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1.
J Cell Mol Med ; 26(16): 4613-4623, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35866203

RESUMO

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti-inflammatory and anti-angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa-like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up-regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down-regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.


Assuntos
Síndrome de Hiperestimulação Ovariana , Serpinas , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Humanos , Camundongos , Síndrome de Hiperestimulação Ovariana/metabolismo , Síndrome de Hiperestimulação Ovariana/prevenção & controle , Gravidez , Serpinas/genética , Serpinas/metabolismo , Serpinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
J Biol Chem ; 296: 100464, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33639162

RESUMO

ß-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent ß-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, ß-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit ß-globin gene HBB2 with high efficiency and generated a ß-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of ß-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a ß-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of ß-thalassemia and associated complications.


Assuntos
Modelos Animais de Doenças , Globinas beta/genética , Talassemia beta/genética , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Coelhos , Globinas beta/metabolismo , Talassemia beta/metabolismo
3.
Biol Reprod ; 107(4): 1125-1138, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-35594452

RESUMO

In mammals, testis development is triggered by the expression of the sex-determining Y-chromosome gene SRY to commit the Sertoli cell (SC) fate at gonadal sex determination in the fetus. Several genes have been identified to be required to promote the testis pathway following SRY activation (i.e., SRY box 9 (SOX9)) in an embryo; however, it largely remains unknown about the genes and the mechanisms involved in stabilizing the testis pathway after birth and throughout adulthood. Herein, we report postnatal males with SC-specific deletion of Raptor demonstrated the absence of SC unique identity and adversely acquired granulosa cell-like characteristics, along with loss of tubular architecture and scattered distribution of SCs and germ cells. Subsequent genome-wide analysis by RNA sequencing revealed a profound decrease in the transcripts of testis genes (i.e., Sox9, Sox8, and anti-Mullerian hormone (Amh)) and, conversely, an increase in ovary genes (i.e., LIM/Homeobox gene 9 (Lhx9), Forkhead box L2 (Foxl2) and Follistatin (Fst)); these changes were further confirmed by immunofluorescence and quantitative reverse-transcription polymerase chain reaction. Importantly, co-immunofluorescence demonstrated that Raptor deficiency induced SCs dedifferentiation into a progenitor state; the Raptor-mutant gonads showed some ovarian somatic cell features, accompanied by enhanced female steroidogenesis and elevated estrogen levels, yet the zona pellucida 3 (ZP3)-positive terminally feminized oocytes were not observed. In vitro experiments with primary SCs suggested that Raptor is likely involved in the fibroblast growth factor 9 (FGF9)-induced formation of cell junctions among SCs. Our results established that Raptor is required to maintain SC identity, stabilize the male pathway, and promote testis development.


Assuntos
Aves Predatórias , Células de Sertoli , Animais , Hormônio Antimülleriano/genética , Estrogênios/metabolismo , Feminino , Fator 9 de Crescimento de Fibroblastos/genética , Folistatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Masculino , Mamíferos/genética , Camundongos , Aves Predatórias/genética , Aves Predatórias/metabolismo , Fatores de Transcrição SOX9/genética , Células de Sertoli/metabolismo , Processos de Determinação Sexual/genética , Testículo/metabolismo , Fatores de Transcrição/genética
4.
Mol Hum Reprod ; 27(3)2021 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-33599278

RESUMO

Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, eight-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.


Assuntos
Blastocisto/enzimologia , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/biossíntese , Zigoto/enzimologia , Blastocisto/patologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Indução Enzimática , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Metilação , Transcrição Gênica , Zigoto/patologia
5.
Biol Reprod ; 103(5): 1121-1131, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32744313

RESUMO

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.


Assuntos
Adenilato Quinase/genética , Metabolismo Energético/fisiologia , Infertilidade Masculina/genética , Motilidade dos Espermatozoides/genética , Adenilato Quinase/metabolismo , Animais , Epididimo/metabolismo , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteômica , Espermátides/metabolismo , Espermatozoides/metabolismo
6.
Arch Gynecol Obstet ; 301(1): 303-308, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31903500

RESUMO

PURPOSE: To identify the optimal time for the frozen embryo transfer (FET) after oocyte retrieval in freeze-all cycles. METHODS: A retrospective analysis of 977 patients was performed. Implantation, clinical pregnancy and live birth rates were analyzed. RESULTS: No significant difference was found between the first FET performed in the first menstrual cycle group and performed within the subsequent menstrual cycle group in terms of implantation, pregnancy and live birth rates. To rule out the effect of endometrial thickness, a hierarchical analysis was performed. There were no differences between groups for pregnancy, multiple pregnancy and live birth rates for all ranges of endometrial thickness. CONCLUSIONS: The first FET should be performed once the endometrial thickness has been prepared well rather than delaying until the subsequent menstrual cycles.


Assuntos
Criopreservação/métodos , Transferência Embrionária/métodos , Recuperação de Oócitos/métodos , Adulto , Feminino , Humanos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Fatores de Tempo
7.
J Biol Chem ; 290(10): 6387-96, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25564616

RESUMO

Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF.


Assuntos
Proteínas de Transporte/genética , Fertilidade/genética , Complexos Multiproteicos/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/genética , Proteínas de Transporte/metabolismo , Feminino , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/patologia , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/patologia , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais , Serina-Treonina Quinases TOR/genética
8.
J Assist Reprod Genet ; 33(5): 581-588, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27052831

RESUMO

PURPOSE: As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos. METHODS: 3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/µl) and guide RNA (gRNA) (50 ng/µl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/µl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/µl) and gRNA (50 ng/µl) and injected into the embryos. RESULTS: By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations. CONCLUSIONS: This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Doenças Genéticas Inatas/genética , Receptores CCR5/genética , Reparo do DNA por Junção de Extremidades , Resistência à Doença/genética , Embrião de Mamíferos , Desenvolvimento Embrionário , Fertilização , Edição de Genes/ética , Doenças Genéticas Inatas/prevenção & controle , Humanos , Análise de Sequência de DNA
9.
Front Endocrinol (Lausanne) ; 15: 1360499, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38455652

RESUMO

Introduction: Males with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive. Methods and results: Herein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis. Discussion: In summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.


Assuntos
Óxido Nítrico , Traumatismos da Medula Espinal , Camundongos , Masculino , Ratos , Humanos , Animais , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Testosterona/metabolismo , Macrófagos/metabolismo , Traumatismos da Medula Espinal/complicações
10.
Nat Cell Biol ; 26(2): 263-277, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38238450

RESUMO

Human in vitro fertilized embryos exhibit low developmental capabilities, and the mechanisms that underlie embryonic arrest remain unclear. Here using a single-cell multi-omics sequencing approach, we simultaneously analysed alterations in the transcriptome, chromatin accessibility and the DNA methylome in human embryonic arrest due to unexplained reasons. Arrested embryos displayed transcriptome disorders, including a distorted microtubule cytoskeleton, increased genomic instability and impaired glycolysis, which were coordinated with multiple epigenetic reprogramming defects. We identified Aurora A kinase (AURKA) repression as a cause of embryonic arrest. Mechanistically, arrested embryos induced through AURKA inhibition resembled the reprogramming abnormalities of natural embryonic arrest in terms of the transcriptome, the DNA methylome, chromatin accessibility and H3K4me3 modifications. Mitosis-independent sequential activation of the zygotic genome in arrested embryos showed that YY1 contributed to human major zygotic genome activation. Collectively, our study decodes the reprogramming abnormalities and mechanisms of human embryonic arrest and the key regulators of zygotic genome activation.


Assuntos
Aurora Quinase A , Multiômica , Humanos , Aurora Quinase A/genética , Blastocisto , Cromatina/genética , Citoesqueleto , Desenvolvimento Embrionário/genética
11.
Dev Cell ; 59(12): 1506-1522.e11, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38582082

RESUMO

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.


Assuntos
Diferenciação Celular , Proteínas de Homeodomínio , Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/citologia , Feminino , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Gravidez , Placenta/metabolismo , Placenta/citologia , Linhagem da Célula , Placentação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Sequências Reguladoras de Ácido Nucleico/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia
12.
J Hazard Mater ; 445: 130544, 2023 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-36493639

RESUMO

Polystyrene microplastics (PS-MPs) can threaten human health, especially male fertility. However, most existing studies have focused on the adulthood stage of male reproduction toxicity caused by relatively short-term PS-MP exposure. This study aimed to investigate the toxic effect of PS-MPs on testicular development and reproductive function upon prenatal and postnatal exposure. Pregnant mice and their offspring were exposed to 0, 0.5 mg/L, 5 mg/L, and 50 mg/L PS-MPs through their daily drinking water from gestational day 1 to postnatal day (PND) 35 or PND70. We found that PS-MP exposure induced testis development disorder by PND35 and spermatogenesis dysfunction by PND70. By combining RNA sequencing results and bioinformatics analysis, the hormone-mediated signaling pathway, G1/S transition of the mitotic cell cycle, coregulation of androgen receptor activity, and Hippo signaling pathway were shown to be involved in testis development on PND35. The meiotic cell cycle, regulation of the immune effector process, neutrophil degranulation, and inflammation mediated by chemokine and cytokine signaling pathways were associated with disturbed spermatogenesis on PND70. These findings show that prenatal and postnatal exposure to PS-MPs resulted in testis development disorder and male subfertility, which may be regulated by the Hippo signaling pathway and involve an immune reaction.


Assuntos
Poliestirenos , Doenças Testiculares , Gravidez , Feminino , Humanos , Criança , Camundongos , Masculino , Animais , Adulto , Poliestirenos/toxicidade , Microplásticos/toxicidade , Plásticos , Deficiências do Desenvolvimento , Fertilidade
14.
J Med Virol ; 83(1): 16-23, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21108335

RESUMO

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.


Assuntos
Infecções por HIV/transmissão , HIV-1/isolamento & purificação , Transmissão Vertical de Doenças Infecciosas , Óvulo/virologia , Espermatozoides/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Animais , Núcleo Celular/virologia , Cricetinae , Feminino , Técnica Direta de Fluorescência para Anticorpo , Proteína do Núcleo p24 do HIV/biossíntese , Infecções por HIV/virologia , HIV-1/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
15.
J Ovarian Res ; 14(1): 51, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789698

RESUMO

BACKGROUND: High-quality single blastocyst transfer (SBT) is increasingly recommended to patients because of its acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared to double blastocyst transfer (DBT). However, there is no consensus on whether this transfer strategy is also suitable for poor-quality blastocysts. Moreover, the effect of the development speed of poor-quality blastocysts on pregnancy outcomes has been controversial. Therefore, this study aimed to explore the effects of blastocyst development speed and morphology on pregnancy and neonatal outcomes during the frozen embryo transfer (FET) cycle of poor-quality blastocysts and to ultimately provide references for clinical transfer strategies. METHODS: A total of 2,038 FET cycles of poor-quality blastocysts from patients 40 years old or less were included from January 2014 to December 2019 and divided based on the blastocyst development speed and number of embryos transferred: the D5-SBT (n = 476), D5-DBT (n = 365), D6-SBT (n = 730), and D6-DBT (n = 467) groups. The SBT group was further divided based on embryo morphology: D5-AC/BC (n = 407), D5-CA/CB (n = 69), D6-AC/BC (n = 580), and D6-CA /CB (n = 150). RESULTS: When blastocysts reach the same development speed, the live birth and multiple pregnancy rates of DBT were significantly higher than those of SBT. Moreover, there was no statistical difference in the rates of early miscarriage and live birth between the AC/BC and CA/CB groups. When patients in the SBT group were stratified by blastocyst development speed, the rates of clinical pregnancy (42.44 % vs. 20.82 %) and live birth (32.35 % vs. 14.25 %) of D5-SBT group were significantly higher than those of D6-SBT group. Furthermore, for blastocysts in the same morphology group (AC/BC or CA/CA group), the rates of clinical pregnancy and live birth in the D5 group were also significantly higher than those of D6 group. CONCLUSIONS: For poor-quality D5 blastocysts, SBT can be recommended to patients because of acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared with DBT. For poor-quality D6, the DBT strategy is recommended to patients to improve pregnancy outcomes. When blastocysts reach the same development speed, the transfer strategy of selecting blastocyst with inner cell mass "C" or blastocyst with trophectoderm "C" does not affect the pregnancy and neonatal outcomes.


Assuntos
Blastocisto/metabolismo , Técnicas de Cultura Embrionária/métodos , Implantação do Embrião/imunologia , Adulto , Feminino , Humanos , Gravidez , Estudos Retrospectivos , Resultado do Tratamento , Vitrificação
16.
Cell Discov ; 7(1): 81, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34489415

RESUMO

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

17.
Aging (Albany NY) ; 13(2): 2727-2749, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33373318

RESUMO

Oxybenzone (OBZ), an ultraviolet light filter that is widely used in sunscreens and cosmetics, is an emerging contaminant found in humans and the environment. Recent studies have shown that OBZ has been detected in women's plasma, urine, and breast milk. However, the effects of OBZ exposure on oocyte meiosis have not been addressed. In this study, we investigated the detrimental effects of OBZ on oocyte maturation and the protective roles of melatonin (MT) in OBZ-exposed mouse models. Our in vitro and in vivo results showed that OBZ suppressed oocyte maturation, while MT attenuated the meiotic defects induced by OBZ. In addition, OBZ facilitated H3K4 demethylation by increasing the expression of the Kdm5 family of genes, elevating ROS levels, decreasing GSH, impairing mitochondrial quality, and disrupting spindle configuration in oocytes. However, MT treatment resulted in significant protection against OBZ-induced damage during oocyte maturation and improved oocyte quality. The mechanisms underlying the beneficial roles of MT involved reduction of oxidative stress, inhibition of apoptosis, restoration of abnormal spindle assembly and up-regulation of H3K4me3. Collectively, our results suggest that MT protects against defects induced by OBZ during mouse oocyte maturation in vitro and in vivo.


Assuntos
Antioxidantes/farmacologia , Benzofenonas/toxicidade , Meiose/efeitos dos fármacos , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Protetores Solares/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Desmetilação , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Peroxidase/genética , Histona Desmetilases/efeitos dos fármacos , Histona Desmetilases/genética , Histonas/efeitos dos fármacos , Histonas/metabolismo , Técnicas In Vitro , Camundongos , Oogênese/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fuso Acromático/efeitos dos fármacos
18.
Cell Death Differ ; 27(1): 227-241, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31114027

RESUMO

Stress granules (SGs) are discrete assemblies of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins (RBPs) mediate their condensation by recruiting populations of mRNPs. However, the cellular and molecular mechanisms underlying the role of ubiquitin-associated protein 2-like (UBAP2L) in the regulation of SG dynamics remain elusive. Here, we show that UBAP2L is required for both SG assembly and disassembly. UBAP2L overexpression nucleated SGs under stress-null conditions. The UBAP2L Arg-Gly-Gly (RGG) motif was required for SG competence, and mediated the recruitment of SG components, including mRNPs, RBPs, and ribosomal subunits. The domain of unknown function (DUF) of UBAP2L-mediated interaction with ras GTPase-activating protein-binding protein (G3BP)1/2, and its deletion caused the cytoplasmic-nuclear transport of UBAP2L and G3BP1/2, thereby compromising SG formation. The protein arginine methyltransferase PRMT1 asymmetrically dimethylated UBAP2L by targeting the RGG motif. Increased arginine methylation blocked, whereas its decrease enhanced UBAP2L interactions with SG components, ablating and promoting SG assembly, respectively. These results provide new insights into the mechanisms by which UBAP2L regulates SG dynamics and RNA metabolism.


Assuntos
Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Arginina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Células HEK293 , Humanos , Metilação , Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
19.
Hum Reprod ; 24(7): 1575-83, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19279032

RESUMO

BACKGROUND: Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function. METHODS: Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed. RESULTS: HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential. CONCLUSIONS: HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.


Assuntos
Antígenos de Superfície da Hepatite B/fisiologia , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Espermatozoides/metabolismo , Anticorpos Monoclonais/química , Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/metabolismo , Relação Dose-Resposta a Droga , Fertilização , Fetuínas , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Ligantes , Masculino , Potenciais da Membrana , Mitocôndrias/metabolismo , Espermatozoides/virologia , Fatores de Tempo , alfa-Fetoproteínas/metabolismo
20.
Int Immunopharmacol ; 71: 336-349, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30952098

RESUMO

OBJECTIVE: UCP2 is involved in the maintenance of mitochondrial function, immune response and regulation of oxidative stress under physiological or pathological conditions. The aim of this study was to investigate the effects of UCP2 on mitochondrial dysfunction, inflammation, and oxidative stress in septic acute kidney injury (AKI). METHODS: We established LPS-induced AKI model in mice and HK-2 cells. In vivo, the UCP2 inhibitor genipin was used to downregulate UCP2 in mouse kidneys. In vitro, UCP2 overexpression or knockdown was achieved by LV5-UCP2 or si-UCP2 transfection, respectively, to characterize the mechanisms of UCP2 in septic AKI. Indicators of renal injury, cell apoptosis, inflammation, oxidative stress, and mitochondrial dysfunction were assessed. RESULTS: Compared to the control group, LPS treatment increased UCP2 expression in vitro and in vivo. In vitro, UCP2 overexpression protected HK-2 cells from LPS-induced injury by suppression of apoptosis, inflammation, oxidative stress, MMP loss and ROS production, increase of ATP production and mtDNA content, and amelioration of damage to the mitochondrial ultrastructure. Additionally, inhibition of UCP2 expression by si-UCP2 resulted in decreased HK-2 cell resistance to LPS toxicity, as shown by increased apoptosis, inflammation, mitochondrial dysfunction and oxidative stress. In vivo, UCP2 downregulation aggravated the LPS-induced renal injury, inflammation, macrophages infiltration, mitochondrial dysfunction, and oxidative stress. CONCLUSION: UCP2 may protect LPS-induced AKI by ameliorating mitochondrial dysfunction, anti-inflammation, and antioxidative activities, ultimately inhibiting tubule epithelial cell apoptosis, and that increasing the UCP2 content in mitochondria constitutes a new therapeutic approach for septic AKI.


Assuntos
Injúria Renal Aguda/metabolismo , Túbulos Renais/patologia , Mitocôndrias/metabolismo , Sepse/metabolismo , Proteína Desacopladora 2/metabolismo , Urotélio/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Inflamação , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Estresse Oxidativo , RNA Interferente Pequeno/genética , Proteína Desacopladora 2/genética , Urotélio/patologia
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