocytes group, and that this was due to decreases in ROS generation, apoptosis, and DNA damage. When melatonin was added during both IVM (10-6 M) and IVC (10-6 M), we observed a cumulative positive influence on the embryonic development and quality; this treatment enhanced the expression level of Oct4 and decreased the levels of ROS, DNA damage, and apoptosis. Together, these findings suggest that the combination of melatonin plus prolonged IVM can improve the quality and development of poor-quality porcine oocytes via anti-oxidative and anti-apoptotic effects.
Subject(s)
Antioxidants/pharmacology , In Vitro Oocyte Maturation Techniques/methods , Melatonin/pharmacology , Oocytes/growth & development , Oocytes/metabolism , Analysis of Variance , Animals , Apoptosis/drug effects , Blastocyst/metabolism , Cells, Cultured , Cumulus Cells/metabolism , DNA Damage/drug effects , Embryonic Development/drug effects , Female , Gene Expression , Membrane Potential, Mitochondrial/drug effects , Octamer Transcription Factor-3/metabolism , Oxidative Stress/drug effects , Pregnancy , Reactive Oxygen Species/metabolism , Receptor, Melatonin, MT1/genetics , SwineABSTRACT
High-protein diets often lead to an increase in urea concentration in follicular fluid of dairy cows, which may reduce oocyte competence. In the present study, maturation media were supplemented with urea (0, 20, 40 mg/dl), and amino acids (AAs) turnover was evaluated in the 24-h spent media of specimens (cell types), bovine cumulus-oocyte complexes (COCs), cumulus cells (CCs), or denuded oocytes (DOs). The main effects of urea and cell type, and their interaction were significant on the individual turnover (expect threonine, glycine, and tyrosine) and total turnover, depletion, and appearance of AAs. The results showed a high level of urea and DOs increased the depletion of all AAs and that of essential and non-AAs, respectively. Sensitivity analysis revealed the highest sensitivity of isoleucine, lysine, and tryptophan to urea, especially in DOs. Principal component analysis (PCA) evaluated the strong correlations between the turnover of: (1) glutamine, aspartic acid or glycine, and developmental competence and fertilization of COCs; (2) serine, isoleucine, valine or glutamic acid, and cleavage rate of DOs; and (3) serine, glutamine, aspartic acid or alanine, and CCs viability. In conclusion, urea significantly changed the turnover of AAs by COCs, CCs and DOs, and reduced the subsequent developmental competence of bovine oocytes.
Subject(s)
Amino Acids/metabolism , Cumulus Cells/metabolism , Urea/metabolism , Animals , Cattle , Cell Culture Techniques/methods , Coculture Techniques/methods , Embryonic Development , Female , Fertilization in Vitro/drug effects , Fertilization in Vitro/veterinary , Oocytes/drug effects , Oocytes/metabolism , Ovarian Follicle/metabolismABSTRACT
Low oocyte quality is a possible causal factor of obesity-induced infertility. High palmitic acid (PA) concentration in follicular fluid is a crucial feature noted in obese women. This study examined how high PA concentration reduced mitochondrial quality in oocytes and investigated a possible countermeasure against mitochondrial dysfunction. Cumulus cell-oocyte complexes were obtained from the ovaries of gilts, and incubated in medium containing PA (0.5 mM) or vehicle (BSA) for 44 h. Culturing oocytes at high PA concentration induced mitochondrial dysfunction determined by high reactive oxygen species and low ATP content in oocytes. Furthermore, high PA levels increased mitochondrial acetylation levels determined by a high degree of co-localization of TOMM20 and acetylated-lysine. In addition, high PA levels reduced the expression of Sirtuin 3 (SIRT3) and phosphorylated AMP-activated protein kinase (AMPK), while the AMPK activator, AICAR, restored mitochondrial function as well as oocyte ability and reduced the acetylation of mitochondrial protein. Supplementation of culture medium with dorsomorphin dihydrochloride (an AMPK inhibitor) reduced mitochondrial function and increased mitochondrial protein acetylation. Treatment of oocytes with LB100 (an inhibitor of AMPK dephosphorylation) reduced mitochondrial acetylation levels and restored mitochondrial function. Furthermore, high PA levels increased ceramide accumulation in oocytes, and addition of ceramide to the culture medium also induced mitochondrial dysfunction and increased mitochondrial acetylation. This detrimental effect of ceramide was diminished by AICAR treatment of oocytes. Our results indicated that PA induces ceramide accumulation and downregulates the AMPK/SIRT3 pathway causing mitochondrial protein hyperacetylation and dysfunction in oocytes.
Subject(s)
Ceramides/metabolism , Mitochondria/drug effects , Mitochondrial Proteins/metabolism , Oocytes/drug effects , Palmitic Acid/pharmacology , AMP-Activated Protein Kinases/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , Cells, Cultured , Cumulus Cells/drug effects , Cumulus Cells/metabolism , Female , Mitochondria/metabolism , Oocytes/metabolism , Oxidative Stress/drug effects , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , Sirtuin 3/metabolism , SwineABSTRACT
The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.
Subject(s)
Follicular Fluid/metabolism , In Vitro Oocyte Maturation Techniques , Lipid Metabolism , Serum/metabolism , Adenosine Triphosphate/metabolism , Animals , Bone Morphogenetic Protein 15/metabolism , Boron Compounds/metabolism , Cell Proliferation , Cumulus Cells/cytology , Cumulus Cells/metabolism , Embryonic Development , Female , Fertilization in Vitro , Fluorescence , Gene Expression Regulation , Glutathione/metabolism , Growth Differentiation Factor 9/metabolism , Lipid Metabolism/genetics , Nuclear Transfer Techniques , Oocytes/cytology , Oocytes/metabolism , Parthenogenesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , SwineABSTRACT
Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.
Subject(s)
Blastocyst/drug effects , Caloric Restriction/veterinary , Cumulus Cells/drug effects , Dairying , Dietary Supplements , Fertilization in Vitro/veterinary , Follicular Fluid/drug effects , Oocytes/drug effects , Propylene Glycol/administration & dosage , Transcriptome/drug effects , Administration, Oral , Animals , Blastocyst/metabolism , Cattle , Cumulus Cells/metabolism , Epigenesis, Genetic/drug effects , Female , Follicular Fluid/metabolism , Gene Expression Profiling/veterinary , Gene Expression Regulation, Developmental/drug effects , Insulin/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Nutritional Status , Oocytes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time FactorsABSTRACT
BACKGROUND: Supplementation of bovine oocyte-cumulus complexes during in vitro maturation (IVM) with 1 µM of docosahexaenoic acid (DHA), C22:6 n-3 polyunsaturated fatty acid, was reported to improve in vitro embryo development. The objective of this paper was to decipher the mechanisms of DHA action. RESULTS: Transcriptomic analysis of 1 µM DHA-treated and control cumulus cells after 4 h IVM showed no significant difference in gene expression. MALDI-TOF mass spectrometry analysis of lipid profiles in DHA-treated and control oocytes and cumulus cells after IVM showed variations of only 3 out of 700 molecular species in oocytes and 7 out of 698 species in cumulus cells (p < 0.01). We showed expression of free fatty acid receptor FFAR4 in both oocytes and cumulus cells, this receptor is known to be activated by binding to DHA. FFAR4 protein was localized close to the cellular membrane by immunofluorescence. Functional studies demonstrated that supplementation with FFAR4 agonist TUG-891 (1 µM or 5 µM) during IVM led to an increased blastocyst rate (39.5% ± 4.1%, 41.3% ± 4.1%), similar to DHA 1 µM treatment (39.2% ± 4.1%) as compared to control (25.2% ± 3.6%). FFAR4 activation via TUG-891 led to beneficial effect on oocyte developmental competence and might explain in part similar effects of DHA. CONCLUSIONS: In conclusion, we suggested that low dose of DHA (1 µM) during IVM might activate regulatory mechanisms without evident effect on gene expression and lipid content in oocyte-cumulus complexes, likely through signaling pathways which need to be elucidated in further studies.
Subject(s)
Cumulus Cells/drug effects , Cumulus Cells/metabolism , Docosahexaenoic Acids/pharmacology , Oocytes/drug effects , Oocytes/metabolism , Animals , Cattle , Embryonic Development/drug effects , Embryonic Development/genetics , Female , Fertilization in Vitro , Gene Expression Profiling , Gene Expression Regulation/drug effects , Immunohistochemistry , In Vitro Oocyte Maturation Techniques , Lipid Metabolism/drug effects , Lipids , Mitogen-Activated Protein Kinase 3/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10-3, 10-5, 10-7, 10-9, and 10-11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10-7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.