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1.
Int J Mol Sci ; 25(18)2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39337686

RESUMO

Ovarian cysts are linked to hormone imbalances and altered gene expressions, but the connection between cysts and ion channel expression is understudied. This study explored the role of TWIK-related acid-sensitive K+ (TASK) channels in bovine ovarian cyst formation. The ovarian follicles were split into small (5 to 10 mm in diameter) and large (>25 mm in diameter) groups. Among the measured K+, Na+, and Cl- concentrations in follicular fluid (FF) obtained from small-sized follicles (SFs) and large-sized follicles (LFs), the K+ concentration was significantly lower in LFFF. Quantitative PCR, Western blot, and immunocytochemistry data revealed that TASK-3 expression levels significantly decreased by approximately 50% in LFs and granulosa cells obtained from LFs (LFGCs) compared to the corresponding controls. The TASK-3 protein was localized to the plasma membranes of GCs. The diameters of LFGCs were larger than those of SFGCs. The cell swelling response to exposure to a hypotonic solution (200 mOsm/L) was highly reduced in TASK-3-overexpressing cells compared to vector-transfected cells. TASK-3-knockdown cells showed arrested growth. Senescence markers were detected in LFGCs and TASK-3-knockdown cells. These findings suggest that reduced TASK-3 expression in LFs is associated with the inhibition of GC growth, leading to senescence and cyst formation.


Assuntos
Senescência Celular , Células da Granulosa , Cistos Ovarianos , Folículo Ovariano , Canais de Potássio de Domínios Poros em Tandem , Animais , Feminino , Bovinos , Células da Granulosa/metabolismo , Cistos Ovarianos/metabolismo , Cistos Ovarianos/patologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Folículo Ovariano/metabolismo , Regulação para Baixo , Líquido Folicular/metabolismo , Potássio/metabolismo
2.
Reprod Fertil Dev ; 35(10): 527-538, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37263290

RESUMO

CONTEXT: Telomerase reverse transcriptase is a key factor responsible for structural and cellular alterations in aged oocytes and changes in the structure of the zona pellucida and mitochondria. Telomerase expression is reduced in aged cumulus oocyte complexes, and its activation or enhanced expression would be beneficial for in vitro oocyte maturation and in vitro embryo development. AIMS: This study aimed to investigate telomerase activation by cycloastragenol and its effect on bovine oocyte in vitro maturation, fertilisation, and early embryo development. METHODS: We used qPCR, Western blot, immunofluorescence, reactive oxygen species (ROS) assay,TUNEL assay, JC-1 assay, and invasion assay to analyse the affect of cycloastragenol (CAG) on bovine oocyte maturation, embryo development, embryo quality and implantation potential. KEY RESULTS: Cycloastragenol treatment of oocytes in in vitro maturation (IVM) media significantly (P <0.05) improved oocyte IVM (90.87%), embryo cleavage (90.78%), blastocyst hatching (27.04%), and embryo implantation potential. Telomerase also interacts with mitochondria, and JC-1 staining results showed significantly (P <0.05) higher mitochondrial membrane potential (ΔΨ m) in the CAG-treated group. Furthermore, the inner cell mass (OCT4 and SOX2) and trophoblasts (CDX2) of the control and CAG groups were examined. Moreover, CAG treatment to primary cultured bovine cumulus cells substantially enhanced telomerase activity. CONCLUSIONS: Telomerase activation via cycloastragenol is beneficial for bovine oocyte IVM and for the production of high-quality bovine embryos. IMPLICATIONS: Cycloastragenol is a natural telomerase activator, and could be useful as a permanent component of oocyte maturation media.


Assuntos
Telomerase , Feminino , Animais , Bovinos , Telomerase/genética , Telomerase/metabolismo , Telomerase/farmacologia , Células do Cúmulo/metabolismo , Oócitos/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Implantação do Embrião , Desenvolvimento Embrionário , Blastocisto
3.
Int J Mol Sci ; 24(23)2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38069370

RESUMO

Embryonic genome activation (EGA) is a critical step during embryonic development. Several transcription factors have been identified that play major roles in initiating EGA; however, this gradual and complex mechanism still needs to be explored. In this study, we investigated the role of nuclear transcription factor Y subunit A (NFYA) in bovine EGA and bovine embryonic development and its relationship with the platelet-derived growth factor receptor-ß (PDGFRß) by using a potent selective activator (PDGF-BB) and inhibitor (CP-673451) of PDGF receptors. Activation and inhibition of PDGFRß using PDGF-BB and CP-673451 revealed that NFYA expression is significantly (p < 0.05) affected by the PDGFRß. In addition, PDGFRß mRNA expression was significantly increased (p < 0.05) in the activator group and significantly decreased (p < 0.05) in the inhibitor group when compared with PDGFRα. Downregulation of NFYA following PDGFRß inhibition was associated with the expression of critical EGA-related genes, bovine embryo development rate, and implantation potential. Moreover, ROS and mitochondrial apoptosis levels and expression of pluripotency-related markers necessary for inner cell mass development were also significantly (p < 0.05) affected by the downregulation of NFYA while interrupting trophoblast cell (CDX2) differentiation. In conclusion, the PDGFRß-NFYA axis is critical for bovine embryonic genome activation and embryonic development.


Assuntos
Receptor beta de Fator de Crescimento Derivado de Plaquetas , Transdução de Sinais , Animais , Bovinos , Becaplermina/metabolismo , Transdução de Sinais/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Diferenciação Celular
4.
Hum Mutat ; 43(10): 1472-1489, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35815345

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.


Assuntos
Aminoacil-tRNA Sintetases , Doença de Charcot-Marie-Tooth , Triptofano-tRNA Ligase , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Éxons , Humanos , Mutação , Linhagem , RNA de Transferência/genética , Síndrome , Triptofano-tRNA Ligase/genética
5.
Reproduction ; 163(4): 219-232, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35129460

RESUMO

Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo's ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.


Assuntos
Vesículas Extracelulares , Junções Íntimas , Animais , Blastocisto/metabolismo , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos , Desenvolvimento Embrionário , Vesículas Extracelulares/metabolismo , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Humanos
6.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673357

RESUMO

The Wnt/ß-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/ß-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/ß-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of ß-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/ß-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/ß-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/ß-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders.


Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas/metabolismo , Doenças Metabólicas/embriologia , PPAR delta/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doenças Metabólicas/patologia , Fator de Transcrição 4/metabolismo
7.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070219

RESUMO

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.


Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Bovinos/genética , Proteínas de Membrana/genética , Prenhez/genética , Telomerase/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Bovinos/fisiologia , Células Cultivadas , Fase de Clivagem do Zigoto/metabolismo , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Expressão Gênica , Células da Granulosa/metabolismo , Proteínas de Membrana/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Gravidez , Prenhez/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
8.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072531

RESUMO

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.


Assuntos
Técnicas de Reprogramação Celular , Clonagem de Organismos , Embrião de Mamíferos , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Apoptose/genética , Biomarcadores , Bovinos , Clonagem de Organismos/métodos , Metilação de DNA , Implantação do Embrião , Epigênese Genética , Feminino , Fibroblastos , Expressão Gênica , Histonas/metabolismo , Gravidez , Sensibilidade e Especificidade , Doadores de Tecidos
9.
Mol Reprod Dev ; 87(10): 1070-1081, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32885880

RESUMO

Increased oxidative stress is one of the main causes of poorly developed embryos in assisted reproductive technologies. Nicotinamide (NAM) has been shown to suppress reactive oxygen species (ROS) production through its potent antioxidative and anti-senescent effects. In the present study, we explored the effects of short-term NAM-treatment (3 and 5 h) during in vitro fertilization (IVF) on the development of bovine embryos. Treatment with 10 mM NAM for 3 h significantly increased the blastocyst formation but extending the treatment to 5 h did not enhance the benefits any further. Immunofluorescence analysis demonstrated that treatment with 10 mM NAM for 3 h decreased the expression of intracellular ROS, 8-oxo-7,8-dihydroguanine, caspase-3, and increased the expression of Sirt1, and incorporation of bromodeoxyuridine in one-cell stage embryos. Similarly, the level of H3K56ac significantly increased in the NAM-treated (3 and 5 h) one-cell stage embryos. Contrastingly, the treatment with 10 mM NAM for 5 h increased the caspase-9 level in blastocysts. Collectively, these findings suggest that NAM possesses antioxidant activity and supplementation of IVF medium with 10 mM NAM for 3 h improves the in vitro developmental competence of bovine embryos.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro , Niacinamida/farmacologia , Animais , Antioxidantes/farmacologia , Bovinos/embriologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Masculino , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
10.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375280

RESUMO

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.


Assuntos
Apoptose , Embrião de Mamíferos/patologia , Mitocôndrias/patologia , Naftoquinonas/toxicidade , Oócitos/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/patologia , Bovinos , Citotoxinas/toxicidade , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Mitocôndrias/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Gravidez , Espécies Reativas de Oxigênio/metabolismo
11.
Int J Mol Sci ; 21(20)2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33066562

RESUMO

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3-4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.


Assuntos
Blastocisto/metabolismo , Meios de Cultivo Condicionados/farmacologia , Exossomos/metabolismo , Mitocôndrias/metabolismo , Oviductos/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Células Cultivadas , Ciclo do Ácido Cítrico , Células Epiteliais/metabolismo , Feminino , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Oviductos/citologia , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismo
12.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906439

RESUMO

CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.


Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas Tirosina Fosfatases/genética , Adulto , Exoma/genética , Feminino , Estudos de Associação Genética , Humanos , Irã (Geográfico) , Mutação com Perda de Função , Masculino , Paquistão , Linhagem , Proteínas Tirosina Fosfatases/metabolismo , Sítios de Splice de RNA/genética , Splicing de RNA
13.
Reprod Fertil Dev ; 31(2): 333-346, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30086822

RESUMO

This study investigated the use of bovine serum albumin (BSA) plus insulin-transferrin-sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P<0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P<0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/administração & dosagem , Insulina/administração & dosagem , Metaloproteinases da Matriz/metabolismo , Soroalbumina Bovina/administração & dosagem , Selenito de Sódio/administração & dosagem , Transferrina/administração & dosagem , Animais , Bovinos , Meios de Cultura , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/fisiologia , Feminino
14.
Int J Mol Sci ; 20(12)2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31212969

RESUMO

Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Bovinos , Núcleo Celular/metabolismo , Células do Cúmulo/efeitos dos fármacos , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810173

RESUMO

The PPARs (peroxisome proliferator-activated receptors) play critical roles in the regulation of lipid and glucose metabolism. PPARδ, a member of the PPARs family, is associated with decreased susceptibility to ectopic lipid deposition and is implicated in the regulation of mitochondrial processes. The current study aimed to determine the role of PPARδ in fatty acid ß-oxidation and its influence on PEPCK for the lipogenic/lipolytic balance during in vitro bovine oocyte maturation and embryo development. Activation of PPARδ by GW501516, but not 2-BP, was indicated by intact embryonic PEPCK (cytosolic) and CPT1 expression and the balance between free fatty acids and mitochondrial ß-oxidation that reduced ROS and inhibited p-NF-κB nuclear localization. Genes involved in lipolysis, fatty acid oxidation, and apoptosis showed significant differences after the GW501516 treatment relative to the control- and 2-BP-treated embryos. GSK3787 reversed the PPARδ-induced effects by reducing PEPCK and CPT1 expression and the mitochondrial membrane potential, revealing the importance of PPARδ/PEPCK and PPARδ/CPT1 for controlling lipolysis during embryo development. In conclusion, GW501516-activated PPARδ maintained the correlation between lipolysis and lipogenesis by enhancing PEPCK and CPT1 to improve bovine embryo quality.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Desenvolvimento Embrionário/genética , PPAR delta/genética , Fosfoenolpiruvato Carboxilase/genética , Animais , Apoptose , Bovinos , Ácidos Graxos não Esterificados/metabolismo , Metabolismo dos Lipídeos/genética , Lipogênese/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Oxirredução , Tiazóis/farmacologia
16.
Int J Mol Sci ; 21(1)2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31905822

RESUMO

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ñ°m), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.


Assuntos
Anticorpos Monoclonais/metabolismo , Blastocisto/metabolismo , Desenvolvimento Embrionário/imunologia , Cromossomo Y , Animais , Bovinos/embriologia , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Genes Ligados ao Cromossomo X , Genes Ligados ao Cromossomo Y , Glucose/metabolismo , Cinética , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias , Fosforilação , Fatores Sexuais , Espermatozoides , Cromossomo X
18.
Mol Reprod Dev ; 85(1): 46-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29219221

RESUMO

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.


Assuntos
Antioxidantes/farmacologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Licopeno/farmacologia , Oócitos/crescimento & desenvolvimento , Acil-CoA Desidrogenase de Cadeia Longa/biossíntese , Animais , Blastocisto/citologia , Proteína Morfogenética Óssea 15/biossíntese , Caspase 3/análise , Caspase 9/análise , Bovinos , Coenzima A Ligases/biossíntese , Fator 9 de Diferenciação de Crescimento/biossíntese , Quinase I-kappa B/biossíntese , NADH Desidrogenase/biossíntese , Superóxido Dismutase/biossíntese
19.
PLoS Pathog ; 11(8): e1005084, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26291525

RESUMO

Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.


Assuntos
Infecções por Adenoviridae/imunologia , Adenovírus Humanos/patogenicidade , Modelos Animais de Doenças , Interferon Tipo I/imunologia , Fator de Transcrição STAT2/deficiência , Infecções por Adenoviridae/patologia , Adenovírus Humanos/imunologia , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Cricetinae , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Mesocricetus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT2/imunologia
20.
Reproduction ; 153(4): 421-431, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28069903

RESUMO

Heat stress has large effects on reproduction including conception rate in cattle. In this study, we examined the effects of coagulansin-A (coa-A), a steroidal lactone, on acquired thermo tolerance during in vitro production of bovine embryos. Oocytes were incubated in in vitro maturation (IVM) media with or without coa-A at two different temperatures, 40.5˚C and 42˚C, for 20 h. The treatment of coa-A significantly improved blastocyst development only at 40.5˚C (P < 0.05). Interestingly, immunofluorescence analysis demonstrated that coa-A induced heat shock protein 70 (HSP70) and phosphatidylinositol-3-kinase (PI3K), but significantly attenuated nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX2). To determine the expression patterns of related genes at the transcription level, qRT-PCR was performed. Expression of HSP70 and PI3K was elevated, whereas expression of NF-κB, COX2 and inducible nitric oxide synthase (iNOS) was significantly (P < 0.05) downregulated in the coa-A-treated group compared with the control group. Moreover, pro-apoptotic genes were downregulated, and antiapoptic genes were upregulated in the coa-A group. We also counted the total cell number and apoptotic nuclei at the blastocyst and found that more cell numbers (143.1 ± 1.5) and less apoptotic damages (6.4 ± 0.5) in the coa-A treatment group comparing to control group (131.4 ± 2.0 and 10.8 ± 0.5), indicating the enhanced embryo quality. In conclusion, our results demonstrate that the coa-A not only improved the blastocyst development in vitro but also increased their resistance to heat stress condition through induction of HSP70/PI3K.


Assuntos
Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Transtornos de Estresse por Calor/prevenção & controle , Vitanolídeos/farmacologia , Animais , Bovinos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Fertilização in vitro/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta/efeitos adversos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Vitanolídeos/química
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