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Objective: Nicotinamide mononucleotide (NMN) is extensively utilized as an anti-aging agent and possesses anti-inflammatory properties. Lipopolysaccharide (LPS) activates Toll-like receptor 4, a process modulated by intracellular signaling pathways such as the Wnt/ß-catenin pathway. This study investigated the impact of NMN on osteogenesis in the presence of LPS. Methods: To elucidate the role of NMN in osteogenesis in the context of Gram-negative bacterial infection after LPS treatment, we cultured a mouse pre-osteoblast cell line (MC3T3-E1) and subsequently incubated it with NMN and/or LPS. We then evaluated osteogenic activity by measuring alkaline phosphatase activity, assessing gene expression and protein levels, and performing Alizarin Red S staining and immunocytochemistry. Results: MC3T3-E1 cells underwent successful differentiation into osteoblasts following treatment with osteogenic induction medium. LPS diminished features related to osteogenic differentiation, which were subsequently partially reversed by treatment with NMN. The restorative effects of NMN on LPS-exposed MC3T3-E1 cells were further substantiated by elucidating the role of Wnt/ß-catenin signaling, as confirmed through immunocytochemistry. Conclusion: This study showed that infection with Gram-negative bacteria disrupted the osteogenic differentiation of MC3T3-E1 cells. This adverse effect was partially reversed by administering a high-dose of NMN. Drawing on these results, we propose that NMN could serve as a viable therapeutic strategy to preserve bone homeostasis in elderly and immunocompromised patients.
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OBJECTIVE: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. METHODS: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. RESULTS: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. CONCLUSION: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.
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Monospermy occurs in the process of normal fertilization where a single sperm fuses with the egg, resulting in the formation of a diploid zygote. During the process of fertilization, the sperm must penetrate the zona pellucida (ZP), the outer layer of the egg, to reach the egg's plasma membrane. Once a sperm binds to the ZP, it undergoes an acrosomal reaction, which involves the release of enzymes from the sperm's acrosome that help it to penetrate the ZP. Ovastacin is one of the enzymes that is involved in breaking down the ZP. Studies have shown that ovastacin is necessary for the breakdown of the ZP and for successful fertilization to occur. However, the activity of ovastacin is tightly regulated to ensure that only one sperm can fertilize the egg. One way in which ovastacin helps to prevent polyspermy (the fertilization of an egg by more than one sperm) is by rapidly degrading the ZP after a sperm has penetrated it. This makes it difficult for additional sperm to penetrate the ZP and fertilize the egg. Ovastacin is also thought to play a role in the block to polyspermy, a mechanism that prevents additional sperm from fusing with the egg's plasma membrane after fertilization has occurred. In summary, the role of ovastacin in monospermic fertilization is to help ensure that only one sperm can fertilize the egg, while preventing polyspermy and ensuring successful fertilization.
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The ultimate goal of human assisted reproductive technology is to achieve a healthy pregnancy and birth, ideally from the selection and transfer of a single competent embryo. Recently, techniques for efficiently evaluating the state and quality of preimplantation embryos using time-lapse imaging systems have been applied. Artificial intelligence programs based on deep learning technology and big data analysis of time-lapse monitoring system during in vitro culture of preimplantation embryos have also been rapidly developed. In addition, several molecular markers of the secretome have been successfully analyzed in spent embryo culture media, which could easily be obtained during in vitro embryo culture. It is also possible to analyze small amounts of cell-free nucleic acids, mitochondrial nucleic acids, miRNA, and long non-coding RNA derived from embryos using real-time polymerase chain reaction (PCR) or digital PCR, as well as next-generation sequencing. Various efforts are being made to use non-invasive evaluation of embryo quality (NiEEQ) to select the embryo with the best developmental competence. However, each NiEEQ method has some limitations that should be evaluated case by case. Therefore, an integrated analysis strategy fusing several NiEEQ methods should be urgently developed and confirmed by proper clinical trials.
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PURPOSE: In women, menopause manifests with a variety of symptoms related to sex-hormone deficiency. Supplementing steroid hormones with pharmacological drugs has been widely practiced. However, considering the possible complications associated with artificial hormone therapy, studies have been conducted to find an alternative to pharmacological hormone replacement therapy. Accordingly, this study aimed to evaluate the efficacy of tissue-based hormone replacement therapy (tHRT) for treating post-menopausal signs and symptoms. MATERIALS AND METHODS: CD-1 mice were ovariectomized, and the ovaries were cryopreserved. Following artificial induction of post-menopausal osteoporosis, cryopreserved ovaries were subcutaneously autografted, and indexes related to bone health were monitored for 12 weeks. Bone mineral density (BMD), bone mineral contents (BMC), total bone volume (BV), and body fat mass were measured by dual energy X-ray absorptiometry. Uterine atrophy was assessed histologically, and bone microstructures were imaged by micro-computed tomography analysis. RESULTS: Regardless of the number of grafted ovaries, the BMC, BMD, and BV values of mice that underwent ovary transplantation were better than those that did not undergo transplantation. The uteruses in these mice were thicker and heavier after auto-transplantation. Furthermore, the bone microstructure recovered after tHRT. CONCLUSION: Recovery of menopause-related bone loss and uterine atrophy was achieved through tHRT. Ovarian tissue cryopreservation and transplantation may be applicable not only in patients wanting to preserve fertility but also in sex hormone-deficient post-menopausal women.
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Terapia de Reposição Hormonal , Menopausa , Absorciometria de Fóton , Animais , Atrofia , Densidade Óssea , Terapia de Reposição de Estrogênios , Feminino , Hormônios , Humanos , Camundongos , Microtomografia por Raio-XRESUMO
OBJECTIVE: As a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte-specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age-dependent oocyte demise in mice. MATERIALS AND METHOD: Tsg101 floxed mice (Tsg101f/f ) were bred with Zp3cre transgenic mice to examine oocyte-specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101. RESULTS: The death of oocytes from Zp3cre /Tsg101f/f (Tsg101d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin-releasing hormone antagonist injections improved the survival rate of oocytes from 5-week-old Tsg101d/d mice. Maturation of oocytes from prepubertal Tsg101d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101d/d mice until 9 h. The depletion of tsg-101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity. CONCLUSIONS: Collectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.
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Complexos Endossomais de Distribuição Requeridos para Transporte , Oócitos , Animais , Proteínas de Ligação a DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Hormônio Liberador de Gonadotropina , Camundongos , Camundongos Transgênicos , Fatores de TranscriçãoRESUMO
Humanity is in the midst of the coronavirus disease 2019 (COVID-19) pandemic, and vaccines-including mRNA vaccines-have been developed at an unprecedented speed. It is necessary to develop guidelines for vaccination for people undergoing treatment with assisted reproductive technology (ART) and for pregnancy-related situations based on the extant laboratory and clinical data. COVID-19 vaccines do not appear to adversely affect gametes, embryos, or implantation; therefore, active vaccination is recommended for women or men who are preparing for ART. The use of intravenous immunoglobulin G (IVIG) for the treatment of immune-related infertility is unlikely to impact the effectiveness of the vaccines, so COVID-19 vaccines can be administered around ART cycles in which IVIG is scheduled. Pregnant women have been proven to be at risk of severe maternal and neonatal complications from COVID-19. It does not appear that COVID-19 vaccines harm pregnant women or fetuses; instead, they have been observed to deliver antibodies against severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) to the fetus. Accordingly, it is recommended that pregnant women receive COVID-19 vaccination. There is no rationale for adverse effects, or clinical cases of adverse reactions, in mothers or neonates after COVID-19 vaccination in lactating women. Instead, antibodies to SARS-CoV-2 can be delivered through breast milk. Therefore, breastfeeding mothers should consider vaccination. In summary, active administration of COVID-19 vaccines will help ensure the safe implementation of ART, pregnancy, and breastfeeding.
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Decorin (DCN) is a proteoglycan belonging to the small leucine-rich proteoglycan family. It is composed of a protein core containing leucine repeats with a glycosaminoglycan chain consisting of either chondroitin sulfate or dermatan sulfate. DCN is a structural component of connective tissues that can bind to type I collagen. It plays a role in the assembly of the extracellular matrix (ECM), and it is related to fibrillogenesis. It can interact with fibronectin, thrombospondin, complement component C1, transforming growth factor (TGF), and epidermal growth factor receptor. Normal DCN expression regulates a wide range of cellular processes, including proliferation, migration, apoptosis, and autophagy, through interactions with various molecules. However, its aberrant expression is associated with oocyte maturation, oocyte quality, and poor extravillous trophoblast invasion of the uterus, which underlies the occurrence of preeclampsia and intrauterine growth restriction. Spatiotemporal hormonal control of successful pregnancy should regulate the concentration and activity of specific proteins such as proteoglycan participating in the ECM remodeling of trophoblastic and uterine cells in fetal membranes and uterus. At the human feto-maternal interface, TGF-ß and DCN play crucial roles in the regulation of trophoblast invasion of the uterus. This review summarizes the role of the proteoglycan DCN as an important and multifunctional molecule in the physiological regulation of oocyte maturation and trophoblast migration. This review also shows that recombinant DCN proteins might be useful for substantiating diverse functions in both animal and in vitro models of oogenesis and implantation.
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Oxygen tension is one of the most critical factors for mammalian embryo development and its survival. The HIF protein is an essential transcription factor that activated under hypoxic conditions. In this study, we evaluated the effect of dynamic oxygen conditions on the expression of embryonic genes and translocation of hypoxia-inducible factor-1α (HIF-1α) in cultured mouse blastocysts. Two-pronuclear (2PN) zygotes harvested from ICR mice were subjected to either high oxygen (HO; 20%), low oxygen (LO; 5%), or dynamic oxygen (DO; 5% to 2%) conditions. In the DO group, PN zygotes were cultured in 5% O2 from days 1 to 3 and then in 2% O2 till day 5 after hCG injection. On day 5, the percentage of blastocysts in the cultured embryos from each group was estimated, and the embryos were also subjected to immunocytochemical and gene expression analysis. We found that the percentage of blastocysts was similar among the experimental groups; however, the percentage of hatching blastocysts in the DO and LO groups was significantly higher than that in the HO group. The total cell number of blastocysts in the DO group was significantly higher than that of both the HO and LO groups. Further, gene expression analysis revealed that the expression of genes related to the embryonic development was significantly higher in the DO group than that in the HO and LO groups. Interestingly, HIF-1α mRNA expression did not significantly differ; however, HIF-1α protein translocation from the cytoplasm to the nucleus was significantly higher in the DO group than in the HO and LO groups. Our study suggests that dynamic oxygen concentrations increase the developmental capacity in mouse preimplantation embryos through activation of the potent transcription factor HIF-1α.
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Blastocisto/citologia , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Contagem de Células , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos ICR , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Advanced maternal age (AMA) is known to be related to the decrease in the quality and quantity of oocytes. Oocyte vitrification is now considered an established assisted reproductive technology for fertility preservation. However, it remains unclear whether the oocytes in older women are more sensitive to various insults during vitrification. Thus, we evaluated whether AMA affects cellular and molecular features and developmental outcomes of oocytes after vitrification in mice. The oocytes were grouped as young fresh (YF), young vitrified/warmed (YV), aged fresh (AF), and aged vitrified/warmed (AV). The survival rate of AV oocytes was significantly lower than that of YV oocytes. The rates of fertilization, cleavage, and blastocyst formation of AV oocytes were significantly lower than those of other groups. AV oocytes were represented as aberrations in mitochondria distribution, microvacuole size, and autophagosome formation, leading to delayed embryo development in mice. This delay was associated with a reduced number of total cells and trophectoderm in the blastocyst developed from AV oocytes. Collectively, AMA exaggerates the vulnerability of oocytes to cryo-damage that occurs during vitrification in mice, suggesting that the current vitrification protocols optimized for oocytes from young females should be modified for oocytes from aged women.
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Blastocisto/metabolismo , Criopreservação , Desenvolvimento Embrionário , Idade Materna , Oócitos/metabolismo , Animais , Feminino , Fertilização in vitro , Masculino , CamundongosRESUMO
OBJECTIVE: Lipopolysaccharide (LPS) from Gram-negative bacteria causes poor uterine receptivity by inducing excessive inflammation at the maternal-fetal interface. This study aimed to investigate the detrimental effects of LPS on the attachment and outgrowth of various types of trophoblastic spheroids on endometrial epithelial cells (ECC-1 cells) in an in vitro model of implantation. METHODS: Three types of spheroids with JAr, JEG-3, and JAr mixed JEG-3 (JmJ) cells were used to evaluate the effect of LPS on early implantation events. ECC-1 cells were treated with LPS to mimic endometrial infection, and the expression of inflammatory cytokines and adhesion molecules was analyzed by quantitative real-time polymerase chain reaction and western blotting. The attachment rates and outgrowth areas were evaluated in the various trophoblastic spheroids and ECC-1 cells treated with LPS. RESULTS: LPS treatment significantly increased the mRNA expression of inflammatory cytokines (CXCL1, IL-8, and IL-33) and decreased the protein expression of adhesion molecules (ITGß3 and ITGß5) in ECC-1 cells. The attachment rates of JAr and JmJ spheroids on ECC-1 cells significantly decreased after treating the ECC-1 cells with 1 and 10 µg/mL LPS. In the outgrowth assay, JAr spheroids did not show any outgrowth areas. However, the outgrowth areas of JEG-3 spheroids were similar regardless of LPS treatment. LPS treatment of JmJ spheroids significantly decreased the outgrowth area after 72 hours of coincubation. CONCLUSION: An in vitro implantation model using novel JmJ spheroids was established, and the inhibitory effects of LPS on ECC-1 endometrial epithelial cells were confirmed in the early implantation process.
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BACKGROUND: Soluble proteins and extracellular vesicles (EVs) are crucial wound repair mediators in cell-based therapy. Previous studies reported that EVs of perivascular cells stimulated migration and proliferation of cell types involved in the dermatological wound healing process. However, these studies only show effects of EVs from perivascular cells (PVCs) for in vitro models. METHODS: EVs were collected from 3D-cultured PVC (PVC-3D-EV) and compared with EVs from 2D-culture PVC (PVC-2D-EV) to investigate effects on wound contraction, angiogenesis, activation of myofibroblast, and collagen deposition. RESULTS: PVC-3D-EV was significantly improved in terms of wound contraction compared with PVC-2D-EV and the control. Activation of myofibroblast and collagen deposition in a rat skin wound model was significantly stimulated by PVC-3D-EV. In addition, angiogenesis and vascular endothelial growth factor expression were also highly stimulated by PVC-3D-EV. These results suggest that PVC-3D-EV was regulated in granulation tissue formation, angiogenesis, and wound contraction in healing of a rat skin wound. These results indicate a pivotal role of PVC-3D-EV in wound healing through multiple mechanisms. CONCLUSIONS: 3D-culture using a polystyrene scaffold is demonstrated to be a better system for providing better physiological conditions than the 2D-culture system, and EVs from 3D-cultured PVC could be a promising option for healing skin wound. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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Vesículas Extracelulares , Fator A de Crescimento do Endotélio Vascular , Animais , Colágeno , Ratos , Pele , CicatrizaçãoRESUMO
The uterus undergoes vascular changes during the reproductive cycle and pregnancy. Steroid hormone deprivation induces macroautophagy/autophagy in major uterine cell types. Herein, we explored the functions of uterine autophagy using the Amhr2-Cre-driven atg7 deletion model. Deletion of Atg7 was confirmed by functional deficit of autophagy in uterine stromal, myometrial, and vascular smooth muscle cells, but not in endothelial cells. atg7d/d uteri exhibited enhanced stromal edema accompanied by dilation of blood vessels. Ovariectomized atg7d/d uteri showed decreased expression of endothelial junction-related proteins, such as CTNNB1/beta-catenin, with increased vascular permeability, and increased expression of VEGFA and NOS1. Nitric oxide (NO) was shown to mediate VEGFA-induced vascular permeability by targeting CTNNB1. NO involvement in maintaining endothelial junctional stability in atg7d/d uteri was confirmed by the reduction in extravasation following treatment with a NOS inhibitor. We also showed that atg7d/d uterine phenotype improved the fetal weight:placental weight ratio, which is one of the indicators of assessing the status of preeclampsia. We showed that autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through the deregulation of VEGFA, NOS1, and CTNNB1.Abbreviations: ACTA2: actin, alpha 2, smooth muscle, aortic; Amhr2: anti-Mullerian hormone type 2 receptor; ANGPT1: angiopoietin 1; ATG: autophagy-related; CDH5: cadherin 5; CLDN5: claudin 5; COL1A1: collagen, type I, alpha 1; CSPG4/NG2: chondroitin sulfate proteoglycan 4; CTNNB1: catenin (cadherin associated protein), beta 1; DES: desmin; EDN1: endothelin 1; EDNRB: endothelin receptor type B; F3: coagulation factor III; KDR/FLK1/VEGFR2: kinase insert domain protein receptor; LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCAM/CD146: melanoma cell adhesion molecule; MYL2: myosin, light polypeptide 2, regulatory, cardiac, slow; MYLK: myosin, light polypeptide kinase; NOS1/nNOS: nitric oxide synthase 1, neuronal; NOS2/iNOS: nitric oxide synthase 2, inducible; NOS3/eNOS: nitric oxide synthase 3, endothelial cell; OVX: ovariectomy; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; POSTN: periostin, osteoblast specific factor; SQSTM1: sequestosome 1; TEK/Tie2: TEK receptor tyrosine kinase; TJP1/ZO-1: tight junction protein 1; TUBB1, tubulin, beta 1 class VI; USC: uterine stromal cell; VEGFA: vascular endothelial growth factor A; VSMC: vascular smooth muscle cell.
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Autofagia , Permeabilidade Capilar , Óxido Nítrico Sintase Tipo I/metabolismo , Artéria Uterina/metabolismo , Útero/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismo , Animais , Microambiente Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Artéria Uterina/fisiologia , Útero/irrigação sanguínea , Útero/fisiologiaRESUMO
OBJECTIVE: We investigated the impact of tyrosine kinase inhibitor (imatinib or dasatinib) coadministration with cyclophosphamide (Cp) on preantral follicle development in an in vitro mouse model. METHODS: Seventy-three female BDF1 mice were allocated into four experimental groups: group A, saline; group B, Cp (25 mg/kg); group C, Cp (25 mg/kg) and imatinib (7.5 mg/kg); and group D, Cp (25 mg/kg) and dasatinib (7.5 mg/kg). Preantral follicles were isolated and cultured in vitro up to 12 days. Final oocyte acquisition and spindle integrity of metaphase II (MII) oocytes were assessed. Levels of 17ß-estradiol and anti-Müllerian hormone (AMH) in the final spent media were measured by enzyme-linked immunosorbent assays, and the mRNA levels of Star, Sod1, Mapk3, and Casp3 in the final follicular cells were quantified by real-time polymerase chain reaction. RESULTS: The percentage of MII oocytes per initiated follicle, the proportion of MII oocytes with normal spindles, and the 17ß-estradiol level were similar in all four groups. The median AMH level in group B (7.74 ng/mL) was significantly lower than that in group A (10.84 ng/mL). However, the median AMH levels in group C (9.96 ng/mL) and group D (9.71 ng/mL) were similar to that in group A. The mRNA expression levels of Star, Sod1, Mapk3, and Casp3 were similar in all four groups. CONCLUSION: Coadministration of imatinib or dasatinib with Cp could preserve AMH production capacity in this in vitro mice preantral follicle culture model, and it did not affect MII oocyte acquisition.
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The mammalian zygote cleaves and develops to blastocyst within the zona pellucida (ZP) in vivo. The presence or absence of ZP may affect the characteristics of the embryo, including blastomere alignment, cell-cell junction, and compaction. This study aimed to compare the morphokinetic characteristics of ZP intact and ZP free mouse pre-implantation embryos with time-lapse monitoring system. Mouse 2-cell embryos were collected 1.5 days post coitum (dpc), and their ZPs were removed by treatment with acid Tyrode's solution. All embryos were cultured in vitro up to the outgrowth stage at 7.5 dpc. In this study, ZP did not influence the cumulative times from 2-cell to further stages and blastulation. Interestingly, ZP free embryos at 4-cell stage have three patterns of blastomere alignment according to the number of contact points between blastomeres. However, blastomere alignment did not lead to any differences in morphokinetic comparisons. Regardless of the presence or absence of ZP, embryos compacted after the 8-cell stage took shorter time to become blastocysts than embryos compacted pre-8-cell stage. Nevertheless, cell-cell junction proteins required for successful compaction were similarly expressed between ZP intact and ZP free embryos. ZP intact embryos compacted post-8-cell stage had higher rate of reaching blastocysts than compacted ZP intact embryos before 8-cell stage while the outgrowth/blastocyst rate was similar. In this study, the presence or absence of ZP did not influence embryonic development and expression of cell surface glycoproteins, whereas compaction timing may be one of the criteria for evaluating embryo quality. ZP free embryos may become an alternative for overcoming cases with ZP problems in a human ART program.
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Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos , Zona Pelúcida/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Caderinas/metabolismo , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos Endogâmicos ICR , beta Catenina/metabolismoRESUMO
The implantation process is highly complex and difficult to mimic in vitro, and a reliable experimental model of implantation has yet to be established. Many researchers have used embryo transfer (ET) to assess implantation potential; however, ET with pseudopregnant mice requires expert surgical skills and numerous sacrificial animals. To overcome those economic and ethical problems, several researchers have tried to use outgrowth models to evaluate the implantation potential of embryos. Many previous studies, as well as our experiments, have found significant correlations between blastocyst outgrowth in vitro and implantation in utero by ET. This review proposes the blastocyst outgrowth model as a possible alternative to animal experimentation involving ET in utero. In particular, the outgrowth model might be a cost- and time-effective alternative method to ET for evaluating the effectiveness of culture conditions or treatments. An advanced outgrowth model and further culture of outgrowth embryos could provide a subtle research model of peri- and postimplantation development, excluding maternal effects, and thereby could facilitate progress in assisted reproductive technologies. Recently, we found that outgrowth embryos secreted extracellular vesicles containing specific microRNAs. The function of microRNAs from outgrowth embryos should be elucidated in further researches.
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BACKGROUND/AIMS: Stress is closely related to the deterioration of digestive disease. Melatonin has potent anti-inflammatory properties. The objective of this study was to determine the effect of water stress (WS) and sleep deprivation (SD) on intestinal microbiota and roles of melatonin in stressful condition. METHODS: We used C57BL/6 mice and specially designed water bath for stress and SD for 10 days. We measured melatonin concentrations in serum, feces, and colon tissues by high-performance liquid chromatography. Genomic DNA was extracted from feces and amplified using primers targeting V3 to V4 regions of bacterial 16S ribosomal RNA genes. RESULTS: Compared to the control, melatonin concentration was lower in the WS and SD. Fecal concentration was 0.132 pg/mL in control, 0.062 pg/mL in WS, and 0.068 pg/mL in SD. In colon tissue, it was 0.45 pg/mL in control, 0.007 pg/mL in WS, and 0.03 pg/mL in SD. After melatonin treatment, melatonin concentrations in feces and colon tissue were recovered to the level of control. Metagenomic analysis of microbiota showed abundance in colitogenic microbiota in WS and SD. Melatonin injection attenuated this harmful effect. WS and SD showed decreased Lactobacillales and increased Erysipelotrichales and Enterobacteriales. Melatonin treatment increased Akkermansia muciniphila and Lactobacillus and decreased Bacteroides massiliensis and Erysipelotrichaceae. CONCLUSIONS: This study showed that stress and SD could affect intestinal dysbiosis and increase colitogenic microbiota, which could contribute to the aggravating digestive disease. Melatonin concentrations in feces and colon tissue decreased under WS and SD. Melatonin treatment brought recovery of melatonin concentration in colon tissue and modulating dysbiosis of intestinal microbiota.
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Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50+/- heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.
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Doença de Charcot-Marie-Tooth , Animais , Doença de Charcot-Marie-Tooth/genética , Camundongos , Camundongos Knockout , Mutação , Nervos Periféricos , Sistema Nervoso PeriféricoRESUMO
OBJECTIVE: We aimed to evaluate the effects of different oxygen conditions (20% [high O2], 5% [low O2] and 5% decreased to 2% [dynamic O2]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. METHODS: The high-O2 and low-O2 groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O2 group, mouse embryos were cultured from the one-cell to the morula stage under 5% O2 for 3 days, followed by culture under 2% O2 to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. RESULTS: The blastocyst formation rate was significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group. The total cell number was significantly higher in the dynamic-O2 group than in the low-O2 and high-O2 groups. Additionally, the apoptotic index was significantly lower in the low-O2 and dynamic-O2 groups than in the high-O2 group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group. CONCLUSION: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.
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Recently, many studies have investigated the role of extracellular vesicles (EVs) on reproductive events, including embryo development and death, oviduct-embryo crosstalk, in vitro fertilization and others. The aim of this study was to demonstrate whether outgrowth embryo-derived EVs function as bioactive molecules and regulate mouse embryonic developmental competence in vitro and implantation potential in utero. The EVs from mouse outgrowth embryos on 7.5 days postcoitum were detected and selectively isolated to evaluate the embryotrophic functions on preimplantation embryos. Developmental outcomes such as the percentage of blastocyst formation, hatching, and trophoblastic outgrowth were assessed. Furthermore, the total cell number and apoptotic index of blastocysts, which were incubated with EVs during the culture period, were evaluated by fluorescence microscopy. Implantation potential in utero was investigated following embryo transfer. The EVs from outgrowth embryo-conditioned media have rounded membrane structures that range in diameter from 20 to 225 nm. Incubation with EVs improved preimplantation embryonic development by increasing cell proliferation and decreasing apoptosis in blastocysts. Moreover, the implantation rates following embryo transfer were significantly higher in EV-supplemented embryos compared with the control. Collectively, EVs from outgrowth embryo could enhance the embryonic developmental competence and even implantation potential in mice.