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In brief: GRK2 deficiency disrupts the early embryonic development in pigs. The regulation of GRK2 on HSP90 and AKT may also play an important role during embryo development and tumor formation. Abstract: Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, G-protein-coupled receptor kinase 2 (GRK2) binds to HSP90 in response to hypoxia or other stresses. In this study, we investigated the effects of GRK2 knockdown and inhibition on porcine embryonic development from the zygote stage. Immunofluorescence and western blotting were used to determine the localization and expression, respectively, of GRK2 and related proteins. First, GRK2 and p-GRK2 were expressed in both the cytoplasm and membrane and co-localized with HSP90 on the membrane. The mRNA level of GRK2 increased until the 8C-morula stage, suggesting that GRK2 may play an essential role during the early development of the porcine embryos. GRK2 knockdown reduced porcine embryo development capacity and led to significantly decreased blastocyst quality. In addition, inhibition of GRK2 also induced poor ability of embryo development at an early stage, indicating that GRK2 is critical for embryonic cleavage in pigs. Knockdown and inhibition of GRK2 reduced HSP90 expression, AKT activation, and cAMP levels. Additionally, GRK2 deficiency increased LC3 expression, suggesting enhanced autophagy during embryo development. In summary, we showed that GRK2 binds to HSP90 on the membrane to regulate embryonic cleavage and AKT activation during embryonic development in pigs.
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Desenvolvimento Embrionário , Quinase 2 de Receptor Acoplado a Proteína G , Proteínas de Choque Térmico HSP90 , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/genética , Suínos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Feminino , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is primarily used as a marker for endoplasmic reticulum stress; however, its other functions have not been well studied. Therefore, in this study, we aimed to investigate the function of GRP78 during porcine embryonic development. The developmental quality of porcine embryos, expression of cell cycle proteins, and function of mitochondria were evaluated by inhibiting the function of GRP78. Porcine oocytes were activated to undergo parthenogenesis, and blastocysts were obtained after 7 days of in vitro culture. GRP78 function was inhibited by adding 20 µM HA15 to the in vitro culture medium. The inhibition in GRP78 function led to a decrease in G proteins release, which subsequently downregulated the cyclic adenosine monophosphate (cAMP)/PKA pathway. Ultimately, inhibition of GRP78 function induced the inhibition of CDK1 and cyclin B expression and disruption of the cell cycle. In addition, inhibition of GRP78 function regulated DRP1 and SIRT1 expression, resulting in mitochondrial dysfunction. This study provides new insights into the role of GRP78 in porcine embryonic development, particularly its involvement in the regulation of the MC4R pathway and downstream cAMP/PKA signaling. The results suggest that the inhibition of GRP78 function in porcine embryos by HA15 treatment may have negative effects on embryo quality and development. This study also demonstrated that GRP78 plays a crucial role in the functioning of MC4R, which releases the G protein during porcine embryonic development.
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Chaperona BiP do Retículo Endoplasmático , Receptor Tipo 4 de Melanocortina , Feminino , Gravidez , Suínos , Animais , Desenvolvimento Embrionário , Partenogênese , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico , Proteínas de Ligação ao GTPRESUMO
Y-box binding protein 1 (YBX1) is a member of the family of DNA- and RNA-binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one-cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four-cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6-methyladenosine (m6A) writer N6-adenosine-methyltransferase 70 kDa subunit (METTL3) and reader insulin-like growth factor 2 mRNA-binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process.
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Proteínas de Ligação a DNA , Desenvolvimento Embrionário , Zigoto , Animais , Adenosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Zigoto/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease that affects women of reproductive age. It is also a significant cause of infertility. Circular RNAs have been found to have a crucial role in the development and progression of reproductive system diseases. In this study, we focused on circ_BECN1 and aimed to investigate its role and mechanism in PCOS, providing a foundation for early diagnosis and treatment of this condition. Our findings revealed an upregulation of circ_BECN1 expression in the ovarian granulosa cells (GCs) of PCOS patients. Additionally, the silencing of circ_BECN1 resulted in inhibited proliferation and enhanced apoptosis of the human ovarian granulosa-like tumor cell line (KGN), therefore implicating circ_BECN1 in the cell cycle process. Through a dual-luciferase reporting assay, we determined that circ_BECN1 acts as a sponge for miR-619-5p and that Rab5b is the target gene of miR-619-5p. Moreover, the expression of Rab5b was found to be upregulated in the ovarian tissue of PCOS patients. Knocking down circ_BECN1 resulted in decreased Rab5b expression, which was then restored by using a miR-619-5p inhibitor. Additionally, rescue experiments demonstrated that overexpressing Rab5b reversed the effects of circ_BECN1 knockdown on cell proliferation and apoptosis in KGN cells. In summary, our findings indicate that circ_BECN1 is upregulated in PCOS GCs and promotes cell growth and cell cycle progression, and reduces cell apoptosis by modulating the miR-619-5p/Rab5b axis. Therefore, circ_BECN1 may serve as a potential therapeutic target for PCOS treatment.
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MicroRNAs , Síndrome do Ovário Policístico , Feminino , Humanos , Apoptose/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , MicroRNAs/genética , Síndrome do Ovário Policístico/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Regulação para CimaRESUMO
Controlling the bandwidth and directionality of thermal emission is important for a broad range of applications, from imaging and sensing to energy harvesting. Here, we propose a new, to the best of our knowledge, type of long-wavelength infrared narrowband thermal emitter that is basically composed of aperiodic Tamm plasmon polariton structures. Compared to the thermal emitter based on periodic structures, more parameters need to be considered. An inverse design algorithm instead of traditional forward methodologies is employed to do the geometric parameter optimization. Both theoretical and experimental results show that the thermal emitter exhibits a narrowband thermal emission peak at the wavelength of 8.6â µm in the normal direction. The angular response of emission properties of the thermal emitter is dependent on the emission angle. We believe that our proposed thermal emitter provides an alternative for low-cost, high-effective narrowband mid-infrared light sources and would have a great potential in many applications.
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The application of plasmonic structure has been demonstrated to improve the performance of infrared photodetectors. However, the successful experimental realization of the incorporation of such optical engineering structure into HgCdTe-based photodetectors has rarely been reported. In this paper, we present a HgCdTe infrared photodetector with integrated plasmonic structure. The experimental results show that the device with plasmonic structure has a distinct narrowband effect with a peak response rate close to 2 A/W, which is nearly 34% higher compared with the reference device. The simulation results are in good agreement with the experiment, and an analysis of the effect of the plasmonic structure is given, demonstrating the crucial role of the plasmonic structure in the enhancement of the device performance.
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N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10â h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.
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Desenvolvimento Embrionário , Epigênese Genética , Animais , Suínos , Temperatura , MamíferosRESUMO
Zinc finger and SCAN domain-containing 4 (ZSCAN4), a DNA-binding protein, maintains telomere length and plays a key role in critical aspects of mouse embryonic stem cells, including maintaining genomic stability and defying cellular senescence. However, the effect of ZSCAN4 in porcine parthenogenetic embryos remains unclear. To investigate the function of ZSCAN4 and the underlying mechanism in porcine embryo development, ZSCAN4 was knocked down via dsRNA injection in the one-cell stage. ZSCAN4 was highly expressed in the four- and five- to eight-cell stages in porcine embryos. The percentage of four-cell stage embryos, five- to eight-cell stage embryos, and blastocysts was lower in the ZSCAN4 knockdown group than in the control group. Notably, depletion of ZSCAN4 induced the protein expression of DNMT1 and 5-Methylcytosine (5mC, a methylated form of the DNA base cytosine) in the four-cell stage. The H3K27ac level and ZGA genes expression decreased following ZSCAN4 knockdown. Furthermore, ZSCAN4 knockdown led to DNA damage and shortened telomere compared with the control. Additionally, DNMT1-dsRNA was injected to reduce DNA hypermethylation in ZSCAN4 knockdown embryos. DNMT1 knockdown rescued telomere shortening and developmental defects caused by ZSCAN4 knockdown. In conclusion, ZSCAN4 is involved in the regulation of transcriptional activity and is essential for maintaining telomere length by regulating DNMT1 expression in porcine ZGA.
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Telômero , Fatores de Transcrição , Animais , Camundongos , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Telômero/genética , Telômero/metabolismo , Encurtamento do Telômero , Proteínas de Ligação a DNA/metabolismo , Zigoto/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.
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Inibidores de Dissociação do Nucleotídeo Guanina/genética , Transporte Proteico , Rede trans-Golgi , Animais , Membrana Celular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Transdução de Sinais , Rede trans-Golgi/metabolismoRESUMO
Thermal infrared camouflage as a kind of counter-surveillance technique has attracted much attention owing to the rapid development of infrared surveillance technology. Various artificial optical structures have been developed for infrared camouflage applications under cold ambient environment (low thermal radiation), but the realization of infrared camouflage under a hot environment (high thermal radiation) is also highly desirable and has been rarely reported. Here, a lithography-free, ultra-thin, high performance long-wavelength infrared (LWIR) selective emitter for thermal infrared camouflage in a high radiation environment is proposed and experimentally demonstrated. Experimental results show that our designed selective emitter exhibits average emissivity higher than 90% over the LWIR range from 8 to 14â µm and low emissivity less than 35% outside this window. Numerical simulations were performed to optimize the geometrical structures and reveal that such a selective emission effect is attributed to the combination of multiple hybrid plasmonic resonances. LWIR thermal images show that the selective emitter can perfectly blend into the high radiation backgrounds. Furthermore, it is found that the sample displays angle-independent emission properties, indicating that our emitter offers great potential for application in evading large-angle detection.
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Tobacco seeds are a valuable food oil resource, and tobacco seed oil is rich in nutrients, especially polyunsaturated fatty acids. The aim of this work was to perform a comprehensive study on the chemical constituents, and the antioxidant, anti-inflammatory, and whitening activities of tobacco seed oils (NC89 and BS4). A GC/MS analysis revealed that NC89 and BS4 had 11 and 6 volatile compounds, respectively. The PUFA contents in NC89 and BS4 were 74.98% and 72.84%, respectively. These two tobacco seed oils also presented good radical scavenging capacities with the neutralization of ABTS, OH-, and superoxide (O2-) radicals in a concentration-dependent manner. Meanwhile, NC89 and BS4 inhibited reactive oxygen species (ROS) accumulation and cell apoptosis, enhanced SOD and CAT activities, and increased the GSH content in H2O2-induced HepG2 cells. In addition, NC89 and BS4 exhibited significant anti-inflammatory activities by inhibiting the expressions of NO, TNF-α, IL-1ß, and IL-6 in LPS-induced RAW.264.7 cells through the regulation of the MAPK signaling pathway. Moreover, NC89 and BS4 expressed whitening activities by inhibiting tyrosinase activity and intracellular melanin production. Therefore, tobacco seed oils could be used as an important oil resource for the development of high value-added products.
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Antioxidantes , Óleos Voláteis , Antioxidantes/química , Nicotiana/metabolismo , Óleos de Plantas/química , Peróxido de Hidrogênio/análise , Sementes/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/análise , Ácidos Graxos Insaturados/análise , Óleos Voláteis/análiseRESUMO
ACSS1/2 converts acetate into acetyl-coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs-Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (-P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta-oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose-dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C-terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA.
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Acetato-CoA Ligase/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Zigoto/enzimologia , Acetato-CoA Ligase/genética , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura Embrionária , Partenogênese , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Sus scrofaRESUMO
The cytoskeleton plays an orchestrating role in polarized cell growth. Microtubules (MTs) not only play critical roles in chromosome alignment and segregation but also control cell shape, division, and motility. A member of the plus-end tracking proteins, end-binding protein 1 (EB1), regulates MT dynamics and plays vital roles in maintaining spindle symmetry and chromosome alignment during mitosis. However, the role of EB1 in mouse oocyte meiosis remains unknown. Here, we examined the localization patterns and expression levels of EB1 at different stages. EB1 protein level was found to be stable during meiosis. EB1 mainly localized along the spindle and had a similar localization pattern as that of α-tubulin. The EB1 protein was degraded with a Trim-Away method, and the results were further confirmed with western blotting and immunofluorescence. At 12 h of culture after EB1 knockdown (KD), a reduced number of mature MII oocytes were observed. EB1 KD led to spindle disorganization, chromosome misalignment, and missegregation; ß-catenin protein binds to actin via the adherens junctional complex, which was significantly reduced in the EB1 KD oocytes. Collectively, we propose that the impairment of EB1 function manipulates spindle formation, thereby promoting chromosomal loss, which is expected to fuel aneuploidy and possibly fertilization failure.
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Meiose , Fuso Acromático , Animais , Cromossomos , Camundongos , Microtúbulos , OócitosRESUMO
Cell cycle of mouse embryo could be delayed by nicotinamide (NAM). Histone H3 lysine 56 (H3K56ac) acetylation plays an important role in mammalian genomic stability and the function of this modification in mouse embryos is not known. Hence, we designed to study the effects of NAM-induced oxidative stress on the developmental ability of mouse embryos, on the acetylation of H3K56ac and the possible functions of this modification related to mouse embryo development. Treatment with NAM (10, 20, or 40 mmol/L for 24 or 48 hr) during in vitro culture significantly decreased developmental rate of blastocyst (24 hr: 90.2 vs. 81.2, 43.2, and 18.2, with p > .05, p < .01, respectively; 48 hr: 89.3 vs. 53.2%, 12.1%, and 0% with p < .05, respectively). NAM treatment (20 mmol/L) for 6 and 31 hr resulted in increased intracellular reactive oxygen species levels in two-cell embryos, and apoptotic cell numbers in blastocysts. Resveratrol (RSV) and I-CBP112 rescued the 20 mmol/L NAM-induced embryo developmental defects. RSV and I-CBP112 increased the level of Sirt1 and decreased the level of H3K56ac induced by NAM in two-cell embryos (p < .05). These data suggest that NAM treatment decreases the expression of Sirt1, which induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced mouse embryo defects, which can be rescued by RSV and I-CBP112.
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Desenvolvimento Embrionário/efeitos dos fármacos , Niacinamida/farmacologia , Oxazepinas/farmacologia , Piperidinas/farmacologia , Resveratrol/farmacologia , Animais , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histonas/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Connexin 43 (CX43) is a component of gap junctions. The lack of functional CX43 induces oxidative stress, autophagy, and apoptosis in somatic cells. However, the role of CX43 in the early development of porcine embryos is still unknown. Thus, the aim of this study was to investigate the role of CX43, and its underlying molecular mechanisms, on the developmental competence of early porcine embryos. We performed CX43 knockdown by microinjecting dsRNA into parthenogenetically activated porcine parthenotes. The blastocyst development rate and the total number of cells in the blastocysts were significantly reduced by CX43 knockdown. Results from FITC-dextran assays showed that CX43 knockdown significantly increased membrane permeability. ZO-1 protein was obliterated in CX43 knockdown blastocysts. Mitochondrial membrane potential and ATP production were significantly reduced following CX43 knockdown. Reactive oxygen species (ROS) levels were significantly increased in the CX43 knockdown group compared to those in control embryos. Moreover, CX43 knockdown induced autophagy and apoptosis. Our findings indicate that CX43 is essential for the development and preimplantation of porcine embryos and maintains mitochondrial function, cell junction structure, and cell homeostasis by regulating membrane permeability, ROS generation, autophagy, and apoptosis in early embryos.
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Conexina 43/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Mitocôndrias/metabolismo , Animais , Apoptose , Autofagia , Blastocisto/metabolismo , Técnicas de Silenciamento de Genes , Junções Intercelulares , Potencial da Membrana Mitocondrial/fisiologia , Oócitos , Estresse Oxidativo , Espécies Reativas de Oxigênio , SuínosRESUMO
Nitrogen molecules are promoted to excited neutral states during femtosecond laser pulse filamentary propagation in atmosphere, leading to a characteristic UV fluorescence. Using a laser-induced fluorescence depletion technique, we measure the formation dynamics of these excited neutral nitrogen molecules with femtosecond time resolution. We find that the excited neutral molecules are formed in an unexpected ultrafast timescale of â¼4 ps at 1 bar and â¼120 ps at 30 mbar pressure. From this observation we deduce that the excitation of neutral N_{2} occurs via multiple collisions with hot free electrons. Numerical simulations based on rate equations reproduce well this ultrafast formation time and its dependence on gas pressure, and thus support this interpretation.
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BACKGROUND: The Poaceae family is of great importance to human beings since it comprises the cereal grasses which are the main sources for human food and animal feed. With the rapid growth of genomic data from Poaceae members, comparative genomics becomes a convinent method to study genetics of diffierent species. The SSRs (Simple Sequence Repeats) are widely used markers in the studies of Poaceae for their high abundance and stability. RESULTS: In this study, using the genomic sequences of 9 Poaceae species, we detected 11,993,943 SSR loci and developed 6,799,910 SSR primer pairs. The results show that SSRs are distributed on all the genomic elements in grass. Hexamer is the most frequent motif and AT/TA is the most frequent motif in dimer. The abundance of the SSRs has a positive linear relationship with the recombination rate. SSR sequences in the coding regions involve a higher GC content in the Poaceae than that in the other species. SSRs of 70-80 bp in length showed the highest AT/GC base ratio among all of these loci. The result shows the highest polymorphism rate belongs to the SSRs ranged from 30 bp to 40 bp. Using all the SSR primers of Japonica, nineteen universal primers were selected and located on the genome of the grass family. The information of SSR loci, the SSR primers and the tools of mining and analyzing SSR are provided in the PSSRD (Poaceae SSR Database, http://biodb.sdau.edu.cn/pssrd/). CONCLUSIONS: Our study and the PSSRD database provide a foundation for the comparative study in the Poaceae and it will accelerate the study on markers application, gene mapping and molecular breeding.
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Genoma de Planta , Repetições de Microssatélites , Poaceae/classificação , Poaceae/genética , Composição de Bases , DNA de Plantas/química , Bases de Dados GenéticasRESUMO
BACKGROUND: Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. RESULTS: In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. CONCLUSION: Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.
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Citocinese/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Crioprotetores/farmacologia , Citocinese/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Etilenoglicol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glicerol/farmacologia , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Injeções de Esperma Intracitoplásmicas , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Craniopharyngiomas originate from the pituitary stalk (PS) and extend along the pituitary-hypothalamic axis. Peripheral retroinfundibular craniopharyngiomas, particularly, may have worse surgery outcomes than other types. This study aims to investigate the advantage of using "one-and-a-half" interdural transcavernous pituitary transposition/rotation to dissect the tumor from the residual stalk and hypophyseal portal system for this subtype of craniopharyngioma. METHODS: From August 2018 to February 2023, patients with peripheral retroinfundibular craniopharyngioma underwent surgical treatment. We analyzed clinical information, surgical records, imaging, and examination findings. The surgical procedure, including "one-and-a-half" interdural transcavernous pituitary transposition and rotation, was explained. Postoperative follow-up included endocrinological tests, MRI examinations, and urination surveys. RESULTS: Among the 52 patients diagnosed with craniopharyngioma who underwent surgical treatment, 9 were classified as peripheral retroinfundibular craniopharyngioma, and they received "one-and-a-half" interdural transcavernous pituitary transposition and stalk rotation. In 6 cases, the residual PS and most of the hypophyseal portal system were preserved. Gross total resection was achieved in 5 patients and near total resection in 1 patient. One patient had a transection of the bilateral inferior hypophyseal arteries and 5 unilaterally. None experienced permanent diabetes insipidus, but varying degrees of anterior pituitary dysfunction postoperatively required hormone replacement therapy, which gradually decreased over time. CONCLUSION: The natural anatomic corridor, "one-and-a-half" interdural transcavernous pituitary transposition, and stalk rotation provide increased working space compared with intradural or extradural pituitary transposition. Simultaneously rotating the tumor and pituitary enables a specific attack angle for lesion dissection after the anteriorly displaced residual stalk is rotated laterally. This approach preserves the residual PS and hypophyseal portal system, avoiding complications of diabetes insipidus and hypopituitarism. In most cases, only one side of the inferior hypophyseal artery needs to be sacrificed, ensuring normal pituitary function.
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Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.