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1.
Anim Sci J ; 92(1): e13608, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34405491

RESUMO

This study aims to investigate the morphology and distribution of mitochondria, spindles, and chromosomes in oocytes of aged mice and examine the effects of SRT1720 on oocyte maturation. C57BL/6J mice were divided into young (4-8 weeks) and aged groups (48-52 weeks). In vitro maturation media contained (0.05, 0.1, and 1.0 µM) SRT1720 and 0.1-µM dimethyl sulfoxide (DMSO control). The rate of chromosome misalignment and spindle misorientation in oocytes of aged mice were significantly higher than that of young mice (P < 0.01). Fluorescence intensity of mitochondria from oocytes of aged mice was significantly lower than that of young mice (P < 0.01). SRT1720 at 0.1 µM significantly improved oocyte maturation, fertilization, and blastocyst formation in aged mice compared with young mice (P < 0.01). Additionally, immunofluorescence intensity of mitochondria, normal spindle morphology, and chromosome alignment were notably enhanced with SRT1720 when compared with the DSMO control group for metaphase II (MII)-stage oocytes matured in vitro (P < 0.01); 0.1-µM SRT1720 enhanced the expression level of SRIT1 in oocytes from aged mice. In summary, the aged mice oocytes showed increased nuclear and cytoplasmic defects, whereas SRT1720 enhanced oocyte maturation and quality. We concluded that 0.1-µM SRT1720 was an appropriate concentration for in vitro maturation media.


Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Animais , Blastocisto , Cromossomos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fertilização/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oócitos/citologia , Oócitos/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/patologia
2.
Cell Physiol Biochem ; 55(S3): 157-170, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34318654

RESUMO

BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.


Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Canais de Potássio KCNQ/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antracenos/farmacologia , Expressão Gênica , Humanos , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetraetilamônio/farmacologia , Xenopus laevis
3.
Toxicol Appl Pharmacol ; 426: 115638, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34242569

RESUMO

Gonadal development begins in the intrauterine phase and females from most species are born with an established oocyte reserve. Exposure to drugs during gestation can compromise the offspring health, also affecting the gametes quality. Nicotine, the main component of cigarettes, is an oxidant agent capable of altering the fertility in men and women. As female gametes are susceptible to oxidative stress, this drug can damage the oolemma and affect oocyte maturation, induce errors during chromosomal segregation and DNA fragmentation. Oocyte mitochondria are particularly susceptible to injuries, contributing to the oocyte quality loss and embryonic development disruption. Thus, considering the high number of women who smoke during pregnancy, while significant events are occurring in the embryo for future fertility of offspring, we seek to verify the quality of the oocytes from adult rats exposed to nicotine during intrauterine phase and breastfeeding. Pregnant Wistar rats received nicotine by osmotic mini-pumps and the female progenies were evaluated in adulthood for oocyte quality (viability, lipid peroxidation, generation of reactive oxygen species and mitochondrial integrity) and reproductive capacity. Embryos (3dpc) and fetuses (20dpc) generated by these rats were also evaluated. The results showed that the dose of 2 mg/kg/day of nicotine through placenta and breast milk does not affect the number of oocytes and the fertility capacity of adult rats. However, it causes some morphological alterations in oocytes, mitochondrial changes, embryonic fragmentation and disruption of fetal development. The malformations in fetuses generated from these gametes can also indicate the occurrence of epigenetic modifications.


Assuntos
Nicotina/toxicidade , Oócitos/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Reprodução/efeitos dos fármacos , Animais , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Lactação , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Troca Materno-Fetal , Mitocôndrias/efeitos dos fármacos , Oócitos/metabolismo , Gravidez , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
4.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299380

RESUMO

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 µM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.


Assuntos
Adenina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Adenina/farmacologia , Animais , Clonagem de Organismos/métodos , Cães , Transferência Embrionária/métodos , Feminino , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Gravidez
5.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207376

RESUMO

A better understanding of the mechanism of primordial follicle activation will help us better understand the causes of premature ovarian insufficiency (POI), and will help us identify new drugs that can be applied to the clinical treatment of infertility. In this study, single oocytes were isolated from primordial and primary follicles, and were used for gene profiling with TaqMan array cards. Bioinformatics analysis was performed on the gene expression data, and Ingenuity Pathway Analysis was used to analyze and predict drugs that affect follicle activation. An ovarian in vitro culture system was used to verify the function of the drug candidates, and we found that curcumin maintains the ovarian reserve. Long-term treatment with 100 mg/kg curcumin improved the ovarian reserve indicators of AMH, FSH, and estradiol in aging mice. Mechanistic studies show that curcumin can affect the translocation of FOXO3, thereby inhibiting the PTEN-AKT-FOXO3a pathway and protecting primordial follicles from overactivation. These results suggest that curcumin is a potential drug for the treatment of POI patients and for fertility preservation.


Assuntos
Curcumina/farmacologia , Proteína Forkhead Box O3/metabolismo , Oócitos/efeitos dos fármacos , Reserva Ovariana , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismo , Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Transdução de Sinais , Análise de Célula Única , Transcriptoma
6.
Aging (Albany NY) ; 13(11): 14829-14842, 2021 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-34091440

RESUMO

Samul-tang (SM), a traditional herbal medicine, is used to treat age-related human conditions, such as infertility and menstrual irregularities. The mechanism underlying the role of SM in ovary function needs elucidation. In this study, the influence of SM administration on the ovarian reserve of aged mice was investigated. Female BALB/c mice (8 and 40 weeks-old) were administered with distilled water (young or old group) or SM for 4 weeks. SM administration prevented age-related ovarian follicle loss in mice. Quality of oocytes and blastocysts were enhanced in SM-administrated mice compared to those of non-treated old mice. Further, SM administration increased the pregnancy rate and number of litters. SM triggered changes in aging-related genes that are linked to the RAS-mediated pathway. Thus, we demonstrate that SM can be used to increase the oocyte yield in aged women, potentially improving age-related cognitive decline in the ovarian reserve.


Assuntos
Envelhecimento/fisiologia , Medicamentos de Ervas Chinesas/farmacologia , Fertilidade/efeitos dos fármacos , Ovário/fisiologia , Transdução de Sinais , Proteínas ras/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Hormônio Antimülleriano/sangue , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Análise por Conglomerados , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Hormônio Foliculoestimulante/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Anotação de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Theranostics ; 11(11): 5197-5213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859742

RESUMO

Rationale: Zearalenone (ZEN), a pollutant in our daily diet, seriously threatens the reproductive health of humans and animals. The primordial follicle (PF) assembly in the mouse occurs during the perinatal period, which determines the whole ovarian reserve in reproductive lifespan. In the current investigation, we administered ZEN orally to perinatal mice from 16.5 days post coitum (dpc) to postnatal day 3 (PD3), and single-cell RNA sequencing (scRNA-seq) was performed on PD0 and PD3 ovarian tissues in the offspring to check ZEN toxic to primordial follicle formation at the single cell level. Methods: Ovarian tissues (in vivo) were examined by single cell RNA sequencing analysis, Immunostaining, and Western blotting. Ovarian tissues (in vitro) were examined by qRT-PCR, Immunostaining, and Western blotting. Results: We found that ZEN exposure altered the developmental trajectory of both germ cells and granulosa cells. Furthermore, after establishing the cell-cell communication network between germ cells and granulosa cells, we found that this was perturbed by ZEN exposure, especially during the Hippo signaling pathway. Conclusions: This study showed that ZEN affected the status of germ cells and granulosa cells through the Hippo signaling pathway and blocked the assembly of PFs. This research contributes to our deeper understanding of the mechanisms of toxicity in different cell types and the disruption of normal intercellular signaling by ZEN exposure.


Assuntos
Folículo Ovariano/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Comunicação Celular/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Anesth Analg ; 132(6): 1756-1767, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33857022

RESUMO

BACKGROUND: The transient receptor potential vanilloid subtype 3 (TRPV3) channel is activated by innocuous temperature and several chemical stimuli. It is proposed to be involved in pathological pain development and is therefore considered a potential target for treating pain. Local anesthetics have been used for patients with both acute and chronic pain. Although blockage of the voltage-gated sodium channel is the primary mechanism by which local anesthetics exert their effects, they cannot be explained by this mechanism alone, especially in pathologic states such as chronic pain. Indeed, the effects of local anesthetics on multiple targets involved in the pain pathway have been reported. It has also been suggested that modulating the function of transient receptor potential (TRP) channels (eg, TRPV1 and transient receptor potential ankyrin 1 [TRPA1]) is one of the mechanisms of action of local anesthetics. However, the effects of local anesthetics on TRPV3 have not been reported. METHODS: We expressed TRPV3 in Xenopus oocytes and investigated the effects of local anesthetics on 2-aminoethoxydiphenyl borate (2APB)-induced currents using 2-electrode voltage-clamp techniques. RESULTS: Clinically used local anesthetics inhibited the 2APB-activated currents from the TRPV3 channel in a concentration-dependent manner at pharmacologically relevant concentrations with half maximal inhibitory concentration (IC50) values of 2.5 (lidocaine), 1.4 (mepivacaine), 0.28 (ropivacaine), and 0.17 (bupivacaine) mmol/L, respectively. Conversely, these local anesthetics also directly induced currents at higher concentrations, although these currents were quite small compared to the 2APB-induced currents. We found that the inhibition of TRPV3 by lidocaine is noncompetitive and independent of intracellular signaling cascades. 2APB-induced TRPV3 currents were reduced by extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) but not by intracellular QX-314 nor benzocaine. Moreover, lidocaine showed a use-dependent block in TRPV3 inhibition. Finally, QX-314 appeared to slightly permeate the activated TRPV3 channel pore based on examination of oocytes coexpressing TRPV3 and a sodium channel. These results suggest that local anesthetics could inhibit TRPV3 channel function by extracellular interactions of their charged forms with the channel pore. CONCLUSIONS: Local anesthetics inhibited TRPV3 2APB-induced currents at pharmacologically relevant concentrations when TRPV3 was expressed in Xenopus oocytes. These effects seem to occur via an extracellular interaction between the charged form of the anesthetic with the TRPV3 channel pore. These results help to elucidate the mechanisms of action of local anesthetics.


Assuntos
Anestésicos Locais/administração & dosagem , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Relação Dose-Resposta a Droga , Feminino , Xenopus laevis
9.
Toxins (Basel) ; 13(4)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917490

RESUMO

Beauvericin (BEA) and deoxynivalenol are toxins produced by Fusarium species that can contaminate food and feed. The aim of this study was to assess the effects of these mycotoxins on the maturation of oocytes from gilts and sows. Furthermore, the antioxidant profiles in the oocytes' environment were assessed. Cumulus-oocyte-complexes (COCs) from gilts and sows were exposed to beauvericin (BEA) or deoxynivalenol (DON) and matured in vitro. As an extra control, these COCs were also exposed to reactive oxygen species (ROS). The maturation was mostly impaired when oocytes from gilts were exposed to 0.02 µmol/L DON. Oocytes from sows were able to mature even in the presence of 5 µmol/L BEA. However, the maturation rate of gilt oocytes was already impaired by 0.5 µmol/L BEA. It was observed that superoxide dismutase (SOD) and glutathione (GSH) levels in the follicular fluid (FF) of gilt oocytes was higher than that from sows. However, the expression of SOD1 and glutathione synthetase (GSS) was higher in the oocytes from sows than in those from gilts. Although DON and BEA impair cell development by diverse mechanisms, this redox imbalance may partially explain the vulnerability of gilt oocytes to these mycotoxins.


Assuntos
Células do Cúmulo/efeitos dos fármacos , Depsipeptídeos/toxicidade , Peróxido de Hidrogênio/metabolismo , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tricotecenos/toxicidade , Ração Animal/microbiologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Células do Cúmulo/metabolismo , Feminino , Microbiologia de Alimentos , Fusarium/metabolismo , Glutationa/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Oócitos/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Sus scrofa
10.
Oxid Med Cell Longev ; 2021: 5573357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33927796

RESUMO

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10-7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.


Assuntos
Metilação/efeitos dos fármacos , Niacinamida/metabolismo , Oócitos/efeitos dos fármacos , Apoptose , Feminino , Humanos , Melatonina , Transdução de Sinais
11.
Genes (Basel) ; 12(2)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672423

RESUMO

Bisphenol S (BPS) is used as an alternative plasticizer to Bisphenol A (BPA), despite limited knowledge of potential adverse effects. BPA exhibits endocrine disrupting effects during development. This article focuses on the impact of bisphenols during oocyte maturation. Connexins (Cx) are gap junctional proteins that may be affected by bisphenols, providing insight into their mechanism during development. Cxs 37 and 43 are crucial in facilitating cell communication between cumulus cells and oocytes. Cumulus-oocyte complexes (COCs), denuded oocytes, and cumulus cells were exposed to 0.05 mg/mL BPA or BPS for 24 h. Both compounds had no effect on Cx43. Cumulus cells exhibited a significant increase in Cx37 expression following BPA (p = 0.001) and BPS (p = 0.017) exposure. COCs treated with BPA had increased Cx37 protein expression, whilst BPS showed no effects, suggesting BPA and BPS act through different mechanisms. Experiments conducted in in vitro cultured cumulus cells, obtained by stripping germinal vesicle oocytes, showed significantly increased expression of Cx37 in BPA, but not the BPS, treated group. BPA significantly increased Cx37 protein expression, while BPS did not. Disrupted Cx37 following BPA exposure provides an indication of possible effects of bisphenols on connexins during the early stages of development.


Assuntos
Compostos Benzidrílicos/farmacologia , Conexina 43/genética , Conexinas/genética , Fenóis/farmacologia , Sulfonas/farmacologia , Animais , Compostos Benzidrílicos/efeitos adversos , Bovinos , Células do Cúmulo/efeitos dos fármacos , Disruptores Endócrinos/efeitos adversos , Disruptores Endócrinos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Fenóis/efeitos adversos , Plastificantes/efeitos adversos , Plastificantes/farmacologia , Sulfonas/efeitos adversos
12.
Toxicology ; 455: 152749, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33771660

RESUMO

Isoniazid (INH), a synthetic first-line tuberculosis antibiotic, has been widely used in clinical treatment. It has been reported to cause toxic effects at multiple tissue sites and also increases the incidence of adverse pregnancy outcomes; but the mechanism of action of INH on the reproductive system of female mammals remains unclear. Here, we demonstrate that oral INH (40 mg/kg/day every other day for 28 days) severely affects oocyte maturation and fertilization, late blastocyst development and fertility. We found that INH could disrupt standard spindle assembly, chromosome arrangement, and actin filament dynamics, which compromised meiotic progression of mouse oocytes. INH treatment increased the level of reactive oxygen species (ROS) and activated the oxidative stress response pathway, Keap1-Nrf2. It also caused apoptosis of oocytes and mitochondrial dysfunction. Our findings demonstrate that oral INH reduces fertility and damages the mammalian reproductive system by altering cytoskeletal dynamics and Juno expression, inducing oxidative stress and apoptosis, and activating the Keap1-Nrf2 signaling pathway in mouse oocytes.


Assuntos
Antituberculosos/toxicidade , Isoniazida/toxicidade , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Antituberculosos/administração & dosagem , Apoptose/efeitos dos fármacos , Feminino , Isoniazida/administração & dosagem , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator 2 Relacionado a NF-E2/metabolismo , Oócitos/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Cell Physiol Biochem ; 55(S3): 1-13, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33656308

RESUMO

BACKGROUND/AIMS: The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor. METHODS: The GluN2B affinity was recorded in radioligand receptor binding studies with the radioligand [3H]ifenprodil. The activity of the ligands was determined in two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNA encoding the GluN1-1a and GluN2B subunits of the NMDA receptor. Docking studies showed the crucial interactions with the NMDA receptor protein. RESULTS: The deconstruction approach showed that removal of the methyl moiety and the phenolic OH moiety in 7-positon resulted in almost the same GluN2B affinity as the parent 3-benzazepine. A considerably reduced GluN2B affinity was found for the 3-benzazepine without further substituents. However, removal of one or both OH moieties led to considerably reduced NMDA receptor inhibition. Introduction of a NO2 moiety or bioisosteric replacement of the phenol by a benzoxazolone resulted in comparable GluN2B affinity, but almost complete loss of inhibitory activity. An O-atom, a carbonyl moiety or a F-atom in the tetramethylene spacer led to 6-7-fold reduced ion channel inhibition. CONCLUSION: The results reveal an uncoupling of affinity and activity for the tested 3-benzazepines. Strong inhibition of [3H]ifenprodil binding by a test compound does not necessarily translate into strong inhibition of the ion flux through the NMDA receptor associated ion channel. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine- 1,7-diol (WMS-1410) shows high GluN2B affinity and strong inhibition of the ion channel. Deconstruction by removal of one or both OH moieties reduced the inhibitory activity proving the importance of the OH groups for ion channel blockade. Reconstruction by introduction of various structural elements into the left benzene ring or into the tetramethylene spacer reduced the NMDA receptor inhibition. It can be concluded that these modifications are not able to translate binding into inhibition.


Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Benzazepinas/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Antagonistas Adrenérgicos alfa/síntese química , Regulação Alostérica , Animais , Benzazepinas/síntese química , Benzoxazóis/química , Sítios de Ligação , Antagonistas de Aminoácidos Excitatórios/síntese química , Humanos , Cinética , Simulação de Acoplamento Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Piperidinas/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Trítio , Xenopus laevis
14.
Cell Physiol Biochem ; 55(S3): 46-64, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33667331

RESUMO

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.


Assuntos
Catequina/análogos & derivados , Canais de Potássio KCNQ/química , Canal de Potássio KCNQ1/química , Artérias Mesentéricas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Chá/química , Animais , Sítios de Ligação , Catequina/química , Catequina/farmacologia , Canais de Potássio KCNQ/agonistas , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/fisiologia , Leite/química , Simulação de Acoplamento Molecular , Miografia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Extratos Vegetais/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Xenopus laevis
15.
PLoS One ; 16(3): e0247518, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667248

RESUMO

This study aimed to evaluate the effect of scriptaid during pre-maturation (PIVM) and/or maturation (IVM) on developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were submitted to PIVM for 6 h in the presence or absence of scriptaid. COCs were distributed into five groups: T1-IVM for 22 h, T2-PIVM for 6 h and IVM for 22 h, T3-PIVM with scriptaid for 6 h and IVM for 22 h, T4-PIVM for 6 h and IVM with scriptaid for 22 h, and T5-PIVM with scriptaid for 6 h and IVM with scriptaid for 22 h. Nuclear maturation, gene expression, cumulus cells (CCs) expansion, and embryo development and quality were evaluated. At the end of maturation, all groups presented the majority of oocytes in MII (P>0.05). Only HAT1 gene was differentially expressed (P<0.01) in oocytes with different treatments. Regarding embryo development at D7, T4 (23%) and T5 (18%) had lower blastocyst rate (P<0.05) than the other treatments (T1 = 35%, T2 = 37% and T3 = 32%). No effect was observed when scriptaid in PIVM was used in less competent oocytes (P>0.05). In conclusion, presence of scriptaid in PIVM and/or IVM did not improve developmental competence or embryo quality.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Hidroxilaminas/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Blastocisto/metabolismo , Bovinos , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histona Acetiltransferases/genética , Meiose/efeitos dos fármacos , Oócitos/fisiologia
16.
FASEB J ; 35(4): e21449, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33724544

RESUMO

Decabromodiphenyl ethane (DBDPE) is a new brominated flame retardant and is widely added to flammable materials to prevent fire. Because it has been continuously detected in a variety of organisms and humans, it is important to reveal the biological toxicity of DBDPE. However, the influence of DBDPE for female reproduction is unclear. In this study, we investigated whether and how DBDPE exposure affects oocyte development. Female mice as a model were orally exposed to DBDPE by 0, 0.05, 0.5, 5, 50 µg/kg bw/day for 30 days (0.05 µg/kg bw/day is close to the environmental exposure concentration). We found that exposure of mice to DBDPE did not affect the first polar body extrusion (PBE) of oocytes. Strikingly, however, asymmetric division of oocytes was markedly impaired in 5 and 50 µg/kg bw/day DBDPE exposed group, which resulted in oocytes with larger polar bodies (PBs). Then, we further explored and found that DBDPE exposure inhibited the spindle migration and membrane protrusion in oocytes during anaphase of meiosis I (anaphase I), thereby impairing asymmetric division. Additionally, we found that DBDPE exposure suppressed the inactivation of cyclin-dependent kinase 1 (Cdk1), resulting in the decrease of cytoplasmic formin2 (FMN2)-mediated F-actin polymerization in oocytes at the onset of anaphase I. Simultaneously, DBDPE exposure damaged the structural integrity of the spindle and the perpendicular relationship between spindle and cortex. These together led to the failure of spindle migration and membrane protrusion required for oocytes asymmetric division. Finally, DBDPE exposure injured the development of blastocysts, leading to blastocyst apoptosis.


Assuntos
Bromobenzenos/toxicidade , Proteína Quinase CDC2/metabolismo , Retardadores de Chama/toxicidade , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Proteína Quinase CDC2/genética , Ciclo Celular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos
17.
FASEB J ; 35(4): e21280, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710673

RESUMO

Ethylene glycol butyl ether (EGBE) is a ubiquitous environmental pollutant that is commonly used in maquillage, industrial, and household products. EGBE has been shown to cause blood toxicity, carcinogenicity, and organ malformations. However, little is known about the impact of EGBE on the female reproductive system, especially oocyte quality. Here, we reported that EGBE influenced oocyte quality by showing the disturbed oocyte meiotic capacity, fertilization potential, and early embryonic development competency. Specifically, EGBE exposure impaired spindle/chromosome structure, microtubule stability, and actin polymerization to result in the oocyte maturation arrest and aneuploidy. In addition, EGBE exposure compromised the dynamics of cortical granules and their component ovastacin, leading to the failure of sperm binding and fertilization. Last, single-cell transcriptome analysis revealed that EGBE-induced oocyte deterioration was caused by mitochondrial dysfunction, which led to the accumulation of ROS and occurrence of apoptosis. Altogether, our study illustrates that mitochondrial dysfunction and redox perturbation is the major cause of the poor quality of oocytes exposed to EGBE.


Assuntos
Etilenoglicóis/toxicidade , Oócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Dano ao DNA , Desenvolvimento Embrionário/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Feminino , Meiose/efeitos dos fármacos , Camundongos , Organelas/efeitos dos fármacos , Organelas/fisiologia , Espécies Reativas de Oxigênio
18.
Aging (Albany NY) ; 13(6): 8849-8864, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742608

RESUMO

HDAC11, the sole member of HDAC class IV family, plays vital roles in activating mitosis and apoptosis of tumor cells, but its functions in meiosis are rarely investigated. In the present study, the effect of HDAC11 on meiosis during porcine oocytes maturation was fully studied. The results showed that HDAC11 inhibition by its specific inhibitor JB-3-22 dramatically decreased the porcine oocyte maturation rate by disturbing spindle organization and chromosomes alignment without affecting the cytoplasmic maturation. Further study indicated that HDAC11 inhibition significantly elevated the acetylation levels of α-tubulin and H4K16, which are crucial for spindle organization and chromosomes alignment. Moreover, immunofluorescence staining results showed that HDAC11 inhibition also disturbed other meiosis-related histone modifications, such as increased H3S10pho, H4K5ac and H4K12ac levels and reduced H3T3pho level. Furthermore, RNA-seq analysis results indicated that HDAC11 inhibition disturbed porcine oocytes transcriptome (157 up-regulation, 106 down-regulation). In addition, HDAC11 inhibition compromised oocytes quality and subsequent development after parthenogenetic activation, which may be caused by the aberrant nuclear maturation and transcriptome expression profile during oocytes maturation. Therefore, our results elucidate the function of HDAC11 in porcine oocytes maturation and embryos development through regulating α-tubulin acetylation, meiosis-related histone modifications and transcriptome.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Oócitos/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Animais , Feminino , Meiose/efeitos dos fármacos , Oócitos/metabolismo , Suínos , Transcriptoma , Regulação para Cima/efeitos dos fármacos
19.
J Med Chem ; 64(3): 1685-1700, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33523678

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are pharmacological targets for the treatment of neuropathic pain, and the α6ß4 subtype has been identified as particularly promising. Rat α6ß4 nAChRs are less sensitive to some ligands than the human homologue potentially complicating the use of rodent α6ß4 receptors for screening therapeutic compounds. We used molecular dynamics simulations coupled with functional assays to study the interaction between α-conotoxin PeIA and α6ß4 nAChRs and to identify key ligand-receptor interactions that contribute to species differences in α-conotoxin potency. Our results show that human and rat α6ß4 nAChRs have distinct ligand-binding motifs and show markedly different sensitivities to α-conotoxins. These studies facilitated the creation of PeIA-5667, a peptide that shows 270-fold higher potency for rat α6ß4 nAChRs over native PeIA and similar potency for the human homologue. Our results may inform the design of therapeutic ligands that target α6ß4 nAChRs for the treatment of neuropathic pain.


Assuntos
Antagonistas Nicotínicos/síntese química , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Conotoxinas/farmacologia , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Neuralgia/tratamento farmacológico , Oócitos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Ratos , Receptores Nicotínicos/química , Xenopus laevis
20.
J Steroid Biochem Mol Biol ; 209: 105826, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33581253

RESUMO

OBJECTIVES: Estrogen plays a critical role in the development and apoptosis of oocytes. Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions including the regulation of reproduction. This study aimed to determine the effect of autophagy regulated by the biologically active form of estrogen (17ß-estradiol) in porcine oocyte maturation in vitro. MATERIALS AND METHODS: We measured the effects of oocyte developmental competencies and autophagic activity in the porcine oocyte regulated by 17ß-estradiol using autophagic inhibitor (Autophinib). In addition, we studied the role of autophagy in reactive oxygen species (ROS) levels, mitochondrial distribution, Ca2+ production, mitochondrial membrane potential (ΔΨm), and early apoptosis by caspase-3, -8 activity in the mature oocytes. RESULTS: The results showed that the oocyte meiotic progression and early embryonic development were gradually decreased with Autophinib treatment, which was improved by 17ß-estradiol. Immunofluorescence experiments revealed that 17ß-estradiol primarily could promote the autophagy in the mature oocytes, and block the reduced-autophagic events by Autophinib. Moreover, 17ß-estradiol improved the Autophinib induced high ROS levels, abnormal mitochondrial distribution and low Ca2+ production in mature oocytes. Analyses of early apoptosis and ΔΨm showed that autophagy inhibition was accompanied by increased cellular apoptosis, and 17ß-estradiol reduced apoptosis rates of mature oocytes. Importantly, autophagy was downregulated by treatment with Autophinib, an activation of caspase-8 and cleaved caspase-3 increased. Those effects were abolished by 17ß-estradiol, which could upregulate autophagy. CONCLUSIONS: Our study have showed important implications that 17ß-estradiol could promote efficacy of the development of porcine oocytes, enhance the autophagy, reduce ROS levels and apoptosis activity in vitro maturation.


Assuntos
Apoptose , Autofagia , Estradiol/farmacologia , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia , Oogênese , Espécies Reativas de Oxigênio/metabolismo , Animais , Desenvolvimento Embrionário , Estrogênios/farmacologia , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Gravidez , Suínos
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